METHODS OF TREATING MUSCULAR DYSTROPHY

Information

  • Patent Application
  • 20190240195
  • Publication Number
    20190240195
  • Date Filed
    December 28, 2018
    6 years ago
  • Date Published
    August 08, 2019
    5 years ago
Abstract
Disclosed herein are α7β1 integrin modulatory agents and methods of using such to treat conditions associated with decreased α7β1 integrin expression or activity, including muscular dystrophy. In one example, methods for treating a subject with muscular dystrophy are disclosed. The methods include administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy. Also disclosed are methods of enhancing muscle regeneration, repair, or maintenance in a subject and methods of enhancing α7β1 integrin expression by use of the disclosed α7β1 integrin modulatory agents. Methods of prospectively preventing or reducing muscle injury or damage in a subject are also disclosed.
Description
FIELD

This disclosure relates to the field of muscular dystrophy and in particular, to compositions and methods for treating muscular dystrophy, such as Duchenne muscular dystrophy, Fukuyama congenital muscular dystrophy or merosin deficient congenital muscular dystrophy type 1A or 1D.


BACKGROUND

Mutations in the α7 integrin gene are responsible for congenital myopathy in man. The α7β1 integrin is also a major modifier of muscle disease progression in various genetic muscle diseases including various types of muscular dystrophy, such as Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) and merosin deficient congenital muscular dystrophy type 1A (MDC1A). However, transcriptional regulation of the α7 integrin gene, including such role in muscular dystrophy (e.g., DMD, FCMD and/or MDC1A), remains poorly understood.


Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disease and the most common form of muscular dystrophy. DMD affects 1 in 3500 live male births with patients suffering from chronic muscle degeneration and weakness. Clinical symptoms are first detected between the ages of 2 and 5 years and, by the time the patient is in their teens, the ability for independent ambulation is lost. Death typically occurs in the patient before they are 30 years old due to cardiopulmonary failure.


Fukuyama congenital muscular dystrophy (FCMD) and MDC1A are congenital muscular dystrophies that are heritable neuromuscular disorders. MDC1A is characterized by muscle weakness at birth or in infancy. Affected infants will present with poor muscle tone and few movements. The quality of life and life span of the child is affected through progressive muscle wasting, respiratory compromise, and spinal rigidity. MDC1A is the most common and severe form of congenital muscular dystrophy, accounting for 30-40% of all congenital muscular dystrophy (CMD) diagnosed cases. MDC1A is characterized by congenital hypotonia, distinct joint contractures, and a lack of independent ambulation. Feeding tube placement and positive pressure ventilation is often required for the respiratory problems that occur. Patients afflicted with MDC1A often die before they reach the age of ten years. FCMD is caused by mutations in the fukutin gene, located at human chromosome 9q31. The disease is inherited in an autosomal recessive manner. FCMD is a type of Limb-Girdle muscular dystrophy. Currently there is no cure for DMD, FCMD or MDC1A.


SUMMARY

The muscular dystrophies are a group of diverse, heritable neuromuscular disorders which represent a group of devastating neuromuscular diseases characterized by primary or secondary skeletal muscle involvement. Currently, there are no cures for such diseases.


Disclosed herein are α7β1 integrin expression modulatory agents and methods of using such to treat a condition associated with impaired α7 integrin expression, such as muscular dystrophy. In one embodiment, a method for treating a subject with muscular dystrophy is disclosed. The method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy (such as MDC1A, MDC1D, LGMD, DMD, FCMD or FHMD).


Also disclosed are methods of enhancing muscle regeneration, repair, or maintenance in a subject. In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject in need of muscle regeneration, repair or maintenance, wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject. In a specific embodiment, the present disclosure provides a method for increasing muscle regeneration in a subject. For example, geriatric subjects, subjects suffering from muscle disorders, and subjects suffering front muscle injury, including activity induced muscle injury, such as injury caused by exercise, may benefit from this embodiment.


In yet further embodiments of the disclosed method, the α7β1 integrin modulatory agent is administered in a preventative manner, such as to prevent or reduce muscular damage or injury (such as activity or exercise induced injury). For example, geriatric subjects, subjects prone to muscle damage, or subjects at risk for muscular injury, such as athletes, may be treated in order to eliminate or ameliorate muscular damage, injury, or disease.


Further disclosed are methods of enhancing α7β1 integrin expression. In some embodiments, the method includes contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof and increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression. The methods of the present disclosure can include administering the α7β1 integrin modulatory agent with one or more additional pharmacological substances, such as a therapeutic agent. In some aspects, the additional therapeutic agent enhances the therapeutic effect of the α7β1 integrin modulatory agent. In further aspects, the therapeutic agent provides independent therapeutic benefit for the condition being treated. In various examples, the additional therapeutic agent is a component of the extracellular matrix, such as an integrin, dystrophin, dystroglycan, utrophin, or a growth factor. In further examples, the therapeutic agent reduces or enhances expression of a substance that enhances the formation or maintenance of the extracellular matrix. In some examples, the therapeutic agent is an additional α7β1 integrin modulatory agent such as laminin-111, a laminin-111 fragment, valproic acid or a valproic acid analog.


In some examples, the α7β1 integrin modulatory agent is applied to a particular area of the subject to be treated. For example, the α7β1 integrin modulatory agent may be injected into a particular area to be treated, such as a muscle. In further examples, the α7β1 integrin modulatory agent is administered such that it is distributed to multiple areas of the subject, such as systemic administration or regional administration.


A α7β1 integrin modulatory agent, can be administered by any suitable method, such as topically, parenterally (such as intravenously or intraperitoneally), or orally. In a specific example, the α7β1 integrin modulatory agent is administered systemically, such as through parenteral administration, such as stomach injection or peritoneal injection.


Although the disclosed methods generally have been described with respect to muscle regeneration, the disclosed methods also may be used to enhance repair or maintenance, or prevent damage to, other tissues and organs. For example, the methods of the present disclosure can be used to treat symptoms of muscular dystrophy stemming from effects to cells or tissue other than skeletal muscle, such as impaired or altered brain function, smooth muscles, or cardiac muscles.


The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.


BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 is a digital image illustrating the results of quantitative real-time PCR used to assess Itga7, Itgb1, and Lama2 transcript levels in C2C12 myoblasts and myotubes treated for 24 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), 10 μM MLS001165937-01 (IED-937). Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD. * denotes a significant difference in relative transcript levels with ** p-value<0.01 and *** p<0.001.



FIG. 2 is a digital image of Western Blots and quantitative analysis of α7 Integrin and GAPDH protein levels in C2C12 myotubes treated for 48 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD. Bands were quantified using Image J software and then graphed as α7 Integrin protein levels relative to GAPDH protein levels. * denotes a significant difference in relative protein levels with ** p<0.01.



FIG. 3 is an image of results (fluorescence relative to DMSO at various concentrations of the agent) from a screen using particular embodiments of the disclosed α7β1 integrin modulatory agents.



FIG. 4A is an image of the relative fluorescence generated by various concentrations of beta-galactosidase from the FDG substrate (FIG. 4A). The effects of DMSO concentrations on α7+/LacZ myoblasts (FIG. 4B) and myotube (FIG. 4C) assays were determined to be unacceptably high at concentrations greater than 1%. (FIG. 4D) The α7+/LacZ myotube assay response with varying levels of FDG in the FDG Buffer,



FIGS. 5A and 5B are graphs illustrating dose-response curves for SU9516 on α7+/LacZ myoblasts (FIG. 5A) or myotubes (FIG. 5B). The non-conventional appearance of the curves is thought to be a mechanism of concentration dependent cellular toxicity.



FIG. 6 is an image of a table providing curve classification results from a primary myoblast screen disclosed herein.



FIGS. 7A-7F provide graphs of myotube dose-response and the corresponding chemical structures [SU9516 (FIG. 7A), MLS000532969 (FIG. 7B), MLS003126425 (FIG. 7C), MLS001060533 (FIG. 7D), MLS000683232 (FIG. 7E), and MLS000683234 (FIG. 7F)] for each compound examined wherein n=6-9 over multiple days for all points.



FIGS. 8A and 8B provide results obtained for C2C12 myotubes treated with DMSO (n=3) or 12 μM SU9516 (n=3), which were examined for α7 Integrin protein levels by western blotting (FIG. 8A) wherein GapDH protein levels were used as a loading normalization control and the α7 Integrin/GapDH levels were quantitated and graphed for both DMSO and SU9516 treatments (FIG. 8B).



FIG. 9 is an image of results obtained for various different compounds disclosed herein and their effects on α7 integrin levels in human DMD myotubes.



FIG. 10 is an image illustrating chemical structures and names, similarity score relative to Stryka-969, and ITGA7+/LacZ myotube dose-response for 4 compounds with similar structures to Stryka-969.



FIG. 11 is an image of a Western blot analysis for α7 Integrin and α-tubulin, quantitated and graphed wherein significance was determined by unpaired t-test with ** p<0.01 and *** p<0.001.



FIG. 12 provides an exemplary synthetic scheme for making specific α7β1 integrin modulatory agents disclosed herein.







DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
I. Overview of Several Embodiments

Disclosed herein are α7β1 integrin expression modulatory agents and methods of using such to treat a condition associated with impaired α7 integrin expression, such as muscular dystrophy.


In one embodiment, a method for treating a subject with muscular dystrophy is disclosed. The method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy.


In some embodiments, a method for treating a subject with muscular dystrophy, comprises administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent comprises a compound having a formula selected from any one of the following




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wherein R1, R2, R, and R3 each independently is as specified in Table 3, Table 4, Table 5, and/or Table 6. In some embodiments, a combination of any of these compounds, or any other α7β1 integrin modulatory agents disclosed herein may be used. The α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy. In some embodiments, the compound may be selected from any of those provided in any one of Tables 1-16 and 18.


In some embodiments, the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1A (MDC1A), merosin deficient congenital muscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).


In some particular embodiments, the muscular dystrophy is DMD, MDC1A or FCMD.


In one particular embodiment, the muscular dystrophy is DMD.


In some embodiments, the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.


In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.


In some embodiments, the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.


In some embodiments, the method further includes selecting a subject with muscular dystrophy.


In some embodiments, the selecting a subject with muscular dystrophy includes diagnosing the subject with muscular dystrophy prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.


In other embodiments, a method of enhancing muscle regeneration, repair, or maintenance in a subject is disclosed.


In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.


In some embodiments, the method includes administering the α7β1 modulatory agent prior to the subject experiencing muscle damage or disease.


In some embodiments, the method is a method of enhancing muscle maintenance in a subject.


In some embodiments, the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.


In some embodiments, the α7β1 integrin modulatory agent is administered to a subject at risk of acquiring a muscle disease or damage, such as an elderly subject.


In some embodiments, the method also includes selecting a subject in need of enhancing muscle regeneration, repair, or maintenance.


In some embodiments, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance includes diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.


In some embodiments, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired production of a component of α7β1 integrin prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.


In some embodiments, the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.


In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.


In some embodiments, the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.


In further embodiments, a method of prospectively preventing or reducing muscle injury or damage in a subject is disclosed.


In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby prospectively preventing or reducing muscle injury or damage in the subject.


In some embodiments, the subject is at risk of developing a muscle injury or damage.


In some embodiments, the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.


In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.


In some embodiments, the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.


In even further embodiments, a method of enhancing α7β1 integrin expression is provided.


In some embodiments, the method includes contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof and increases α7β1 integrin expression in the treated cell relative α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression.


In some embodiments, the cell is a muscle cell.


In some embodiments, the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.


II. Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.


It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.


Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).


Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.


A person of ordinary skill in the art would recognize that the above definitions are not intended to include impermissible substitution patterns (e,g., methyl substituted with 5 different groups, pentavalent carbon, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art.


All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All sequences provided in the disclosed Genbank Accession numbers are incorporated herein by reference as available on Aug. 11, 2011. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.


In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:


Administration: To provide or give a subject one or more agents, such as an agent that increases α7β1 expression and/or treats one or more symptoms associated with muscular dystrophy, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.


Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject with a muscular dystrophy).


In some examples, an agent can act directly or indirectly to alter the expression and/or activity of α7β1. In a particular example, a therapeutic agent significantly increases the expression and/or activity of α7β1 (which is a muscular dystrophy associated molecule) thereby treating one or more signs or symptoms associated with muscular dystrophy. An example of a therapeutic agent is one that can increase the expression and/or activity of the α7β1 gene or gene product, for example as measured by a clinical response (such as a decrease in one or more signs or symptoms associated with the muscular dystrophy, an improvement in muscular health, regeneration, repair or maintenance of a muscle cell or tissue). “Improving muscular health” refers to an improvement in muscular health compared with a preexisting state or compared with a state which would occur in the absence of treatment. For example, improving muscular health may include enhancing muscle regeneration, maintenance, or repair. Improving muscular health may also include prospectively treating a subject to prevent or reduce muscular damage or injury, “Regeneration” refers to the repair of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, following injury or damage to at least partially restore the muscle or tissue to a condition similar to which the cells or tissue existed before the injury or damage occurred. Regeneration also refers to facilitating repair of cells or tissue in a subject having a disease affecting such cells or tissue to eliminate or ameliorate the effects of the disease. In more specific examples, regeneration places the cells or tissue in the same condition or an improved physiological condition as before the injury or damage occurred or the condition which would exist in the absence of disease. “Maintenance” of cells or tissue, such as muscle cells or tissue organs) which includes muscle cells, refers to maintaining the cells or tissue in at least substantially the same physiological condition, such as maintaining such condition even in the presence of stimulus which would normally cause damage, injury, or disease. “Repair” of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, refers to the physiological process of healing damage to the cells or tissue following damage or other trauma.


A “pharmaceutical agent” is a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when administered to a subject, alone or in combination with another therapeutic agent(s) or pharmaceutically acceptable carriers. In a particular example, a pharmaceutical agent significantly increases the expression and/or activity of α7β1 thereby treating a condition or disease associated with decreased α7β1 expression/activity, such as muscular dystrophy.


Acyl: H—C(O)—, alkyl-C(O)—, substituted alkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substituted heteroaryl-C(O)—, heterocyclyl-C(O)—, and substituted heterocyclyl-C(O)—.


Acylamino: —NRaC(O)alkyl, —NRaC(O)substituted alkyl. —NRaC(O)cycloalkyl, —NRaC(O)substituted cycloalkyl —NRaC(O)cycloalkenyl, —NRaC(O)substituted cycloalkenyl, —NRaC(O)alkenyl, —NRaC(O)substituted alkenyl, —NRaC(O)alkynyl, —NRaC(O)substituted alkynyl, —NRaC(O)aryl, —NRaC(O)substituted aryl, —NRaC(O)heteroaryl, —NRaC(O)substituted heteroaryl, —NRaC(O)heterocyclyl, and —NRaC(O)substituted heterocyclyl, wherein Ra is selected from hydrogen, alkyl, aryl, and cycloalkyl.


Acyloxy: alkyl-C(O)O—, substituted alkyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—, heterocyclyl-C(O)O—, and substituted heterocyclyl-C(O)O—.


Acylalkyloxy: alkyl-C(O)alkylO—, substituted alkyl-C(O)alkylO—, aryl-C(O)alkylO—, substituted aryl-C(O)alkylO—, cycloalkyl-C(O)alkylO—, substituted cycloalkyl-C(O)alkylO—, heteroaryl-C(O)alkylO—, substituted heteroaryl-C(O)alkylO—, heterocyclyl-C(O)alkylO—, and substituted heterocyclyl-C(O)alkylO—.


Alkyl: A saturated or unsaturated monovalent hydrocarbon having a number of carbon atoms ranging from one to ten (e.g., C-10alkyl), which is derived from removing one hydrogen atom from one carbon atom of a parent compound (e.g., alkane, alkene, alkyne). An alkyl group may be branched or straight-chain.


Alkenyl: A unsaturated monovalent hydrocarbon having a number of carbon atoms ranging from one to ten (e.g., C2-10alkenyl), which has at least one carbon-carbon double bond and is derived from removing one hydrogen atom from one carbon atom of a parent alkene. An alkenyl group may be branched, straight-chain, cyclic, cis, or trans (e.g., E or Z).


Alkynyl: A unsaturated monovalent hydrocarbon having a number of carbon atoms ranging from one to ten (e.g., C2-10alkynyl), which has at least one carbon-carbon triple bond and is derived from removing one hydrogen atom from one carbon atom of a parent alkyne. An alkynyl group may be branched, straight-chain, or cyclic.


Alkoxy: —O-alkyl (e.g., methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy).


Alkylthio: —S-alkyl, wherein alkyl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-alkyl, or —S(O)2-alkyl.


Amino: —NH2.


Aminocarbonyl: —C(O)N(Rb)2, wherein each Rb independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each Rb may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both Rb are not both hydrogen.


Aminocarbonylalkyl: -alkylC(O)N(Rb)2, wherein each Rb independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each Rb may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both Rb are not both hydrogen.


Aminocarbonylamino: —NRaC(O)N(Rb)2, wherein Ra and each Rb are as defined herein.


Aminodicarbonylamino: —NRaC(O)C(O)N(Rb)2, wherein Ra and each Rb are as defined herein.


Aminocarbonyloxy: —O—C(O)N(Rb)2, wherein each Rb independently is as defined herein.


Aminosulfonyl: —SO2N(Rb)2, wherein each Rb independently is as defined herein.


Analog or Derivative: A compound which is sufficiently homologous to a compound such that it has a similar functional activity for a desired purpose as the original compound. Analogs or derivatives refers to a form of a substance, such as cholestan, which has at least one functional group altered, added, or removed, compared with the parent compound. In some examples, examples of an analog are provided in Tables 1-6, for example. “Functional group” refers to a radical, other than a hydrocarbon radical, that adds a physical or chemical property to a substance.


Aryl: a monovalent aromatic carbocyclic group of from 6 to 15 carbon atoms having a single ring (e,g., phenyl) or multiple condensed rings (e.g., naphthyl), which condensed rings may or may not be aromatic provided that the point of attachment is through an atom of the aromatic aryl group.


Aryloxy —O-aryl.


Arylthio —S-aryl, wherein aryl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-aryl, or —S(O)2-aryl.


Biological activity: The beneficial or adverse effects of an agent on living matter. When the agent is a complex chemical mixture, this activity is exerted by the substance's active ingredient or pharmacophore, but can be modified by the other constituents. Activity is generally dosage-dependent and it is not uncommon to have effects ranging from beneficial to adverse for one substance when going from low to high doses. In one example, the agent significantly increases the biological activity of α7β1 which reduces one or more signs or symptoms associated with the muscular dystrophy.


Contacting: Placement in direct physical association, including both a solid and liquid form. Contacting an agent with a cell can occur in vitro by adding the agent to isolated cells or in vivo by administering the agent to a subject.


Control: A sample or standard used for comparison with a test sample, such as a biological sample obtained from a patient (or plurality of patients) without a particular disease or condition, such as a muscular dystrophy. In some embodiments, the control is a sample obtained from a healthy patient (or plurality of patients) (also referred to herein as a “normal” control), such as a normal biological sample. In some embodiments, the control is a historical control or standard value (e.g., a previously tested control sample or group of samples that represent baseline or normal values (e.g., expression values), such as baseline or normal values of a particular gene such as a α7β1 gene, gene product in a subject without a muscular dystrophy). In some examples, the control is a standard value representing the average value (or average range of values) obtained from a plurality of patient samples (such as an average value or range of values of the gene or gene products, such as the α7β1 gene or gene products, in the subjects without a muscular dystrophy).


Carboxyl: —COON or salts thereof.


Carboxyester: —C(O)O-alkyl, —C(O)O— substituted alkyl, —C(O)O-aryl, —C(O)O-substituted —C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclyl, and —C(O)O-substituted heterocyclyl.


(Carboxyester)amino: —NRa—C(O)O-alkyl, —NRa—C(O)O— substituted alkyl, —NRa—C(O)O-aryl, —NRa—C(O)O-substituted aryl, —NRa—C(O)O-cycloalkyl, —NRa—C(O)O-substituted cycloalkyl, —NRa—C(O)O-heteroaryl, —NRa—C(O)O-substituted heteroaryl, —NRa—C(O)O-heterocyclyl, and —NRa—C(O)O-substituted heterocyclyl, wherein Ra is as recited herein.


(Carboxyester)oxy: —O—C(O)O-alkyl, —O—C(O)O— substituted alkyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substituted cycloalkyl, —O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclyl, and —O—C(O)O-substituted heterocyclyl.


Cyano: —CN.


Cycloalkyl: cyclic alkyl (or alkenyl, or alkynyl) groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems (e.g., cyclopropyl, cyclobutyl, etc.)


(Cycloalkyl)oxy: —O-cycloalkyl.


(Cycloalkyl)thio: —S-cycloalkyl. This term also encompasses oxidized forms of sulfur, such as —S(O)-cycloalkyl, or —S(O)2-cycloalkyl.


Decrease: To reduce the quality, amount, or strength of something. In one example, a therapy decreases one or more symptoms associated with the muscular dystrophy, for example as compared to the response in the absence of the therapy.


Diagnosis: The process of identifying a disease, such as muscular dystrophy, by its signs, symptoms and results of various tests. The conclusion reached through that process is also called “a diagnosis.” Forms of testing commonly performed include blood tests, medical imaging, urinalysis, and biopsy.


Effective amount: An amount of agent that is sufficient to generate a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. When administered to a subject, a dosage will generally be used that will achieve target tissue/cell concentrations. In some examples, an “effective amount” is one that treats one or more symptoms and/or underlying causes of any of a disorder or disease. In some examples, an “effective amount” is a therapeutically effective amount in which the agent alone with an additional therapeutic agent(s) (for example anti-pathogenic agents), induces the desired response such as treatment of a muscular dystrophy, such as DMD, FCMD or MDC1A.


In particular examples, it is an amount of an agent capable of increasing α7β1 gene expression or activity by least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the disease to a point beyond detection).


In some examples, an effective amount is an amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response.


In one example, a desired response is to increase the subject's survival time by slowing the progression of the disease, such as slowing the progression of muscular dystrophy. The disease does not need to he completely inhibited for the pharmaceutical preparation to be effective. For example, a pharmaceutical preparation can decrease the progression of the disease by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%. as compared to the progression typical in the absence of the pharmaceutical preparation.


In another or additional example, it is an amount sufficient to partially or completely alleviate symptoms of the muscular dystrophy within the subject. Treatment can involve only slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently.


Effective amounts of the agents described herein can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the muscular dystrophy in the subject or measuring the expression level of one or more molecules known to be associated with the muscular dystrophy. Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.


The disclosed therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount can be dependent on the source applied (for example a nucleic acid molecule isolated from a cellular extract versus a chemically synthesized and purified nucleic acid), the subject being treated, the severity and type of the condition being treated, and the manner of administration.


Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation. RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced. In an example, expression, such as expression of α7β1 , can be regulated to treat one or more signs or symptoms associated with muscular dystrophy.


The expression of a nucleic acid molecule can be altered relative to a normal (wild type) nucleic acid molecule. Alterations in gene expression, such as differential expression, include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression. Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein.


Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few (such as no more than 10-20) amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues (such as at least 20 residues), such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein compared to a control or standard amount; (5) expression of a decreased amount of the protein compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not he, or alternatively is not expressed when it nominally would be); (8) alteration in stability of a protein through increased longevity in the time that the protein remains localized in a cell; and (9) alteration of the localized (such as organ or tissue specific or subcellular localization) expression of the protein (such that the protein is not expressed where it would normally be expressed or is expressed where it normally would not be expressed), each compared to a control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1A) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.


Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.


Extracellular matrix: An extracellular structure of a tissue or a layer thereof, including the arrangement, composition, and forms of one or more matrix components, such as proteins, including structural proteins such as collagen and elastin, proteins such as fibronectin and laminins, and proteoglycans. The matrix may comprise fibrillic collagen, having a network of fibers. In some examples, the extracellular matrix is connected to cells through the costameric protein network.


Halogen or Halo: fluoro, chloro, bromo, and iodo.


Heteroaryl: an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), wherein the condensed rings may or may not be aromatic and/or contain a heteroatom, provided that the point of attachment is through an atom of the aromatic heteroaryl group. In one embodiment, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties.


Heteroaryloxy: —O-heteroaryl.


Heteroarylthio: —S-heteroaryl. This term also encompasses oxidized forms of sulfur, such as —S(O)-heteroaryl, or —S(O)2-heteoaryl.


Heterocyclyl: a saturated, unsaturated group, or combinations thereof, having a single ring or multiple condensed rings, including fused bridged and Spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 heteroatoms, selected from nitrogen, sulfur, or oxygen. These groups may be substituted with one or more of the substituents disclosed herein for substituted aryl and/or substituted alkyl. These groups encompass, for example, a saturated heterocyclyl fused with one or more aromatic hydrocarbons or heteroaryl groups.


Heterocyclyloxy: —O-heterocycyl.


Heterocyclylthio: —S-heterocycyl. This term also encompasses oxidized forms of sulfur, such as —S(O)-heterocyclyl, or —S(O)2-heterocyclyl.


Hydroxyl or Hydroxy: —OH.


Imino: —N═Rc wherein Rc may be selected from hydrogen, aminocarbonylalkyloxy, substituted aminocarbonylalkyloxy, aminocarbonylalkylamino, and substituted aminocarbonylalkylamino.


Increase: To enhance the quality, amount, or strength of something. In one example, an agent increases the activity or expression of α7β1, for example relative to an absence of the agent. In a particular example, an agent increases the activity or expression of α7β1 by at least 10%, at least 20%, at least 50%, or even at least 90%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Such increases can be measured using the methods disclosed herein.


In a particular example, a therapy increases (also known as up-regulates) the expression of α7β1, such as an increase of at least 10%, at least 20%, at least 50%, or even at least 90% in α7β1 expression, thereby treating/alleviating one or more signs or symptoms associated with muscular dystrophy. In some examples, an increase in expression refers to an increase in a α7β1 gene product. An α7β1 gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein.


Gene upregulation includes any detectable increase in the production of a α7β1 gene product. In certain examples, production of a α7β1 gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal cell). In one example, a control is a relative amount of α7 gene expression or protein expression in a biological sample taken from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1A. Such increases can be measured using the methods disclosed herein. For example, “detecting or measuring expression of α7β1” includes quantifying the amount of the gene, gene product or modulator thereof present in a sample. Quantification can be either numerical or relative. Detecting expression of the gene, gene product or modulators thereof can be achieved using any method known in the art or described herein, such as by measuring nucleic acids by PCR (such as RT-PCR) and proteins by ELISA. In primary embodiments, the change detected is an increase or decrease in expression as compared to a control, such as a reference value or a healthy control subject. In some examples, the detected increase or decrease is an increase or decrease of at least two-fold compared with the control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1A) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.


Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.


In other embodiments of the methods, the increase or decrease is of a diagnostically significant amount, which refers to a change of a sufficient magnitude to provide a statistical probability of the diagnosis.


The level of expression in either a qualitative or quantitative manner can detect nucleic acid or protein. Exemplary methods include microarray analysis, RT-PCR, Northern blot, Western blot, and mass spectrometry.


Inhibiting a disease or condition: A phrase referring to reducing the development of a disease or condition, for example, in a subject who is at risk for a disease or who has a particular disease. Particular methods of the present disclosure provide methods for inhibiting muscular dystrophy.


Integrin: A cell surface transmembrane glycoprotein receptor. Integrins are involved in many biological processes such as wound healing, blood clot formation, gene regulation, and immune responses. Integrins can regulate tissue specific cell adhesion molecules. Integrins are heterodimeric non-covalently associated glycoproteins composed of two subunits. The subunits, which are designated α and beta, have approximate molecular weights of 150-180 kilodaltons and 90-110 kilodaltons, respectively.


The α7β1 integrin is a major laminin receptor expressed in skeletal muscle. The α7β1 integrin plays a role in the development of neuromuscular and myotendinous junctions. In the adult, the α7β1 integrin is concentrated at junctional sites and found in extrajunctional regions where it mediates the adhesion of the muscle fibers to the extracellular matrix. Mice that lack the α7 chain develop muscular dystrophy that affects the myotendinous junctions. The absence of α7 integrin results in defective matrix deposition at the myotendinous junction. Loss of the α7 integrin in γ-sarcoglycan mice results in severe muscle pathology. Absence of the α7 integrin in mdx mice also results in severe muscular dystrophy, confirming that the α7β1 integrin serves as a major genetic modifier for Duchenne and other muscular dystrophies.


Mutations in the α7 gene are responsible for muscular dystrophy in humans. A screen of 117 muscle biopsies from patients with undefined muscle disease revealed 3 which lacked the α7 integrin chain and had reduced levels of β1D integrin chain. These patients exhibited delayed developmental milestones and impaired mobility consistent with the role for the α7β1 integrin in neuromuscular and myotendinous junction development and function.


Several lines of evidence suggest the α7 integrin may be important for muscle regeneration. For example, during embryonic development, the α7β1 integrin regulates myoblast migration to regions of myofiber formation. It has been found that MyoD (myogenic determination protein) transactivates α7 integrin gene expression in vitro, which would increase α7 integrin levels in activated satellite cells. Human, mouse and rat myoblast cell lines derived from satellite cells express high levels of α7 integrin. Elevated α7 integrin mRNA and protein are detected in the skeletal muscle of 5 week old mdx mice, which correlates with the period of maximum muscle degeneration and regeneration. In addition, the α7β1 integrin associates with muscle specific β1-integrin binding protein (MIBP), which regulates laminin deposition in C2C12 myoblasts. Laminin provides an environment that supports myoblast migration and proliferation. Finally, enhanced expression of the α7 integrin in dystrophic skeletal muscle results in increased numbers of satellite cells.


The sequences for α7β1 integrin subunits are publicly available on GenBank, see, for example Gene Accession No. NM_001144116 (human) and NM_008398.2 (mouse) for α7 integrin, and Gene Accession No. NM_002211 for β1 integrin (also known as CD29), each of which is herein incorporated by reference as available on Sep. 8, 2011. Exemplary α7β1 integrin modulatory agents are disclosed herein, such as a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.


A α7β1 integrin-associated condition is a condition associated with altered α7β1 integrin expression or activity, including muscular dystrophy, such as DMD, FCMD, LGMD, FHMD, Beckers muscular dystrophy and/or MDC1A.


Laminin: Any of the family of glycoproteins that are typically involved in the formation and maintenance of extracellular matrices. Laminin is a heterotrimers formed from an α chain, a β chain, and a γ chain. The various chains of a particular laminin can affect the properties of the molecule. In some aspects of the present disclosure, fragments, derivatives, or analogs of various laminins can be used, such as laminins having at least a portion at least substantially homologous to the laminin α1 chain. A “fragment of laminin,” as used herein, refers to a portion of a substance, such as laminin. A fragment may be, in some examples, a particular domain or chain of a protein. For example, particular embodiments of the present disclosure involve administering a fragment of laminin-1 corresponding to at least a portion of (or all of) the laminin al chain. Fragments may be synthetic or may be derived from larger parent substances.


In some aspects, laminins may be administered as a mixture of laminins, including fragments, analogs, and derivatives thereof. Suitable methods for preparing analogs of laminin domains are disclosed in U.S. Pat. No. 6,933,280, incorporated by reference herein to the extent not inconsistent with this disclosure.


The laminin materials or compositions of the present disclosure may be delivered as discrete molecules or may be complexed with, or conjugated to, another substance. For example, the laminin may be combined with a carrier, such as to aid in delivery of the laminin to a site of interest or to increase physiological uptake or incorporation of the laminin.


In specific examples, the laminin administered includes or consists of laminin-1 (LAM-111), which includes the chains α1β1γ1. In further examples, the laminin administered includes or consists of laminin-2, which includes the chains α2β1γ1. In yet further examples, the laminin administered includes or consists of laminin-4, which includes the chains α2β2γ1.


Laminins may be obtained from any suitable source. For example, laminin-1 may be obtained from placental tissue or from Engelbreth-Holm-Swarm murine sarcoma. Suitable methods of isolating various laminins are disclosed in U.S. Pat. No. 5,444,158, incorporated by reference herein to the extent not inconsistent with the present disclosure.


Muscle: Any myoblast, myocyte, myofiber, myotube or other structure composed of muscle cells. Muscles or myocytes can be skeletal, smooth, or cardiac. Muscle may also refer to, in particular implementations of the present disclosure, cells or other materials capable of forming myocytes, such as stem cells and satellite cells.


Muscular dystrophy: A term used to refer to a group of genetic disorders that lead to progressive muscle weakness. Muscular dystrophy can result in skeletal muscle weakness and defects in skeletal muscle proteins, leading to a variety of impaired physiological functions. No satisfactory treatment of muscular dystrophy exists. Existing treatments typically focus on ameliorating the effects of the disease and improving the patient's quality of life, such as through physical therapy or through the provision of orthopedic devices.


Mutated genes associated with muscular dystrophy are responsible for encoding a number of proteins associated with the costameric protein network. Such proteins include laminin-2, collagen, dystroglycan, integrins, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin, plectin, BPAG1b, muscle LIM protein, desmin, actinin-associated LIM protein, α-actin, titin, telethonin, cypher, myotilin, and the sarcoglycan/sarcospan complex.


The most common form of muscular dystrophy is DMD, affecting 1 in 3,500 live male births. DMD is an X-linked recessive disorder characterized by a mutation in the gene that codes for dystrophin. Dystrophin is a cytoskeletal protein about 430 kDa in size. This protein works to connect the cell's cytoskeleton and extracellular matrix. The loss of dystrophin in DMD patients leads to a loss of muscle fiber attachment at the extracellular matrix during contraction, which ultimately leads to progressive fiber damage, membrane leakage and a loss of muscle function. Most patients die before they reach the age of 30 due to respiratory or cardiac failure.


Beckers muscular dystrophy (also known as Benign pseudohypertrophic muscular dystrophy) is related to DMD in that both result from a mutation in the dystrophin gene, but in DMD no functional dystrophin is produced making DMD much more severe than BMD. BMD is an X-linked recessive inherited disorder characterized by slowly progressive muscle weakness of the legs and pelvis. BMD is a type of dystrophinopathy, which includes a spectrum of muscle diseases in which there is insufficient dystrophin produced in the muscle cells, results in instability in the structure of muscle cell membrane. This is caused by mutations in the dystrophin gene, which encodes the protein dystrophin. The pattern of symptom development of BMD is similar to DMD, but with a later, and much slower rate of progression.


Congenital muscular dystrophies are caused by gene mutations, FCMD and MDC1A are examples of congenital muscular dystrophies. MDC1A is a congential muscular dystrophy due to a genetic mutation in the LAMA2 gene which results in lack of or complete loss of laminin-α2 protein. This loss of laminin-α2 leads to an absence of laminins-211/221. Laminins-211/221 are major components of the extracellular matrix and play a key role in muscle cell development. During muscle cell differentiation laminin binds to the α7β1 integrin. Without laminin-α2, muscle fibers are unable to adhere to the basement membrane and myotubes undergo apotosis. Muscle regeneration also fails, leading to a loss of muscle repair and an increase in muscle fibrosis and inflammation. This chronic tissue injury is a major cause of morbidity and mortality in MDC1A.


Congenital Muscular Dystrophies (CMD) and Limb-Girdle muscular dystrophy (LGMD) are common forms of highly heterogeneous muscular dystrophies which can be distinguished by their age at onset. In CMD, onset of symptoms is at birth or within the first 6 months of life; in LGMD onset of symptoms is in late childhood, adolescence or even adult life. Inheritance in LGMD can be autosomal dominant (LGMD type 1) or autosomal recessive (LGMD type 2), CMD is recessively inherited. CMD and LGMD can overlap both clinically and genetically


MDC1A is a progressive muscle wasting disease that results in children being confirmed to a wheelchair, requiring ventilator assistance to breathe and premature death. Symptoms are detected at birth with poor muscle tone and “floppy” baby syndrome. DMD, BMD and LGMD are progressive muscle degenerative diseases usually diagnosed at 3-5 years of age when children show developmental delay including ability to walk and climb stairs. The disease is progressive and children are usually confined to a wheelchair in their teens and require ventilator assistance.


Fukuyama congenital muscular dystrophy (FCMD) is an inherited condition that predominantly affects the muscles, brain, and eyes. Congenital muscular dystrophies are a group of genetic conditions that cause muscle weakness and wasting (atrophy) beginning very early in life. Fukuyama congenital muscular dystrophy affects the skeletal muscles, which are muscles the body uses for movement. The first signs of the disorder appear in early infancy and include a weak cry, poor feeding, and weak muscle tone (hypotonia). Weakness of the facial muscles often leads to a distinctive facial appearance including droopy eyelids (ptosis) and an open mouth. In childhood, muscle weakness and joint deformities (contractures) restrict movement and interfere with the development of motor skills such as sitting, standing, and walking. Fukuyama congenital muscular dystrophy also impairs brain development. People with this condition have a brain abnormality called cobblestone lissencephaly, in which the surface of the brain develops a bumpy, irregular appearance (like that of cobblestones). These changes in the structure of the brain lead to significantly delayed development of speech and motor skills and moderate to severe intellectual disability. Social skills are less severely impaired. Most children with Fukuyama congenital muscular dystrophy are never able to stand or walk, although some can sit without support and slide across the floor in a seated position. More than half of all affected children also experience seizures. Other signs and symptoms of Fukuyama congenital muscular dystrophy include impaired vision, other eye abnormalities, and slowly progressive heart problems after age 10. As the disease progresses, affected people may develop swallowing difficulties that can lead to a bacterial lung infection called aspiration pneumonia. Because of the serious medical problems associated with Fukuyama congenital muscular dystrophy, most people with the disorder live only into late childhood or adolescence.


Fukuyama congenital muscular dystrophy is seen almost exclusively in Japan, where it is the second most common form of childhood muscular dystrophy (after Duchenne muscular dystrophy). Fukuyama congenital muscular dystrophy has an estimated incidence of 2 to 4 per 100,000 Japanese infants.


Fukuyama congenital muscular dystrophy is caused by mutations in the FKTN gene which encodes fukutin. The most common mutation in the FKTN gene reduces the amount of fukutin produced within cells. A shortage of fukutin likely prevents the normal modification of α-dystroglycan, which disrupts that protein's normal function. Without functional α-dystroglycan to stabilize muscle cells, muscle fibers become damaged as they repeatedly contract and relax with use. The damaged fibers weaken and die over time, leading to progressive weakness and atrophy of the skeletal muscles.


Defective α-dystroglycan also affects the migration of neurons during the early development of the brain. Instead of stopping when they reach their intended destinations, some neurons migrate past the surface of the brain into the fluid-filled space that surrounds it. Because Fukuyama congenital muscular dystrophy involves a malfunction of α-dystroglycan, this condition is described as a dystroglycanopathy.


Facioscapulohumeral muscular dystrophy (FHMD) is a form of muscular dystrophy associated with progressive muscle weakness and loss of muscle tissue. Unlike DMD and BMD which mainly affect the lower body, FSHD affects the upper body mainly the face, shoulder and upper arm muscles. However, it can affect muscles around the pelvis, hips, and lower leg. Symptoms for FSHD often do not appear until age 10-26, but it is not uncommon for symptoms to appear much later. In some cases, symptoms never develop. Symptoms are usually mild and very slowly become worse. Facial muscle weakness is common, and may include eyelid drooping, inability to whistle, decreased facial expression, depressed or angry facial expression, difficulty pronouncing words, shoulder muscle weakness (leading to deformities such as pronounced shoulder blades (scapular winging) and sloping shoulders), weakness of the lower, hearing loss and possible heart conditions.


Oxo: (═O).


Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more 001 modulatory agents.


In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.


Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, and autopsy material. In one example, a sample includes muscle biopsy, such as from a subject with DMD, FCMD, or MDC1A.


Signs or symptoms: Any subjective evidence of disease or of a subject's condition, e.g., such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state. A “sign” is any abnormality indicative of disease, discoverable on examination or assessment of a subject. A sign is generally an objective indication of disease. Signs include, but are not limited to any measurable parameters such as tests for detecting muscular dystrophy, including measuring creatine kinase levels, electromyography (to determine if weakness is caused by destruction of muscle tissue rather than by damage to nerves) or immunohistochemistry/immunoblotting/immunoassay (e.g., ELISA) to measure muscular dystrophy-associated molecules, such as α7β1 integrin. In one example, reducing or inhibiting one or more symptoms or signs associated with muscular dystrophy, includes increasing the activity or expression of α7β1 integrin by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to the activity and/or expression in the absence of the treatment. Symptoms of muscular dystrophy include, but are not limited to, muscle weakness and loss, difficulty running, difficulty hopping, difficulty jumping, difficulty walking, difficulty breathing, fatigue, skeletal deformities, muscle deformities (contractions of heels; pseudohypertrophy of calf muscles), heart disease (such as dilated cardiomyopathy), elevated creatine phosphokinase (CK) levels in blood or combinations thereof.


Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.


Substituted Alkyl: an alkyl (or alkenyl, or alkynyl) group having from 1 to 5 hydrogen atoms replaced with substituents selected alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, acylalkyloxy, amino, substituted amino, aminocarbonyl, aminocarbonylamnino, aminocarbonyloxy, aminodicarbonylamino, aminocarbonylalkyl, aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, aminodiacylamino, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted hetemcyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, imino, oxo, sulfonylamino, nitro, SO3H, sulfonyl, thiol, imino, substituted imino, alkylthio, and substituted alkylthio. The alkyl may be substituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which are defined herein.


Substituted Alkoxy: —O-(substituted alkyl).


Substituted Alkylthio: —S-(substituted alkyl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted alkyl, or —S(O)2-substituted alkyl.


Substituted Amino: —N(Rb)2, wherein each Rb independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each Rb may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both Rb are not both hydrogen.


Substituted Aryl: aryl groups having 1 to 5 hydrogens replaced with substituents independently selected from alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, sulfonyl, thiol, alkylthio, and substituted alkylthio. The amyl group may be substituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which are defined herein.


Substituted Aryloxy: —O-(substituted aryl).


Substituted Arylthio: —S-(substituted aryl), wherein substituted aryl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted aryl, or —S(O)2-substituted aryl.


Substituted Cycloalkyl: cycloalkyl, cycloalkenyl, or cycloalkynyl group having from 1 to 5 substituents selected from the group consisting of oxo, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, sulfonyl, thiol, alkylthio, and substituted alkylthio. The aryl group may be substituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which are defined herein. In some embodiments, the cycloalkyl group may have multiple condensed rings (e.g. tetrahydronaphthyl or tetrahydroanthacenyl), provided that the point of attachment is through an atom of the nonaromatic ring.


Substituted (Cycloalkyl)oxy: —O-(substituted cycloalkyl).


Substituted (Cycloalkyl)thio: refers to —S-(substituted cycloalkyl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted cycloalkyl, or —S(O)2-substituted cycloalkyl.


Substituted Heteroaryl: heteroaryl groups that are substituted with from 1 to 5 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.


Substituted Heteroaryloxy: —O-(substituted heteroaryl).


Substituted Heteroarylthio: —S-(substituted heteroaryl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted heteroaryl, or —S(O)2-substituted heteoaryl.


Substituted Heterocycyloxy: —O-(substituted heterocyclyl) wherein the heterocyclyl group is substituted with one or more of the substituents recited for substituted alkyl.


Substituted Heterocycylthio: —S-(substituted heterocycyl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted heterocyclyl, or —S(O)2-substituted heterocyclyl.


Sulfonyl: —SO2-alkyl, —SO2-substituted alkyl, —SO2-cycloalkyl, —SO2-substituted cycloalkyl, —SO2-aryl, —SO2-substituted aryl, —SO2-heteroaryl, —SO2-substituted heteroaryl, —SO2-hetemcyclyl, and —SO2-substituted heterocyclyl.


Sulfonylamino: —NR1SO2alkyl, —NR1SO2substituted alkyl, —NR1SO2cycloalkyl, —NR1SO2substituted cycloalkyl, —NR1SO2aryl, —NR1SO2substituted aryl, —NR1SO2heteroaryl, —NR1SO2substituted heteroaryl, ——NR1SO2heterocyclyl, —NR1SO2substituted heterocyclyl, wherein each Ra independently is as defined herein.


Thiol: —SH.


Thiocarbonyl: (═S)


Tissue: An aggregate of cells, usually of a particular kind, together with their intercellular substance that form one of the structural materials of an animal and that in animals include connective tissue, epithelium, muscle tissue, and nerve tissue.


Treating a disease: A therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to a muscular dystrophy, such as a sign or symptom of muscular dystrophy. Treatment can induce remission or cure of a condition or slow progression, for example, in sonic instances can include inhibiting the fill development of a disease, for example preventing development of a muscular dystrophy. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.


Treating a disease can be a reduction in severity of sonic or all clinical symptoms of the disease or condition, a reduction in the number of relapses of the disease or condition, an improvement in the overall health or well-being of the subject, by other parameters well known in the art that are specific to the particular disease or condition, and combinations of such factors.


Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity. In one example, includes administering a disclosed agent to a subject sufficient to allow the desired activity. In particular examples, the desired activity is increasing the expression or activity of α7β1.


III. Compounds for Treating Muscular Dystrophy

Disclosed herein are compounds that may be used as α7β1 integrin modulatory agents in methods disclosed herein. In particular disclosed embodiments, the compound is effective in treating muscular dystrophy. The compound is a small-molecule therapeutic. In particular disclosed embodiments, the small-molecule therapeutic is a cyclic compound comprising a heteroatom-containing skeleton. In other disclosed embodiments, the small-molecule therapeutic is a cyclic compound comprising an all-carbon skeleton. In certain disclosed embodiments, the cyclic compound comprising a heteroatom-containing skeleton has a formula illustrated below:




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wherein each R1 independently is selected from C1-10alkyl, substituted C1-10alkyl, C1-10alkoxy, substituted C1-10alkoxy, acyl, acylamino, acyloxy, acylC1-10alkyloxy, amino, substituted amino, aminoacyl, aminocarbonylC1-10alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C6-15aryl, substituted C6-15aryl, C6-15aryloxy, substituted C6-15aryloxy, C6-15arylthio, substituted C6-15arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C3-8cycloalkyl, substituted C3-8cycloalkyl, (C3-8cycloalkyl)oxy, substituted (C3-8cycloalkyl)oxy, (C3-8cycloalkyl)thio, substituted (C3-8cycloalkyl)thio, halo, hydroxyl, C3-10heteroaryl, substituted C1-10heteroaryl, C1-10heteroaryloxy, substituted C1-10heteroaryloxy, C1-10heteroarylthio, substituted C1-10heteroarylthio, C2-10heterocyclyl, C2-10substituted heterocyclyl, C2-10heterocyclyloxy, substituted C2-10heterocyclyloxy, C2-10heterocyclylthio, substituted C2-10heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C1-10alkylthio, and substituted C1-10alkythio, thiocarbonyl; or


two R1 substituents, together with the atom to which each is bound, may form ring selected from a C6-15aryl, substituted C6-15aryl, C3-8cycloalkyl, substituted C3-8cycloalkyl, C1-10heteroaryl, substituted C1-10heteroaryl, C2-10substituted heterocyclyl, and C2-10heterocyclyloxy, substituted;


each of A, B, C, D, and E independently may be selected from carbon, nitrogen, oxygen, and sulfur; and


n may be zero, 1, 2, 3, 4, or 5.


In other embodiments, the cyclic compound comprising a heteroatom-containing moiety has a formula illustrated below:




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wherein each R2 independently is selected from C1-10alkyl, substituted C1-10alkyl, C1-10alkoxy, substituted C1-10alkoxy, acyl, acylamino, acyloxy, acylC1-10alkyloxy, amino, substituted amino, aminoacyl, aminocarbonylC1-10alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C6-15aryl, substituted C6-15aryl, C6-15aryloxy, substituted C6-15aryloxy, C6-15arylthio, substituted C6-15arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C3-8cycloalkyl, substituted C3-8cycloalkyl, (C3-8cycloalkyl)oxy, substituted (C3-8cycloalkyl)oxy, (C3-8cycloalkyl)thio, substituted (C3-8cycloalkyl)thio, halo, hydroxyl, C1-10heteroaryl, substituted C1-10heteroaryl, C1-10heteroaryloxy, substituted C1-10heteroaryloxy, C1-10heteroarylthio, substituted C1-10heteroarylthio, C2-10heterocyclyl, C2-10substituted heterocyclyl, C2-10heterocyclyloxy, substituted C2-10heterocyclyloxy, C2-10heterocyclylthio, substituted C2-10heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C1-10alkylthio, and substituted C1-10alkythio, thiocarbonyl; or


two R2 substituents, together with the atom to which each is bound, may form ring selected from a C6-15aryl, substituted C6-15aryl, C3-8cycloalkyl, substituted C3-8cycloalkyl, C1-10heteroaryl, substituted C1-10heteroaryl, C2-10substituted heterocyclyl, and C2-10heterocyclyloxy, substituted;


each of A, B, C, D, E, and F independently may be selected from carbon, nitrogen, oxygen, and sulfur; and


n may be zero, 1, 2, 3, 4, or 5.


In particular disclosed embodiments, the cyclic compound comprising an all-carbon skeleton may have a general formula provided below:




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wherein R3 and R4 independently may be selected from hydroxyl, hydrogen, C1-10alkyl, substituted C1-10alkyl, carboxyl, acyl, aminoacyl, acylamino, amino, substituted amino, C6-15aryl, substituted C6-15aryl, and C1-10alkoxy; R5 is selected from amino, substituted amino, oxo, hydroxyl, C1-10alkoxy, and imino; and n may be zero, 1, 2, 3, 4, or 5. A person of ordinary skill in the art will recognize that the dashed lines indicate optional bonds which may be present in certain compounds and not present in others.


In particular disclosed embodiments, rings A and B are connected via the optional bonds to form a steroid-based skeleton. In embodiments wherein rings A and B are connected, R5 may be bound to ring A via a double bond or a single bond, a feature that is indicated with the optional dashed bond in Formula 13. For example, if R5 is amino, hydroxyl, substituted amino, or C1-10alkoxy, then R5 is attached to ring A via a single bond, whereas if R5 is oxo or imino, then R5 is attached to ring A via a double bond.


In particular disclosed embodiments. C6-15aryl may be selected from phenyl, biphenyl, naphthalene, anthracene, and the like; substituted C6-15aryl may be selected from phenyl, biphenyl, naphthalene, and anthracene substituted with one or more substituents as defined herein; C1-10alkyl may be selected from C1-10alkane, C2-10alkene, and C2-10alkyne; more typically from methyl, ethyl, propyl, butyl, pentyl, hexyl, and the like; ethylene, propylene, butylene, and the like; and ethyne, propyne, butyne, and the like; substituted C1-10alkyl may be selected from C1-10alkane, C2-10alkene, and C2-10alkyne substituted with one or of the substituents as provided herein.


Exemplary embodiments concerning hetercyclyl and heteroaryl substitutents include, but are not limited to, epoxy, pyrrolyl, imidazole, pyrazole, pyridinyl, pyrazine, pyrimidine, oxanyl, thianyl, dioxanyl, dithianyl, coumarin, pyridazine, indolizine, isoindole, indolyl, indolinyl (or dihydroindole), indazole, purine, isoquinoline, quinoline, benzo[d]pyridazine, naphthyridine, quinoxaline, quinazoline, benzopyridazine, pteridine, carbazole, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, oxazolidinyl, oxazolyl, thiophenyl, isooxazolidinyl, and tetrahydrofuranyl.


Exemplary substituents wherein at least two R1 groups have been joined together include the following:




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Particular disclosed embodiments concern cyclic compounds comprising a five-membered heteroatom-containing skeleton having a formula selected from those provided below.




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With reference to Formulas 4-6, R1 and n are as recited herein, and each X independently may be selected from carbon, oxygen, nitrogen, and sulfur.


In yet other embodiments, the cyclic compound comprising a five-membered heteroatom-containing skeleton may have any one of the following formulas




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wherein R1 is as recited herein.


Exemplary compounds are provided below.









TABLE 1





Exemplary Compounds









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Particular embodiments concern cyclic compounds comprising a six-membered heteroatom-containing skeleton having any one of the formulas provided below:




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wherein R2 and n are as recited herein, Z may be selected from carbon and nitrogen, Y may be selected from nitrogen and oxygen, and each X independently may be selected from nitrogen and carbon. A person of ordinary skill in the art will recognize that the dashed lines indicate variable bonds which may or may not be present, depending on the valency of the atom to which each variable bond is attached. For example, if the variable bond indicated in Formula 11 is present, X typically is carbon, as a carbon atom can accommodate four bonds. X may be nitrogen in such a compound; however, a person of ordinary skill in the art would recognize that the nitrogen atom would be positively charged due to the fact that its lone pairs are used to accommodate a fourth bond.


Exemplary compounds are provided below solely as illustrative examples.









TABLE 2





Exemplary Compounds









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Particular embodiments concern compounds comprising an all-carbon, steroidal skeleton having a formula as illustrated below.




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An exemplary compound is provided below.




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In other disclosed embodiments of the compound comprising an all-carbon skeleton, ring A is not connected with ring B and exists as an aryl compound having a formula illustrated below.




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Exemplary compounds are illustrated below.




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Other compounds suitable for use in the methods disclosed herein are provided below in Tables 3, 4, 5, and 6. Any of the compounds provided in Tables 3-6, and any other compounds disclosed herein, can be made using synthetic methods well known in the art of chemical synthesis.









TABLE 3









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R1
R1
R2
R3





C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-Ph
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


H
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)O(CH2)0-5CH3
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)OH
NHR3, or N[(CH2)0-5CH3]2
Halo
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NR[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


H
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)O(CH2)0-5CH3
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)OH
NHR3, or N[(CH2)0-5CH3]2
OH
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
NH2
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NR-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
—(CH2)0-5CH3
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
NO2
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
CF3
H or —(CH2)0-5CH3


C(O)NH2
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)N[(CH2)0-5CH3]2
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NHPh
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NH-2,4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NH-4-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NH-3-fluorophenyl
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NH-4-morpholinylphenyl
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NH-4-piperazinyllphenyl
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3


C(O)NH-3-thiomethylphenyl
NHR3, or N[(CH2)0-5CH3]2
H
H or —(CH2)0-5CH3
















TABLE 4









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R1
R2






C(O)NH2
—Ph-Halo



C(O)N[(CH2)0-5CH3]2
—Ph-Halo



C(O)NHPh
—Ph-Halo



C(O)NH-2,4-fluorophenyl
—Ph-Halo



C(O)NH-4-fluorophenyl
—Ph-Halo



C(O)NH-3-fluorophenyl
—Ph-Halo



C(O)NH-4-
—Ph-Halo



morpholinylphenyl




C(O)NH-4-
—Ph-Halo



piperazinyllphenyl




C(O)NH-3-
—Ph-Halo



thiomethylphenyl




H
—Ph-Halo



C(O)O(CH2)0-5CH3
—Ph-Halo



C(O)OH
—Ph-Halo



Ph—OH
—Ph-Halo



Ph—NH2
—Ph-Halo



Ph-Halo
—Ph-Halo



Ph—O(CH2)0-5CH3
—Ph-Halo



Ph—N[(CH2)0-5CH3]2
—Ph-Halo



C(O)NH2
—Ph—OH



C(O)N[(CH2)0-5CH3]2
—Ph—OH



C(O)NHPh
—Ph—OH



C(O)NH-2,4-fluorophenyl
—Ph—OH



C(O)NH-4-fluorophenyl
—Ph—OH



C(O)NH-3-fluorophenyl
—Ph—OH



C(O)NH-4-
—Ph—OH



morpholinylphenyl




C(O)NH-4-
—Ph—OH



piperazinyllphenyl




C(O)NH-3-
—Ph—OH



thiomethylphenyl




H
—Ph—OH



C(O)O(CH2)0-5CH3
—Ph—OH



C(O)OH
—Ph—OH



Ph—OH
—Ph—OH



Ph—NH2
—Ph—OH



Ph-Halo
—Ph—OH



Ph—O(CH2)0-5CH3
—Ph—OH



Ph—N[(CH2)0-5CH3]2
—Ph—OH



C(O)NH2
—Ph—O(CH2)0-5CH3



C(O)N[(CH2)0-5CH3]2
—Ph—O(CH2)0-5CH3



C(O)NHPh
—Ph—O(CH2)0-5CH3



C(O)NH-2,4-fluorophenyl
—Ph—O(CH2)0-5CH3



C(O)NH-4-fluorophenyl
—Ph—O(CH2)0-5CH3



C(O)NH-3-fluorophenyl
—Ph—O(CH2)0-5CH3



C(O)NH-4-
—Ph—O(CH2)0-5CH3



morpholinylphenyl




C(O)NH-4-
—Ph—O(CH2)0-5CH3



piperazinyllphenyl




C(O)NH-3-
—Ph—O(CH2)0-5CH3



thiomethylphenyl




H
—Ph—O(CH2)0-5CH3



C(O)O(CH2)0-5CH3
—Ph—O(CH2)0-5CH3



C(O)OH
—Ph—O(CH2)0-5CH3



Ph—OH
—Ph—O(CH2)0-5CH3



Ph—NH2
—Ph—O(CH2)0-5CH3



Ph-Halo
—Ph—O(CH2)0-5CH3



Ph—O(CH2)0-5CH3
—Ph—O(CH2)0-5CH3



Ph—N[(CH2)0-5CH3]2
—Ph—O(CH2)0-5CH3



C(O)NH2
—Ph—NH2



C(O)N[(CH2)0-5CH3]2
—Ph—NH2



C(O)NHPh
—Ph—NH2



C(O)NH-2,4-fluorophenyl
—Ph—NH2



C(O)NH-4-fluorophenyl
—Ph—NH2



C(O)NH-3-fluorophenyl
—Ph—NH2



C(O)NH-4-
—Ph—NH2



morpholinylphenyl




C(O)NH-4-
—Ph—NH2



piperazinyllphenyl




C(O)NH-3-
—Ph—NH2



thiomethylphenyl




H
—Ph—NH2



C(O)O(CH2)0-5CH3
—Ph—NH2



C(O)OH
—Ph—NH2



Ph—OH
—Ph—NH2



Ph—NH2
—Ph—NH2



Ph-Halo
—Ph—NH2



Ph—O(CH2)0-5CH3
—Ph—NH2



Ph—N[(CH2)0-5CH3]2
—Ph—NH2



C(O)NH2
—Ph—N[(CH2)0-5CH3]2



C(O)N[(CH2)0-5CH3]2
—Ph—N[(CH2)0-5CH3]2



C(O)NHPh
—Ph—N[(CH2)0-5CH3]2



C(O)NH-2,4-fluorophenyl
—Ph—N[(CH2)0-5CH3]2



C(O)NH-4-fluorophenyl
—Ph—N[(CH2)0-5CH3]2



C(O)NH-3-fluorophenyl
—Ph—N[(CH2)0-5CH3]2



C(O)NH-4-
—Ph—N[(CH2)0-5CH3]2



morpholinylphenyl




C(O)NH-4-
—Ph—N[(CH2)0-5CH3]2



piperazinyllphenyl




C(O)NH-3-
—Ph—N[(CH2)0-5CH3]2



thiomethylphenyl




H
—Ph—N[(CH2)0-5CH3]2



C(O)O(CH2)0-5CH3
—Ph—N[(CH2)0-5CH3]2



C(O)OH
—Ph—N[(CH2)0-5CH3]2



Ph—OH
—Ph—N[(CH2)0-5CH3]2



Ph—NH2
—Ph—N[(CH2)0-5CH3]2



Ph-Halo
—Ph—N[(CH2)0-5CH3]2



Ph-O(CH2)0-5CH3
—Ph—N[(CH2)0-5CH3]2



Ph—N[(CH2)0-5CH3]2
—Ph—N[(CH2)0-5CH3]2



C(O)NH2
—Ph—(CH2)0-5CH3



C(O)N[(CH2)0-5CH3]2
—Ph—(CH2)0-5CH3



C(O)NHPh
—Ph—(CH2)0-5CH3



C(O)NH-2,4-fluorophenyl
—Ph—(CH2)0-5CH3



C(O)NH-4-fluorophenyl
—Ph—(CH2)0-5CH3



C(O)NH-3-fluorophenyl
—Ph—(CH2)0-5CH3



C(O)NH-4-
—Ph—(CH2)0-5CH3



morpholinylphenyl




C(O)NH-4-
—Ph—(CH2)0-5CH3



piperazinyllphenyl




C(O)NH-3-
—Ph—(CH2)0-5CH3



thiomethylphenyl




H
—Ph—(CH2)0-5CH3



C(O)O(CH2)0-5CH3
—Ph—(CH2)0-5CH3



C(O)OH
—Ph—(CH2)0-5CH3



Ph—OH
—Ph—(CH2)0-5CH3



Ph—NH2
—Ph—(CH2)0-5CH3



Ph-Halo
—Ph—(CH2)0-5CH3



Ph—O(CH2)0-5CH3
—Ph—(CH2)0-5CH3



Ph—N[(CH2)0-5CH3]2
—Ph—(CH2)0-5CH3



C(O)NH2
—Ph—S[(CH2)0-5CH3]2



C(O)N[(CH2)0-5CH3]2
—Ph—S[(CH2)0-5CH3]2



C(O)NHPh
—Ph—S[(CH2)0-5CH3]2



C(O)NH-2,4-fluorophenyl
—Ph—S[(CH2)0-5CH3]2



C(O)NH-4-fluorophenyl
—Ph—S[(CH2)0-5CH3]2



C(O)NH-3-fluorophenyl
—Ph—S[(CH2)0-5CH3]2



C(O)NH-4-
—Ph—S[(CH2)0-5CH3]2



morpholinylphenyl




C(O)NH-4-
—Ph—S[(CH2)0-5CH3]2



piperazinyllphenyl




C(O)NH-3-
—Ph—S[(CH2)0-5CH3]2



thiomethylphenyl




H
—Ph—S[(CH2)0-5CH3]2



C(O)O(CH2)0-5CH3
—Ph—S[(CH2)0-5CH3]2



C(O)OH
—Ph—S[(CH2)0-5CH3]2



Ph—OH
—Ph—S[(CH2)0-5CH3]2



Ph—NH2
—Ph—S[(CH2)0-5CH3]2



Ph-Halo
—Ph—S[(CH2)0-5CH3]2



Ph—O(CH2)0-5CH3
—Ph—S[(CH2)0-5CH3]2



Ph—N[(CH2)0-5CH3]2
—Ph—S[(CH2)0-5CH3]2



C(O)NH2
—Ph—NO2



C(O)N[(CH2)0-5CH3]2
—Ph—NO2



C(O)NHPh
—Ph—NO2



C(O)NH-2,4-fluorophenyl
—Ph—NO2



C(O)NH-4-fluorophenyl
—Ph—NO2



C(O)NH-3-fluorophenyl
—Ph—NO2



C(O)NH-4-
—Ph—NO2



morpholinylphenyl




C(O)NH-4-
—Ph—NO2



piperazinyllphenyl




C(O)NH-3-
—Ph—NO2



thiomethylphenyl




H
—Ph—NO2



C(O)O(CH2)0-5CH3
—Ph—NO2



C(O)OH
—Ph—NO2



Ph—OH
—Ph—NO2



Ph—NH2
—Ph—NO2



Ph-Halo
—Ph—NO2



Ph—O(CH2)0-5CH3
—Ph—NO2



Ph—N[(CH2)0-5CH3]2
—Ph—NO2



C(O)NH2
—Ph—CF3



C(O)N[(CH2)0-5CH3]2
—Ph—CF3



C(O)NHPh
—Ph—CF3



C(O)NH-2,4-fluorophenyl
—Ph—CF3



C(O)NH-4-fluorophenyl
—Ph—CF3



C(O)NH-3-fluorophenyl
—Ph—CF3



C(O)NH-4-
—Ph—CF3



morpholinylphenyl




C(O)NH-4-
—Ph—CF3



piperazinyllphenyl




C(O)NH-3-
—Ph—CF3



thiomethylphenyl




H
—Ph—CF3



C(O)O(CH2)0-5CH3
—Ph—CF3



C(O)OH
—Ph—CF3



Ph—OH
—Ph—CF3



Ph—NH2
—Ph—CF3



Ph-Halo
—Ph—CF3



Ph—O(CH2)0-5CH3
—Ph—CF3



Ph—N[(CH2)0-5CH3]2
—Ph—CF3



C(O)NH2
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)N[(CH2)0-5CH3]2
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)NHPh
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)NH-2,4-fluorophenyl
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)NH-4-fluorophenyl
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)NH-3-fluorophenyl
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)NH-4-
NH2, NH(CH2)0-5CH3, or



morpholinylphenyl
N[(CH2)0-5CH3]2



C(O)NH-4-
NH2, NH(CH2)0-5CH3, or



piperazinyllphenyl
N[(CH2)0-5CH3]2



C(O)NH-3-
NH2, NH(CH2)0-5CH3, or



thiomethylphenyl
N[(CH2)0-5CH3]2



H
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)O(CH2)0-5CH3
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



C(O)OH
NH2, NH(CH2)0-5CH3 or




N[(CH2)0-5CH3]2



Ph—OH
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



Ph—NH2
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



Ph-Halo
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



Ph—O(CH2)0-5CH3
NH2, NH(CH2)0-5CH3, or




N[(CH2)0-5CH3]2



Ph—N[(CH2)0-5CH3]2
NH2, NH(CH)0-5CH3 or




N[(CH2)0-5CH3]2



C(O)NH2
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)N[(CH2)0-5CH3]2
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)NHPh
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)NH-2,4-fluorophenyl
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)NH-4-fluorophenyl
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)NH-3-fluorophenyl
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)NH-4-
NHC(O)H or



morpholinylphenyl
NHC(O)(CH2)0-5CH3



C(O)NH-4-
NHC(O)H or



piperazinyllphenyl
NHC(O)(CH2)0-5CH3



C(O)NH-3-
NHC(O)H or



thiomethylphenyl
NHC(O)(CH2)0-5CH3



H
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)O(CH2)0-5CH3
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)OH
NHC(O)H or




NHC(O)(CH2)0-5CH3



Ph—OH
NHC(O)H or




NHC(O)(CH2)0-5CH3



Ph—NH2
NHC(O)H or




NHC(O)(CH2)0-5CH3



Ph-Halo
NHC(O)H or




NHC(O)(CH2)0-5CH3



Ph—O(CH2)0-5CH3
NHC(O)H or




NHC(O)(CH2)0-5CH3



Ph—N[(CH2)0-5CH3]2
NHC(O)H or




NHC(O)(CH2)0-5CH3



C(O)NH2
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)N[(CH2)0-5CH3]2
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)NHPh
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)NH-2,4-fluorophenyl
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)NH-4-fluorophenyl
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)NH-3-fluorophenyl
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)NH-4-
NHS(O)2H or



morpholinylphenyl
NHS(O)2(CH2)0-5CH3



C(O)NH-4-
NHS(O)2H or



piperazinyllphenyl
NHS(O)2(CH2)0-5CH3



C(O)NH-3-
NHS(O)2H or



thiomethylphenyl
NHS(O)2(CH2)0-5CH3



H
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)O(CH2)0-5CH3
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)OH
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



Ph—OH
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



Ph—NH2
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



Ph-Halo
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



Ph—O(CH2)0-5CH3
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



Ph—N[(CH2)0-5CH3]2
NHS(O)2H or




NHS(O)2(CH2)0-5CH3



C(O)NH2
OH or —O(CH2)0-5CH3



C(O)N[(CH2)0-5CH3]2
OH or —O(CH2)0-5CH3



C(O)NHPh
OH or —O(CH2)0-5CH3



C(O)NH-2,4-fluorophenyl
OH or —O(CH2)0-5CH3



C(O)NH-4-fluorophenyl
OH or —O(CH2)0-5CH3



C(O)NH-3-fluorophenyl
OH or —O(CH2)0-5CH3



C(O)NH-4-
OH or —O(CH2)0-5CH3



morpholinylphenyl




C(O)NH-4-
OH or —O(CH2)0-5CH3



piperazinyllphenyl




C(O)NH-3-
OH or —O(CH2)0-5CH3



thiomethylphenyl




H
OH or —O(CH2)0-5CH3



C(O)O(CH2)0-5CH3
OH or —O(CH2)0-5CH3



C(O)OH
OH or —O(CH2)0-5CH3



Ph—OH
OH or —O(CH2)0-5CH3



Ph—NH2
OH or —O(CH2)0-5CH3



Ph-Halo
OH or —O(CH2)0-5CH3



Ph—O(CH2)0-5CH3
OH or —O(CH2)0-5CH3



Ph—N[(CH2)0-5CH3]2
OH or —O(CH2)0-5CH3



C(O)NH2
1-pyrrolidyl



C(O)N[(CH2)0-5CH3]2
1-pyrrolidyl



C(O)NHPh
1-pyrrolidyl



C(O)NH-2,4-fluorophenyl
1-pyrrolidyl



C(O)NH-4-fluorophenyl
1-pyrrolidyl



C(O)NH-fluorophenyl
1-pyrrolidyl



C(O)NH-4-
1-pyrrolidyl



morpholinylphenyl




C(O)NH-4-
1-pyrrolidyl



piperazinyllphenyl




C(O)NH-3-
1-pyrrolidyl



thiomethylphenyl




H
1-pyrrolidyl



C(O)O(CH2)0-5CH3
1-pyrrolidyl



C(O)OH
1-pyrrolidyl



Ph—OH
1-pyrrolidyl



Ph—NH2
1-pyrrolidyl



Ph-Halo
1-pyrrolidyl



Ph—O(CH2)0-5CH3
1-pyrrolidyl



Ph—N[(CH2)0-5CH3]2
1-pyrrolidyl



C(O)NH2
1-piperidinyl



C(O)N[(CH2)0-5CH3]2
1-piperidinyl



C(O)NHPh
1-piperidinyl



C(O)NH-2,4-fluorophenyl
1-piperidinyl



C(O)NH-4-fluorophenyl
1-piperidinyl



C(O)NH-3-fluorophenyl
1-piperidinyl



C(O)NH-4-
1-piperidinyl



morpholinylphenyl




C(O)NH-4-
1-piperidinyl



piperazinyllphenyl




C(O)NH-3-
1-piperidinyl



thiomethylphenyl




H
1-piperidinyl



C(O)O(CH2)0-5CH3
1-piperidinyl



C(O)OH
1-piperidinyl



Ph—OH
1-piperidinyl



Ph—NH2
1-piperidinyl



Ph-Halo
1-piperidinyl



Ph—O(CH2)0-5CH3
1-piperidinyl



Ph—N—[(CH2)0-5CH3]2
1-piperidinyl



C(O)NH2
4-morpholinyl



C(O)N[(CH2)0-5CH3]2
4-morpholinyl



C(O)NHPh
4-morpholinyl



C(O)NH-2,4-fluorophenyl
4-morpholinyl



C(O)NH-4-fluorophenyl
4-morpholinyl



C(O)NH-3-fluorophenyl
4-morpholinyl



C(O)NH-4-
4-morpholinyl



morpholinylphenyl




C(O)NH-4-
4-morpholinyl



piperazinyllphenyl




C(O)NH-3-
4-morphylinyl



thiomethylphenyl




H
4-morpholinyl



C(O)O(CH2)0-5CH3
4-morpholinyl



C(O)OH
4-morpholinyl



Ph—OH
4-morpholinyl



Ph—NH2
4-morpholinyl



Ph-Halo
4-morpholinyl



Ph—O(CH2)0-5CH3
4-morpholinyl



Ph—N[(CH2)0-5CH3]2
4-morpholinyl



C(O)NH2
—(CH2)0-5CH3



C(O)N[(CH2)0-5CH3]2
—(CH2)0-5CH3



C(O)NHPh
—(CH2)0-5CH3



C(O)NH-2,4-fluorophenyl
—(CH2)0-5CH3



C(O)NH-4-fluorophenyl
—(CH2)0-5CH3



C(O)NH-3-fluorophenyl
—(CH2)0-5CH3



C(O)NH-4-
—(CH2)0-5CH3



morpholinylphenyl




C(O)NH-4-
—(CH2)0-5CH3



piperazinyllphenyl




C(O)NH-3-
—(CH2)0-5CH3



thiomethylphenyl




H
—(CH2)0-5CH3



C(O)O(CH2)0-5CH3
—(CH2)0-5CH3



C(O)OH
—(CH2)0-5CH3



Ph—OH
—(CH2)0-5CH3



Ph—NH2
—(CH2)0-5CH3



Ph-Halo
—(CH2)0-5CH3



Ph—O(CH2)0-5CH3
—(CH2)0-5CH3



Ph—N[(CH2)0-5CH3]2
—(CH2)0-5CH3



C(O)NH2
H



C(O)N[(CH2)0-5CH3]2
H



C(O)NHPh
H



C(O)NH-2,4-fluorophenyl
H



C(O)NH-4-fluorophenyl
H



C(O)NH-3-fluorophenyl
H



C(O)NH-4-
H



morpholinylphenyl




C(O)NH-4-
H



piperazinyllphenyl




C(O)NH-3-
H



thiomethylphenyl




H
H



C(O)O(CH2)0-5CH3
H



C(O)OH
H



Ph—OH
H



Ph—NH2
H



Ph-Halo
H



Ph—O(CH2)0-5CH3
H



Ph—N[(CH2)0-5CH3]2
H
















TABLE 5









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embedded image














R1
R2
R3





pyrrolidinyl
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


piperidinyl
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


indole
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


NH2
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


—NHPh
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


Ph
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


OPh
H, NO2, CN, or
NH2, NH(CH2)0-5CH3, or



SO2CF3
N[(CH2)0-5CH3]2


pyrrolidinyl
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


piperidinyl
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


indole
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


NH2
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


—NHPh
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


Ph
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


OPh
H, NO2, CN, or
NHC(O)H or



SO2CF3
NHC(O)(CH2)0-5CH3


pyrrolidinyl
H, NO2, CN, or
NHS(O)2H or



SO2CH3
NHS(O)2(CH2)0-5CH3


piperidinyl
H, NO2, CN, or
NHS(O)2H or



SO2CF3
NHS(O)2(CH2)0-5CH3


indole
H, NO2, CN, or
NHS(O)2H or



SO2CF3
NHS(O)2(CH2)0-5CH3


NH2
H, NO2, CN, or
NHS(O)2H or



SO2CF3
NHS(O)2(CH2)0-5CH3


—NHPh
H, NO2, CN, or
NHS(O)2H or



SO2CF3
NHS(O)2(CH2)0-5CH3


Ph
H, NO2, CN, or
NHS(O)2H or



SO2CF3
NHS(O)2(CH2)0-5CH3


OPh
H, NO2, CN, or
NHS(O)2H or



SO2CF3
NHS(O)2(CH2)0-5CH3


pyrrolidinyl
H, NO2, CN, or
H



SO2CF3



piperidinyl
H, NO2, CN, or
H



SO2CF3



indole
H, NO2, CN, or
H



SO2CF3



NH2
H, NO2, CN, or
H



SO2CF3



—NHPh
H, NO2, CN, or
H



SO2CF3



Ph
H, NO2, CN, or
H



SO2CF3



OPh
H, NO2, CN, or
H



SO2CF3



pyrrolidinyl
H, NO2, CN, or
—NHPh



SO2CF3



piperidinyl
H, NO2, CN, or
—NHPh



SO2CF3



indole
H, NO2, CN, or
—NHPh



SO2CF3



NH2
H, NO2, CN, or
—NHPh



SO2CF3



—NHPh
H, NO2, CN, or
—NHPh



SO2CF3



Ph
H, NO2, CN, or
—NHPh



SO2CF3



OPh
H, NO2, CN, or
—NHPh



SO2CF3



pyrrolidinyl
H, NO2, CN, or
OH



SO2CF3



piperidinyl
H, NO2, CN, or
OH



SO2CF3



indole
H, NO2, CN, or
OH



SO2CF3



NH2
H, NO2, CN, or
OH



SO2CF3



—NHPh
H, NO2, CN, or
OH



SO2CF3



Ph
H, NO2, CN, or
OH



SO2CF3



OPh
H, NO2, CN, or
OH



SO2CF3



pyrrolidinyl
H, NO2, CN or ,
1-pyrrolidyl



SO2CF3



piperidinyl
H, NO2, CN, or
1-pyrrolidyl



SO2CF3



indole
H, NO2, CN, or
1-pyrrolidyl



SO2CF3



NH2
H, NO2, CN, or
1-pyrrolidyl


—NHPh
H, NO2, CN, or
1-pyrrolidyl



SO2CF3



Ph
H, NO2, CN, or
1-pyrrolidyl



SO2CF3



OPh
H, NO2, CN, or
1-pyrrolidyl



SO2CF3



pyrrolidinyl
H, NO2, CN, or
1-piperidinyl



SO2CF3



piperidinyl
H, NO2, CN or
1-piperidinyl



SO2CF3



indole
H, NO2, CN , or
1-piperidinyl



SO2CF3



NH2
H, NO2, CN, or
1-piperidinyl



SO2CF3



—NHPh
H, NO2, CN, or
1-piperidinyl



SO2CF3



Ph
H, NO2, CN, or
1-piperidinyl



SO2CF3



OPh
H, NO2, CN, or
1-piperidinyl



SO2CF3



pyrrolidinyl
H, NO2, CN, or
Ph



SO2CF3



piperidinyl
H, NO2, CN, or
Ph



SO2CF3



indole
H, NO2, CN, or
Ph



SO2CF3



NH2
H, NO2, CN, or
Ph



SO2CF3



—NHPh
H, NO2, CN, or
Ph



SO2CF3



Ph
H, NO2, CN, or
Ph



SO2CF3



OPh
H, NO2, CN, or
Ph



SO2CF3



pyrrolidinyl
H, NO2, CN, or
—Ph-Halo



SO2CF3



piperidinyl
H, NO, CN, or
—Ph-Halo



SO2CF3



indole
H, NO2, CN, or
—Ph-Halo



SO2CF3



NH2
H, NO2, CN, or
—Ph-Halo



SO2CF3



—NHPh
H, NO2, CN, or
—Ph-Halo



SO2CF3



Ph
H, NO2, CN, or
—Ph-Halo



SO2CF3



OPh
H, NO2, CN, or
—Ph-Halo



SO2CF3
















TABLE 6









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R1
R3
R2 (Selected from one of the following)














OC(O)NH2
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
Halo
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
Halo
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
OH
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
OH
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
—O(CH2)0-5CH3
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)(CH3)3
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
—O(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
NH2
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
NH2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
N[(CH2)0-5CH3]2
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
N[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
—(CH2)0-5CH3
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
—(CH2)0-5CH3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
S[(CH2)0-5CH3]2
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
S[(CH2)0-5CH3]2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
NO2
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)O(CH2)0-5CH3
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
NO2
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)NH2
CF3
C(O)NH22
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)N[(CH2)0-5CH3]2
CF3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OH
CF3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OP(O)(OH)2
CF3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)C(CH3)3
CF3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)OC(O)NMe2
CF3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


O(CO)O(CH2)0-5CH3
CF3
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3


OC(O)(CH2)0-5CH3
CF
C(O)NH2
C(O)N[(CH2)0-5CH3]2
C(O)O(CH2)0-5CH3










Certain embodiments can use any one or more of the following compounds:




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III. Methods of Use

i. Methods of Treating Muscular Dystrophy


The α7β1 integrin has been shown to be a major modifier of disease progression in patients with muscular dystrophy. Increased expression of the α7 integrin in muscle can alleviate muscle disease in mouse models of muscular dystrophy. By use of a muscle cell-based assay, the inventors identified molecules that up-regulate α7β1 integrin expression in muscle: laminin-111; valproic acid; ciclopirox ethanolamine; deferoxamine; 2,2-dipyridyl; 5α-cholestan-3β-ol-6-one; Compound ID #1001; Compound ID #1002; Compound ID #1003; and analogs of cholestan. Based on these observations, methods of treatment of muscular dystrophy by increasing the expression or activity of α7β1 integrin with additional suitable compounds are disclosed.


In particular, methods are disclosed herein for treating muscular dystrophy, such as DMD, FCMD, LGMD, FHMD, BMD, MDC1A or MDC1D. In one example, the method includes administering an effective amount of a α7β1 integrin modulatory agent to a subject with muscular dystrophy or suspected of having or developing muscular dystrophy, in which the agent increases the biological activity or expression of α7β1 integrin and thereby, treating the muscular dystrophy in the subject. In some example, the method of treatment inhibits or reduces one or more signs or symptoms associated with muscular dystrophy in the subject.


In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In some examples, an analog is synthesized according to the synthesis pathway shown in the Schemes below. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1, For examples, in some examples, the α7β1 integrin modulatory agent includes one or more molecules provided by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.


The disclosed α7β1 integrin modulatory agents can alter the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).


In a particular example, the subject is a human.


In additional aspects, the method involves selecting a subject with muscular dystrophy. In some example, a subject is selected for treatment following diagnosing the subject with muscular dystrophy. For example, the method can include diagnosing the subject as suffering from muscular dystrophy, such as DMD, MDC1A, MDC1D, LGMD, DMD, FCMD or FHMD.


Methods of diagnosing a subject with muscular dystrophy are known to those of skillin the art and include, but are not limited to, muscle biopsies and measuring serum creatine kinase levels. Additionally, alterations in biomarker known to be associated with muscular dystrophy may be detected by measuring such levels in serum or urine sample.


In a further implementation, the method involves diagnosing the subject as suffering from a disease, disorder, or condition characterized by a mutation in the gene encoding α7 integrin. In another implementation, the method involves diagnosing the subject as suffering from a disease, disorder, or condition characterized by a decreased level of α7 integrin expression.


Alterations in the expression can be measured at the nucleic acid level (such as by real time quantitative polymerase chain reaction or microarray analysis) or at the protein level (such as by Western blot analysis or ELISA). These methods are known to those of skill in the art.


In some examples, following the measurement of the expression levels of α7 integrin expression or serum creatine kinase levels, the assay results, findings, diagnoses, predictions and/or treatment recommendations are recorded and communicated to technicians, physicians and/or patients, for example. In certain embodiments, computers are used to communicate such information to interested parties, such as, patients and/or the attending physicians. The therapy selected for administered is then based upon these results.


In one embodiment, the results and/or related information is communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to a test subject by any means of communication, including writing, such as by providing a written report, electronic forms of communication, such as email, or telephone. Communication may be facilitated by use of a computer, such as in case of email communications. In certain embodiments, the communication containing results of a diagnostic test and/or conclusions drawn from and/or treatment recommendations based on the test, may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in U.S. Pat. No. 6,283,761; however, the present disclosure is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the disclosure, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.


In several embodiments, identification of a subject as having muscular dystrophy, such as DMD, LGMD, FHMD, BMD, FCMD, MDC1D or MDC1A, results in the physician treating the subject, such as prescribing one or more disclosed α7β1 agents for inhibiting or delaying one or more signs and symptoms associated with muscular dystrophy. In additional embodiments, the dose or dosing regimen is modified based on the information obtained using the methods disclosed herein.


ii. Methods of Enhancing Muscle Regeneration, Repair, or Maintenance


Also disclosed are methods of enhancing muscle regeneration, repair or maintenance in a subject. In some examples, the method includes administering an effective amount of an α7β1 integrin modulatory agent to a subject in need of muscle regeneration, repair or maintenance, wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.


In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In some examples, an analog is synthesized according to the synthesis pathway provided in the Examples below. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. For examples, in some examples, the α7β1 integrin modulatory agent includes one or more molecules provided in a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.


The disclosed α7β1 integrin modulatory agents can increase the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).


Muscle regeneration may benefit, for example, geriatric or other patient populations with reduced muscle repair capability, or simply speed the muscle repair process for otherwise physiologically unimpaired patients. In particular implementations, administration of a α7β1 integrin modulatory agent can aid muscle repair, or reduction of muscle damage, in athletes or others having activity-induced muscle injury or damage. In yet further implementations, muscle repair in patients suffering from muscle damage, such as through accident or injury, can be augmented by administration of a α7β1 integrin modulatory agent.


In some examples, α7β1 modulatory agent is administered prior to the subject experiencing muscle damage or disease. In some examples, the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.


In some examples, the method further includes selecting a subject in need of enhancing muscle regeneration, repair, or maintenance. For example, in some instances, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject. Methods for diagnosing and selecting a subject in need of muscle regeneration, repair or maintenance are known to those of ordinary skill in the art and include those provided described herein (including those in the Methods of Treatment of Muscular Dystrophy). As stated above, subjects may be selected based upon their life style (e.g., engaged in moderate to intense exercise or physical activities), age (e.g., elderly population at more risk of experiencing muscle degeneration or injury) or pre-disposition to muscle degeneration or injury (e.g., aenetics or previous muscle injury).


iii. Methods of Prospectively Preventing or Reducing Muscle Injury or Damage


Also disclosed are methods prospectively preventing or reducing muscle injury or damage in a subject. In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby prospectively preventing or reducing muscle injury or damage in the subject.


In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. For examples, in some examples, the α7β1 integrin modulatory agent includes one or more molecules provided by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. An exemplary synthetic scheme for making certain α7β1 integrin modulatory agent disclosed herein is provided in Schemes 1 and 2 as illustrated in FIG. 12. A person of ordinary skill in the art would recognize that derivatives of such compounds can be obtained using methods known in the art, such as functionalizing the core structure using suitable reagents and conditions. Exemplary groups that can be modified to produce various analogs are indicated in the Schemes shown in FIG. 12; and in some embodiments, the core of the molecule can be modified to include one or more additional heteroatoms and/or to replace an existing heteroatom with a different suitable heteroatom.


The disclosed α7β1 integrin modulatory agents can increase the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).


In some examples, the method further includes selecting a subject at risk for developing a muscle injury or damage. In some examples, the α7β1 integrin modulatory agent is administered to a subject prior to the subject exercising.


In some examples, the method further includes selecting a subject at risk for developing a muscle injury or damage. Methods for selecting such s subject are known to those of ordinary skill in the art and include those provided described herein. As stated above, subjects may be selected based upon their life style (e.g., engaged in moderate to intense exercise or physical activities), age (elderly population at more risk of experiencing muscle degeneration or injury) or pre-disposition to muscle degeneration or injury (e,g., genetics or previous muscle injury).


iv. Methods of Enhancing α7β1 Integrin Expression


Also disclosed herein are methods of enhancing α7β1 integrin expression. In some examples, these methods include contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof and increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression. In some examples, the cell is a muscle cell, such as a skeletal muscle cell. In some examples, the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.


In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. In some examples, the α7β1 integrin modulatory agent includes one or more molecules encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.


In some examples, the disclosed α7β1 integrin modulatory agents can increase the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).


Administration of an Effective Amount of an α7β1 Integrin Modulatory Agent

For any of the disclosed methods, an effective amount of α7β1 integrin modulatory agent is one when administered by a particular route and concentration induces the desired response (e.g., treatment of muscular dystrophy, enhancing muscle regeneration, repair or maintenance, preventing or reducing muscle injury or damage, or enhancing α7β1 integrin expression).


i. Administration Routes, Formulations and Concentrations


Methods of administration of the disclosed α7β1 integrin modulatory agents are routine, and can be determined by a skilled clinician. The disclosed α7β1 integrin modulatory agents or other therapeutic substance are in general administered topically, nasally, intravenously, orally, intracranially, intramuscularly, parenterally or as implants, but even rectal or vaginal use is possible in principle. The disclosed α7β1 integrin modulatory agents also may be administered to a subject using a combination of these techniques.


Suitable solid or liquid pharmaceutical preparation forms are, for example, aerosols, (micro)capsules, creams, drops, drops or injectable solution in ampoule form, emulsions, granules, powders, suppositories, suspensions, syrups, tablets, coated tablets, and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as binders, coating agents, disintegrants, flavorings, lubricants, solubilizers, sweeteners, or swelling agents are customarily used as described above. The pharmaceutical agents are suitable for use in a variety of drug delivery systems. For a brief review of various methods for drug delivery, see Langer, “New Methods of Drug Delivery,” Science 249:1527-1533 (1990), incorporated by reference herein to the extent not inconsistent with the present disclosure.


The disclosed α7β1 integrin modulatory agents or other therapeutic agents of the present disclosure can be formulated into therapeutically-active pharmaceutical agents that can be administered to a subject parenterally or orally. Parenteral administration routes include, but are not limited to epidermal, intraarterial, intramuscular (IM and depot IM), intraperitoneal (IP), intravenous (IV), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, injection into the stomach, subcutaneous injections (subcutaneous (SQ and depot SQ), transdermal, topical, and ophthalmic.


The disclosed α7β1 integrin modulatory agents or other therapeutic agents can be mixed or combined with a suitable pharmaceutically acceptable excipients to prepare pharmaceutical agents. Pharmaceutically acceptable excipients include, but are not limited to, alumina, aluminum stearate, buffers (such as phosphates), glycine, ion exchangers (such as to help control release of charged substances), lecithin, partial glyceride mixtures of saturated vegetable fatty acids, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes such as cellulose-based substances, colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols, such as polyethylene glycol, polyethylene-polyoxypropylene-block polymers, polyvinyl pyrrolidone, potassium hydrogen phosphate, protamine sulfate, group 1 halide salts such as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat, and zinc salts, for example, Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers.


Upon mixing or addition of one or more disclosed α7β1 integrin modulatory agents and/or or other therapeutic agents, the resulting mixture may be a solid, solution, suspension, emulsion, or the like. These may be prepared according to methods known to those of ordinary skill in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier. Pharmaceutical carriers suitable for administration of the disclosed α7β1 integrin modulatory agents or other therapeutic agents include any such carriers known to b suitable for the particular mode of administration. In addition, the disclosed α7β1 integrin modulatory agents or other therapeutic substance can also be mixed with other inactive or active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.


Methods for solubilizing may be used where the agents exhibit insufficient solubility in a carrier. Such methods are known and include, but are not limited to, dissolution in aqueous sodium bicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), and using surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Del.).


The disclosed α7β1 integrin modulatory agents or other therapeutic agents can be prepared with carriers that protect them against rapid elimination from the body, such as coatings or time-release formulations. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. A disclosed α7β1 integrin modulatory agents or other therapeutic agent is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect, typically in an amount to avoid undesired side effects, on the treated subject. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated condition. For example, mouse models of muscular dystrophy may be used to determine effective amounts or concentrations that can then be translated to other subjects, such as humans, as known in the art.


Injectable solutions or suspensions can be formulated, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as 1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer's solution, saline solution, or water; or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid; a naturally occurring vegetable oil such as coconut oil, cottonseed oil, peanut oil, sesame oil, and the like; glycerine; polyethylene glycol; propylene glycol; or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; buffers such as acetates, citrates, and phosphates, chelating agents such as ethylenediaminetetraacetic acid (EDTA); agents for the adjustment of tonicity such as sodium chloride and dextrose; and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required. Where administered intravenously, suitable carriers include physiological saline, phosphate-buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.


For topical application, one or more disclosed α7β1 integrin modulatory agents, or other therapeutic agent may be made up into a cream, lotion, ointment, solution, or suspension in a suitable aqueous or non-aqueous carrier. Topical application can also be accomplished by transdermal patches or bandages which include the therapeutic substance. Additives can also be included, e.g., buffers such as sodium metabisulphite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride, or chlorhexidine; and thickening agents, such as hypromellose.


If the disclosed α7β1 integrin modulatory agent, or other therapeutic agent is administered orally as a suspension, the pharmaceutical agents can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain a suspending agent, such as alginic acid or sodium alginate, bulking agent, such as microcrystalline cellulose, a viscosity enhancer, such as methylcellulose, and sweeteners/flavoring agents. Oral liquid preparations can contain conventional additives such as suspending agents, gelatin, glucose syrup, hydrogenated edible fats, methyl cellulose, sorbitol, and syrup; emulsifying agents, e.g., acacia, lecithin, or sorbitan monooleate; non-aqueous carriers (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring or coloring agents. When formulated as immediate release tablets, these agents can contain dicalcium phosphate, lactose, magnesium stearate, microcrystalline cellulose, and starch and/or other binders, diluents, disintegrants, excipients, extenders, and lubricants.


If oral administration is desired, one or more disclosed α7β1 integrin modulatory agents, or other therapeutic substances can be provided in a composition that protects it from the acidic environment of the stomach. For example, he disclosed α7β1 integrin modulatory agents or other therapeutic agents can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The disclosed α7β1 integrin modulatory agents, or other therapeutic agent can also be formulated in combination with an antacid or other such ingredient.


Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, one or more of the disclosed α7β1 integrin modulatory agents, or other therapeutic substances can be incorporated with excipients and used in the form of capsules, tablets, or troches. Pharmaceutically compatible adjuvant materials or binding agents can be included as part of the composition.


The capsules, pills, tablets, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, acacia, corn starch, gelatin, gum tragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate, polyethylene glycol, silica, or talc; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.


When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier, such as a fatty oil. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. One or more of the disclosed α7β1 integrin modulatory agents, or other therapeutic agent can also be administered as a component of an elixir, suspension, syrup, wafer, tea, chewing gum, or the like. A syrup may contain, in addition to the active compounds, sucrose or glycerin as a sweetening agent and certain preservatives, dyes and colorings, and flavors.


When administered orally, the compounds can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds need to be administered less frequently.


In some examples, one or more of the disclosed α7β1 integrin modulatory agents and/or a therapeutic agent is injected into the stomach of a subject is incorporated systemically in the subject, such as in diverse muscle groups. Examples of methods and compositions for administering therapeutic substances which include proteins include those discussed in Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems 2ed. (2005); Mahato, Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids (2004); McNally, Protein Formulation and Delivery, 2ed. (2007); and Kumar et al., “Novel Delivery Technologies for Protein and Peptide Therapeutics,” Current Pharm. Biotech., 7:261-276 (2006); each of which is incorporated by reference herein to the extent not inconsistent with the present disclosure.


In some implementations, the effective amount of one or more of the disclosed α7β1 integrin modulatory agents is administered as a single dose per time period, such as every three or four months, month, week, or day, or it can be divided into at least two unit dosages for administration over a period. Treatment may be continued as long as necessary to achieve the desired results. For instance, treatment may continue for about 3 or 4 weeks up to about 12-24 months or longer, including ongoing treatment. The compound can also be administered in several doses intermittently, such as every few days (for example, at least about every two, three, four, five, or ten days) or every few weeks (for example at least about every two, three, four, five, or ten weeks).


Particular dosage regimens can be tailored to a particular subject, condition to be treated, or desired result. For example, when the methods of the present disclosure are used to treat muscular dystrophy or similar conditions, an initial treatment regimen can be applied to arrest the condition. Such initial treatment regimen may include administering a higher dosage of one or more of the disclosed α7β1 integrin modulatory agents, or administering such material more frequently, such as daily. After a desired therapeutic result has been obtained, such as a desired level of muscle regeneration, a second treatment regimen may be applied, such as administering a lower dosage of one or more of the disclosed α7β1 integrin modulatory agents or administering such material less frequently, such as monthly, bi-monthly, quarterly, or semi-annually. In such cases, the second regimen may serve as a “booster” to restore or maintain a desired level of muscle regeneration. Similar treatment regimens may be used for other subjects with reduced or impaired muscle regeneration capabilities, such as geriatric subjects.


When particular methods of the present disclosure are used to prevent or mitigate muscle damage, such as damage caused by exertion or injury, the subject is typically treated a sufficient period of time before the exertion or injury in order to provide therapeutic effect. For example, the subject may be treated at least about 24 hours before the expected activity or potential injury, such as at least about 48 hours, about 72 hours, about 1 week, about 2 weeks, about three weeks, or about 4 weeks or more prior.


When embodiments of the method of the present disclosure are used to prevent or treat a muscle injury, one or more of the disclosed α7β1 integrin modulatory agents or other therapeutic substance can be applied directly to, or proximately to, the area to be treated. For example, the substance can be injected into or near the area. In further examples, the substance can be applied topically to the area to be treated. Treatment is typically initiated prior to the injury to several weeks following the injury. In more specific implementations, the treatment is initiated between about 12 and about 72 hours following injury, such as between about 24 and about 48 hours following injury. In some cases, a single administration of the substance is effective to provide the desired therapeutic effect. In further examples, additional administrations are provided in order to achieve the desired therapeutic effect.


Amounts effective for various therapeutic treatments of the present disclosure may, of course, depend on the severity of the disease and the weight and general state of the subject, as well as the absorption, inactivation, and excretion rates of the therapeutically-active compound or component, the dosage schedule, and amount administered, as well as other factors known to those of ordinary skill in the art. It also should be apparent to one of ordinary skill in the art that the exact dosage and frequency of administration will depend on the particular α7β1 integrin modulatory agent, or other therapeutic substance being administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular subject, and other medication the subject may be taking. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. For example, mouse models of muscular dystrophy may be used to determine effective dosages that can then be translated to dosage amount for other subjects, such as humans, as known in the art. Various considerations in dosage determination are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa. (1990), each of which is herein incorporated by reference to the extent not inconsistent with the present disclosure.


In specific examples, the one or more disclosed α7β1 integrin modulatory agents is administered to a subject in an amount sufficient to provide a dose of the agent of between about 10 fmol/g and about 500 nmol/g, such as between about 2 nmol/g and about 20 nmol/g or between about 2 nmol/g and about 10 mmol/g. In additional examples, the α7β1 integrin modulatory agent is administered to a subject in an amount sufficient to provide a dose of between about 0.01 μg/kg and about 1000 mg/kg or between about 0.1 mg/kg and about 1000 mg/kg, in particular examples this amount is provided per day or per week. In another example, the disclosed α7β1 integrin modulatory agent is administered to a subject in an amount sufficient to provide a dose of agent of between about 0.2 mg/kg and about 2 mg/kg. In further examples, the α7β1 integrin modulatory agent is administered to a subject in an amount sufficient to provide a concentration of α7β1 integrin modulatory agent in the administrated material of between about 5 nM and about 500 nM, such as between about 50 nM and about 200 nm, or about 100 nM. In other examples, the α7β1 integrin modulatory agent is administered to a subject between about 500 μg/ml and about 1 μg/ml, such as about 300 μg/ml and about 3 μg/ml, about 200 μg/ml and about 20 μg/ml, including 500 μg/ml, 400 μg/ml, 300 μg/ml, 250 μg/ml, 200 μg/ml, 150 μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μμg/ml, 2.5 μg/ml and 1.25 μg/ml.


ii. Desired Response


One or more disclosed α7β1 integrin modulatory agents and/or additional therapeutic agents are administered by a specific route and/or concentration to generate a desired response. In some examples, a desired response refers to an amount effective for lessening, ameliorating, eliminating, preventing, or inhibiting at least one symptom of a disease, disorder, or condition treated and may be empirically determined. In various embodiments of the present disclosure, a desired response is muscle regeneration, reductions or prevention of muscle degeneration, promotion of muscle maintenance, reduction or prevention of muscle injury or damage, reduction or prevention in one more signs or symptoms associated with muscular dystrophy.


In particular, indicators of muscular health, such as muscle cell regeneration, maintenance, or repair, can be assessed through various means, including monitoring markers of muscle regeneration, such as transcription factors such as Pax7, Pax3, MyoD, MRF4, and myogenin. For example, increased expression of such markers can indicate that muscle regeneration is occurring or has recently occurred. Markers of muscle regeneration, such as expression of embryonic myosin heavy chain (eMyHC), can also be used to gauge the extent of muscle regeneration, maintenance, or repair. For example, the presence of eMyHC can indicate that muscle regeneration has recently occurred in a subject.


Muscle cell regeneration, maintenance, or repair can also be monitored by determining the girth, or mean cross sectional area, of muscle cells or density of muscle fibers. Additional indicators of muscle condition include muscle weight and muscle protein content. Mitotic index (such as by measuring BrdU incorporation) and myogenesis can also be used to evaluate the extent of muscle regeneration.


In particular examples, the improvement in muscle condition, such as regeneration, compared with control is at least about 10%, such as at least about 30%, or at least about 50% or more, including an at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, including a 10% to 90% decrease, 20% to 80% increase, 30% to 70% increase or a 40% to 60% increase (e a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more increase)


iii. Additional Treatments or Therapeutic Agents


In particular examples, prior to, during, or following administration of an effective amount of an agent that reduces or inhibits one or more signs or symptoms associated with muscular dystrophy, the subject can receive one or more other therapies. In one example, the subject receives one or more treatments prior to administration of a disclosed α7β1 modulatory agent. Examples of such therapies include, but are not limited to, laminin-111 protein therapy, which works to stabilize the sarcolemma and reduce muscle degeneration. In some examples, a source of muscle cells can be added to aid in muscle regeneration and repair. In some aspects of the present disclosure, satellite cells are administered to a subject in combination with laminin therapy. U.S. Patent Publication 2006/0014287, incorporated by reference herein to the extent not inconsistent with the present disclosure, provides methods of enriching a collection of cells in myogenic cells and administering those cells to a subject. In further aspects, stem cells, such as adipose-derived stem cells, are administered to the subject, Suitable methods of preparing and administering adipose-derived stem cells are disclosed in U.S. Patent Publication 2007/0025972, incorporated by reference herein to the extent not inconsistent with the present disclosure. Additional cellular materials, such as fibroblasts, can also be administered, in some examples.


Additional therapeutic agents include agents which enhance the effect of the disclosed α7β1 modulatory agents, such as a component of the extracellular matrix, such as an integrin, dystrophin, dystroglycan, utrophin, or a growth factor. In some examples, the additional therapeutic agent reduces or enhances expression of a substance that enhances the formation or maintenance of the extracellular matrix. In some examples, the additional substance can include aggrecan, angiostatin, cadherins, collagens (including collagen I, collagen III, or collagen IV), decorin, elastin, ena.ctin, endostatin, fibrin, fibronectin, osteopontin, tenascin, thrombospondin, vitronectin, and combinations thereof. Biglycans, glycosaminoglycans (such as heparin), glycoproteins (such as dystroglycan), proteoglycans (such as heparan sulfate), and combinations thereof can also be administered.


In some examples, growth stimulants such as cytokines, polypeptides, and growth factors such as brain-derived neurotrophic factor (BDNF), CNF (ciliary neurotrophic factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), glial growth factor (GGF), glial maturation factor (GMF) glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), insulin, insulin-like growth factors, kerotinocyte growth factor (KGF), nerve growth factor (NGF), neurotropin-3 and -4, PDGF (platelet-derived growth factor), vascular endothelial growth factor (VEGF), and combinations thereof may be administered with one of the disclosed methods.


IV. Clinical Trials

To obtain regulatory approval for the use of one or more of the disclosed α7β1 modulatory agents to treat a muscular disorder, clinical trials are performed. As is known in the art, clinical trials progress through phases of testing, which are identified as Phases I, II, III, and IV.


Initially the disclosed α7β1 modulatory agent is evaluated in a Phase I trial. Typically Phase I trials are used to determine the best mode of administration (for example, by pill or by injection), the frequency of administration, and the toxicity for the compounds. Phase I studies frequently include laboratory tests, such as blood tests and biopsies, to evaluate the effects of the potential therapeutic in the body of the patient. For a Phase I trial, a small group of patients with a muscular disorder are treated with a specific dose of a disclosed α7β1 modulatory agent. During the trial, the dose is typically increased group by group in order to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) associated with the compound. This process determines an appropriate dose to use in a subsequent Phase II trial.


A Phase II trial can be conducted to further evaluate the effectiveness and safety of the disclosed α7β1 modulatory agent. In Phase H trials, a disclosed α7β1 modulatory agent is administered to groups of patients with a muscular disorder using the dosage found to be effective in Phase I trials.


Phase III trials focus on determining how a disclosed α7β1 modulatory agent compares to the standard, or most widely accepted, treatment. In Phase III trials, patients are randomly assigned to one of two or more “arms”. In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will receive a disclosed α7β1 modulatory agent treatment (investigational group).


Phase IV trials are used to further evaluate the long-term safety and effectiveness of a disclosed α7β1 modulatory agent. Phase IV trials are less common than Phase I, II and III trials and take place after a disclosed α7β1 modulatory agent has been approved for standard use.


Eligibility of Patients for Clinical Trials

Participant eligibility criteria can range from general (for example, age, sex, type of disease) to specific (for example, type and number of prior treatments, disease characteristics, blood cell counts, organ function). In one embodiment, eligible patients have been diagnosed with a muscular disorder. Eligibility criteria may also vary with trial phase. Patients eligible for clinical trials can also be chosen based on objective measurement of a muscular disorder and failure to respond to other muscular disorder treatments. For example, in Phase I and II trials, the criteria often exclude patients who may be at risk from the investigational treatment because of abnormal organ function or other factors. In Phase II and III trials additional criteria are often included regarding disease type and stage, and number and type of prior treatments.


Phase I trials usually include 15 to 30 participants for whom other treatment options have not been effective. Phase II trials typically include up to 100 participants who have already received drug therapy, but whom the treatment has not been effective.


Participation in Phase III trials is often restricted based on the previous treatment received. Phase III trials usually include hundreds to thousands of participants. This large number of participants is necessary in order to determine whether there are true differences between the effectiveness of a disclosed α7β1 modulatory agent and the standard treatment. Phase III can include patients ranging from those newly diagnosed with a muscular disorder to those with re-occurring signs and/or symptoms associated with a muscular disorder or a muscular disorder that did not respond to prior treatment.


One skilled in the art will appreciate that clinical trials should be designed to be as inclusive as possible without making the study population too diverse to determine whether the treatment might be as effective on a more narrowly defined population. The more diverse the population included in the trial, the more applicable the results could be to the general population, particularly in Phase III trials. Selection of appropriate participants in each phase of clinical trial is considered to be within the ordinary skills of a worker in the art.


Assessment of Patients Prior to Treatment

Prior to commencement of the study, several measures known in the art can be used to first classify the patients. Patients can first be assessed, for example by determining serum creatine kinase (CK) levels or other indicators of a muscle disorder, such as increased levels of muscle inflammation, apoptosis, muscle loss, myotube hypertrophy, and/or decreased myofibers stability and cell survival.


Administration of a Disclosed α7β1 Modulatory Agent in Clinical Trials

A disclosed α7β1 modulatory agent is typically administered to the trial participants orally. A range of doses of the agent can be tested. Provided with information from preclinical testing, a skilled practitioner can readily determine appropriate dosages of agent for use in clinical trials. In one embodiment, a dose range is from about 100 μg/kg and about 5000 mg/kg of the subject's weight, such as 1 mg/kg and about 2000 mg/kg of the subject's weight, about 100 mg/kg and about 1500 mg/kg of the subject's weight, about 100 μg/kg and about 2000 mg/kg of the subject's weight, about 200 mg/kg and about 1000 mg/kg of the subject's weight, about 200 mg/kg and about 750 mg/kg of the subject's weight, about 250 mg/kg and about 500 mg/kg of the subject's weight, about 100 μm and about 500 mM. In some embodiments, subjects are given a disclosed α7β1 modulatory agent orally at 10 to 60 mg/kg of body weight per day. For example, 10-15 mg/kg of a disclosed α7β1 modulatory agent is administered for two weeks and if well tolerated the dose is increased by 5-10 mg/kg/week to achieve optimal clinical response. In some examples, the daily dose does not exceed 60 mg/kg of body weight and is given for a minimum of 6 months with liver function monitored every two weeks to monthly.


Pharmacokinetic Monitoring

To fulfill Phase I criteria, distribution of the disclosed α7β1 modulatory agent is monitored, for example, by chemical analysis of samples, such as blood, collected at regular intervals. For example, samples can be taken at regular intervals up until about 72 hours after the start of treatment.


If analysis is not conducted immediately, the samples can be placed on dry ice after collection and subsequently transported to a freezer to be stored at −70° C. until analysis can be conducted. Samples can be prepared for analysis using standard techniques known in the art and the amount of the disclosed α7β1 modulatory agent present can be determined, for example, by high-performance liquid chromatography (HPLC). Pharmacokinetic data can be generated and analyzed in collaboration with an expert clinical pharmacologist and used to determine, for example, clearance, half-life and maximum plasma concentration,


Monitoring of Patient Outcome

The endpoint of a clinical trial is a measurable outcome that indicates the effectiveness of a compound under evaluation. The endpoint is established prior to the commencement of the trial and will vary depending on the type and phase of the clinical trial. Examples of endpoints include, for example, decline in serum CK levels, inflammation, apoptosis, and muscle loss. For example, at least a 10% reduction in serum CK levels indicates the patient is responsive to the treatment.


The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.


EXAMPLES
Example 1

This example demonstrates the results of quantitative real-time PCR used to assess Itga7, Itgb1, and Lama2 transcript levels in C2C12 myoblasts and myotubes treated for 24 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), 10 μM MLS001165937-01 (IED-937), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD (FIG. 1). FIG. 2 is a digital image of Western Blots and quantitative analysis of α7 Integrin and GAPDH protein levels in C2C12 myotubes treated for 48 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD. Bands were quantified using Image J software and then graphed as α7 Integrin protein levels relative to GAPDH protein levels. * denotes a significant difference in relative protein levels with ** p<0.01.


Example 2

The additional structures provided below provide additional compounds that may be used in the methods disclosed herein increasing α7 integrin expression in muscle (see Table 7). In some examples, the analogs were made in 5 mg quantities, salt form (e.g., hydrochloride salt), as a dry powder, at an at least 90% purity as measured by HPLC. In some embodiments, physical properties (kinetic, solubility, PAMPA permeability, clog P) and metabolic stability (plasma and microsomal stability, metabolite identification) of all synthesized molecules, as well as measure pharmacokinetics properties, compound levels and distribution of selected compounds are determined. Besides systematic substitution for exploring selected chemotypes, classical medicinal chemistry parameters (MW, number of hydrogen donors and acceptors, tPSA, clogP, flexibility of the molecule) as well as in house-measured physical and metabolic properties of analog molecules are used to guide the selection of potent, efficacious and non-toxic compounds having suitable pharmacokinetic properties for use in the disclosed methods.









TABLE 7





Exemplary Compounds


















embedded image


Compound 1





Potency: 1.122



Efficacy: 198.146








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Compound 2





Potency: 3.5481



Efficacy: 228.085








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Compound 3





Potency: 2.2387



Efficacy: 193.433








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Compound 4





Potency: 2.8184



Efficacy: 129.298








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Compound 5





Potency: 1.122



Efficacy: 92.4838








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Compound 6





Potency: 1.122



Efficacy: 128.12








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Compound 7





Potency: 4.4668



Efficacy: 94.9208








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Compound 8





Potency: 2.8184



Efficacy: 111.685








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Compound 9





Potency: 2.8184



Efficacy: 122.703








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Compound 10





Potency: 3.1623



Efficacy: 102.022








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Compound 11





Potency: 3.9811



Efficacy: 101.893








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Compound 12





Potency: 2.8184



Efficacy: 92.9057








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Compound 13





Potency: 4.4668



Efficacy: 121.996








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Compound 14





Potency: 2.2387



Efficacy: 84.7736








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Compound 15





Potency: 3.1623



Efficacy: 91.3808








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Compound 16





Potency: 2.2387



Efficacy: 115.349








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Compound 17





Potency: 2.5119



Efficacy: 96.1948








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Compound 18





Potency: 10



Efficacy: 1751.56








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Compound 19





Potency: 1.4125



Efficacy: 81.6138








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Compound 20





Potency: 3.5481



Efficacy: 101.579








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Compound 21





Potency: 5.6234



Efficacy: 92.2825








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Compound 22





Potency: 4.4668



Efficacy: 83.4252








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Compound 23





Potency: 3.9811



Efficacy: 85.1623








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Compound 24





Potency: 3.1623



Efficacy: 76.9771








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Compound 25





Potency: 2.2387



Efficacy: 82.8995








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Compound 26





Potency: 1.9953



Efficacy: 95.7363








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Compound 27





Potency: 1.9953



Efficacy: 81.8506








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Compound 28





Potency: 2.8184



Efficacy: 92.0171








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Compound 29





Potency: 3.9811



Efficacy: 81.1658








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Compound 30





Potency: 3.9811



Efficacy: 92.7977








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Compound 31





Potency: 2.5119



Efficacy: 86.4354








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Compound 32





Potency: 2.8184



Efficacy: 88.796








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Compound 33





Potency: 1.9953



Efficacy: 83.3258








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Compound 34





Potency: 1.4125



Efficacy: 70.4537








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Compound 35





Potency: 2.5119



Efficacy: 72.7515








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Compound 36





Potency: 3.1623



Efficacy: 75.7047








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Compound 37





Potency: 2.5119



Efficacy: 77.0458








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Compound 38





Potency: 4.4668



Efficacy: 75.5605








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Compound 39





Potency: 3.5481



Efficacy: 78.5404








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Compound 40





Potency: 14.1254



Efficacy: 436.691








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Compound 41





Potency: 14.1254



Efficacy: 447.862








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Compound 42





Potency: 14.1254



Efficacy: 475.262








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Compound 43





Potency: 3.9811



Efficacy: 148.01








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Compound 44





Potency: 11.2202



Efficacy: 328.277








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Compound 45





Potency: 14.1254



Efficacy: 426.535








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Compound 46





Potency: 7.9433



Efficacy: 231.415








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Compound 47





Potency: 5.6234



Efficacy: 141.317








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Compound 48





Potency: 7.0795



Efficacy: 214.145








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Compound 49





Potency: 5.6234



Efficacy: 150.998








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Compound 50





Potency: 7.9433



Efficacy: 181.323








embedded image


Compound 51





Potency: 10



Efficacy: 273.425








embedded image


Compound 52





Potency: 4.4668



Efficacy: 165.293








embedded image


Compound 53





Potency: 7.0795



Efficacy: 163.55








embedded image


Compound 54





Potency: 12.5893



Efficacy: 222.708








embedded image


Compound 55





Potency: 12.5893



Efficacy: 271.246








embedded image


Compound 56





Potency: 3.9811



Efficacy: 88.764








embedded image


Compound 57





Potency: 4.4668



Efficacy: 115.732








embedded image


Compound 58





Potency: 3.5481



Efficacy: 137.991








embedded image


Compound 59





Potency: 10



Efficacy: 188.981








embedded image


Compound 60





Potency: 4.4668



Efficacy: 128.904








embedded image


Compound 61





Potency: 5.0119



Efficacy: 140.322








embedded image


Compound 62





Potency: 7.0795



Efficacy: 186.257








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Compound 63





Potency: 10



Efficacy: 200.43








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Compound 64





Potency: 5.0119



Efficacy: 134.919








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Compound 65





Potency: 19.9526



Efficacy: 491.985








embedded image


Compound 66





Potency: 12.5893



Efficacy: 215.25








embedded image


Compound 67





Potency: 10



Efficacy: 157.616








embedded image


Compound 68





Potency: 12.5893



Efficacy: 239.617








embedded image


Compound 69





Potency: 3.9811



Efficacy: 90.5081








embedded image


Compound 70





Potency: 4.4668



Efficacy: 115.234








embedded image


Compound 71





Potency: 7.9433



Efficacy: 124.512








embedded image


Compound 72





Potency: 8.9125



Efficacy: 138.663








embedded image


Compound 73





Potency: 12.5893



Efficacy: 170.328








embedded image


Compound 74





Potency: 4.4668



Efficacy: 109.274








embedded image


Compound 75





Potency: 3.9811



Efficacy: 71.4431








embedded image


Compound 76





Potency: 6.3096



Efficacy: 114.425








embedded image


Compound 77





Potency: 4.4668



Efficacy: 121.35








embedded image


Compound 78





Potency: 10



Efficacy: 152.941








embedded image


Compound 79





Potency: 4.4668



Efficacy: 85.208








embedded image


Compound 80





Potency: 10



Efficacy: 175.516








embedded image


Compound 81





Potency: 12.5893



Efficacy: 213.226








embedded image


Compound 82





Potency: 3.1623



Efficacy: 96.4736








embedded image


Compound 83





Potency: 11.2202



Efficacy: 175.054








embedded image


Compound 84





Potency: 6.3096



Efficacy: 122.242








embedded image


Compound 85





Potency: 5.0119



Efficacy: 99.705








embedded image


Compound 86





Potency: 4.4668



Efficacy: 94.2018








embedded image


Compound 87





Potency: 5.0119



Efficacy: 66.4058








embedded image


Compound 88





Potency: 8.9125



Efficacy: 88.9864








embedded image


Compound 89





Potency: 5.0119



Efficacy: 86.1979








embedded image


Compound 90





Potency: 11.2202



Efficacy: 140.713








embedded image


Compound 91





Potency: 11.2202



Efficacy: 139.097








embedded image


Compound 92





Potency: 4.4668



Efficacy: 75.211








embedded image


Compound 93





Potency: 11.2202



Efficacy: 117.921








embedded image


Compound 94





Potency: 10



Efficacy: 128.349








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Compound 95





Potency: 7.0795



Efficacy: 98.2729








embedded image


Compound 96





Potency: 4.4668



Efficacy: 83.068








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Compound 97





Potency: 5.6234



Efficacy: 78.6154








embedded image


Compound 98





Potency: 10



Efficacy: 125.681








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Compound 99





Potency: 10



Efficacy: 121.073








embedded image


Compound 100





Potency: 3.9811



Efficacy: 95.5318








embedded image


Compound 101





Potency: 8.9125



Efficacy: 131.53








embedded image


Compound 102





Potency: 12.5893



Efficacy: 149.767








embedded image


Compound 103





Potency: 6.3096



Efficacy: 119.897








embedded image


Compound 104





Potency: 10



Efficacy: 137.595








embedded image


Compound 105





Potency: 10



Efficacy: 96.3714








embedded image


Compound 106





Potency: 7.0795



Efficacy: 81.3792








embedded image


Compound 107





Potency: 4.4668



Efficacy: 95.5906








embedded image


Compound 108





Potency: 12.5893



Efficacy: 139.967








embedded image


Compound 109





Potency: 12.5893



Efficacy: 166.495








embedded image


Compound 110





Potency: 12.5893



Efficacy: 180.783








embedded image


Compound 111





Potency: 7.0795



Efficacy: 99.2205








embedded image


Compound 112





Potency: 5.6234



Efficacy: 73.9187








embedded image


Compound 113





Potency: 10



Efficacy: 111.758








embedded image


Compound 114





Potency: 11.2202



Efficacy: 138.65








embedded image


Compound 115





Potency: 7.0795



Efficacy: 85.5436








embedded image


Compound 116





Potency: 8.9125



Efficacy: 104.576








embedded image


Compound 117





Potency: 7.9433



Efficacy: 88.998








embedded image


Compound 118





Potency: 5.0119



Efficacy: 82.7908








embedded image


Compound 119





Potency: 11.2202



Efficacy: 127.904








embedded image


Compound 120





Potency: 10



Efficacy: 114.827








embedded image


Compound 121





Potency: 8.9125



Efficacy: 92.062








embedded image


Compound 122





Potency: 10



Efficacy: 127.132








embedded image


Compound 123





Potency: 8.9125



Efficacy: 92.918








embedded image


Compound 124





Potency: 8.9125



Efficacy: 123.416








embedded image


Compound 125





Potency: 11.2202



Efficacy: 136.36








embedded image


Compound 126





Potency: 8.9125



Efficacy: 124.439








embedded image


Compound 127





Potency: 12.5893



Efficacy: 171.242








embedded image


Compound 128





Potency: 14.1254



Efficacy: 138.869








embedded image


Compound 129





Potency: 11.2202



Efficacy: 145.936








embedded image


Compound 130





Potency: 11.2202



Efficacy: 119.808








embedded image


Compound 131





Potency: 14.1254



Efficacy: 175.002








embedded image


Compound 132





Potency: 12.5893



Efficacy: 120.609








embedded image


Compound 133





Potency: 10



Efficacy: 100.712








embedded image


Compound 134





Potency: 10



Efficacy: 95.4818








embedded image


Compound 135





Potency: 10



Efficacy: 91.1222








embedded image


Compound 136





Potency: 8.9125



Efficacy: 89.1179








embedded image


Compound 137





Potency: 10



Efficacy: 81.023








embedded image


Compound 138





Potency: 11.2202



Efficacy: 135.363








embedded image


Compound 139





Potency: 11.2202



Efficacy: 67.3145








embedded image


Compound 140





Potency: 10



Efficacy: 81.9056








embedded image


Compound 141





Potency: 10



Efficacy: 100.669








embedded image


Compound 142





Potency: 8.9125



Efficacy: 89.9548








embedded image


Compound 143





Potency: 10



Efficacy: 76.5877








embedded image


Compound 144





Potency: 12.5893



Efficacy: 94.0383








embedded image


Compound 145





Potency: 10



Efficacy: 69.9943








embedded image


Compound 146





Potency: 10



Efficacy: 96.4095








embedded image


Compound 147





Potency: 11.2202



Efficacy: 90.1504








embedded image


Compound 148





Potency: 11.2202



Efficacy: 138.741








embedded image


Compound 149





Potency: 8.9125



Efficacy: 84.3701








embedded image


Compound 150





Potency: 10



Efficacy: 67.6116








embedded image


Compound 151





Potency: 10



Efficacy: 91.5246








embedded image


Compound 152





Potency: 12.5893



Efficacy: 97.2406








embedded image


Compound 153





Potency: 7.9433



Efficacy: 95.7207








embedded image


Compound 154





Potency: 3.1623



Efficacy: 93.8685








embedded image


Compound 155





Potency: 10



Efficacy: 93.0585








embedded image


Compound 156





Potency: 10



Efficacy: 67.8359








embedded image


Compound 157





Potency: 10



Efficacy: 70.1776








embedded image


Compound 158





Potency: 10



Efficacy: 71.256








embedded image


Compound 159





Potency: 11.2202



Efficacy: 109.576








embedded image


Compound 160





Potency: 11.2202



Efficacy: 103.065








embedded image


Compound 161





Potency: 3.9811



Efficacy: 97.9916








embedded image


Compound 162





Potency: 5.6234



Efficacy: 87.0645








embedded image


Compound 163





Potency: 10



Efficacy: 79.1483








embedded image


Compound 164





Potency: 10



Efficacy: 90.988








embedded image


Compound 165





Potency: 11.2202



Efficacy: 128.695








embedded image


Compound 166





Potency: 12.5893



Efficacy: 93.7937








embedded image


Compound 167





Potency: 7.0795



Efficacy: 86.8199








embedded image


Compound 168





Potency: 8.9125



Efficacy: 83.6415








embedded image


Compound 169





Potency: 10



Efficacy: 84.5587








embedded image


Compound 170





Potency: 10



Efficacy: 84.3344








embedded image


Compound 171





Potency: 10



Efficacy: 59.61








embedded image


Compound 172





Potency: 10



Efficacy: 65.8043








embedded image


Compound 173





Potency: 11.2202



Efficacy: 85.4536








embedded image


Compound 174





Potency: 11.2202



Efficacy: 104.045








embedded image


Compound 175





Potency: 5.6234



Efficacy: 116.69








embedded image


Compound 176





Potency: 10



Efficacy: 115.567








embedded image


Compound 177





Potency: 10



Efficacy: 70.6131








embedded image


Compound 178





Potency: 11.2202



Efficacy: 107.982








embedded image


Compound 179





Potency: 5.6234



Efficacy: 84.0774








embedded image


Compound 180





Potency: 10



Efficacy: 77.6845








embedded image


Compound 181





Potency: 12.5893



Efficacy: 80.5239








embedded image


Compound 182





Potency: 10



Efficacy: 99.1066








embedded image


Compound 183





Potency: 10



Efficacy: 98.6809








embedded image


Compound 184





Potency: 10



Efficacy: 86.1315








embedded image


Compound 185





Potency: 10



Efficacy: 117.72








embedded image


Compound 186





Potency: 3.9811



Efficacy: 93.5129








embedded image


Compound 187





Potency: 8.9125



Efficacy: 120.104








embedded image


Compound 188





Potency: 10



Efficacy: 88.0312








embedded image


Compound 189





Potency: 8.9125



Efficacy: 76.0346








embedded image


Compound 190





Potency: 11.2202



Efficacy: 116.431








embedded image


Compound 191





Potency: 11.2202



Efficacy: 115.598








embedded image


Compound 192





Potency: 11.2202



Efficacy: 74.0117








embedded image


Compound 193





Potency: 11.2202



Efficacy: 89.7146








embedded image


Compound 194





Potency: 8.9125



Efficacy: 76.8665








embedded image


Compound 195





Potency: 10



Efficacy: 99.4569








embedded image


Compound 196





Potency: 10



Efficacy: 67.0912








embedded image


Compound 197





Potency: 8.9125



Efficacy: 83.2038








embedded image


Compound 198





Potency: 10



Efficacy: 78.0861








embedded image


Compound 199





Potency: 12.5893



Efficacy: 96.0151








embedded image


Compound 200





Potency: 8.9125



Efficacy: 98.6316








embedded image


Compound 201





Potency: 11.2202



Efficacy: 94.7942








embedded image


Compound 202





Potency: 10



Efficacy: 83.9426








embedded image


Compound 203





Potency: 7.0795



Efficacy: 106.69








embedded image


Compound 204





Potency: 10



Efficacy: 70.2778








embedded image


Compound 205





Potency: 7.0795



Efficacy: 98.0553








embedded image


Compound 206





Potency: 11.2202



Efficacy: 77.9394








embedded image


Compound 207





Potency: 10



Efficacy: 75.3009








embedded image


Compound 208





Potency: 10



Efficacy: 83.7154








embedded image


Compound 209





Potency: 15.8489



Efficacy: 130.232








embedded image


Compound 210





Potency: 12.5893



Efficacy: 101.354








embedded image


Compound 211





Potency: 11.2202



Efficacy: 109.939








embedded image


Compound 212





Potency: 14.1254



Efficacy: 119.538








embedded image


Compound 213





Potency: 11.2202



Efficacy: 110.24








embedded image


Compound 214





Potency: 11.2202



Efficacy: 94.2928








embedded image


Compound 215





Potency: 10



Efficacy: 73.527








embedded image


Compound 216





Potency: 10



Efficacy: 90.7389








embedded image


Compound 217





Potency: 10



Efficacy: 56.3855








embedded image


Compound 218





Potency: 10



Efficacy: 58.0835








embedded image


Compound 219





Potency: 11.2202



Efficacy: 83.9475








embedded image


Compound 220





Potency: 11.2202



Efficacy: 86.9112








embedded image


Compound 221





Potency: 11.2202



Efficacy: 59.5046








embedded image


Compound 222





Potency: 12.5893



Efficacy: 56.9925
















TABLE 8





Compound data


























Fit
Fit

Fit Infinite
Fit Zero
Fit




Curve
LogA
Hill
Fit
Activity
Activity
Curve


No.
Score
Description
C50
Slope
R2
[%]
[%]
Class





1
94
Complete
−5.95
3.132
0.9985
195.171
−2.9752
1.1




curve; high




efficacy


2
91
Complete
−5.45
1.4163
0.9999
231.285
3.1995
1.1




curve; high




efficacy


3
89
Complete
−5.65
3.0654
0.9999
158.124
−35.3084
1.1




curve; high




efficacy


4
87
Complete
−5.55
1.8617
0.9998
136.905
7.6069
1.1




curve; high




efficacy


5
86
Complete
−5.95
2.5334
0.9999
87.4201
−5.0637
1.1




curve; high




efficacy


6
85
Complete
−5.95
1.3437
0.9999
69.589
−58.5311
1.1




curve; high




efficacy


7
85
Complete
−5.35
1.6924
0.9999
107.421
12.5
1.1




curve; high




efficacy


8
85
Complete
−5.55
4.9549
0.9996
96.0327
−15.6523
1.1




curve; high




efficacy


9
85
Complete
−5.55
1.3723
0.9999
92.2022
−30.5005
1.1




curve; high




efficacy


10
85
Complete
−5.5
2.8473
1
102.022
0
1.1




curve; high




efficacy


11
85
Complete
−5.4
2.5334
0.9997
101.384
−0.509
1.1




curve; high




efficacy


12
85
Complete
−5.55
1.4781
0.9999
102.305
9.3996
1.1




curve; high




efficacy


13
84
Complete
−5.35
3.9295
0.9991
94.7101
−27.2857
1.1




curve; high




efficacy


14
84
Complete
−5.65
2.3332
0.9995
62.8391
−21.9345
1.1




curve; high




efficacy


15
84
Complete
−5.5
1.6259
0.9999
75.2612
−16.1195
1.1




curve; high




efficacy


16
84
Complete
−5.65
1.3443
0.9999
77.492
−37.8568
1.1




curve; high




efficacy


17
84
Complete
−5.6
1.2475
0.9998
71.4307
−24.7641
1.1




curve; high




efficacy


18
84
Partial
−5
4.9549
0.9995
1760.36
8.7984
2.1




curve; high




efficacy


19
84
Complete
−5.85
1.3437
0.9992
52.7174
−28.8963
1.1




curve; high




efficacy


20
84
Complete
−5.45
2.3031
0.9999
83.8587
−17.7204
1.1




curve; high




efficacy


21
84
Complete
−5.25
4.4495
0.9992
93.4556
1.1731
1.1




curve; high




efficacy


22
84
Complete
−5.35
4.9549
0.9999
83.1353
−0.2899
1.1




curve; high




efficacy


23
84
Complete
−5.4
2.9023
0.9998
98.4745
13.3122
1.1




curve; high




efficacy


24
84
Complete
−5.5
2.1211
0.9996
82.7045
5.7274
1.1




curve; high




efficacy


25
84
Complete
−5.65
1.6604
0.9995
64.1141
−18.7854
1.1




curve; high




efficacy


26
84
Complete
−5.7
1.1
1
59.5556
−36.1807
1.1




curve; high




efficacy


27
83
Complete
−5.7
2.2526
0.9994
54.9511
−26.8995
1.1




curve; high




efficacy


28
83
Complete
−5.55
2.5334
0.9999
55.3257
−36.6914
1.1




curve; high




efficacy


29
83
Complete
−5.4
1.7885
0.9997
55.7271
−25.4387
1.1




curve; high




efficacy


30
83
Complete
−5.4
2.1876
0.9999
73.6408
−19.157
1.1




curve; high




efficacy


31
83
Complete
−5.6
1.9673
0.9999
62.1733
−24.2621
1.1




curve; high




efficacy


32
83
Complete
−5.55
2.1211
1
60.2337
−28.5623
1.1




curve; high




efficacy


33
82
Complete
−5.7
1.1
0.9991
33.1603
−50.1655
1.1




curve; high




efficacy


34
65
Complete
−5.85
1.8851
0.9995
76.3552
5.9015
1.2




curve; partial




efficacy


35
64
Complete
−5.6
3.132
0.9995
72.5763
−0.1751
1.2




curve; partial




efficacy


36
64
Complete
−5.5
1.4163
0.9997
81.2047
5.5
1.2




curve; partial




efficacy


37
64
Complete
−5.6
2.4064
0.9991
74.4523
−2.5935
1.2




curve; partial




efficacy


38
63
Complete
−5.35
4.9549
0.9992
73.5449
−2.0156
1.2




curve; partial




efficacy


39
63
Complete
−5.45
1.7529
0.9998
69.5378
−9.0026
1.2




curve; partial




efficacy


40
48
Partial
−4.85
4.5045
0.9986
433.479
−3.212
2.1




curve; high




efficacy


41
48
Partial
−4.85
4.5045
1
456.749
8.8874
2.1




curve; high




efficacy


42
48
Partial
−4.85
4.9549
0.9987
484.883
9.6207
2.1




curve; high




efficacy


43
47
Partial
−5.4
0.9
0.9999
148.01
0
2.1




curve; high




efficacy


44
47
Partial
−4.95
4.095
0.9997
310.669
−17.6073
2.1




curve; high




efficacy


45
47
Partial
−4.85
4.5045
0.9989
419.509
−7.0263
2.1




curve; high




efficacy


46
46
Partial
−5.1
1.01
0.9992
195.813
−35.6022
2.1




curve; high




efficacy


47
46
Partial
−5.25
1.331
0.9993
153.844
12.5272
2.1




curve; high




efficacy


48
46
Partial
−5.15
1.7137
0.9992
178.694
−35.4511
2.1




curve; high




efficacy


49
46
Partial
−5.25
1.9282
0.9996
153.971
2.973
2.1




curve; high




efficacy


50
46
Partial
−5.1
1.8851
0.9989
186.381
5.0579
2.1




curve; high




efficacy


51
46
Partial
−5
4.9549
0.9988
249.6
−23.8252
2.1




curve; high




efficacy


52
46
Partial
−5.35
1.4781
0.9998
137.586
−27.7073
2.1




curve; high




efficacy


53
45
Partial
−5.15
0.8
0.9991
152.283
−11.267
2.1




curve; high




efficacy


54
45
Partial
−4.9
1.1341
1
262.045
39.3368
2.1




curve; high




efficacy


55
45
Partial
−4.9
4.9549
0.9985
252.825
−18.4203
2.1




curve; high




efficacy


56
45
Partial
−5.4
1.5095
0.9999
110.65
21.8862
2.1




curve; high




efficacy


57
45
Partial
−5.35
1.7529
0.9999
115.173
−0.5596
2.1




curve; high




efficacy


58
45
Partial
−5.45
0.9
0.999
95.6282
−42.3624
2.1




curve; high




efficacy


59
45
Partial
−5
3.5722
0.9991
185.351
−3.6299
2.1




curve; high




efficacy


60
45
Partial
−5.35
1.7529
0.9992
116.389
−12.5146
2.1




curve; high




efficacy


61
45
Partial
−5.3
1.1705
0.9991
136.382
−3.94
2.1




curve; high




efficacy


62
45
Partial
−5.15
1.9673
0.9992
162.393
−23.864
2.1




curve; high




efficacy


63
45
Partial
−5
3.1925
0.9993
198.688
−1.7425
2.1




curve; high




efficacy


64
45
Partial
−5.3
2.4729
0.9996
129.946
−4.9734
2.1




curve; high




efficacy


65
45
Partial
−4.7
4.095
0.9991
501.215
9.2299
2.1




curve; high




efficacy


66
45
Partial
−4.9
4.5045
0.9991
227.168
11.9175
2.1




curve; high




efficacy


67
44
Partial
−5
1.4163
0.9999
162.351
4.7344
2.1




curve; high




efficacy


68
44
Partial
−4.9
4.5045
0.9986
221.625
−17.9914
2.1




curve; high




efficacy


69
44
Partial
−5.4
1.3987
0.9999
88.0721
−2.436
2.1




curve; high




efficacy


70
44
Partial
−5.35
1.2876
0.9999
96.1281
−19.1057
2.1




curve; high




efficacy


71
44
Partial
−5.1
1.3443
0.9999
122.331
−2.1806
2.1




curve; high




efficacy


72
44
Partial
−5.05
2.3332
0.9987
136.354
−2.3089
2.1




curve; high




efficacy


73
44
Partial
−4.9
4.095
0.9993
186.64
16.3113
2.1




curve; high




efficacy


74
44
Partial
−5.35
1.8617
1
86.169
−23.105
2.1




curve; high




efficacy


75
44
Partial
−5.4
1.5579
0.9999
98.3982
26.9551
2.2




curve; partial




efficacy


76
44
Partial
−5.2
1.3443
0.9997
105.907
−8.518
2.1




curve; high




efficacy


77
44
Partial
−5.35
1.8617
1
87.1172
−34.2328
2.1




curve; high




efficacy


78
44
Partial
−5
1.7137
0.9999
162.598
9.6566
2.1




curve; high




efficacy


79
44
Partial
−5.35
1.3437
0.9999
84.8433
−0.3646
2.1




curve; high




efficacy


80
44
Partial
−5
1.4163
0.9999
169.032
−6.484
2.1




curve; high




efficacy


81
44
Partial
−4.9
4.5045
0.9998
216.419
3.1931
2.1




curve; high




efficacy


82
44
Partial
−5.5
1.1341
1
89.9991
−6.4745
2.1




curve; high




efficacy


83
44
Partial
−4.95
2.0937
1
162.065
−12.9886
2.1




curve; high




efficacy


84
44
Partial
−5.2
1.2221
0.9999
114.278
−7.9639
2.1




curve; high




efficacy


85
44
Partial
−5.3
1.8617
0.9999
102.938
3.2331
2.1




curve; high




efficacy


86
44
Partial
−5.35
0.8
0.9999
94.6981
0.4963
2.1




curve; high




efficacy


87
44
Partial
−5.3
2.1211
0.9997
87.756
21.3502
2.2




curve; partial




efficacy


88
43
Partial
−5.05
1.01
0.9999
98.4045
9.418
2.1




curve; high




efficacy


89
43
Partial
−5.3
1.6924
0.999
75.3022
−10.8957
2.2




curve; partial




efficacy


90
43
Partial
−4.95
4.5045
0.999
146.447
5.7331
2.1




curve; high




efficacy


91
43
Partial
−4.95
4.095
0.9986
122.391
−16.7058
2.1




curve; high




efficacy


92
43
Partial
−5.35
1.6259
0.9999
76.9712
1.7602
2.2




curve; partial




efficacy


93
43
Partial
−4.95
1.7529
1
114.284
−3.6365
2.1




curve; high




efficacy


94
43
Partial
−5
4.9549
0.9996
138.031
9.6823
2.1




curve; high




efficacy


95
43
Partial
−5.15
1.01
0.9993
83.3763
−14.8966
2.2




curve; partial




efficacy


96
43
Partial
−5.35
1.9887
0.9998
74.9858
−8.0822
2.2




curve; partial




efficacy


97
43
Partial
−5.25
3.132
0.9994
79.8262
1.2108
2.2




curve; partial




efficacy


98
43
Partial
−5
4.5045
1
125.633
−0.0479
2.1




curve; high




efficacy


99
43
Partial
−5
2.1211
0.9999
121.073
0
2.1




curve; high




efficacy


100
43
Partial
−5.4
1.01
0.9992
64.0202
−31.5116
2.1




curve; high




efficacy


101
43
Partial
−5.05
1.6604
0.9989
115.659
−15.8708
2.1




curve; high




efficacy


102
43
Partial
−4.9
4.5045
0.9999
160.171
10.4042
2.1




curve; high




efficacy


103
43
Partial
−5.2
1.8265
0.9986
96.8922
−23.0045
2.1




curve; high




efficacy


104
43
Partial
−5
3.6772
0.9988
128.477
−9.1182
2.1




curve; high




efficacy


105
43
Partial
−5
3.5117
0.9995
113.218
16.8467
2.1




curve; high




efficacy


106
43
Partial
−5.15
1.21
1
79.2242
−2.155
2.2




curve; partial




efficacy


107
43
Partial
−5.35
1.4781
0.9998
76.3701
−19.2206
2.1




curve; high




efficacy


108
43
Partial
−4.9
4.5045
0.9999
142.05
2.0834
2.1




curve; high




efficacy


109
43
Partial
−4.9
4.9549
0.9994
174.079
7.5839
2.1




curve; high




efficacy


110
43
Partial
−4.9
4.5045
0.9999
162.565
−18.2174
2.1




curve; high




efficacy


111
43
Partial
−5.15
1.21
0.9999
101.431
2.2101
2.1




curve; high




efficacy


112
43
Partial
−5.25
3.132
1
74.0416
0.1229
2.2




curve; partial




efficacy


113
43
Partial
−5
3.9295
0.9994
105.867
−5.8907
2.1




curve; high




efficacy


114
43
Partial
−4.95
4.5045
0.9989
134.828
−3.8223
2.1




curve; high




efficacy


115
43
Partial
−5.15
1.8265
0.9997
80.6607
−4.8829
2.2




curve; partial




efficacy


116
43
Partial
−5.05
1.5579
0.9999
94.7519
−9.8246
2.1




curve; high




efficacy


117
43
Partial
−5.1
1.6604
0.9998
88.7778
−0.2202
2.1




curve; high




efficacy


118
43
Partial
−5.3
1.6924
0.9992
74.8733
−7.9176
2.2




curve; partial




efficacy


119
43
Partial
−4.95
4.9549
0.9997
111.883
−16.0213
2.1




curve; high




efficacy


120
43
Partial
−5
0.8
0.9996
126.76
11.933
2.1




curve; high




efficacy


121
43
Partial
−5.05
1.8851
0.9997
97.7753
5.7133
2.1




curve; high




efficacy


122
43
Partial
−5
1.2475
0.9999
127.656
0.5248
2.1




curve; high




efficacy


123
43
Partial
−5.05
1.3443
0.9999
96.9016
3.9836
2.1




curve; high




efficacy


124
43
Partial
−5.05
1.2475
0.9999
102.977
−20.4393
2.1




curve; high




efficacy


125
43
Partial
−4.95
1.1705
0.9999
133.669
−2.6906
2.1




curve; high




efficacy


126
43
Partial
−5.05
1
1
125.112
0.673
2.1




curve; high




efficacy


127
43
Partial
−4.9
4.5045
0.9995
152.332
−18.9102
2.1




curve; high




efficacy


128
43
Partial
−4.85
4.5045
0.9991
153.542
14.6735
2.1




curve; high




efficacy


129
43
Partial
−4.95
4.095
0.9985
128.069
−17.8675
2.1




curve; high




efficacy


130
43
Partial
−4.95
4.5045
0.9998
127.62
7.8122
2.1




curve; high




efficacy


131
43
Partial
−4.85
4.9549
0.9997
183.761
8.7591
2.1




curve; high




efficacy


132
43
Partial
−4.9
4.9549
0.9996
133.42
12.8108
2.1




curve; high




efficacy


133
42
Partial
−5
2.7202
0.9992
73.1459
−27.5656
2.1




curve; high




efficacy


134
42
Partial
−5
4.4495
0.9993
72.9186
−22.5632
2.1




curve; high




efficacy


135
42
Partial
−5
4.9549
0.9997
61.8199
−29.3022
2.1




curve; high




efficacy


136
42
Partial
−5.05
0.8
0.9997
85.8101
−3.3079
2.2




curve; partial




efficacy


137
42
Partial
−5
4.9549
0.9999
61.7369
−19.2861
2.1




curve; high




efficacy


138
42
Partial
−4.95
1.3437
1
107.998
−27.3652
2.1




curve; high




efficacy


139
42
Partial
−4.95
4.5045
0.9999
82.0264
14.712
2.1




curve; high




efficacy


140
42
Partial
−5
2.7868
0.9991
77.8665
−4.0391
2.2




curve; partial




efficacy


141
42
Partial
−5
4.9549
0.9998
82.7722
−17.8968
2.1




curve; high




efficacy


142
42
Partial
−5.05
2.4064
0.9988
71.1773
−18.7775
2.1




curve; high




efficacy


143
42
Partial
−5
4.4495
0.9999
81.2596
4.6719
2.1




curve; high




efficacy


144
42
Partial
−4.9
4.095
0.9995
103.816
9.7776
2.1




curve; high




efficacy


145
42
Partial
−5
4.9549
0.9999
70.4611
0.4668
2.2




curve; partial




efficacy


146
42
Partial
−5
4.9549
0.9999
75.4854
−20.924
2.1




curve; high




efficacy


147
42
Partial
−4.95
2.0937
0.9999
91.2034
1.0531
2.1




curve; high




efficacy


148
42
Partial
−4.95
2.3531
0.9997
109.969
−28.7719
2.1




curve; high




efficacy


149
42
Partial
−5.05
1.111
0.9999
82.4023
−1.9678
2.2




curve; partial




efficacy


150
42
Partial
−5
4.5045
0.9999
67.4005
−0.2112
2.2




curve; partial




efficacy


151
42
Partial
−5
3.132
0.9999
65.1432
−26.3814
2.1




curve; high




efficacy


152
42
Partial
−4.9
1.1341
0.9999
88.7605
−8.4801
2.2




curve; partial




efficacy


153
42
Partial
−5.1
1.5095
0.9998
74.4536
−21.2671
2.1




curve; high




efficacy


154
42
Partial
−5.5
0.9
0.9999
47.5155
−46.353
2.1




curve; high




efficacy


155
42
Partial
−5
3.99
0.9998
60.936
−32.1225
2.1




curve; high




efficacy


156
42
Partial
−5
2.5334
0.9999
67.8023
−0.0336
2.2




curve; partial




efficacy


157
42
Partial
−5
3.6272
0.9989
68.2216
−1.9559
2.2




curve; partial




efficacy


158
42
Partial
−5
1.9282
0.9994
70.7327
−0.5234
2.2




curve; partial




efficacy


159
42
Partial
−4.95
4.095
0.9995
106.672
−2.9033
2.1




curve; high




efficacy


160
42
Partial
−4.95
4.095
0.9998
100.584
−2.4804
2.1




curve; high




efficacy


161
42
Partial
−5.4
1.1
0.9998
55.0498
−42.9418
2.1




curve; high




efficacy


162
42
Partial
−5.25
2.2526
1
55.2524
−31.8121
2.1




curve; high




efficacy


163
42
Partial
−5
4.045
0.9999
78.2931
−0.8551
2.2




curve; partial




efficacy


164
42
Partial
−5
4.5045
0.9998
91.825
0.8371
2.1




curve; high




efficacy


165
42
Partial
−4.95
4.5045
0.9999
108.575
−20.12
2.1




curve; high




efficacy


166
42
Partial
−4.9
1.3987
0.9998
104.163
10.3691
2.1




curve; high




efficacy


167
42
Partial
−5.15
1.7885
0.9995
48.5966
−38.2233
2.1




curve; high




efficacy


168
42
Partial
−5.05
1.9673
0.9989
82.861
−0.7805
2.2




curve; partial




efficacy


169
42
Partial
−5
4.4495
0.9987
64.8483
−19.7104
2.1




curve; high




efficacy


170
42
Partial
−5
4.4495
0.9988
87.8075
3.4732
2.1




curve; high




efficacy


171
42
Partial
−5
3.132
0.9999
69.5258
9.9159
2.2




curve; partial




efficacy


172
42
Partial
−5
2.3332
0.9987
73.8043
8
2.2




curve; partial




efficacy


173
42
Partial
−4.95
0.9
0.9987
93.0488
7.5952
2.2




curve; partial




efficacy


174
42
Partial
−4.95
1.8579
0.9993
83.6585
−20.386
2.1




curve; high




efficacy


175
42
Partial
−5.25
1.3443
0.9985
54.0029
−62.687
2.1




curve; high




efficacy


176
42
Partial
−5
4.9549
0.9985
96.131
−19.4361
2.1




curve; high




efficacy


177
42
Partial
−5
4.9549
0.9998
71.5284
0.9153
2.2




curve; partial




efficacy


178
42
Partial
−4.95
1.1341
0.9994
85.3274
−22.6542
2.1




curve; high




efficacy


179
42
Partial
−5.25
2.2526
0.9998
48.386
−35.6913
2.1




curve; high




efficacy


180
42
Partial
−5
3.1925
0.9991
76.0607
−1.6238
2.2




curve; partial




efficacy


181
42
Partial
−4.9
4.9549
1
93.0239
12.5
2.1




curve; high




efficacy


182
42
Partial
−5
2.2481
0.9998
98.2495
−0.8571
2.1




curve; high




efficacy


183
42
Partial
−5
4.4495
0.9997
99.728
1.0471
2.1




curve; high




efficacy


184
42
Partial
−5
2.9023
0.9998
85.5304
−0.6011
2.1




curve; high




efficacy


185
42
Partial
−5
4.9549
0.9994
94.2492
−23.4705
2.1




curve; high




efficacy


186
42
Partial
−5.4
0.9
0.9999
47.9827
−45.5302
2.1




curve; high




efficacy


187
42
Partial
−5.05
1.3437
0.9992
65.7373
−54.3669
2.1




curve; high




efficacy


188
42
Partial
−5
4.5045
0.9999
88.2262
0.195
2.1




curve; high




efficacy


189
42
Partial
−5.05
0.8
0.9999
84.5737
8.5392
2.2




curve; partial




efficacy


190
42
Partial
−4.95
2.0937
0.9991
87.524
−28.9068
2.1




curve; high




efficacy


191
42
Partial
−4.95
4.9549
0.9998
96.2795
−19.3188
2.1




curve; high




efficacy


192
42
Partial
−4.95
4.095
0.9992
76.4906
2.4789
2.2




curve; partial




efficacy


193
42
Partial
−4.95
4.095
0.9997
92.2616
2.547
2.1




curve; high




efficacy


194
42
Partial
−5.05
1.7137
0.9999
73.9084
−2.9581
2.2




curve; partial




efficacy


195
42
Partial
−5
4.045
0.9987
89.5777
−9.8792
2.1




curve; high




efficacy


196
42
Partial
−5
2.8473
0.9999
66.5821
−0.5091
2.2




curve; partial




efficacy


197
42
Partial
−5.05
0.9
0.9999
84.7038
1.5
2.2




curve; partial




efficacy


198
42
Partial
−5
4.9549
0.9993
79.1775
1.0914
2.2




curve; partial




efficacy


199
42
Partial
−4.9
1.2876
0.9988
96.2012
0.1862
2.2




curve; partial




efficacy


200
42
Partial
−5.05
1.4781
0.9999
56.5654
−42.0662
2.1




curve; high




efficacy


201
42
Partial
−4.95
2.3531
0.9987
74.9126
−19.8816
2.1




curve; high




efficacy


202
42
Partial
−5
1.8617
0.9999
80.5565
−3.3861
2.2




curve; partial




efficacy


203
42
Partial
−5.15
1.4781
0.9998
59.3901
−47.2995
2.1




curve; high




efficacy


204
42
Partial
−5
3.5722
0.9999
70.7642
0.4864
2.2




curve; partial




efficacy


205
42
Partial
−5.15
1.4781
0.9999
57.7575
−40.2978
2.1




curve; high




efficacy


206
42
Partial
−4.95
4.095
0.9985
91.8921
13.9528
2.1




curve; high




efficacy


207
42
Partial
−5
2.5334
0.9998
75.4708
0.1699
2.2




curve; partial




efficacy


208
42
Partial
−5
3.1925
0.9988
74.5741
−9.1413
2.2




curve; partial




efficacy


209
42
Partial
−4.8
4.095
0.9997
142.41
12.1779
2.1




curve; high




efficacy


210
42
Partial
−4.9
4.9549
0.9986
113.27
11.9156
2.1




curve; high




efficacy


211
42
Partial
−4.95
1.1341
0.9991
109.674
−0.265
2.1




curve; high




efficacy


212
42
Partial
−4.85
4.5045
0.9993
128.55
9.0119
2.1




curve; high




efficacy


213
42
Partial
−4.95
1.2876
0.9999
109.228
−1.0122
2.1




curve; high




efficacy


214
42
Partial
−4.95
2.0937
0.9997
108.196
13.9034
2.1




curve; high




efficacy


215
42
Partial
−5
2.1211
0.9999
87.4293
13.9023
2.1




curve; high




efficacy


216
42
Partial
−5
0.8
0.9996
94.4089
3.67
2.2




curve; partial




efficacy


217
42
Partial
−5
3.0654
0.9995
66.1813
9.7958
2.2




curve; partial




efficacy


218
42
Partial
−5
3.6272
0.9998
71.8402
13.7566
2.2




curve; partial




efficacy


219
42
Partial
−4.95
1.5936
0.9993
76.3682
−7.5793
2.2




curve; partial




efficacy


220
42
Partial
−4.95
3.2975
0.9995
95.08
8.1688
2.1




curve; high




efficacy


221
41
Partial
−4.95
4.095
0.9994
66.5046
7
2.2




curve; partial




efficacy


222
41
Partial
−4.9
4.095
0.9999
66.8444
9.8519
2.2




curve; partial




efficacy


















Max
Activity
Activity
Activity
Activity



Excl'd
Response
at 0.307
at 1.530
at 7.660
at 38.30


No.
Points
[%]
uM [%] **
uM [%] **
uM [%] **
uM [%] **





1
0 0 0 0
190.551
0
142.678
198.344
190.551


2
0 0 0 0
222.818
9.5199
56.9884
175.022
222.818


3
0 0 0 0
158.59
−34.2446
9.5695
153.966
158.59


4
0 0 0 0
136.632
9.8511
38.314
118.549
136.632


5
0 0 0 0
87.1241
−1.7993
58.4455
87.2456
87.1241


6
0 0 0 0
68.1048
−39.7851
19.1903
60.5602
68.1048


7
0 0 0 0
104.699
13.4132
25.2727
80.6487
104.699


8
0 0 0 0
94.3383
−15.3725
−10.6082
96.4184
94.3383


9
0 0 0 0
88.6572
−24.4743
6.2893
68.0567
88.6572


10
0 0 0 0
101.819
0
11.3964
94.1328
101.819


11
0 0 0 0
101.791
0
7.5981
83.8704
101.791


12
0 0 0 0
100.299
12.3078
36.4535
84.576
100.299


13
0 0 0 0
95.6495
−24.7665
−27.3506
80.6874
95.6495


14
0 0 0 0
61.7295
−21.6121
3.5416
59.0823
61.7295


15
0 0 0 0
73.6236
−13.6594
4.7051
58.0277
73.6236


16
0 0 0 0
75.2018
−30.2295
5.6414
58.1985
75.2018


17
0 0 0 0
67.6004
−18.3073
9.3675
52.6808
67.6004


18
0 0 0 0
1767.43
7.745
30.584
357.845
1767.43


19
0 0 0 0
52.0677
−20.1458
14.9627
43.8719
52.0677


20
0 0 0 0
82.8998
−18.1348
−4.4619
69.3278
82.8998


21
0 0 0 0
93.2691
2.856
0
75.5731
93.2691


22
0 0 0 0
82.8991
0
0
78.2915
82.8991


23
0 0 0 0
98.87
12.6255
18.8414
87.3421
98.87


24
0 0 0 0
81.3384
5.8865
19.6198
73.0848
81.3384


25
0 0 0 0
63.0097
−16.5079
11.1354
54.5336
63.0097


26
0 0 0 0
56.0664
−25.4416
4.5885
41.9493
56.0664


27
0 0 0 0
53.7617
−26.1631
3.3138
51.6127
53.7617


28
0 0 0 0
54.6586
−35.9928
−20.5758
48.8507
54.6586


29
0 0 0 0
54.9055
−23.8063
−13.4709
35.9591
54.9055


30
0 0 0 0
73.4371
−18.3179
−9.0684
55.4601
73.4371


31
0 0 0 0
62.3312
−22.9164
−0.943
53.2493
62.3312


32
0 0 0 0
59.833
−28.0565
−9.4589
51.0458
59.833


33
0 0 0 0
31.0963
−41.8046
−14.3237
16.9691
31.0963


34
0 0 0 0
75.3947
9.4093
43.8029
74.3361
75.3947


35
0 0 0 0
71.7515
0
12.0467
71.5303
71.7515


36
0 0 0 0
78.5607
7.6747
25.7129
65.0516
78.5607


37
0 0 0 0
72.867
−1.7745
15.279
70.7278
72.867


38
0 0 0 0
72.6726
−0.6248
−2.8833
69.1705
72.6726


39
0 0 0 0
69.0926
−7.7641
5.5069
52.9348
69.0926


40
0 0 0 0
427.494
−13.7066
5.577
25.2218
427.494


41
0 0 0 0
453.124
8.7547
9.7092
34.1487
453.124


42
0 0 0 0
481.034
20.1308
0
32.0577
481.034


43
0 0 0 0
131.214
13.6807
43.9438
94.2627
131.214


44
0 0 0 0
308.204
−13.4703
−18.7598
38.1168
308.204


45
0 0 0 0
413.716
−16.6135
0.0514
18.6133
413.716


46
0 0 0 0
156.151
−29.6685
4.4407
77.5979
156.151


47
0 0 0 0
142.448
17.1767
32.1719
97.3497
142.448


48
0 0 0 0
165.458
−32.0109
−23.2923
76.5902
165.458


49
0 0 0 0
150.952
2.3042
15.4629
102.178
150.952


50
0 0 0 0
176.497
7.9011
9.4014
94.1426
176.497


51
0 0 0 0
249.102
−27.9345
−17.6035
31.4284
249.102


52
0 0 0 0
130.29
−25.6891
1.731
86.3564
130.29


53
0 0 0 0
118.6
0
28.0603
71.9463
118.6


54
0 0 0 0
213.045
42.0153
58.6176
119.863
213.045


55
0 0 0 0
252.32
−24.5803
−12.1219
3.459
252.32


56
0 0 0 0
107.846
23.9844
38.0808
86.6615
107.846


57
0 0 0 0
112.914
1.0034
14.5781
82.1622
112.914


58
0 0 0 0
82.8269
−29.6769
3.3459
49.0869
82.8269


59
0 0 0 0
183.88
0
−6.1928
48.4893
183.88


60
0 0 0 0
115.466
−10.9556
4.4512
78.2781
115.466


61
0 0 0 0
126.28
−0.1233
25.8337
82.3962
126.28


62
0 0 0 0
154.66
−21.3534
−18.0137
77.9021
154.66


63
0 0 0 0
195.944
−3.9508
2.2361
57.8488
195.944


64
0 0 0 0
128.914
−6.3997
3.462
95.0937
128.914


65
0 0 0 0
464.087
8.214
2.5941
27.1667
464.087


66
0 0 0 0
225.365
16.1816
8.6821
32.7652
225.365


67
0 0 0 0
141.667
6.398
14.4975
69.8045
141.667


68
0 0 0 0
219.866
−11.823
−22.0487
4.4563
219.866


69
0 0 0 0
84.3602
0
16.1931
62.7677
84.3602


70
0 0 0 0
89.5048
−15.4839
4.1098
57.1687
89.5048


71
0 0 0 0
109.03
0
9.6296
58.7582
109.03


72
0 0 0 0
132.126
0.9098
−2.4849
54.5396
132.126


73
0 0 0 0
185.158
13.4604
18.6219
35.6621
185.158


74
0 0 0 0
84.2199
−22.8223
−9.7273
56.677
84.2199


75
0 0 0 0
95.9047
28.0842
39.9836
79.7093
95.9047


76
0 0 0 0
96.9841
−5.8134
5.6525
55.1076
96.9841


77
0 0 0 0
85.0608
−33.466
−19.7149
54.711
85.0608


78
0 0 0 0
148.085
10.1341
15.4808
69.8478
148.085


79
0 0 0 0
80.1706
1.5242
16.235
57.3461
80.1706


80
0 0 0 0
145.969
−4.7753
4.5639
65.9307
145.969


81
0 0 0 0
214.702
1.8272
5.1171
24.2861
214.702


82
0 0 0 0
84.7449
0
23.1094
63.7317
84.7449


83
0 0 0 0
149.231
−13.5366
−10.5766
41.5268
149.231


84
0 0 0 0
102.4
−5.2905
11.0012
59.7194
102.4


85
0 0 0 0
100.92
3.2508
13.2564
71.6469
100.92


86
0 0 0 0
80.4147
10.3775
28.9147
57.3965
80.4147


87
0 0 0 0
86.5444
20.8761
26.4772
69.1414
86.5444


88
0 0 0 0
81.7691
12.5888
21.7607
50.5291
81.7691


89
0 0 0 0
74.1053
−9.9886
−0.2967
45.5813
74.1053


90
0 0 0 0
146.154
2.8256
8.1166
27.152
146.154


91
0 0 0 0
121.42
−19.1946
−13.3374
6.5712
121.42


92
0 0 0 0
74.9657
2.5679
13.3008
54.89
74.9657


93
0 0 0 0
101.857
−3.4693
0
36.266
101.857


94
0 0 0 0
137.756
11.4997
8.6578
36.5266
137.756


95
0 0 0 0
67.941
−12.4138
3.0575
36.2186
67.941


96
0 0 0 0
73.1974
−7.8942
1.1034
54.4732
73.1974


97
0 0 0 0
79.2643
1.9074
1.3725
59.0705
79.2643


98
0 0 0 0
125.383
−0.0399
0.1051
28.903
125.383


99
0 0 0 0
114.004
0.7442
1.7316
44.4339
114.004


100
0 0 0 0
56.3462
−24.5207
−4.9865
30.1418
56.3462


101
0 0 0 0
104.197
−14.2124
−11.5581
43.2955
104.197


102
0 0 0 0
158.9
10.2684
11.2981
24.3058
158.9


103
0 0 0 0
91.3206
−22.0036
−16.3773
50.2037
91.3206


104
0 0 0 0
127.457
−6.4868
−11.6559
27.3282
127.457


105
0 0 0 0
112.32
15.3847
17.8982
44.2423
112.32


106
0 0 0 0
70.115
−0.495
9.0228
40.4026
70.115


107
0 0 0 0
73.3496
−17.2197
−3.2434
45.9779
73.3496


108
0 0 0 0
140.923
2.0308
3.1422
15.3462
140.923


109
0 0 0 0
173.731
5.1999
10.0589
20.86
173.731


110
0 0 0 0
161.275
−18.9356
−17.8813
0
161.275


111
0 0 0 0
89.9207
4.1224
15.4494
54.5855
89.9207


112
0 0 0 0
73.7713
0
1.5319
54.0265
73.7713


113
0 0 0 0
105.027
−4.9426
−7.7278
23.4771
105.027


114
0 0 0 0
134.559
−6.7171
−1.2468
17.1799
134.559


115
0 0 0 0
77.6085
−3.9401
0
40.2449
77.6085


116
0 0 0 0
84.9032
−9.1033
−3.9425
36.7288
84.9032


117
0 0 0 0
82.2017
0
5.0693
43.6203
82.2017


118
0 0 0 0
73.5555
−8.1247
2.294
48.7391
73.5555


119
0 0 0 0
111.66
−17.1068
−14.6664
0.6285
111.66


120
0 0 0 0
97.3577
17.804
33.8372
63.3531
97.3577


121
0 0 0 0
92.5902
6.8559
8.4589
45.0425
92.5902


122
0 0 0 0
107.455
2.4969
11.4724
54.0343
107.455


123
0 0 0 0
85.4512
5.1227
12.1485
45.1238
85.4512


124
0 0 0 0
85.8569
−19.2219
−7.3929
35.4594
85.8569


125
0 0 0 0
107.624
0
9.2558
50.3725
107.624


126
0 0 0 0
101.717
5.1764
18.9708
58.0571
101.717


127
0 0 0 0
151.123
−20.0375
−16.1406
−2.9507
151.123


128
0 0 0 0
152.324
17.3412
12.5761
22.6146
152.324


129
0 0 0 0
127.052
−21.7078
−15.591
8.9584
127.052


130
0 0 0 0
127.365
7.1779
9.1804
26.1033
127.365


131
0 0 0 0
182.302
10.6925
7.1202
16.169
182.302


132
0 0 0 0
133.154
11.1461
13.8268
23.024
133.154


133
0 0 0 0
70.2432
−25.994
−28.4428
5.985
70.2432


134
0 0 0 0
72.8618
−20.9617
−23.9361
−0.0686
72.8618


135
0 0 0 0
62.005
−28.5367
−29.3616
−11.1775
62.005


136
0 0 0 0
65.1019
2.0231
14.5105
38.0502
65.1019


137
0 0 0 0
61.7759
−19.2435
−19.3578
−1.6699
61.7759


138
0 0 0 0
85.8466
−26.0685
−19.0518
23.5861
85.8466


139
0 0 0 0
81.696
15.171
14.5765
24.988
81.696


140
0 0 0 0
75.9628
−2.7644
−4.956
22.8114
75.9628


141
0 0 0 0
82.663
−17.1706
−18.2787
2.6033
82.663


142
0 0 0 0
68.9039
−17.1048
−18.6659
16.7527
68.9039


143
0 0 0 0
80.844
4.2822
4.8849
22.4204
80.844


144
0 0 0 0
102.992
8.5445
11.1102
20.4218
102.992


145
0 0 0 0
70.5123
0.3023
0.1807
15.6647
70.5123


146
0 0 0 0
75.4881
−20.8468
−20.9197
0
75.4881


147
0 0 0 0
84.9194
0.4502
2.7027
29.0697
84.9194


148
0 0 0 0
102.444
−30.0492
−26.8413
12.5122
102.444


149
0 0 0 0
68.0415
−0.1259
8.5917
37.0328
68.0415


150
0 0 0 0
67.1116
0
0.1599
15.1419
67.1116


151
0 0 0 0
63.6617
−26.5586
−25.9538
2.045
63.6617


152
0 0 0 0
67.3709
−6.7254
0
26.2892
67.3709


153
0 0 0 0
66.0451
−20.7719
−14.2846
26.1958
66.0451


154
0 0 0 0
38.9061
−36.1249
−14.4105
17.8641
38.9061


155
0 0 0 0
60.5343
−32.0766
−31.7653
−9.1234
60.5343


156
0 0 0 0
65.3359
0
0
22.9027
65.3359


157
0 0 0 0
67.4685
−0.7059
−3.266
17.7416
67.4685


158
0 0 0 0
65.8674
0.6705
0.6338
26.3415
65.8674


159
0 0 0 0
105.826
−4.2541
−1.9112
17.1057
105.826


160
0 0 0 0
99.7859
−2.4977
−3.3563
16.0042
99.7859


161
0 0 0 0
48.073
−37.0265
−17.738
22.4249
48.073


162
0 0 0 0
54.2827
−31.8337
−27.5288
26.3243
54.2827


163
0 0 0 0
78.1035
−0.8037
−0.3894
19.4261
78.1035


164
0 0 0 0
91.6418
1.1551
0.0123
22.043
91.6418


165
0 0 0 0
107.713
−20.5064
−20.4325
0
107.713


166
0 0 0 0
87.6791
10.2974
15.5578
41.6567
87.6791


167
0 0 0 0
43.999
−38.1941
−33.4689
9.5564
43.999


168
0 0 0 0
77.8049

0
35.7996
77.8049


169
0 0 0 0
64.3252
−18.1947
−21.5953
0.301
64.3252


170
0 0 0 0
87.6323
2.3641
5.3324
22.1704
87.6323


171
0 0 0 0
68.362
10.0449
10.2067
28.1342
68.362


172
0 0 0 0
71.1503
8.9457
7.2239
31.1775
71.1503


173
0 0 0 0
72.2674
10.3627
21.0562
42.1173
72.2674


174
0 0 0 0
74.1998
−21.7408
−16.656
13.5733
74.1998


175
0 0 0 0
48.1163
−59.8895
−45.4644
5.3909
48.1163


176
0 0 0 0
95.8578
−22.0943
−16.8813
5.2636
95.8578


177
0 0 0 0
71.1427
1.3136
0.2953
16.2008
71.1427


178
0 0 0 0
64.2561
−21.1531
−11.2645
18.8833
64.2561


179
0 0 0 0
46.8236
−35.3915
−32.1742
20.8832
46.8236


180
0 0 0 0
75.0675
0
−2.3977
21.2822
75.0675


181
0 0 0 0
92.8383
12.3946
12.9169
18.8847
92.8383


182
0 0 0 0
93.5709
0
0
34.7191
93.5709


183
0 0 0 0
99.5289
0
2.0346
24.4291
99.5289


184
0 0 0 0
83.8533
0
−0.5506
26.0285
83.8533


185
0 0 0 0
94.548
−21.6443
−24.0045
0
94.548


186
0 0 0 0
37.2481
−37.158
−17.69
15.0968
37.2481


187
0 0 0 0
51.5472
−53.6324
−42.397
−1.7858
51.5472


188
0 0 0 0
88.0501
0
0.7946
19.9555
88.0501


189
0 0 0 0
66.7349
13.1405
23.7073
43.9763
66.7349


190
0 0 0 0
79.582
−30.5111
−25.2626
6.3098
79.582


191
0 0 0 0
96.0338
−18.0752
−19.9657
−4.197
96.0338


192
0 0 0 0
76.1007
0.8898
2.9636
15.9079
76.1007


193
0 0 0 0
91.5294
3.7365
1.9996
17.8759
91.5294


194
0 0 0 0
68.0475
−2.4257
0
30.6498
68.0475


195
0 0 0 0
89.3989
−7.8811
−12.0365
15.2586
89.3989


196
0 0 0 0
65.0912
0
0
20.6484
65.0912


197
0 0 0 0
66.8554
5.2461
15.7031
40.796
66.8554


198
0 0 0 0
79.0861
2.3908
0
17.574
79.0861


199
0 0 0 0
78.1385
0
7.6087
32.1891
78.1385


200
0 0 0 0
46.2423
−40.8885
−35.7652
1.5723
46.2423


201
0 0 0 0
69.7901
−21.5136
−17.1084
8.0866
69.7901


202
0 0 0 0
74.538
−3.4095
−0.6635
28.063
74.538


203
0 0 0 0
51.7897
−45.9905
−36.9465
8.4225
51.7897


204
0 0 0 0
70.2936
0.8815
0.1572
20.0962
70.2936


205
0 0 0 0
50.1627
−39.3964
−31.6886
12.0722
50.1627


206
0 0 0 0
91.1628
12.1077
15.2718
28.5203
91.1628


207
0 0 0 0
73.3009
0.545
0
25.4562
73.3009


208
0 0 0 0
73.4157
−7.8355
−10.7764
16.4066
73.4157


209
0 0 0 0
138.801
12.9601
10.613
18.5675
138.801


210
0 0 0 0
113.044
13.8203
9.4299
20.2893
113.044


211
0 0 0 0
87.88
0.2296
11.6146
43.0472
87.88


212
0 0 0 0
127.53
11.1275
7.371
15.9241
127.53


213
0 0 0 0
90.1221
0
6.4924
41.084
90.1221


214
0 0 0 0
101.307
13.239
16.0649
43.8718
101.307


215
0 0 0 0
83.2597
14.2367
15.1789
40.8331
83.2597


216
0 0 0 0
71.6749
8.2756
20.8299
43.9905
71.6749


217
0 0 0 0
65.2014
10.407
9.2853
27.4661
65.2014


218
0 0 0 0
71.4891
13.9594
13.1115
29.7899
71.4891


219
0 0 0 0
66.0085
−8.2231
−3.0469
21.4195
66.0085


220
0 0 0 0
93.7673
7.1434
9.5135
27.6356
93.7673


221
0 0 0 0
66.1162
6.3785
7.8673
16.8498
66.1162


222
0 0 0 0
66.4815
9.4439
9.7686
16.5469
66.4815









Example 3

In this example, embodiments of small molecules that increase α7 integrin in skeletal muscle are disclosed. Using myogenic cells from mice in which the LacZ reporter gene was inserted into exon 1 of the mouse α7 integrin gene, 403,000 compounds were screened and more than 1500 hits that increased the β-galactosidase reporter were identified. Further evaluation identified 6 compounds that increased α7 integrin at least 1.5-fold in myotubes. Compounds were classified as iron chelating compounds, cell cycle inhibitors and compounds with undefined function. Compounds identified from this screen represent novel molecular probes that can be used to further elucidate regulation of α7β1 integrin expression and signaling in skeletal muscle and may serve as potential therapeutics for the treatment of DMD.


Several therapeutic approaches have been developed with the aim of restoring dystrophin expression and shown efficacy in animal models of DMD. These include virally mediated delivery and expression of dystrophin, myoblast cell transfer and engraftment, exon-skipping and stop-codon read-through. Currently, none of these methods have been approved as therapy for DMD patients. An alternative approach is to target and enhance levels of proteins which modify disease progression and act to partially compensate for the absence of dystrophin. These disease modifiers include utrophin, α7β1 integrin, GalNac, nNos and Adam12.


The α7β1 integrin is a laminin receptor in skeletal muscle that serves to link laminin-211/221 in the basal lamina and the actin cytoskeleton of muscle. The α7β1 integrin has structural and signaling functions that contribute to muscle development and physiology and was originally identified as a marker for muscle differentiation. Studies have shown that enhanced transgenic expression of the α7 integrin in skeletal and cardiac muscle can ameliorate dystrophic pathology and extend the lifespan of mdx/utr−/− mice more than three-fold. Multiple mechanisms appear to contribute to α7 integrin mediated rescue of dystrophin deficient muscle including maintenance of myotendinous and neuromuscular junctions, enhanced muscle hypertrophy and regeneration, and decreased apoptosis and cardiomyopathy. Enhanced α7 integrin also protects muscles against exercise-induced damage. Conversely loss of the α7 integrin in mdx mice results in more severe muscle disease. Together these observations support the idea that the α7β1 integrin is a major disease modifier in DMD.


To translate transgenic mouse studies into potential therapies for DMD, a drug discovery program to identify chemical probes that increase α7 integrin in skeletal muscle was initiated. A report on the generation and characterization of an α7 integrin knockout line of mice in which the LacZ gene is inserted into intron 1, downstream of the endogenous α7 integrin promoter has been reported. Thus, β-galactosidase functions as a reporter for α7 integrin expression in these animals. Primary myogenic cells were isolated from a heterozygous mouse (α7βgal+/−) so that the cells express α7 integrin and also report for transcription of the integrin. The myogenic reporter cells were designated α7βgal+/− and were used to identify two molecules, valproic acid and laminin-111, in preliminary screens and have been successfully tested in mouse models of DMD.


To identify further small molecules that increase α7 integrin in skeletal muscle, a muscle cell-based assay to screen 403,000 compounds including FDA approved drugs and the large compound libraries at the National Chemical and Genomics Center (NCGC) was used. Several compounds that increase the α7 integrin with known mechanisms of action including iron chelators, microtubule inhibitors, cell cycle inhibitors and steroid-like molecules were identified. Additionally, several small molecules with unknown biological activities were identified. Overall, the results identified novel small molecules that increase the α7 integrin in cultured muscle cells and may serve as molecular probes to further dissect signaling pathways that regulate the α7β1 integrin in skeletal muscle. These small molecules could potentially be developed as novel therapeutics in the treatment of Duchenne and other fatal muscular dystrophies.


Cell Culture: C2C12 myoblasts and myotubes were grown as previously described. α7+/LacZ myoblasts were originally isolated and maintained as described. Briefly, myoblasts were grown and maintained in DMEM without phenol red (Sigma) containing 20% FBS (Atlanta Biologicals), 1% Penicillin/Streptomycin (P/S) (GIBCO)+L-Glutamine (GIBCO). Myoblasts were maintained below 70% confluence until use in assay. Myoblasts were differentiated into myotubes in DMEM without phenol red, 1% horse-serum (Atalanta Biologicals), and 1% P/S+L-Glutamine. All cells were incubated at 37° C. with 5% CO2. Assays were performed on myoblasts and myotubes between passages 8 and 14.


Compound Libraries: Four compound libraries were screened using our muscle cell based assay: 1) Prestwick Chemical and Microsource Spectrum Libraries (BioFocus DPI, Leiden Netherlands with facilities in UK, Basel, Heidelberg) (Overington et al., 2006). 2) DIVERSet library (Chembridge Corp., San Diego, Calif.) and compounds from the ChemDiv library. 3) LOPAC library (Sigma-RPI) consists of 1280 pharmaceutically active compounds. 4) MLSMR-Molecular Libraries Small Molecule Repository.


Myoblast α7+/LacZ integrin FDG assay: A total of 5000 α7βgal+/− myoblasts were dispensed in 1.00 μL growth media using a 12-well multi-pipette (Rainin) onto Nunc black sided TC coated 96-well plate. After 24 hours up to 1 μl of compound in DMSO was added to the myoblast plates from pre-made working drug plates using a 1 μl 96-well pin tool or using an 8-well automatic multichannel pipette. Each working drug plate contained a column of a positive control (sodium butyrate (Fluka) or SU9516 (Tocris)) and at least one column containing DMSO alone. After incubating for 48 hours the media was aspirated, and cells were lysed with 50 μL of Mammalian Protein Extraction Reagent (MPER) (Thermo) per well followed by incubation at room temperature for 10 minutes. β-galactosidase (βgal) activity in each well was quantified by adding 50 μL of FDG assay solution (20% 0.1M sodium phosphate buffer pH 7.0 (Sigma), 0.2% 1M MgCl2 (Sigma), 0.2% 20 mM fluorescein di-galactoside (FDG) (Marker Gene Technologies)) and incubating the plates in the dark for 20 minutes at room temperature. Stop solution (2×TE) was then added (100 μl/well) and plates were read for fluorescence on the Victor V (Perkin-Elmer) with an excitation filter at 485 nm, an emission filter at 535 nm, and a 0.1 s/well count time.


Myotube α7 integrin FDG assay: A total of 25,000 α7βgal+/− myoblasts were dispensed in 100 μL growth media. After 24 hours, growth media was aspirated, wells were washed with 200 μL PBS, and 100 μL/well of differentiation media was added. Differentiation media was changed daily between 72 and 120 hours, and up to 1 μL compounds in DMSO were added as previously described once wells contained differentiated myotubes. The FDG fluorescence assay was performed as described in the myoblast screen with the one notable exception being the incubation after FDG solution addition being shortened from 20 minute to 5 minutes at RT due to the higher levels of βgal in myotubes.


qHTS of the LOPAC library and MLSMR: A total of 250 α7LacZ+/− myoblasts at passage 13 were dispensed using a mulitdrop (Thermo) into black low base tissue culture treated microclear aurora plates in 6 μl media containing DMEM without phenol, 5% FBS, 1× GluMax and 1× Penicillin/Streptomycin. The plates were incubated 16-24 hours at 37C., 5% CO2, 95% humidity covered with low evaporation stainless steel lids from Kalypsys. Compounds were then dispensed using a Kalypsys pintool to deliver 23 nl/well compounds in DMSO (diluted into 6 μl resulting in a 1:260 dilution of compound). The positive control compound used was the cdk2 inhibitor SU9516 (Tocris) (identified in the initial LOPAC screen). The plates were incubated for 48 hours at 37C., 5% CO2, 95% humidity using the same stainless steel lids. After incubation with compound, 5 μl of the media was aspirated using the Kalypsys washer/dispenser and 3 μl of the Mammalian Protein Extraction Reagent (MPER) lysis buffer (Thermo Fisher) was added. The plates were spun at 2000 rpm to remove bubbles and an initial capture was acquired on the Viewlux (Perkin Elmer) with excitation at 480 mn and emission at 540 nm for 25 seconds to omit any auto fluorescent compounds. The plates were then incubated for 10 minutes at room temperature. After incubation, 3 μl of 125 μM FDG (Marker Gene Technologies) diluted in PBS with 2 mM MgCl2 and 0.2% β-mercaptoethanol (BME) was added, the plates were then centrifuged at 2000 rpms and incubated for 30 minutes at room temperature. The plates were again read on the Viewlux with excitation at 480 nm and emission at 540 nm for 25 seconds.


CMV-LacZ Secondary Screen: In order to determine compounds that acted to stabilize β-gal in myogenic cells, a CMV-LacZ C2C12 cell line was used in a secondary screen. The EGFP gene from the pEGFP (Clontech) vector was replaced by LACZ PCR product with digests of the vector/PCR product performed using EcoRI (NEB) and NodI (NEB) and ligated using T4 DNA Ligase (NEB) following standard procedures to produce the CMV-LACZ vector. The LACZ gene was generated by PCR from the pBK-RSV (Stratagene) vector using primers LACZ EcoRI F- and LACZ NotI R-. The CMV-LACZ vector was then digested with AfiII (NEB) to linearize the construct, which was then transfected in C2C12 myoblasts using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were selected in growth medium containing G418 (Life Technologies) at 1.5 mg/ml. Clonal lines were selected and screened for βgal levels using the myoblast FDG assay. C2C12 cell lines with the highest βgal expression were used to screen cherry picked compounds, following the same procedures defined above for the myoblast and myotube screens. Compounds were determined to have failed this secondary assay if signal was elevated above the average DMSO+2× standard deviation (˜30%) for two separate data points.


Western Blotting: C2C12 myotubes were treated for 48 hours with each hit compound, washed with PBS, then scraped and pelleted using standard procedures. Cell pellets were resuspended in RIPA buffer and protein concentration determined by BCA. Approximately 10 μg of protein was loaded per lane and separated on 8% SDS-PAGE gels and transferred to nitrocellulose using standard conditions. Blots were probed using α7A and α7B integrin specific rabbit polyclonal antibody as previously described and normalized to α-tubulin.


Statistical analysis and Curve-fitting: Statistical analysis was performed using Graphpad Prism software and unpaired t-test comparison against the DMSO control treatment group. Graphpad prism software was also used to fit curves using nonlinear regression analysis with log (agonist) vs. response with a variable slope. A constraint equal to 1 was placed on the bottom of the curve and either 2 or 2.5 at the top (when needed) in order to produce appropriate EC50 values.


Generation and validation of α7+/LacZ integrin myoblast assay: In order identify α7 Integrin enhancing compounds, an assay based on α7+/LacZ mouse derived primary myoblasts and myotubes was developed. Heterozygous myoblasts were used in order to maintain a copy of the Itga7 gene for normal myogenic cell adhesion and signaling. The second Itga7 gene allele had exon 1 replaced by the LacZ gene, providing a reporter for α7 integrin transcription and maintaining the endogenous promoter, enhancer, and chromatin environment. By deriving primary muscle cells, myogenic cells capable of differentiating into myotubes and in which the μ-gal reporter levels accurately mimicked the α7 integrin protein levels during this developmental process were produced. Several rounds of pre-plating were performed with the myogenic line used for the screen in order to remove fibroblasts, leaving a relatively pure population of myogenic cells.


α7 integrin muscle cell-based assay: Assessing the growth rate of myogenic cells in a 96-well format was carried out. For the myoblast assay, it was determined that plating 5000 α7+/LacZ cells led to a confluence of ˜80% at the time of fluorescence quantification. Limiting the confluence to less than 80% helped prevent entry into a differentiated state in this assay which may affect LacZ expression. For the myotube assay, it was determined that plating 25,000 myoblasts led to a confluence of around 95% after 24 hours allowing myogenic differentiation to proceed. Next, the linearity of fluorescence for the assay was determined for various amounts of purified E. coli β-galactosidase using the FDG assay solution (FIG. 4A). This displayed a linear response that was comparable to levels observed in the α7+/LacZ myoblasts. Finally, as library compounds are normally solubilized in DMSO, the effects of DMSO on our assay were examined for both myoblasts and myotubes (FIG. 4B). In myoblasts, DMSO concentrations of up to 0.5% had no significant effect on the assay compared to cells without DMSO addition (FIG. 4C). At 1% DMSO, there was a 10% decrease in overall fluorescent signal in the assay, which was considered to be within acceptable range (FIG. 4C). The effect of DMSO on the myotube assay was more robust, where 0.5% DMSO decreased fluorescence by ˜12% and 1% DMSO displayed ˜25% decrease in overall fluorescence (FIG. 4B). Greater than 1% DMSO resulted in reduced fluorescence and therefore 1% DMSO in media was determined to be the maximum acceptable level for myotubes. The levels of FDG were optimized to 125 μM which was used in the MLSMR screen and subsequent verification assays (FIG. 4D).


For our preliminary screens, sodium butyrate was determined to increase β-galactosidase levels with treatments of ˜1.5 mM. In initial studies with sodium butyrate a Z′ factor of 0.6, which is considered a strong assay for drug discovery, was calculated and thus a myotube screen of the Prestwick Chemical at 0.2 ug/mL and Microsource Spectrum Libraries at 10 μM was initiated. As these libraries are relatively small, myotubes were initially screened because they are the ideal therapeutic target for α7 Integrin elevation. Using a 1.2 fold increase over DMSO control as the minimum cutoff, 24 compounds in the Prestwick chemical library and 30 compounds in the Microsource spectrum library were identified. These corresponded to a hit percentage of 2.1% and 1.5% of the libraries. After secondary and counter screens, the compounds that increased α7 integrin were classified as either iron chelators or a cholesterol analog. The iron chelators identified in the screen were Ciclopirox ethanolamine, deferoxamine, and 2,2-Dipyridyl which all displayed positive dose-response curves in myotubes. The cholesterol analog compound. 5alpha-cholestan-3β-ol-6-one, also displayed a positive dose-response curve.


This initial success led us to attempt a larger scale high-throughput screen (HTS) using the DIVERSet library of compounds with sodium butyrate as a positive control. Due to the number of compounds in the DIVERSet library (˜50,000), a myoblast screen was performed. This screen generated several more “hits” which upon secondary screens were narrowed down to three compounds annotated: 1001, 1002, and 1003. After further studies, these compounds and analogs were either less robust or too toxic to move forward with further studies.


Primary screen of LOPAC library and the MLSMR: The assay was adapted for a high-throughput screen with the fully automated Kalypsys robotic system using 1536 well plates at NCGC. A preliminary screen of the LOPAC library with 1001 (DIVERSet) as a positive control was used to develop the new conditions for this assay, comparing offline to online results and reducing myoblasts plated to 2000 cells/well for the 384-well format. This screen produced several “hit” compounds including Sodium Nitroprusside dihydrate, a Nitric oxide donor, and two cdk2 inhibitors SU9516 and CK2 inhibitor 2. SU9516 gave a relatively robust response of around 2.1-fold, relative to DMSO alone. There was still a relatively high variability in DMSO background signal (˜30%) even using SU9516 as a positive control. However, as SU9516 was the most reproducibly active drug that had been identified it was selected as a positive control for the qHTS. To this end a dose-response curve SU9516 treatment of myoblasts and myotubes was generated (FIGS. 5A and 5B).


Using SU9516 as a positive plate control and DMSO as a negative control, the assay was then used to screen the Molecular Libraries Small Molecule Repository (MLSMR) of 368,680 compounds at NIH Chemical Genomics Center (NCGC) using concentrations at 0.08 μM, 0.4 μM, 2 μM, and 10 μM. Around 1500 compounds were cherry picked as “hits” based on previously defined curve classifications (FIG. 6).


Offline confirmation and β-galactosidase stability secondary screen: From the 1500 cherry picked “hits” two pools were chosen for further off-line myoblast, myotube and secondary screening. The first pool consisted of 166 compounds, which were still active in a subsequent online myoblast screening of the original 1500 cherry picks (Tables 9-12). The second pool consisted of the top 197 compounds, which had not been repeatedly active in subsequent screens (Tables 9-12). Finally, as SU9516 had proven to be a highly reliable positive control, 44 analogs, based on the SU9516 platform, were selected from the MLSMR library or from separate stock compounds and screened. The secondary screen consisted of a CMV-LACZ stably transfected C2C12 myoblast line with consistent β-Galactosidase activity (Tables 9-16, and Table 17). The assays were used the original 96-well format and new dose-response curves for myoblasts (N=3-6 replicates for each data point), myotubes (N=3-6 replicates for data point), and CMV-LacZ secondary assay (N=2 replicates for each data point) using concentrations of 0.5 μM, 1 μM, 10 μM, 20 μM, and 40 μM (Tables 9-16). A cutoff of greater than 25% relative to DMSO was used to exclude compounds (Table 17). Almost one fifth of the compounds from the initial 197 top “hits” worked exclusively in myotubes assay and not in myoblasts. Together, this data suggested that during the primary screen some of the cells in the assay had differentiated into myotubes. This trend was not observed in the 166 NCGC compounds where almost one third worked exclusively in myoblasts. Again, during the transition from on-line to off-line screening many of the compounds from both groups (54% and 69%, Table 17) failed to achieve the minimum cutoff of 25% increase over DMSO levels in either myoblasts or myotubes. In addition the majority of compounds, which showed activity in the β-galactosidase stabilizing secondary screen, did not show activity in either of the α7+/LacZ based assays (Table 17, full results in Tables 9-12). These results suggest some of these compounds may not be actually stabilizing or catalyzing the P-galactosidase enzymatic reaction but instead may be activating the CMV promoter in the C2C12 cells.


As SU9516 was used as our positive control, it was used to potentially represent a platform that could be modified to produce other “hit” compounds off-line screens with 44 SU9516 analogs were performed. The myoblast, myotube, and secondary CMV-LacZ assays were performed as previously described and results from this screen are summarized in Table 17 (Full results in Tables 13-16). Surprisingly, many of them, as with SU9516 itself, showed activity in the C2C12 CMV-LACZ myoblasts and myotubes. Most of the compounds had activity in the β-galactosidase stabilizing screen also had no response in the primary screen suggesting that they were not β-galactosidase stabilizers but instead activators of CMV or the region of CMV-LACZ stable integration within this cell line. This result also suggests that SU9516 was likely activating both the ITGA7 promoter and the CMV-promoter by inhibition or activation of separate signaling pathways. With reference to Tables 9-16 below, values of less than about 0.9 correspond to less desirable compounds as compounds with such values were toxic at the indicated concentration value; values of 0.9 to about 1.1 indicate compounds with some activity (substantially similar to the DMSO background); and values greater than about 1.2 indicate compounds that have desirable activity as these values indicate compounds exhibiting an activity about 20% above the background.









TABLE 9







Burkin Assay in Myoblasts














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000591667-01
1.1351
0.86235
1.088
1.13163
1.03356
0.99007


MLS000568234-01
1.01115
1.10558
1.37321
1.13643
1.03188
1.09422


MLS000689562-01
0.88167
0.95366
1.22402
1.01263
1.014
1.07412


MLS000732652-01
0.87916
1.0346
1.2648
0.97202
1.19119
1.17557


MLS001240181-01
1.19808
1.15299
1.07353
1.4585
1.08072
1.08233


MLS001211139-01
1.21551
1.218
0.96826
1.06491
1.13586
1.2392


MLS001030268-01
0.60686
0.72598
0.87162
1.48967
1.14329
1.23909


MLS000912699-01
0.43922
0.6724
0.9298
0.93216
1.03847
1.11521


MLS001125260-01
0.90571
1.02411
0.96159
0.98381
1.11006
1.20955


MLS000717689-01
0.98659
0.95999
1.03846
1.03203
1.04195
1.15829


MLS001197665-01
1.18868
1.09447
0.95234
1.01544
1.06912
1.06224


MLS001075922-01
0.64637
0.56776
0.2869
0.60386
1.29157
1.2085


MLS001124046-01
1.07367
1.10106
1.01243
1.07966
1.15704
1.06118


MLS001197220-01
1.0836
1.11129
0.92184
1.84318
1.28622
1.2811


MLS001221318-01
1.07461
1.05387
0.99442
1.00961
1.28345
1.11823


MLS000947910-01
1.01847
1.04651
1.0429
1.03277
1.18767
1.02381


MLS001215795-01
0.71873
0.74097
0.77762
0.93196
1.20673
1.14257


MLS002163670-01
0.7779
0.89724
0.94682
1.01239
1.23366
1.11688


MLS001200149-01
0.98756
1.02136
0.99804
1.0782
1.15573
1.03945


MLS001359861-01
0.91135
0.87253
0.93239
0.94838
0.93763
0.93526


MLS000710669-01
0.82425
1.84768
0.99409
1.0722
1.04969
1.02525


MLS001035690-01
1.09083
1.05474
0.98721
1.18564
1.15072
1.05147


MLS001030621-01
1.00997
1.00129
1.16138
1.15044
1.15833
1.00359


MLS001083082-01
0.94677
1.01412
1.02292
1.05814
0.95159
0.90356


MLS000045588-01
0.89188
1.00313
0.91698
1.07484
1.10703
1.01584


MLS001216939-01
0.78651
0.86335
0.98283
1.11114
1.2827
1.12283


MLS001163859-01
0.92799
0.92082
1.02427
1.05488
1.09001
0.97855


MLS000683232-01
1.65393
1.82061
1.77787
1.6445
1.19868
1.23351


MLS001170856-01
0.71333
0.92168
1.24442
0.97435
0.86511
0.94996


MLS002667707-01
0.83137
0.93474
1.23469
1.01997
0.91716
1.0156


MLS001200665-01
1.02863
1.14332
1.24505
1.00302
0.8539
0.89824


MLS002161853-01
0.9354
1.07259
1.1978
0.93857
0.82784
0.85687


MLS002163101-01
0.97754
0.94568
1.25085
0.96995
0.94422
0.95515


MLS000062431-01
0.9038
1.78859
1.20925
1.01602
0.95817
0.9542


MLS000028160-01
0.85354
1.10681
0.85212
0.98053
0.98728
1.03731


MLS002248819-01
0.76886
0.81835
1.0849
1.06786
1.11265
1.12574


MLS000080654-01
0.91656
1.11208
0.94286
0.92349
0.92297
0.97875


MLS000760876-01
0.95041
1.10218
0.91131
1.81188
0.98558
1.0287


MLS000677675-01
0.95543
1.00416
1.02813
0.98734
1.06053
1.09282


MLS000113985-01
0.12198
0.17793
0.48041
0.78982
1.01847
1.06463


MLS001182368-01
0.89517
1.00108
0.89449
0.91403
1.21452
1.08093


MLS001212882-01
1.04785
1.06823
0.9069
1.04855
1.06975
1.0132


MLS001004364-01
0.93735
0.97287
0.95313
1.03485
1.10452
1.0917


MLS000736846-01
0.95628
1.0406
1.01151
1.06976
1.2858
1.21826


MLS001098105-01
1.13456
1.07472
0.97405
0.99364
1.02871
0.94487


MLS000678673-01
1.13357
1.17252
0.96866
1.04367
1.05388
0.93249


MLS000925023-01
0.41794
0.77654
0.79566
0.95232
1.03462
0.99596


MLS001212319-01
0.78199
0.85293
0.88263
0.94115
1.02595
1.04963


MLS000779126-01
0.93895
0.91244
0.81956
0.92292
1.11482
1.01879


MLS000948055-01
0.58675
0.82128
0.8986
1.13571
1.05347
1.32788


MLS000110418-01
0.8889
1.00365
1.11988
1.24866
1.07925
1.3095


MLS000693704-01
0.80188
0.84732
0.94181
1.1427
1.15286
1.42821


MLS001225512-01
1.28384
1.10796
0.88323
1.17276
0.91313
1.1596


MLS001006798-01
1.01583
1.11843
1.00833
1.13293
0.92818
1.19095


MLS000711491-01
0.57934
0.79492
1.02377
1.02034
1.0159
1.21405


MLS000582947-01
0.77374
0.9417
0.98066
0.94304
0.96323
1.23612


MLS000531177-01
0.80079
1.01406
1.62515
1.06657
0.92304
0.88259


MLS001202389-01
0.25563
0.58161
1.1004
1.00911
0.94807
0.9323


MLS000536064-01
0.45011
0.54402
1.04652
0.73332
0.80395
0.80279


MLS000586245-01
1.02068
1.03337
1.24403
1.11633
1.04937
0.99033


MLS001061374-01
0.67743
0.87297
1.16907
0.98264
0.84475
0.84766


MLS000675441-01
0.89965
0.90155
1.24817
1.04359
0.8764
0.93971


MLS001200396-01
0.4794
0.48269
1.1321
0.53784
0.92449
0.88271


MLS001165937-01
0.68201
1.03268
1.70065
1.27472
0.89489
0.86889


MLS000325736-01
0.88542
0.91197
1.16957
1.42277
0.78755
0.77572


MLS001215357-01
0.53275
0.9203
1.48859
0.88374
0.93
1.03703


MLS000588210-01
0.96107
1.13586
0.98164
0.90019
0.97069
1.05483


MLS000764729-01
0.72603
0.9581
0.96935
0.98159
1.0582
1.08805


MLS000689492-01
0.96521
1.08264
0.94309
0.93998
0.90795
1.07297


MLS001000299-01
0.77383
0.93971
0.97497
0.95516
0.99529
1.08932


MLS000393762-01
0.46958
0.59613
0.69566
0.77991
0.94283
0.97379


MLS001130011-01
0.91554
1.03734
0.97619
1.37412
0.89472
0.97145


MLS001229477-01
0.90194
0.96342
0.94542
1.36703
0.82957
0.87031


MLS000707378-01
1.49252
1.31687
1.07579
1.02429
1.04559
1.00348


MLS000573208-01
0.62351
1.63233
0.81207
0.95231
1.01493
1.03104


MLS001167281-01
0.86182
0.88689
0.97761
1.07478
1.18016
1.08931


MLS000053342-01
1.02205
1.0354
0.86681
0.97655
1.08832
1.22195


MLS002171615-01
0.61782
0.70899
0.88587
0.97469
0.94068
1.05229


MLS001005712-01
0.76395
0.87627
0.87827
0.92598
1.17003
1.1047


MLS001176153-01
1.12011
1.14544
0.87665
0.91849
1.04463
1.06004


MLS000735021-01
0.99412
1.02509
0.97651
0.99818
0.92462
0.94018


MLS000767397-01
0.97544
1.61335
0.93339
1.47579
0.83903
0.88624


MLS001196572-01
0.9345
0.93622
0.94826
1.22354
0.76263
0.88263


MLS000393966-01
0.96498
0.99041
1.07573
1.79279
0.96263
0.94947


MLS001034810-01
0.84068
0.8996
1.19071
1.37466
0.99747
1.02099


MLS001165394-01
1.00709
1.03548
0.95105
1.15909
1.20071
1.14858


MLS000089464-01
0.39111
0.56309
0.7825
0.93396
0.98019
0.99651


MLS000698617-01
0.89657
0.84622
0.93172
1.06001
1.00295
1.00774


MLS001175021-01
0.95749
0.97466
0.90603
0.88063
0.98034
0.92001


MLS001166758-01
0.61737
0.72753
0.82445
0.97363
0.73944
0.82402


MLS001008109-01
0.83059
0.82603
0.86263
1.00193
0.78003
0.84815


MLS001181936-01
0.32845
0.50999
0.82189
0.85734
0.75618
0.84427


MLS000560266-01
0.55892
0.83206
1.13014
1.02573
0.85564
0.95298


MLS001215074-01
0.59739
0.73389
0.97384
0.94307
0.9004
1.02349


MLS001215123-01
0.52524
0.665
1.15451
1.1241
0.97081
1.07545


MLS001033255-01
0.82795
0.98503
1.12416
1.01684
0.93725
1.01302


MLS001160611-01
0.92674
0.97495
1.11549
1.00379
0.97673
1.1058


MLS001006302-01
0.94141
0.97305
1.17248
0.95352
0.93033
1.12331


MLS001123876-01
0.88436
0.93393
1.09536
1.43977
0.82637
1.04105


MLS001122698-01
0.83886
0.9061
0.96519
1.00842
0.85728
0.98289


MLS000755214-01
0.86097
0.84475
0.96348
0.97565
1.02077
1.07667


MLS000731285-01
0.60769
0.60402
0.7344
0.77267
0.92346
1.06384


MLS000776409-01
0.34443
0.50867
0.98382
0.93888
1.00738
1.1194


MLS001221908-01
0.56836
1.43328
0.88118
0.93061
0.95314
1.05089


MLS000419286-01
0.71995
0.7945
0.8951
0.91739
1.00244
1.16649


MLS000554416-01
0.82485
1.08704
0.9591
0.99152
1.0679
1.21133


MLS000073150-01
1.06802
1.13888
0.90898
0.92622
1.0875
1.26179


MLS000663185-01
0.83278
0.92216
0.93123
1.39854
0.97437
1.10863


MLS001078811-01
0.74109
1.68071
0.89103
0.95834
0.99261
0.9376


MLS002694363-01
0.70661
0.75811
1.08986
1.04452
1.06141
1.06241


MLS000689218-01
1.03748
1.07006
1.05082
1.13165
1.17068
1.06835


MLS001215294-01
0.20458
0.20253
0.61411
0.76405
1.00264
0.97828


MLS001183575-01
0.53384
0.69163
0.79465
0.89805
1.00931
0.96443


MLS000393567-01
0.87054
0.83339
0.93251
0.97236
1.05083
1.03233


MLS000546316-01
1.02675
1.02594
1.12291
1.16412
1.22254
1.15942


MLS000912258-01
0.37249
0.63513
0.84251
1.2722
0.90885
0.84356


MLS000850522-01
0.88047
0.8743
1.00276
1.30679
0.93316
0.79932


MLS001197779-01
0.75302
0.84665
0.98695
1.28053
1.0032
0.91613


MLS001095705-01
0.72839
0.79174
0.969
1.29549
0.87876
0.78726


MLS000912562-01
0.55515
0.69112
0.92532
1.03872
0.92904
0.76968


MLS000092785-01
0.6509
0.78329
0.93433
1.1703
1.03537
0.95442


MLS000418650-01
0.94454
0.91042
0.96326
1.12987
1.06885
0.91564


MLS000860966-01
1.11657
1.24227
1.12552
1.22473
0.95061
0.8092


MLS001180929-01
1.1941
1.10157
1.05028
1.04982
1.0555
1.07667


MLS000564564-01
0.97011
0.94044
1.0834
1.04244
0.94502
0.99186


MLS001164432-01
0.66217
0.7137
1.06663
1.09125
1.1813
1.24825


MLS000621451-01
1.08838
1.0713
1.11408
1.06613
1.01829
1.06548


MLS000078709-01
0.26162
0.51714
0.80516
0.81574
0.996
1.06264


MLS000688267-01
1.10743
1.0466
0.91328
1.35814
1.10445
1.09233


MLS001006516-01
0.48019
0.69276
0.79316
0.8796
1.13422
1.09738


MLS001095033-01
1.0618
1.12466
0.96135
0.96322
1.18613
1.28147


MLS000072290-01
0.22377
0.39842
0.6517
0.73337
1.19579
1.40542


MLS000036988-01
0.65813
0.90234
0.82559
0.86568
1.0825
1.18898


MLS001217935-01
0.58698
0.80022
0.89296
0.91061
1.07572
1.16224


MLS000777780-01
0.72314
0.91334
1.24779
0.94395
1.05647
1.13609


MLS001174740-01
0.54139
0.96576
1.18066
0.85119
1.19298
1.22104


MLS000392555-01
1.22568
1.31366
0.97342
1.30012
1.10052
1.2055


MLS000693795-01
1.24042
1.18741
0.97277
1.02032
1.24179
1.1137


MLS000684034-01
0.30855
0.42247
0.62584
0.78721
1.01964
0.99899


MLS001172822-01
0.9597
0.98696
0.90137
0.96623
1.35133
1.23301


MLS000052969-01
0.75911
0.82915
0.83559
0.96455
1.35174
1.24959


MLS001217212-01
1.06402
0.99668
0.99332
0.97848
1.17844
1.09519


MLS001004864-01
0.88826
1.10454
1.02341
1.10852
1.19287
1.1396


MLS001116535-01
1.26741
1.23894
0.93856
0.99796
1.11141
1.09197


MLS001165424-01
1.22991
1.17612
0.95477
0.99659
1.2222
1.15184


MLS001116079-01
1.34036
1.24425
0.9749
1.02479
1.08671
1.02952


MLS001198271-01
1.0038
1.08798
1.03423
1.29177
1.00045
1.03412


MLS001167798-01
1.00561
1.05069
1.06388
1.27705
1.16574
1.05836


MLS000710288-01
0.59627
0.86808
1.1778
1.3928
1.30459
1.16807


MLS000734270-01
0.98726
0.97392
0.95501
1.21022
1.04793
1.0135


MLS000858981-01
1.00476
1.0113
1.01009
1.18572
0.999
0.95297


MLS000698826-01
1.02313
0.99365
0.95981
1.24627
1.00831
1.01911


MLS001000874-01
0.95449
1.00725
0.86772
1.07723
1.03283
1.17509


MLS000682750-01
1.10263
1.12414
1.07554
1.22927
1.02005
1.20347


MLS001090787-01
1.22203
1.26522
1.45989
1.26261
0.97343
1.14195


MLS002636056-01
0.82025
0.93768
1.22389
1.03743
1.06589
1.15719


MLS002170630-01
0.67353
0.78253
1.21616
1.0982
0.97082
1.12201


MLS002162890-01
0.49245
0.694
1.26109
1.1338
1.03367
1.10651


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0.86044
0.96531
1.00963
0.96419
0.85974


MLS000707281-01
0.79693
0.83587
0.88542
0.89346
0.97563
0.95067


MLS000591198-01
0.47304
0.77654
0.95931
1.03702
0.92511
1.00578


MLS000714175-01
0.96263
0.99361
1.06
1.1625
0.97498
0.53831


MLS002163386-01
0.93876
1.01374
0.88796
1.03415
0.9023
1.0508


MLS000761297-01
0.95256
1.07174
1.05527
1.12127
1.07133
1.0693


MLS002245351-01
0.86569
0.90007
0.97044
1.03385
1.03691
1.08244


MLS000718886-01
0.81389
0.91243
0.96613
1.08727
0.93943
1.05198


MLS002156485-01
1.02644
1.05206
0.87521
0.92779
0.9382
1.05917


MLS001140657-01
0.79783
0.85676
0.87677
0.95283
0.96198
0.9918


MLS002157024-01
1.08144
1.09694
0.85235
0.96094
0.97128
1.01144


MLS000721730-01
0.93424
0.82698
0.88242
0.81574
0.81973
0.83765


MLS000705922-01
0.87835
0.88922
0.72832
0.72819
1.02
0.96273


MLS000724709-01
0.55365
0.48598
0.77902
0.73891
0.95538
0.98992


MLS002161757-01
1.15126
1.00861
0.91351
0.91126
0.89196
0.96138


MLS002164687-01
0.81941
0.83778
0.84902
0.69489
0.81935
0.77498


MLS001060533-01
1.21969
1.39744
1.10126
1.03286
0.99152
0.90492


MLS000685139-01
0.98483
0.98933
0.77595
0.84163
0.6285
0.82342


MLS001217286-01
1.06759
1.00563
0.82726
0.76705
0.8506
0.74275


MLS001221619-01
0.89756
0.96481
0.67637
0.73674
0.76333
0.68552


MLS001219621-01
0.54233
0.78837
0.75418
0.72486
0.80633
0.86421


MLS001166156-01
0.34807
0.75715
0.76012
0.75432
0.90852
0.8005


MLS000534926-01
0.42215
0.59957
0.88325
0.85863
0.94733
1.52217


MLS000548725-01
0.07694
0.51687
0.76446
0.89891
0.88938
0.91317


MLS000374261-01
0.83211
0.83763
0.71381
0.72479
0.77583
0.7676


MLS000123454-01
1.01341
1.51865
0.86209
0.78618
0.8234
0.8934


MLS000625140-01
0.68287
0.75893
0.78335
0.87481
0.8709
0.88394


MLS001214443-01
0.9313
0.9132
0.81732
0.7787
0.80738
0.92033
















TABLE 10







Burkin Assay in Myotubes














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000591667-01
0.69646
0.67806
0.99863
1.07156
1.1434
1.00531


MLS000568234-01
0.97051
0.86942
1.08971
0.96419
1.16742
1.07977


MLS000689562-01
0.85465
0.72357
1.0159
0.97883
1.03466
1.08068


MLS000732652-01
0.73092
0.67974
1.14125
1.1938
1.11599
1.09214


MLS001240181-01
0.79047
0.88714
0.96827
1.64271
1.214
1.17431


MLS001211139-01
0.75468
0.82834
0.94255
1.00287
1.28848
1.16726


MLS001030268-01
0.76063
0.62773
0.92494
1.21276
1.16147
1.07576


MLS000912699-01
0.65181
0.52169
0.93901
0.92519
0.93756
1.17498


MLS001125260-01
0.74508
0.76341
1.09105
0.96768
1.20801
1.28483


MLS000717689-01
0.71346
0.82829
1.07589
1.04848
0.90065
1.11085


MLS001197665-01
0.95828
1.00065
0.99899
0.97302
1.06784
1.11547


MLS001075922-01
0.82425
1.06745
0.62896
0.65563
1.14344
1.29247


MLS001124046-01
0.91979
0.89276
1.01603
1.41708
1.09565
1.14517


MLS001197220-01
0.83097
0.90004
1.00438
1.25409
1.3061
1.32659


MLS001221318-01
0.7186
0.80714
0.95914
0.92662
1.08244
1.13276


MLS000947910-01
0.95151
0.96285
1.03459
0.99998
1.10147
1.13447


MLS001215795-01
0.88728
0.9147
0.91066
0.86305
1.19109
1.29108


MLS002163670-01
0.73497
0.85264
1.07165
0.97782
1.29363
1.22297


MLS001200149-01
0.83633
0.92859
1.09373
1.06886
1.07233
1.26026


MLS001359861-01
0.80395
1.0337
0.876
0.90881
1.18375
1.09515


MLS000710669-01
1.0073
0.99667
0.90001
1.05381
1.16308
1.04515


MLS001035690-01
0.92376
0.97197
0.93577
1.07274
1.07159
1.11088


MLS001030621-01
0.72029
0.7954
0.94292
0.99904
0.94925
0.97239


MLS001083082-01
0.93703
0.89872
1.05034
1.07008
0.94106
1.03453


MLS000045588-01
0.95463
1.06301
1.03566
1.135
1.09854
1.07688


MLS001216939-01
0.47431
0.85294
1.06002
1.06991
1.18531
1.19276


MLS001163859-01
1.05788
1.11822
1.24934
1.38468
1.28077
1.13173


MLS000683232-01
1.31969
1.4149
1.44782
1.27158
1.2778
1.13572


MLS001170856-01
1.02912
0.77626
1.06447
0.91001
0.74357
0.82959


MLS002667707-01
0.7617
0.80494
0.95401
0.89392
1.01022
0.99551


MLS001200665-01
0.88772
0.83653
1.12552
1.09065
0.84612
1.03137


MLS002161853-01
0.93534
0.91896
1.15981
0.98887
0.80923
0.87612


MLS002163101-01
0.97609
0.77823
1.30157
1.10149
0.90746
1.04549


MLS000062431-01
0.88412
0.76433
1.1989
1.17804
0.84151
0.95709


MLS000028160-01
0.81118
0.90493
0.95982
0.91617
1.10188
1.08464


MLS002248819-01
0.85083
0.92428
1.24039
1.06554
1.19767
1.11701


MLS000080654-01
0.62169
0.74219
0.97069
0.90438
1.09159
1.06711


MLS000760876-01
0.89931
0.86336
0.9755
1.24882
1.25578
1.2427


MLS000677675-01
0.68734
0.66754
1.04369
1.08103
1.20496
1.27844


MLS000113985-01
0.66792
0.76552
0.99254
0.99189
1.03688
0.98825


MLS001182368-01
0.91934
0.90974
1.02027
1.2117
1.0792
1.04351


MLS001212882-01
0.91073
0.84206
0.8877
0.90721
1.02311
0.98143


MLS001004364-01
1.05525
0.88751
0.86169
0.90474
1.10504
1.09276


MLS000736846-01
0.96729
0.87764
0.93964
1.00375
1.14446
1.13093


MLS001098105-01
0.84588
0.81422
0.83539
0.91598
1.05323
1.05204


MLS000678673-01
0.78099
0.86554
0.96062
0.97673
0.98018
0.97725


MLS000925023-01
0.73184
0.80043
0.8784
0.89694
1.23995
1.16045


MLS001212319-01
0.63105
0.73964
0.92007
0.94764
1.13867
1.13422


MLS000779126-01
0.77678
0.88319
0.87581
0.91457
1.01779
0.98357


MLS000948055-01
0.75145
0.98002
0.8264
0.90529
1.05415
1.06716


MLS000110418-01
0.88548
0.99394
0.943
1.01658
1.0975
1.07264


MLS000693704-01
0.82627
0.9587
0.92299
1.00259
1.0787
0.93149


MLS001225512-01
0.74015
0.84309
0.82346
0.97987
1.05854
0.92901


MLS001006798-01
0.88856
0.97456
0.89057
1.05957
1.04218
0.93455


MLS000711491-01
0.57898
0.67103
0.94615
0.9629
1.06241
1.06977


MLS000582947-01
0.79101
1.10221
0.97595
1.01969
1.15528
0.90028


MLS000531177-01
0.90051
0.80069
1.16345
1.05071
0.73291
0.82589


MLS001202389-01
0.55936
0.64125
1.05346
0.99362
0.93925
0.9181


MLS000536064-01
0.7073
0.76421
1.13143
0.95394
0.95554
0.90458


MLS000586245-01
1.02629
0.94257
1.17916
1.04311
0.9584
1.00329


MLS001061374-01
0.81546
0.73205
1.18149
1.00437
1.09204
1.05776


MLS000675441-01
0.71678
0.74757
1.11902
0.95908
1.07053
0.85516


MLS001200396-01
0.71138
0.6408
1.26379
0.98293
0.94875
0.93009


MLS001165937-01
0.72485
1.00559
1.52391
1.21196
1.16352
0.96798


MLS000325736-01
0.69718
0.6582
1.16824
1.02677
0.80167
0.91355


MLS001215357-01
0.69286
0.75605
0.88397
0.90573
0.81367
0.9366


MLS000588210-01
0.95653
0.97629
0.91553
0.94662
0.95586
0.94246


MLS000764729-01
0.60751
0.68098
0.97198
1.00794
1.08392
1.05496


MLS000689492-01
0.75436
0.88357
0.92917
0.93108
1.13846
1.15571


MLS001000299-01
0.55782
0.793
0.91625
0.87644
1.00592
0.92249


MLS000393762-01
0.70455
0.77047
1.00696
0.97947
0.92497
0.94021


MLS001130011-01
0.7512
0.78831
1.0699
0.88339
0.99521
0.95531


MLS001229477-01
0.8339
0.83764
0.90149
0.97022
0.93677
0.80147


MLS000707378-01
1.05105
1.04623
1.24093
0.88992
0.85116
0.87159


MLS000573208-01
0.59664
0.82393
1.04515
0.97999
1.00715
0.94525


MLS001167281-01
0.83453
0.82926
1.14202
1.07436
1.05393
1.06349


MLS000053342-01
1.11028
1.04947
1.03003
1.00173
1.01051
1.05896


MLS002171615-01
0.83027
0.84318
0.99471
0.97089
1.16975
1.10717


MLS001005712-01
0.74045
0.78848
0.93903
0.94721
1.17289
0.87044


MLS001176153-01
0.98183
0.92376
1.10591
0.95378
1.02681
1.00708


MLS000735021-01
0.85229
0.84443
1.00585
0.91367
1.00886
1.01265


MLS000767397-01
1.01365
0.99933
1.0608
1.00428
0.94806
0.92939


MLS001196572-01
0.71815
0.98051
0.92943
1.14827
0.88824
0.92363


MLS000393966-01
1.01539
1.23304
0.95462
1.30422
0.84361
0.85472


MLS001034810-01
0.75928
1.00003
1.16544
1.40257
1.06959
1.03516


MLS001165394-01
1.03397
1.18823
0.98536
1.23914
1.13467
1.06778


MLS000089464-01
0.83732
0.95449
0.99169
1.12764
1.02877
1.06322


MLS000698617-01
0.79633
1.09199
0.94496
1.13858
1.07472
1.19659


MLS001175021-01
0.87883
0.97988
0.99128
1.05123
1.0167
1.06135


MLS001166758-01
0.87545
0.98563
0.89787
0.98565
1.02671
1.0327


MLS001008109-01
0.61033
0.82635
0.93355
1.18189
0.91465
0.92469


MLS001181936-01
0.73871
0.58684
1.02387
0.98561
0.76335
0.91972


MLS000560266-01
0.60594
0.67057
1.18475
1.03662
0.88245
0.98908


MLS001215074-01
0.84879
0.75046
1.13207
1.04996
0.83074
0.78865


MLS001215123-01
0.816
0.69382
1.13745
1.04903
0.90099
0.94663


MLS001033255-01
1.23171
0.83368
1.24617
1.08579
0.81878
0.90997


MLS001160611-01
1.23985
0.83969
1.16888
1.00621
0.86282
0.94515


MLS001006302-01
1.07828
0.78349
1.21228
1.03871
0.93734
0.98217


MLS001123876-01
1.00321
0.71141
1.23169
1.1689
0.81926
0.86603


MLS001122698-01
0.85722
0.92091
1.03016
0.95745
0.90389
0.91311


MLS000755214-01
0.8312
0.96553
1.0353
0.93209
0.96607
1.00954


MLS000731285-01
0.19528
0.68996
0.94092
0.86954
0.96101
0.85441


MLS000776409-01
0.80563
0.89489
0.92835
0.88195
1.02936
0.89249


MLS001221908-01
0.74062
0.81797
0.96928
0.88901
0.95887
1.19709


MLS000419286-01
0.193
0.45703
0.94796
0.87898
0.8124
0.9511


MLS000554416-01
0.49152
0.68156
0.93336
0.83662
0.9261
0.91456


MLS000073150-01
0.85596
0.83687
0.94757
0.9467
0.92051
1.11147


MLS000663185-01
1.11786
0.77913
1.0708
0.97669
0.81366
0.96799


MLS001078811-01
0.45984
0.63069
0.8773
0.98157
0.90642
0.91635


MLS002694363-01
0.41079
0.66943
0.87872
0.91782
1.01024
0.99095


MLS000689218-01
0.96764
1.00798
0.98295
0.96996
1.08276
0.96628


MLS001215294-01
0.8079
0.80622
0.82158
0.89346
1.10597
1.04694


MLS001183575-01
0.68676
0.73778
0.79376
0.81721
0.96813
1.0887


MLS000393567-01
0.92366
0.8607
0.88565
0.90647
0.99533
0.88817


MLS000546316-01
1.17565
0.85707
1.09939
1.06571
1.17392
1.13749


MLS000912258-01
0.71931
0.87695
0.96307
1.14382
0.85708
0.92635


MLS000850522-01
0.93236
0.94157
0.92633
1.10981
0.90575
0.9439


MLS001197779-01
0.91301
0.82374
0.92492
1.10225
0.96099
1.03803


MLS001095705-01
0.79366
0.94108
0.8798
1.07342
0.94627
0.9916


MLS000912562-01
0.70243
0.88725
0.8748
0.92895
0.85822
0.89803


MLS000092785-01
0.82253
0.82933
0.85855
1.02029
0.96616
1.02722


MLS000418650-01
0.75932
0.75808
0.96539
1.03182
1.00364
1.08596


MLS000860966-01
0.89269
1.05975
1.10725
1.1137
0.82114
0.92753


MLS001180929-01
1.13472
0.86586
1.02995
0.96426
0.92563
0.89896


MLS000564564-01
1.08968
0.80854
1.05291
0.94262
0.94023
0.95865


MLS001164432-01
0.84639
0.71209
0.99848
0.98784
0.95093
0.89821


MLS000621451-01
1.02447
0.83274
1.07136
0.92535
0.8932
0.94711


MLS000078709-01
0.48942
0.63113
0.98985
0.95354
0.82363
0.83742


MLS000688267-01
1.13079
0.88079
1.10089
1.13158
0.83733
0.9011


MLS001006516-01
0.71268
0.71266
0.90855
0.92575
0.87139
0.92612


MLS001095033-01
0.91577
0.89377
0.92979
0.94401
0.94882
0.99586


MLS000072290-01
0.17841
0.47499
0.84049
0.90961
1.12146
1.10865


MLS000036988-01
0.10401
0.41718
0.82226
0.80994
0.75767
0.85532


MLS001217935-01
0.52464
0.78867
1.08057
0.88116
1.01763
0.90754


MLS000777780-01
0.77559
0.88145
0.93933
0.86968
0.98371
1.04663


MLS001174740-01
0.70642
0.78226
0.90166
0.85739
0.86868
0.90778


MLS000392555-01
0.93005
0.93498
0.97413
1.10516
0.96637
0.96465


MLS000693795-01
0.95128
1.00214
0.93842
0.9381
0.91713
0.83969


MLS000684034-01
0.73851
0.74026
0.80757
0.83826
0.95363
0.97308


MLS001172822-01
0.78582
0.84656
0.86099
0.85134
0.93148
0.94964


MLS000052969-01
0.55483
0.66209
0.876
0.91836
1.05823
0.91311


MLS001217212-01
0.73655
0.76634
0.87723
0.88308
0.83859
0.82329


MLS001004864-01
0.37599
0.60748
0.96185
0.97835
1.02518
0.949


MLS001116535-01
0.83664
0.89931
0.85528
0.89286
1.00307
0.95003


MLS001165424-01
0.76414
0.77905
0.83154
0.89381
0.85679
0.87714


MLS001116079-01
0.96857
0.90267
0.90118
0.95586
0.95017
0.95237


MLS001198271-01
0.82169
0.90557
0.98564
1.11296
0.9334
1.0083


MLS001167798-01
0.72315
0.79831
0.93824
1.11791
0.92426
1.05071


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1.17995
0.97031
0.93451


MLS001163121-01
1.09034
1.07048
1.17333
0.93955
0.98945
1.05191


MLS001060561-01
0.93292
0.85568
0.99768
0.98322
0.93293
0.80472


MLS001139515-01
1.01273
1.12444
0.94878
1.01346
1.00945
0.9636


MLS001149811-01
0.54317
0.84563
0.90865
0.94337
0.99933
0.95702


MLS000773700-01
0.47196
0.98949
0.95155
0.71177
1.01875
1.00839


MLS001177045-01
0.96779
1.10985
1.0851
1.02356
0.81454
1.04675


MLS000693747-01
1.0456
1.12188
0.91107
1.43271
0.71944
1.06136


MLS001175556-01
0.75145
0.90095
0.90127
1.09994
1.02814
0.94901


MLS001175473-01
0.79712
1.04117
0.99773
0.8872
0.91773
0.95559


MLS002156278-01
1.02456
0.97186
0.99244
0.69084
0.9637
1.00503


MLS000707281-01
1.14087
1.15567
0.75052
1.05935
0.65371
1.01094


MLS000591198-01
0.70144
0.98525
0.85477
1.00046
0.88105
1.01695


MLS000714175-01
1.02244
1.11863
1.01289
1.13111
1.06141
1.08575


MLS002163386-01
1.01995
1.06351
1.06415
1.11206
1.04751
1.10384


MLS000761297-01
1.09327
1.04516
1.03348
1.15364
1.02167
1.0906


MLS002245351-01
1.03102
1.06127
1.042
1.10724
0.94631
1.04658


MLS000718886-01
0.97849
0.93799
0.99814
1.06782
0.95909
0.98188


MLS002156485-01
1.15603
1.05728
1.0687
1.2005
0.98067
1.0551


MLS001140657-01
0.97076
0.9948
0.99812
1.08308
0.95059
1.03407


MLS002157024-01
1.07674
1.18645
0.93233
1.12188
0.88418
1.01348


MLS000721730-01
1.07097
0.87695
1.02458
0.85627
1.06447
0.91949


MLS000705922-01
1.03921
0.90721
0.95439
0.85107
1.13665
0.89838


MLS000724709-01
0.97675
0.885
1.00234
1.04022
1.46739
0.99608


MLS002161757-01
1.14604
0.97951
1.06023
1.09241
1.06958
0.8459


MLS002164687-01
1.0594
1.01681
1.36762
1.23385
1.00235
0.85316


MLS001060533-01
1.11677
1.34224
1.51976
1.31756
1.20754
0.94638


MLS000685139-01
1.26695
1.10202
1.24049
1.12834
0.92023
0.87033


MLS001217286-01
0.5964
1.09526
1.21873
1.19269
0.99106
0.76168


MLS001221619-01
1.03393
0.86027
1.01242
0.94404
0.95568
0.82601


MLS001219621-01
0.45369
0.648
0.86159
0.8883
0.86027
0.9394


MLS001166156-01
0.44609
0.82177
0.75916
1.07035
0.85209
1.00131


MLS000534926-01
0.85648
1.33769
0.9622
1.00584
0.94445
1.02592


MLS000548725-01
0.22187
1.03126
0.84619
0.9206
0.84182
0.86975


MLS000374261-01
0.44896
0.42298
0.92885
1.13089
0.83858
0.81441


MLS000123454-01
0.88519
0.68413
1.08253
1.04655
0.89626
1.06673


MLS000625140-01
0.65137
0.67934
0.91312
1.11353
0.84537
0.84836


MLS001214443-01
1.08326
1.15407
1.19298
0.94422
0.90181
0.82616
















TABLE 11







CMV-LACZ in Myoblasts














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000591667-01
1.0593
0.8030
1.1592
1.1587
0.7850
0.9491


MLS000568234-01
0.7322
0.8819
0.9894
0.9043
0.9372
0.9556


MLS000689562-01
1.0619
0.9919
1.0858
0.8060
0.7409
0.8453


MLS000732652-01
0.7856
0.9374
1.1235
1.1327
0.7744
0.6700


MLS001240181-01
0.8907
1.0367
1.1645
0.8408
1.0441
0.9550


MLS001211139-01
0.8744
1.0659
0.8608
0.9500
0.7347
0.9691


MLS001030268-01
0.5556
0.7467
1.1787
1.1347
1.1550
1.0809


MLS000912699-01
0.5204
0.8826
1.0462
0.9111
0.8744
0.7147


MLS001125260-01
1.2270
1.0563
0.9131
1.0114
1.0584
1.1581


MLS000717689-01
0.8996
1.0407
1.0936
1.1129
1.0203
1.0125


MLS001197665-01
1.0267
0.9556
0.8403
0.9214
0.8347
0.8584


MLS001075922-01
1.3037
0.9881
1.2361
1.6332
1.3028
0.9434


MLS001124046-01
1.0807
0.9881
0.8501
0.9307
1.2650
0.8409


MLS001197220-01
1.2844
0.9567
1.1383
0.9355
1.1569
1.2028


MLS001221318-01
1.5641
1.0181
0.9955
1.0999
1.0538
0.8528


MLS000947910-01
1.0500
1.0870
1.0379
0.9452
0.9250
1.0094


MLS001215795-01
0.8256
0.8196
0.8628
1.0974
1.1375
0.8003


MLS002163670-01
1.4715
0.8611
0.9725
0.8671
0.8738
1.1806


MLS001200149-01
1.1552
0.9363
1.0523
0.9489
0.9203
1.1234


MLS001359861-01
1.0330
0.9074
0.8064
0.9009
0.9184
0.9444


MLS000710669-01
0.8778
1.1319
1.0823
1.0325
0.9975
1.0969


MLS001035690-01
0.9178
1.3585
1.2020
0.9910
1.0406
0.9819


MLS001030621-01
0.9063
1.0307
1.1844
1.0494
1.0531
0.7081


MLS001083082-01
0.8144
0.9615
1.1713
1.0409
0.9234
0.8591


MLS000045588-01
1.2167
0.8056
1.0611
1.0245
0.8372
0.7647


MLS001216939-01
1.0644
1.1863
0.9452
1.0913
1.1134
1.0991


MLS001163859-01
0.8104
0.7593
1.1812
1.2009
1.1684
1.1113


MLS000683232-01
0.8237
0.6119
0.5224
0.5638
0.7131
0.8043


MLS001170856-01
0.8389
0.8881
1.0004
0.7938
0.9857
0.7337


MLS002667707-01
1.1226
1.0044
1.0803
1.1897
0.6763
1.1434


MLS001200665-01
0.9937
0.7152
1.0036
0.9398
0.9783
1.1043


MLS002161853-01
0.9348
1.0922
0.9436
0.8819
0.8734
1.0131


MLS002163101-01
1.1556
0.9644
1.0313
1.0262
1.0317
0.9991


MLS000062431-01
1.0993
1.0467
1.1632
0.9848
1.1631
0.9557


MLS000028160-01
0.8600
0.8967
1.2759
0.9378
0.9029
1.0663


MLS002248819-01
1.0270
1.7330
1.1575
1.2604
1.2206
0.9206


MLS000080654-01
0.8815
1.0174
0.9345
1.0703
1.4314
1.1851


MLS000760876-01
0.8504
0.9396
0.8973
0.8293
1.0546
0.9069


MLS000677675-01
1.1578
1.0763
1.0373
0.9905
1.1214
1.0343


MLS000113985-01
0.1841
0.2126
0.9187
1.0836
1.0049
0.9809


MLS001182368-01
1.1615
1.2322
0.9225
1.0528
0.8783
0.9571


MLS001212882-01
1.0326
1.3230
1.0086
0.9792
1.1291
1.1409


MLS001004364-01
1.0441
1.0263
0.9011
1.0438
1.0643
0.9117


MLS000736846-01
1.0174
1.3459
1.1101
1.0269
1.0386
1.1009


MLS001098105-01
0.8704
1.0381
0.9421
0.9282
0.8054
1.4689


MLS000678673-01
0.9656
0.8674
0.8991
1.0561
1.1914
1.2106


MLS000925023-01
0.3278
0.5504
0.8397
0.9762
0.9629
1.0703


MLS001212319-01
1.0259
1.2056
0.8600
1.0840
1.2040
0.9129


MLS000779126-01
0.7441
0.9944
0.7610
0.9637
0.9994
0.9751


MLS000948055-01
0.5522
0.5407
1.0993
1.0787
0.9674
0.7540


MLS000110418-01
0.7141
0.8926
0.9455
1.0913
0.9989
0.8871


MLS000693704-01
1.0567
0.7996
0.9977
0.9522
1.1157
0.8989


MLS001225512-01
1.0111
0.8237
1.4266
1.1655
1.0843
0.9597


MLS001006798-01
0.8263
0.9622
1.0189
1.1509
1.0326
0.8334


MLS000711491-01
0.7800
1.5063
1.0199
0.9088
1.1060
1.0889


MLS000582947-01
0.8819
0.7811
1.0067
1.1273
0.8991
0.9691


MLS000531177-01
0.9422
1.2952
0.8958
1.0150
1.5065
1.0785


MLS001202389-01
0.5081
0.6115
0.8920
1.0084
1.1108
1.0237


MLS000536064-01
2.4896
2.5396
1.3388
2.0402
0.7412
0.9105


MLS000586245-01
0.7674
0.7726
1.0505
0.9258
0.8985
0.9034


MLS001061374-01
1.1037
0.9744
0.9793
1.0601
1.1702
0.7117


MLS000675441-01
0.7437
0.8656
0.8690
0.8800
0.9289
1.0332


MLS001200396-01
0.9011
0.7252
1.2107
1.0212
0.9157
1.0489


MLS001165937-01
0.4007
0.4526
0.7441
0.8816
0.7788
0.7295


MLS000325736-01
1.0537
1.0111
1.0696
0.9758
0.9135
0.7862


MLS001215357-01
0.4719
1.2467
1.0378
0.8575
1.2686
1.1560


MLS000588210-01
0.9000
1.0307
0.9316
0.7808
0.9782
1.2908


MLS000764729-01
0.7504
1.1200
0.8880
1.0259
0.9520
0.9865


MLS000689492-01
1.0026
1.0141
0.8653
1.0863
1.0252
0.9363


MLS001000299-01
1.0107
1.1522
0.8248
0.9901
1.2745
1.4188


MLS000393762-01
0.4467
0.3793
1.0198
1.0114
0.9351
0.9560


MLS001130011-01
0.8311
0.7837
1.1138
1.0011
0.7695
1.1702


MLS001229477-01
0.8800
1.0674
1.1311
1.1078
1.1345
1.0283


MLS000707378-01
0.6548
0.8667
0.8072
0.8309
1.0883
1.0175


MLS000573208-01
0.9289
0.6626
0.8678
1.1030
0.9594
1.0197


MLS001167281-01
0.7467
0.9256
1.1951
0.9684
1.1289
1.0114


MLS000053342-01
0.9930
0.9811
0.8785
1.0865
1.0397
1.0289


MLS002171615-01
0.6726
1.2159
0.9905
0.9273
1.3505
1.0498


MLS001005712-01
1.0230
1.0241
0.9125
0.8934
1.2385
1.0188


MLS001176153-01
1.0763
0.9322
0.9140
0.8976
1.4588
1.2729


MLS000735021-01
0.9767
1.2130
0.9907
0.9756
1.1646
0.8683


MLS000767397-01
1.1433
0.8485
0.9687
0.9910
1.0646
1.2748


MLS001196572-01
1.0778
1.0767
0.9456
1.0605
1.3172
1.0105


MLS000393966-01
0.9156
1.0411
0.8087
1.1744
1.0625
0.9249


MLS001034810-01
0.7030
1.1193
0.9459
1.0948
0.9538
0.9698


MLS001165394-01
0.9570
0.8663
0.9792
1.0941
1.0015
1.0655


MLS000089464-01
0.5274
0.7500
1.1969
0.9372
0.9160
0.9117


MLS000698617-01
1.0356
1.1670
1.0603
1.1399
0.9852
1.3409


MLS001175021-01
1.0907
1.1070
0.9395
0.9282
1.0729
1.1772


MLS001166758-01
0.3481
0.6126
1.0661
1.1998
1.0175
0.9357


MLS001008109-01
0.7619
0.8730
1.2178
0.9228
1.1138
0.9542


MLS001181936-01
0.6130
0.7863
0.8296
0.7292
0.9273
0.8487


MLS000560266-01
1.0484
1.1095
0.9991
0.9405
0.8123
0.9030


MLS001215074-01
1.1432
1.1277
1.0294
1.1754
0.7800
0.7877


MLS001215123-01
1.0098
1.2639
1.1943
1.0888
0.8837
1.0530


MLS001033255-01
0.7905
1.1919
1.0219
0.8766
1.0723
0.7590


MLS001160611-01
1.0389
0.8144
1.0340
1.0117
0.8157
1.2070


MLS001006302-01
0.7646
0.9502
1.0767
0.8911
0.7663
0.9343


MLS001123876-01
0.7333
0.9618
0.9871
0.9251
1.0263
0.8260


MLS001122698-01
1.0639
1.0404
0.8608
0.7759
1.1003
1.1513


MLS000755214-01
0.8172
1.0835
1.0994
0.9281
1.1650
1.0190


MLS000731285-01
0.6572
0.9582
0.8436
1.0851
1.0853
1.1747


MLS000776409-01
0.2105
1.3344
0.9052
0.8929
0.9103
1.1220


MLS001221908-01
0.6789
0.9726
0.7908
0.9357
0.7607
0.9663


MLS000419286-01
0.6295
0.9368
0.8022
0.9701
0.7960
0.9960


MLS000554416-01
0.8611
1.1077
1.1123
0.8594
1.3983
1.1310


MLS000073150-01
0.9000
0.9491
0.9954
0.8193
1.8970
1.0753


MLS000663185-01
0.9347
0.9211
0.8567
0.9688
1.1477
1.1390


MLS001078811-01
0.9014
0.8393
0.6873
0.7927
1.2290
1.2397


MLS002694363-01
0.5698
1.0123
1.0392
0.7616
1.2840
1.1110


MLS000689218-01
1.6337
1.2835
1.0813
0.8689
1.2273
1.1977


MLS001215294-01
0.4116
0.3302
1.3412
0.8138
1.2817
1.2983


MLS001183575-01
0.8856
1.2825
0.7762
0.8972
1.2440
1.1603


MLS000393567-01
1.1319
1.0175
0.8148
0.6593
1.1373
1.2117


MLS000546316-01
1.5049
1.3463
1.2440
0.8090
1.1373
1.0623


MLS000912258-01
0.8621
1.1074
1.0171
0.9071
1.1693
1.1073


MLS000850522-01
1.0091
0.8358
0.8671
0.8809
1.3763
0.8880


MLS001197779-01
1.1267
0.9846
0.6490
0.7604
1.2563
0.8987


MLS001095705-01
1.1723
1.1400
0.7229
0.6476
1.2293
1.2060


MLS000912562-01
0.8747
1.0302
0.6246
0.7103
0.9150
0.8680


MLS000092785-01
0.3235
1.1004
0.7351
0.9882
1.1653
0.8600


MLS000418650-01
1.1323
1.1523
0.8344
1.0080
0.9533
0.9237


MLS000860966-01
0.7600
0.6232
0.8424
0.7918
1.0900
0.9370


MLS001180929-01
0.6163
0.9811
0.8753
0.8251
0.9288
0.8571


MLS000564564-01
0.8844
1.0422
0.9091
0.9399
1.0092
0.9117


MLS001164432-01
1.1289
1.1059
1.3183
1.1387
0.7842
0.9600


MLS000621451-01
1.1926
1.2493
1.3023
0.9255
1.1579
1.0388


MLS000078709-01
0.4830
0.6919
1.0163
0.9270
0.7008
0.8167


MLS000688267-01
1.0774
1.1407
0.8935
0.9129
1.1579
1.0950


MLS001006516-01
0.6589
0.4956
0.9076
0.9829
0.8858
1.0038


MLS001095033-01
0.9811
0.9359
1.0269
0.9043
0.9179
0.9488


MLS000072290-01
0.2915
0.3911
1.3303
1.4390
0.9000
1.0579


MLS000036988-01
0.6578
0.8796
1.0435
0.9702
1.0846
1.0767


MLS001217935-01
0.5381
0.7859
1.0465
1.0175
1.1167
1.1729


MLS000777780-01
0.9463
1.3393
1.0126
0.9893
0.9063
0.8779


MLS001174740-01
0.4578
0.4293
0.9490
1.0046
0.9721
1.2750


MLS000392555-01
1.2007
0.9559
0.9799
0.9342
0.7638
0.8954


MLS000693795-01
0.8730
0.8526
0.9207
0.9554
1.1104
0.9100


MLS000684034-01
0.3100
0.5374
1.0568
1.2045
1.1492
1.2525


MLS001172822-01
1.2767
0.9285
1.0904
1.1375
1.0617
0.9304


MLS000052969-01
0.6800
1.1522
1.0444
0.9976
1.0529
0.8950


MLS001217212-01
0.9459
1.1137
1.0626
1.0711
0.9179
1.1804


MLS001004864-01
0.9933
1.2930
1.0417
1.2276
0.9288
1.2650


MLS001116535-01
1.1263
1.1815
1.0580
1.0944
1.1779
1.0167


MLS001165424-01
1.2074
1.2730
1.0078
1.0607
0.9796
1.1754


MLS001116079-01
0.9078
1.0448
1.0228
0.9306
1.0492
1.0950


MLS001198271-01
1.2089
0.9944
0.8516
1.2105
1.0883
1.0204


MLS001167798-01
1.1452
1.2196
1.1494
1.1822
0.7750
1.0342


MLS000710288-01
0.9063
1.1193
1.2515
1.0737
0.9325
1.0338


MLS000734270-01
0.9548
1.0122
0.9468
0.9337
0.8379
0.9296


MLS000858981-01
0.9696
0.9985
0.9009
1.1687
0.9696
0.9229


MLS000698826-01
1.2519
1.2944
0.9013
0.8824
0.9717
0.9942


MLS001000874-01
1.1100
1.0556
1.0295
0.8886
1.0442
1.1029


MLS000682750-01
1.0404
1.1433
0.9934
0.8717
1.0467
0.9679


MLS001090787-01
0.5907
0.7533
0.8594
0.9700
0.9754
0.9196


MLS002636056-01
0.8270
1.1173
1.2571
1.0394
0.9021
0.9392


MLS002170630-01
0.7387
0.8233
0.9628
0.9890
0.8054
0.9954


MLS002162890-01
0.9507
0.7603
0.9939
0.9701
0.8713
0.8392


MLS001105912-01
0.3873
0.5323
0.9597
1.0003
1.0321
1.0725


MLS001007892-01
0.4953
0.6390
1.1495
1.0511
0.8108
0.9338


MLS000089748-01
0.4437
0.3140
0.9905
1.0665
0.8592
1.2129


MLS000912726-01
1.0643
1.0257
1.0325
1.0182
0.9692
1.1004


MLS000086970-01
0.8880
1.0197
0.9068
0.8994
1.0504
0.9925


MLS000420298-01
1.1930
0.9807
1.0482
0.8414
0.9383
1.2808


MLS001147478-01
0.8747
1.0513
1.2999
1.2105
0.8808
1.0721


MLS000090135-01
0.3653
0.6017
0.9296
0.9731
0.9421
1.0896
















TABLE 12







CMV1: AB305V-LACZ in Myotubes














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000591667-01
0.8800
0.8929
1.0152
1.0824
1.0726
0.8600


MLS000568234-01
0.9141
0.9918
0.9074
1.0764
1.2622
1.3844


MLS000689562-01
0.9788
0.9824
0.8105
1.0300
0.8652
0.9474


MLS000732652-01
1.0329
0.8518
1.0697
1.0614
0.8859
1.1615


MLS001240181-01
1.1376
1.0976
0.9955
1.0895
1.0511
0.9519


MLS001211139-01
1.0224
0.8259
1.0196
1.1755
0.8948
0.9281


MLS001030268-01
0.9506
0.9365
1.1128
1.1318
1.1711
0.8830


MLS000912699-01
0.5365
0.9235
1.1310
0.9419
0.9859
0.8089


MLS001125260-01
0.9212
1.0000
1.0994
0.9614
1.0993
1.0074


MLS000717689-01
0.9129
0.9576
0.9569
0.9686
1.0956
0.8415


MLS001197665-01
1.0741
1.0047
1.2593
0.9771
0.8756
0.9415


MLS001075922-01
0.8388
1.0282
1.2173
1.2947
1.0533
1.0222


MLS001124046-01
0.9812
1.0906
1.0202
0.8395
1.0156
1.0193


MLS001197220-01
1.0271
0.9659
0.9289
0.9682
0.9356
0.8896


MLS001221318-01
0.8576
1.2212
0.9944
1.1337
1.4059
1.0037


MLS000947910-01
1.0082
1.0165
0.8930
1.2332
0.8348
1.0333


MLS001215795-01
1.1035
1.3929
1.0250
0.9309
1.0207
0.9363


MLS002163670-01
1.1235
1.1659
0.8898
0.9788
1.2044
0.9237


MLS001200149-01
0.9424
0.8318
0.8176
0.9974
0.9933
0.8378


MLS001359861-01
0.9412
0.9976
1.1230
0.9547
0.9533
0.8978


MLS000710669-01
1.1824
0.8682
1.2372
0.9690
0.8881
0.9341


MLS001035690-01
1.0976
1.1976
1.1317
1.1552
0.8644
1.0474


MLS001030621-01
1.0141
0.9024
1.0546
0.9872
0.7970
0.7844


MLS001083082-01
1.1165
1.0282
0.9776
0.9984
1.0326
0.9615


MLS000045588-01
0.9424
1.0494
0.9776
1.2294
0.8607
0.9059


MLS001216939-01
0.9659
1.1518
1.2568
1.1826
0.9748
1.0311


MLS001163859-01
1.2482
1.0965
1.1727
1.0856
1.1326
0.8756


MLS000683232-01
0.8365
0.8835
1.0086
0.8743
0.7752
0.8200


MLS001170856-01
0.8753
1.1518
1.0562
1.0074
0.9105
1.1095


MLS002667707-01
1.1071
1.1576
1.0227
0.9449
1.0590
0.8905


MLS001200665-01
1.2706
1.1800
0.9662
1.1274
0.9695
1.1543


MLS002161853-01
1.0647
1.2341
0.9103
1.1122
0.8352
1.0524


MLS002163101-01
1.5447
0.9212
0.8407
0.8302
1.1400
1.0533


MLS000062431-01
1.2047
0.8812
0.9690
0.9004
0.9743
0.9133


MLS000028160-01
1.1976
1.0471
0.9249
1.0348
1.0362
1.0524


MLS002248819-01
1.1929
0.9847
1.1534
1.1171
0.9505
1.0819


MLS000080654-01
1.4259
1.1871
1.0399
1.0335
1.0086
1.1229


MLS000760876-01
1.2565
0.9482
0.9789
1.1074
1.1010
1.0248


MLS000677675-01
1.2494
0.9918
1.0091
1.0578
0.9962
0.8619


MLS000113985-01
1.0482
1.0541
1.0241
1.0031
1.0638
1.0629


MLS001182368-01
1.0318
0.9953
0.8690
0.8931
1.0619
0.8667


MLS001212882-01
1.3024
1.1471
1.1133
1.0963
0.8705
1.0238


MLS001004364-01
1.1824
1.2824
0.9480
0.8978
1.0095
0.9210


MLS000736846-01
1.2788
1.1000
1.1737
1.0250
0.9714
0.8438


MLS001098105-01
1.2553
0.9671
1.3183
0.9211
1.2543
0.9248


MLS000678673-01
1.1835
0.9635
0.9367
0.7918
1.0286
1.1229


MLS000925023-01
1.1106
1.1565
0.8859
1.1638
1.1333
0.7505


MLS001212319-01
1.0706
0.9965
0.9253
0.9525
0.8152
1.1524


MLS000779126-01
1.1671
0.9988
1.0467
1.0257
1.2162
0.9495


MLS000948055-01
1.0753
1.2212
1.1022
1.2519
0.9638
1.0895


MLS000110418-01
1.3647
1.0459
0.9599
0.9617
0.9000
1.0333


MLS000693704-01
1.0600
1.1647
1.0812
1.1170
0.8695
0.8571


MLS001225512-01
0.8871
1.2482
1.0077
0.9459
1.0381
1.0162


MLS001006798-01
1.0976
0.9953
1.0267
1.2236
0.9648
1.0419


MLS000711491-01
0.9929
1.1318
0.8645
0.9993
0.9095
0.9495


MLS000582947-01
1.0247
1.0729
0.9127
0.9280
0.9610
0.8848


MLS000531177-01
1.1522
0.9244
0.8917
0.8757
1.0411
1.1989


MLS001202389-01
0.9311
1.0900
1.1312
1.0432
1.1821
1.1558


MLS000536064-01
1.0233
1.2344
1.1027
0.8894
1.0389
0.9705


MLS000586245-01
1.2656
1.2267
1.1338
0.9511
1.0800
1.0611


MLS001061374-01
1.0489
1.1689
0.8498
1.1237
1.1000
1.2568


MLS000675441-01
0.9822
0.9578
1.0337
1.0460
1.1032
1.0074


MLS001200396-01
0.9733
0.9644
0.7738
0.8987
0.8442
0.9137


MLS001165937-01
0.7222
0.7678
0.7827
0.8619
0.9663
0.9305


MLS000325736-01
0.9889
0.8244
0.9324
0.9630
0.8968
0.9263


MLS001215357-01
1.0667
1.3622
1.1192
1.1407
1.1400
1.1547


MLS000588210-01
1.1656
1.1322
1.0815
0.9182
1.0853
1.1263


MLS000764729-01
1.1900
0.9656
0.9634
1.0779
1.1432
1.0358


MLS000689492-01
0.9267
1.0444
1.0940
1.0504
1.2400
1.0853


MLS001000299-01
1.0744
0.9544
0.9029
1.0658
1.3158
0.6411


MLS000393762-01
1.0578
1.0800
1.0049
1.0005
0.9326
0.8653


MLS001130011-01
1.0156
0.8367
0.8998
0.9057
1.0211
0.9358


MLS001229477-01
0.9611
0.9622
0.9587
0.9250
1.1095
1.0126


MLS000707378-01
0.9167
0.9689
1.0300
0.9294
1.3937
1.0537


MLS000573208-01
0.9478
0.9633
0.8913
1.1765
1.3021
0.9547


MLS001167281-01
0.9722
1.1089
0.9727
0.8948
1.0789
1.0663


MLS000053342-01
1.1344
1.3044
0.9187
0.9033
0.9863
0.9947


MLS002171615-01
1.0711
1.0111
0.9582
0.9989
0.7874
0.9863


MLS001005712-01
1.3167
1.1133
0.8075
0.9623
1.0379
1.0684


MLS001176153-01
1.0878
1.0256
0.9388
0.7944
0.9832
0.8316


MLS000735021-01
1.0778
1.0378
0.9616
0.9548
1.0221
1.0105


MLS000767397-01
1.0067
1.0144
0.9519
0.9425
1.0368
0.8737


MLS001196572-01
1.1422
1.1456
1.0223
0.9853
0.9379
0.9632


MLS000393966-01
1.1167
1.0378
1.1877
0.8958
1.3516
0.9358


MLS001034810-01
1.1344
1.0578
1.0965
0.9578
1.7389
1.1000


MLS001165394-01
0.9544
1.0344
0.9045
0.9589
1.1558
0.9232


MLS000089464-01
1.0178
1.3067
0.9529
0.9400
1.3716
0.9716


MLS000698617-01
1.0111
1.0011
0.9614
0.9014
1.0842
1.1221


MLS001175021-01
0.9733
1.1378
0.7979
0.9049
0.9621
1.0611


MLS001166758-01
0.9867
1.1044
0.9444
0.9780
0.7474
0.9958


MLS001008109-01
1.0600
1.0756
0.9289
0.9839
1.0168
1.0621


MLS001181936-01
1.0280
0.9020
1.0274
0.9246
0.9886
1.0705


MLS000560266-01
0.9470
1.0520
1.0224
1.0253
1.0000
0.9724


MLS001215074-01
0.9930
0.8700
0.8583
0.8364
0.9238
0.8752


MLS001215123-01
1.1030
1.0710
1.2929
1.1153
1.1457
1.1657


MLS001033255-01
0.7740
0.9930
1.0297
0.9525
1.1438
1.1038


MLS001160611-01
0.9190
1.0240
0.9661
0.8306
1.2419
0.9762


MLS001006302-01
0.9720
0.9010
1.0122
0.9398
0.9143
1.0229


MLS001123876-01
1.0300
0.9880
0.9279
0.9002
1.4943
0.8962


MLS001122698-01
1.0110
1.0340
1.1482
1.2038
1.0133
1.0019


MLS000755214-01
1.0190
0.9280
1.0591
1.0763
0.8600
0.9543


MLS000731285-01
0.6450
1.0310
1.3012
1.4794
1.0562
1.2048


MLS000776409-01
1.1540
1.1040
1.1035
1.0186
0.9752
1.0552


MLS001221908-01
0.8850
1.0770
1.1838
0.9745
0.9171
0.8562


MLS000419286-01
0.7830
0.8530
1.1581
1.0268
1.1867
0.9990


MLS000554416-01
0.7060
0.8760
0.9798
0.8969
0.9629
1.0019


MLS000073150-01
1.0390
0.9750
1.1088
1.2058
1.0848
1.0752


MLS000663185-01
1.1270
0.9540
0.8722
0.9322
0.9800
1.0924


MLS001078811-01
0.6710
1.0510
0.9981
1.1571
0.9086
0.8486


MLS002694363-01
0.7460
0.9870
0.9775
1.1420
1.1800
1.0695


MLS000689218-01
1.7990
1.4900
1.2135
1.3684
0.9514
0.9657


MLS001215294-01
1.4470
1.2800
1.4324
1.1438
1.0800
0.8838


MLS001183575-01
1.1230
0.8230
0.9136
0.7726
0.8657
0.9648


MLS000393567-01
0.9100
1.0160
0.9858
0.9322
0.9524
1.0400


MLS000546316-01
1.2640
1.0190
1.0825
1.0040
1.0019
1.2590


MLS000912258-01
1.0350
1.0760
1.2253
1.1251
0.9162
0.9743


MLS000850522-01
1.0200
1.0580
1.0751
1.1090
0.8933
0.8848


MLS001197779-01
1.0540
1.0630
1.0983
1.0456
1.1705
1.1286


MLS001095705-01
1.0800
1.2020
1.3164
1.1898
0.9933
0.9486


MLS000912562-01
1.2780
1.2090
1.1674
0.9602
0.8924
1.0905


MLS000092785-01
1.0500
1.0400
1.0738
1.0185
1.0143
0.9105


MLS000418650-01
1.2260
1.1180
1.1746
1.0198
1.2819
1.0086


MLS000860966-01
0.7920
0.9190
0.8601
0.9632
1.0743
0.8229


MLS001180929-01
1.0274
1.1347
1.0084
1.0533
0.7876
0.8848


MLS000564564-01
1.0411
0.9779
1.1848
1.0689
0.9924
0.8267


MLS001164432-01
1.1179
1.1358
1.2418
0.9500
1.3543
0.9248


MLS000621451-01
0.9695
0.9853
1.1398
1.0177
0.9295
1.1114


MLS000078709-01
1.7737
1.0853
0.8743
0.9348
0.9257
1.0133


MLS000688267-01
1.1505
0.9505
1.2248
0.9634
1.1333
0.8648


MLS001006516-01
0.9484
0.9032
0.8542
0.9283
0.9257
0.9581


MLS001095033-01
1.0358
1.0326
1.0031
1.0230
1.0867
1.2895


MLS000072290-01
1.0653
1.3484
1.1459
1.3265
1.1495
0.9362


MLS000036988-01
0.5021
0.7116
1.0868
0.8979
0.8905
0.9867


MLS001217935-01
0.9495
1.0547
0.9648
1.1473
1.1381
0.9705


MLS000777780-01
1.0474
0.9316
0.8808
1.0471
0.8838
0.8914


MLS001174740-01
0.8863
1.0568
1.0520
0.9101
0.9267
0.8533


MLS000392555-01
0.9326
1.1537
0.8568
1.0094
0.9676
1.0257


MLS000693795-01
1.0716
1.0453
1.0774
1.1780
1.0314
0.9771


MLS000684034-01
1.0105
1.2347
1.1903
1.1407
0.9810
1.0962


MLS001172822-01
0.6358
1.1768
1.1819
1.2505
1.0514
1.2590


MLS000052969-01
0.9726
0.9800
0.8406
1.0651
1.1714
0.9152


MLS001217212-01
1.0105
1.1842
1.0832
0.9795
0.9610
1.0190


MLS001004864-01
0.8884
0.9653
0.9501
1.0697
0.9610
0.9657


MLS001116535-01
1.0011
0.9800
1.2856
1.0648
0.8771
1.2552


MLS001165424-01
0.8558
1.0316
1.1758
1.0571
1.1248
0.9152


MLS001116079-01
1.0811
0.9063
1.1374
1.0227
0.8152
1.3543


MLS001198271-01
1.0053
1.1053
0.9957
0.9907
1.0686
0.9933


MLS001167798-01
1.2800
1.3337
1.2238
1.1987
0.9295
0.9143


MLS000710288-01
0.8905
0.9400
1.1225
1.0717
1.1133
1.0371


MLS000734270-01
0.9095
1.1158
1.0885
1.1042
1.0038
1.0019


MLS000858981-01
1.0000
1.0421
0.9781
1.0786
0.8419
1.0257


MLS000698826-01
1.0789
1.0505
0.9634
1.1232
1.0200
1.2010


MLS001000874-01
1.0947
1.1484
0.9960
1.1124
1.1590
1.0943


MLS000682750-01
1.0579
1.1189
1.0939
1.1285
0.9638
1.0248


MLS001090787-01
0.9450
0.8990
1.1178
1.0126
0.9055
1.0009


MLS002636056-01
1.1160
1.4090
1.1613
1.0269
1.2636
0.9855


MLS002170630-01
0.8580
0.9710
0.9248
1.2068
1.0036
0.8136


MLS002162890-01
1.0170
1.0140
1.0530
1.1237
1.0191
1.0682


MLS001105912-01
1.8130
1.1910
1.0857
0.9313
0.9618
1.1391


MLS001007892-01
1.0540
1.2170
0.9543
1.0066
0.9291
1.1673


MLS000089748-01
1.2820
1.3800
0.9813
1.1104
0.9809
0.9527


MLS000912726-01
1.0150
1.2060
1.1041
0.9111
0.9264
0.8527


MLS000086970-01
0.9880
0.9870
0.9738
1.1136
0.8855
0.9673


MLS000420298-01
1.1250
1.3660
1.2388
1.0338
0.8527
0.9573


MLS001147478-01
0.9510
1.0810
1.2959
0.8937
1.3827
1.3536


MLS000090135-01
1.1840
1.1100
1.0114
0.7503
1.1036
1.0155


MLS001179717-01
0.48286
0.57476
0.81333
1.084


MLS000683234-01
0.93
0.69524
0.81111
0.82044


MLS000695955-01
0.93
0.99238
0.896
0.78933


MLS001125488-01
0.87238
1.1819
0.96756
0.74711


MLS000768008-01
0.94286
1.02762
0.96444
0.82756


MLS000913117-01
0.65381
0.59381
0.82622
0.80356


MLS000860538-01
0.96667
1.01667
0.96133
1.00133


MLS001177259-01
1.10048
0.85429
0.73156
0.94622


MLS000861434-01
0.8381
0.84286
0.80267
0.88444


MLS000047918-01
1.05
1.03762
0.8
0.93733


MLS000389484-01
2.78095
1.11571
0.83867
0.85244


MLS001217673-01
0.93524
1.16095
0.94089
0.936


MLS000389677-01
0.76095
1.01857
0.74178
0.81422


MLS001208858-01
0.92381
0.94714
0.80178
0.944


MLS000333610-01
0.99476
0.94286
0.78667
0.832


MLS001117351-01
0.89524
1.1481
1.08756
1.01644


MLS000682883-01
0.89667
1.08667
1.08356
0.93333


MLS001095231-01
0.62286
0.9181
0.74089
1.03511


MLS000721584-01
1.1019
1.13333
0.90711
0.95733


MLS001183429-01
0.58095
0.91048
0.944
0.84356


MLS002158881-01
0.80952
0.86429
0.82667
0.79244


MLS001165749-01
1.03762
1.02429
0.92844
0.82711


MLS001237320-01
1.08238
0.98952
0.75689
0.95822


MLS000763405-01
0.80238
0.82429
0.876
1.04978


MLS000538580-01
0.27048
0.38381
0.57289
0.66133


MLS001033202-01
1.19952
0.82143
0.99333
0.96578


MLS001216260-01
0.83667
1.08952
1.03111
0.85156


MLS000085522-01
0.95095
0.77333
0.98044
1.092


MLS000702680-01
1.18143
1.23286
1.10711
1.00756


MLS001212998-01
0.78333
0.9719
0.90844
0.78


MLS001160885-01
0.8319
0.90952
0.90089
1.16711


MLS001122718-01
0.89
0.95667
0.96622
0.88622


MLS000027478-01
0.97905
0.94429
0.924
0.94889


MLS001177364-01
0.97429
1.05286
0.95778
1.26222


MLS001179695-01
1.04714
1.17524
1.09467
1.18311


MLS002251986-01
0.97762
0.82381
0.96089
0.98311


MLS001166704-01
1.0685
1.4595
1.1553
1.08848


MLS001196422-01
0.856
0.8295
0.89309
0.89631


MLS001179624-01
0.4965
0.452
0.8788
0.86037


MLS001223425-01
1.0095
0.979
1.03502
0.95622


MLS001117140-01
1.1745
1.3785
1.12442
0.95069


MLS001110618-01
0.4765
1.0555
0.93364
0.89539


MLS001223482-01
0.8095
0.858
0.79309
0.90691


MLS000680049-01
0.4315
1.4405
1.10599
1.12719


MLS001212498-01
0.8475
0.749
0.78387
0.84977


MLS001124732-01
0.972
1.07
1.01567
1.02488


MLS000526364-01
1.1865
1.1735
0.953
0.95069


MLS000767227-01
1.1815
0.9975
1.09954
1.02396


MLS000703499-01
0.769
0.689
0.99677
0.93134


MLS001167169-01
1.103
1.085
1.00876
0.93226


MLS001198693-01
1.112
1.025
0.92581
1.09171


MLS001219345-01
1.0255
1.0075
0.86313
1.18802


MLS001211651-01
0.977
0.8465
0.84332
0.87143


MLS000806880-01
1.0655
0.9145
1.12995
0.96544


MLS001223567-01
1.012
1.241
0.89309
1.00184


MLS001005283-01
1.1425
1.032
0.90507
0.90046


MLS001218427-01
0.6965
34.571
15.1793
1.20922


MLS001139288-01
1.0825
1.096
1.04654
1.24654


MLS000696445-01
0.924
1.122
1.01106
1.26774


MLS001218795-01
0.989
1.2115
1.02258
0.9553


MLS000419555-01
1.22
1.2165
1.21475
1.21982


MLS001225507-01
0.8805
0.8655
0.99401
1.04009


MLS000663651-01
0.861
0.949
0.9788
0.96359


MLS000706349-01
1.1355
1.0245
0.9553
0.94654


MLS000393110-01
0.8895
1.0745
0.96728
1.10323


MLS000574647-01
1.0095
0.962
0.93641
0.91659


MLS000532969-01
0.8385
0.8525
0.88295
0.77051


MLS001125260-01
1.175
1.0765
1.06498
0.96452


MLS000122749-01
1.0025
0.808
1.06267
1.02166


MLS001150751-01
0.889
0.992
0.83364
0.91336


MLS001221867-01
1.0745
1.0515
0.98341
0.99539


MLS001147727-01
1.1835
1.2365
0.90691
1.11982


MLS000688437-01
1.05088
1.02982
0.93462
1.23248


MLS001211976-01
1.01404
0.96974
0.99786
0.8547


MLS002161350-01
1.04561
0.88465
0.96795
0.90598


MLS001077207-01
0.78246
0.86623
1.0359
0.95214


MLS001209245-01
0.99518
0.96623
0.96752
0.85684


MLS000737953-01
0.90965
0.94781
1.22393
0.82735


MLS000552080-01
0.80439
0.95219
1.0688
1.19658


MLS000737204-01
0.3807
0.57719
0.80684
1.02906


MLS000579238-01
0.9443
0.83026
0.87179
0.81453


MLS001181671-01
0.93772
1.03026
0.92009
0.88034


MLS001167424-01
0.99254
1.15702
0.97179
0.74915


MLS000094770-01
0.84211
0.94123
0.9406
0.80812


MLS001123810-01
0.93465
1.15921
0.81496
0.8812


MLS000532078-01
0.86228
0.9443
0.89145
0.79103


MLS000585616-01
0.87939
0.90965
0.7906
0.90256


MLS000553673-01
0.91272
1.00526
0.96026
0.80214


MLS001175592-01
1.01842
0.96491
1.03077
0.94701


MLS001033255-01
1.23553
1.01272
1.04487
0.84231


MLS000733703-01
0.79649
0.95833
0.86538
1.1188


MLS001096269-01
0.77544
0.9864
0.74017
1.08974


MLS001162872-01
0.97939
1.06096
1.08376
0.78547


MLS000584511-01
0.74781
0.87105
0.8735
0.83547


MLS001166325-01
0.87851
0.89693
0.97949
0.77265


MLS000122180-01
1.00702
0.8136
0.88077
0.77735


MLS001157804-01
1.06974
1.05044
0.98419
0.70299


MLS000693729-01
0.87895
0.90307
1.05684
0.97222


MLS001220669-01
1.00044
1.0114
0.77009
0.82735


MLS001214461-01
0.95658
0.91667
0.93632
0.81368


MLS000081838-01
0.96974
1.01842
0.88889
0.91752


MLS002402866-01
0.7307
0.83202
0.79658
0.87393


MLS001200980-01
1.0886
0.92719
0.95684
0.87821


MLS001174719-01
1.10789
0.92544
0.83205
0.85513


MLS001175449-01
0.86711
0.88904
0.90342
0.85342


MLS001172577-01
0.96272
1.02895
0.86197
0.89444


MLS001122792-01
0.72544
0.99693
0.96752
0.81368


MLS001216405-01
1.07982
1.34474
0.80085
0.91923


MLS000673766-01
1.04045
1.10955
0.96316
0.98469


MLS000912614-01
1.01227
0.97
1.16603
1.01818


MLS001194551-01
0.78091
1.04682
0.94593
0.82727


MLS001214704-01
0.91364
0.93273
1.27847
0.88517


MLS001219159-01
1.02318
0.96364
0.93158
0.93541


MLS001060549-01
1.075
0.88136
0.89522
1.32153


MLS000850824-01
0.94591
0.99455
0.99234
0.88517


MLS000879190-01
0.46273
0.84955
1.04737
0.99091


MLS001163140-01
1.18955
0.91591
1.06124
1.02871


MLS000046123-01
1.07636
1.14455
1.08421
0.91244


MLS000086970-01
0.82727
0.93955
1.18565
0.95407


MLS000388722-01
0.86682
1.08455
1.06699
0.95694


MLS000676974-01
0.46
1.08273
0.45742
1.07656


MLS000772580-01
1.08773
1.02909
1.0933
0.89856


MLS000698686-01
0.88636
0.88636
0.97273
0.83636


MLS001162337-01
1.05
0.84909
0.95933
0.99043


MLS001204005-01
0.78136
1.01818
1.13636
0.94019


MLS000721525-01
0.91273
0.84091
1.03636
0.86603


MLS000525404-01
0.74955
0.84136
0.87273
0.76029


MLS000772194-01
0.99682
1.05636
0.92344
1.06842


MLS000775793-01
1.02136
0.93227
1.01196
0.85502


MLS000710130-01
0.93364
0.98636
0.86459
0.81435


MLS001146463-01
1.14091
1.06045
0.93923
1.07416


MLS000712769-01
0.85818
0.91409
1.07129
0.96507


MLS000334464-01
0.96364
0.98773
1.07751
1.30144


MLS000862690-01
1.01818
0.93227
0.89426
0.86842


MLS001179267-01
0.55545
0.61409
0.8244
0.90909


MLS000683174-01
1.23773
1.035
1.0866
1.15646


MLS000913052-01
0.92
0.91545
1.28708
1.11531


MLS001080869-01
1.01045
0.99273
0.91531
0.93493


MLS000332693-01
0.93045
0.88045
0.97943
1.24402


MLS001141113-01
1.91273
0.98227
1.38278
1.13397


MLS001176611-01
0.785
1.03364
1.23541
0.92871


MLS001202627-01
0.735
0.93455
0.9555
0.92344


MLS000765108-01
0.97
0.99273
1.09856
1.17656


MLS000937079-01
0.96818
0.96409
0.99234
1.08708


MLS001215742-01
0.98509
0.92544
1.1034
1.02596


MLS001217045-01
0.91272
0.92719
0.98213
0.96128


MLS001196946-01
3.9614
2.7886
1.12979
0.9817


MLS001216714-01
3.63333
1.41711
0.98
0.91617


MLS000772430-01
1.03684
0.89561
0.90936
0.8183


MLS000693370-01
1.1136
0.99474
0.96213
0.9783


MLS000769322-01
1.09079
1.11842
1.01957
0.86085


MLS000721030-01
0.79167
0.92763
0.96553
1.11106


MLS001176897-01
1.04649
1.12149
0.79702
1.00851


MLS000774940-01
1.00526
1.17105
0.86043
0.95404


MLS001030746-01
0.99123
1.09781
0.87574
0.97915


MLS003126425-01
0.71228
0.74298
0.89021
1.35447


MLS001217697-01
0.73947
0.76404
0.86468
1.20043


MLS000516719-01
1.07193
1.10307
1.11404
0.88936


MLS001165323-01
1.48772
1.31535
0.9
1.07234


MLS001220803-01
1.04956
1.09079
1.06426
1.11489


MLS001163121-01
0.92105
0.87807
0.82936
0.85064


MLS001060561-01
0.64254
0.93728
1.00213
0.93957


MLS001139515-01
0.96009
0.92368
0.76043
0.94043


MLS001149811-01
1.65044
1.12193
1.04766
0.9566


MLS000773700-01
0.40921
0.92895
1.1166
1.12553


MLS001177045-01
1.11798
1.05833
1.11021
0.92383


MLS000693747-01
1.04298
0.9693
0.95234
0.99617


MLS001175556-01
1.10263
0.91579
1.05277
1.11277


MLS001175473-01
1.06579
0.95921
0.85872
0.89617


MLS002156278-01
1.03114
0.96667
1.09574
0.86936


MLS000707281-01
0.95263
0.94737
0.92553
0.87064


MLS000591198-01
0.45307
0.83377
0.93191
0.96809


MLS000714175-01
0.97105
1.12412
1.09745
0.93191


MLS002163386-01
1.06623
0.94649
1.19617
0.97489


MLS000761297-01
0.88289
0.99298
0.81064
0.99106


MLS002245351-01
0.9307
1.00921
1.00128
0.83064


MLS000718886-01
0.83553
0.87982
0.98043
0.97872


MLS002156485-01
0.94825
1.11491
0.95234
0.94681


MLS001140657-01
1.075
1.05044
1.04596
1.05319


MLS002157024-01
0.83333
1.05921
0.85532
1.14638


MLS000721730-01
1.03067
0.93111
0.92069
0.85043


MLS000705922-01
0.99467
0.93022
0.9306
0.87759


MLS000724709-01
1.12533
1.05378
0.88276
0.84224


MLS002161757-01
1.256
1.15289
1.10216
0.91897


MLS002164687-01
1.11333
0.968
0.92198
0.7625


MLS001060533-01
0.68711
0.77156
0.77888
0.83578


MLS000685139-01
0.93867
1.03111
1.03621
0.93621


MLS001217286-01
0.36044
0.39911
1.22672
0.95991


MLS001221619-01
1.78133
1.51333
1.32069
1.17155


MLS001219621-01
1.29022
1.132
1.06293
0.94914


MLS001166156-01
0.52267
0.77956
0.91983
0.87069


MLS000534926-01
0.844
0.95467
0.96897
0.97241


MLS000548725-01
0.332
0.87289
1.12629
0.97888


MLS000374261-01
0.50356
0.448
0.8319
0.82457


MLS000123454-01
1.05467
0.87956
0.93233
0.93534


MLS000625140-01
1.016
1.10667
1.03621
0.91724


MLS001214443-01
0.90222
0.95067
1.07241
0.97371
















TABLE 13







Burkin Assay in Myoblasts














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000763074-01
0.60851
0.65857
1.13427
0.843
1.06668
1.19903


MLS000564846-01
0.75904
0.74063
0.87038
0.80671
0.84376
0.83593


MLS001202366-01
0.24561
0.48734
0.74576
0.87622
0.96981
1.00673


MLS001182278-01
0.84246
0.76628
1.17046
1.06287
1.0742
1.21321


MLS001196427-01
0.87626
0.81017
0.97767
0.93838
1.03303
1.01962


MLS001202425-01
0.34448
0.74647
0.86448
0.96729
1.21932
1.2214


MLS001214276-01
0.02694
0.20219
0.32354
0.62344
1.22246
1.32758


MLS000682748-01
0.24723
0.46691
0.765
0.89229
1.16212
1.259


MLS000834756-01
1.08092
1.06424
1.17192
1.22657
1.09843
1.05646


MLS001202402-01
0.32496
0.64813
1.08015
0.92555
0.90338
1.05088


MLS001214264-01
0.23942
0.78788
0.70087
0.74298
0.84644
0.8908


MLS000391588-01
0.30047
0.78036
0.81758
0.91191
1.01537
1.06962


MLS001013431-01
0.33625
0.45216
0.53107
0.70768
1.00039
1.20211


MLS001163848-01
0.14441
0.29703
0.31406
0.48542
0.89062
1.00231


MLS000327715-01
0.57757
0.90154
1.02899
1.14772
1.09422
1.37572


MLS001214300-01
0.02593
0.18472
0.35298
0.80412
0.93656
1.21103


MLS000834755-01
0.79619
1.19253
1.15647
1.08036
1.07782
1.27365


MLS001163860-01
0.23471
0.17222
0.49218
0.57932
0.84566
0.94942


MLS000710233-01
0.17433
0.17232
0.56369
0.80665
1.15918
1.17028


MLS002702497-01
0.04886
0.05588
0.18885
0.17496
0.55454
0.78041


MLS000546982-01
0.88835
1.05267
1.07821
1.27207
1.15498
0.87993


MLS001197838-01
0.86478
0.89105
1.04838
1.05318
1.19792
1.25617


MLS001202354-01
0.42755
0.51598
1.07323
1.03576
1.02304
1.07717


MLS001202330-01
0.64675
0.83048
0.93405
0.95578
1.02782
0.97384


MLS001182377-01
0.61764
0.87441
0.90613
0.91503
1.04505
1.17882


MLS000834757-01
1.02067
1.11712
0.96506
1.12452
1.00317
1.0102


MLS001202634-01
0.93072
0.99395
1.31203
1.14099
1.10045
1.20777


MLS001194544-01
0.50277
0.79143
1.14522
1.02036
0.99063
1.04429


MLS000554109-01
0.89366
0.9611
1.09535
1.06742
1.04915
0.99289


NCGC00263072-01
0.98868
1.10983
1.04886
1.01335
1.24172
1.27253


NCGC00263071-01
0.53023
0.69803
0.78353
0.9185
1.13222
1.35429


NCGC00263039-01
0.81077
0.99561
1.02563
1.02082
0.91594
1.1697


NCGC00164631-03
0.01283
0.01408
0.01373
0.32241
1.03203
0.83726


NCGC00179302-02
0.80668
0.84305
0.71633
0.60599
1.0859
1.06101


NCGC00241113-01
0.9168
0.95936
0.86618
0.9062
1.05675
1.01086


NCGC00094381-04
0.64658
0.77957
0.63974
0.7317
1.05336
1.03555


NCGC00015546-04
0.14966
0.19368
0.21399
0.37707
0.92282
0.89229


NCGC00094381-03
0.52027
0.6668
0.65793
0.73271
0.9952
0.93685


NCGC00094381-05
0.75776
0.88787
0.7145
0.75321
0.98039
0.94327


NCGC00015545-02
0.18236
0.30858
0.31898
0.59598
1.32546
1.17517


NCGC00164631-04
0.01361
0.01476
0.08696
0.52898
1.01135
1.02287


NCGC00094112-04
0.34285
0.45094
0.49426
0.57218
0.99267
1.01949


NCGC00242500-01
0.01945
0.01841
0.58791
0.87045
1.07607
1.05199


NCGC00015545-07
0.242
0.30684
0.56798
0.84629
1.12041
1.07252
















TABLE 14







Burkin Assay in Myotubes














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000763074-01
0.26221
0.19215
0.90703
1.04266
1.12824
1.0274


MLS000564846-01
0.75294
0.70065
1.07066
1.00195
0.95569
0.88356


MLS001202366-01
0.51046
0.52721
0.91937
1.08888
1.09876
1.11392


MLS001182278-01
0.64876
0.56532
1.41234
1.40178
1.11835
1.1969


MLS001196427-01
0.93648
0.79418
1.13545
1.02154
1.01999
0.91943


MLS001202425-01
0.35401
0.51782
0.80333
0.9499
1.20487
1.14828


MLS001214276-01
0.07736
0.23625
0.69453
0.9019
1.19651
1.16185


MLS000682748-01
0.62062
0.74301
1.08485
1.14662
1.21063
0.98015


MLS000834756-01
0.91886
0.94946
0.9214
1.09476
1.18855
1.07853


MLS001202402-01
0.37173
0.59069
1.0295
0.96293
0.89618
0.96279


MLS001214264-01
0.71731
0.71648
1.05756
0.90726
0.89746
0.95761


MLS000391588-01
0.66403
0.76307
0.93558
0.89942
0.95626
1.07272


MLS001013431-01
0.70692
0.7376
0.85055
0.85067
1.0097
1.23758


MLS001163848-01
0.48759
0.68784
0.74688
0.95866
1.04499
1.0964


MLS000327715-01
1.11892
1.19159
1.11494
1.30229
1.02522
0.88377


MLS001214300-01
0.31784
0.74468
0.87335
1.09139
1.03917
1.01853


MLS000834755-01
0.81603
0.96865
0.98675
1.00334
1.23201
1.26329


MLS001163860-01
1.20854
0.86198
1.13792
0.92233
0.94035
1.00928


MLS000710233-01
0.62947
0.69332
0.86883
0.93307
1.033
1.11028


MLS002702497-01
0.38389
0.45669
0.78137
0.76924
0.74901
0.84142


MLS000546982-01
0.71115
0.85558
0.99622
1.14451
1.03799
1.04819


MLS001197838-01
0.99815
0.86987
1.09145
1.00443
0.98105
1.00877


MLS001202354-01
0.57849
0.5824
1.06858
0.95451
0.90225
0.84594


MLS001202330-01
0.62071
0.63028
1.01709
0.91234
0.91736
0.97095


MLS001182377-01
0.91488
0.86542
0.9389
0.93717
1.02095
1.01535


MLS000834757-01
0.94642
0.99887
0.95369
1.02962
0.94297
1.01879


MLS001202634-01
1.28943
0.94727
1.13854
0.9675
1.1156
0.99278


MLS001194544-01
0.76105
0.754
1.06983
1.00203
0.9995
0.96112


MLS000554109-01
1.04819
0.84256
0.98744
0.96115
1.05362
0.88389


NCGC00263072-01
0.88975
0.93047
0.95374
0.96945
1.00581
0.98195


NCGC00263071-01
0.65683
0.68367
0.77685
0.86036
0.89218
0.86806


NCGC00263039-01
0.59697
0.74735
0.86326
0.98314
0.95574
0.96891


NCGC00164631-03
0.07758
0.09031
0.18808
0.55055
0.94454
0.95119


NCGC00179302-02
0.68712
0.68199
0.79125
0.81812
0.93769
0.93491


NCGC00241113-01
0.63117
0.74575
0.76777
0.84894
1.10523
1.07764


NCGC00094381-04
0.93082
0.76798
0.87417
0.83691
0.94176
0.8006


NCGC00015546-04
0.79646
0.78811
0.82527
0.85291
1.05081
0.9443


NCGC00094381-03
1.15097
0.94969
1.02446
0.90148
0.93337
0.8564


NCGC00094381-05
1.13784
1.05028
1.31892
1.04722
0.93095
0.92337


NCGC00015545-02
0.35192
0.49594
0.55162
0.69213
0.91177
0.91301


NCGC00164631-04
0.08093
0.08218
0.21321
0.77386
0.99976
0.97606


NCGC00094112-04
0.34997
0.68129
0.62896
0.7596
0.81588
0.74019


NCGC00242500-01
0.21319
0.652
0.75336
0.94215
0.93914
0.99743


NCGC00015545-07
0.49755
0.66539
0.74573
0.87208
1.08023
1.12579
















TABLE 15







CMV-LACZ in Myoblasts














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000763074-01
1.6644
1.2981
1.1966
1.1447
0.8256
0.8431


MLS000564846-01
1.0885
1.2363
1.3619
0.9379
0.8172
0.9922


MLS001202366-01
0.3322
0.7222
1.3214
1.0533
0.6431
0.7656


MLS001182278-01
0.5959
1.0519
0.9778
0.9823
0.9491
0.7466


MLS001196427-01
0.9563
0.7144
1.1811
0.9957
1.0356
0.7219


MLS001202425-01
0.6233
0.9081
1.1386
0.8675
1.0919
1.0625


MLS001214276-01
0.1781
1.2922
1.2231
1.3014
0.8731
0.9516


MLS000682748-01
0.2419
0.3893
0.9959
1.0215
0.9666
0.9075


MLS000834756-01
1.1889
0.8022
1.0917
1.2652
1.0422
0.8709


MLS001202402-01
0.4819
0.7204
0.7598
0.8771
1.0106
1.0546


MLS001214264-01
0.4856
0.3870
0.9233
1.3058
1.1383
1.0657


MLS000391588-01
1.1119
1.4307
1.0968
0.9885
0.7411
1.0009


MLS001013431-01
0.6670
0.5904
0.6673
0.8366
1.0640
0.9906


MLS001163848-01
2.2215
2.1030
1.0762
0.9336
0.9620
1.1146


MLS000327715-01
1.0648
1.3181
1.0060
0.9664
1.1731
1.2294


MLS001214300-01
0.1793
0.4007
1.1047
0.7949
0.9403
1.0446


MLS000834755-01
0.9133
0.9007
0.9834
0.9591
1.0400
1.0317


MLS001163860-01
0.6488
0.3425
1.1417
0.9610
0.8230
0.9350


MLS000710233-01
0.8779
0.8530
0.8509
0.8263
0.8263
1.0173


MLS002702497-01
0.2779
0.2593
3.6076
2.6344
1.0213
0.8643


MLS000546982-01
1.3937
0.9512
1.0785
0.9273
1.2640
1.0877


MLS001197838-01
0.9437
1.1615
1.1118
1.0010
0.9954
0.7904


MLS001202354-01
0.7437
0.8378
0.9170
0.9669
0.9521
1.2275


MLS001202330-01
0.7619
0.9511
0.9082
1.0099
0.9325
0.7417


MLS001182377-01
0.4170
0.4441
0.9294
0.9113
1.1646
1.1229


MLS000834757-01
1.1026
0.8115
1.0942
0.9101
0.9300
1.0367


MLS001202634-01
0.9217
1.0817
1.0990
1.1287
1.0071
1.0500


MLS001194544-01
0.7383
0.8983
1.0363
0.9068
0.7213
0.8975


MLS000554109-01
0.9650
0.9853
0.9457
0.9003
1.1863
1.1083


NCGC00263072-01
1.2880
1.1540
1.1158
1.0035
0.9983
1.2383


NCGC00263071-01
0.6430
0.7803
0.9908
1.2012
1.2813
1.0800


NCGC00263039-01
0.9543
0.9740
0.9335
1.0522
1.1700
1.0746


NCGC00164631-03
0.1767
0.1830
0.2763
0.7817

0.8658


NCGC00179302-02
1.2873
1.0247
1.6628
2.4679

1.1867


NCGC00241113-01
3.3097
2.7827
1.1352
1.1634

1.4142


NCGC00094381-04
0.9697
0.9330
0.9770
1.1366

1.0763


NCGC00015546-04
0.5873
0.8083
1.2439
1.4889

1.1442


NCGC00094381-03
1.0253
0.7777
1.0089
0.8667

0.9504


NCGC00094381-05
0.8220
0.7147
0.8606
0.9614

0.8383


NCGC00015545-02
0.2480
0.2907
0.8475
0.9440

1.0179


NCGC00164631-04
0.1827
0.2323
0.4405
0.6842

0.8225


NCGC00094112-04
0.3627
0.4403
1.0782
1.0309
1.1779
1.1721


NCGC00242500-01
0.4237
0.3570
2.4909
1.6977
1.1446
1.1021


NCGC00015545-07
0.3383
0.5257
0.8603
0.7896
1.1950
1.2967
















TABLE 16







CMV-LACZ in Myotubes














40 uM
20 uM
10 uM
5 uM
1 uM
0.5 uM

















MLS000763074-01
1.9682
1.7671
0.9645
1.0487
0.8593
1.0089


MLS000564846-01
1.0094
1.2012
0.8711
1.1234
1.0444
0.8304


MLS001202366-01
1.0847
0.8329
0.9927
0.8072
1.0570
0.9844


MLS001182278-01
1.1212
1.0224
1.0297
1.0327
1.0126
0.9674


MLS001196427-01
0.9000
0.9918
0.9282
0.8423
1.0704
0.9363


MLS001202425-01
0.9976
0.9918
1.0423
1.1496
1.0185
0.8793


MLS001214276-01
0.4976
0.5259
11.1000
2.0151
0.9141
0.7733


MLS000682748-01
1.1071
1.1753
0.9205
0.8790
0.8459
0.9356


MLS000834756-01
2.2953
2.0788
1.1713
1.1994
1.0059
0.9859


MLS001202402-01
0.8129
0.8847
0.8015
0.9237
0.8695
0.9200


MLS001214264-01
1.4482
1.2424
1.1534
0.9411
1.1905
1.1210


MLS000391588-01
0.9094
0.9212
0.9740
1.0019
1.0010
0.8400


MLS001013431-01
1.2059
1.0682
0.9162
0.9388
1.1257
0.8638


MLS001163848-01
6.8624
6.7729
4.2586
1.1937
0.9638
1.2133


MLS000327715-01
1.4871
1.2200
0.9983
1.1289
1.0562
1.1762


MLS001214300-01
0.7118
6.2635
1.0956
1.0550
0.8924
0.9819


MLS000834755-01
0.8789
1.2989
1.1048
1.0514
1.2863
0.5547


MLS001163860-01
1.4490
1.3880
1.2757
1.1250
1.1562
1.0352


MLS000710233-01
1.1380
1.1130
1.0698
0.9060
0.9410
0.8781


MLS002702497-01
0.6550
0.6520
6.3598
7.0726
2.2829
1.1181


MLS000546982-01
1.3290
1.0160
1.1265
1.0159
0.8467
1.0438


MLS001197838-01
1.0063
0.8547
1.1637
0.9701
1.0038
1.0305


MLS001202354-01
0.7863
0.7000
1.0340
0.8910
0.9200
1.0676


MLS001202330-01
0.8242
0.9168
0.8717
0.9600
0.9562
0.8314


MLS001182377-01
1.0263
1.0305
1.0589
0.9608
0.8105
0.7762


MLS000834757-01
1.3600
1.0789
1.1047
1.0546
0.8695
1.0238


MLS001202634-01
0.9340
0.9750
0.9661
1.2348
1.0073
0.9582


MLS001194544-01
0.9400
0.9800
0.9739
0.9683
1.1318
1.0764


MLS000554109-01
0.9950
0.9100
1.1355
1.1559
0.9491
1.2682


NCGC00263072-01
1.0230
0.9190
1.0660
1.0396
0.8536
1.0945


NCGC00263071-01
0.9160
1.0840
0.9779
1.0165
1.2909
0.9845


NCGC00263039-01
1.0510
0.8580
0.9982
0.9481
0.9727
0.9645


NCGC00164631-03
0.7680
0.6690
0.3964
0.8420
1.2736
1.2682


NCGC00179302-02
1.1410
1.0030
0.9196
1.1533
0.9736
0.9618


NCGC00241113-01
0.9520
0.9790
1.0724
0.9410
1.3227
0.8627


NCGC00094381-04
8.1220
3.8710
1.1010
1.2849
1.4927
1.0845


NCGC00015546-04
1.0700
1.0230
1.1513
1.0553
1.1427
1.1555


NCGC00094381-03
18.6030
10.9800
1.5444
1.1125
1.2227
0.9227


NCGC00094381-05
18.0610
6.8950
2.8305
1.2069
0.8773
1.0045


NCGC00015545-02
0.4770
0.5490
0.5058
0.7586
0.9536
0.8627


NCGC00164631-04
0.6690
0.6490
0.3804
0.8803
0.9164
1.1336


NCGC00094112-04
0.5420
0.7190
0.7933
0.8123
0.8864
1.0409


NCGC00242500-01
7.0800
5.2430
3.3039
2.2432
1.9682
1.2482


NCGC00015545-07
0.5470
0.7010
0.8393
0.9434
1.1036
1.0736
















TABLE 17







407 compounds chosen from the initial 1500 MLSMR “hit” compounds and the


results from the Burkin lab rescreening in myoblasts (MB), myotubes (MT),


and β-Gal stabilizing assay















Both MB and
β-Gal
Less than 25%


407 compounds
MB >25%
MT >25%
MT >25%
Stabilizing or
max increase in


chosen for Burkin
increase
increase over
increase over
CMV
both MB and


Lab evaluation
over DMSO
DMSO
DMSO
activating
MT




















NCGC repeatedly
52
(31%)
12
(7.2%)
10
(6%)
2*
(1.2%)
90
(54%)


active (166)












Original top hits
6
(3%)
37
(18.8%)
12
(6.1%)
6**
(3%)
136
(69%)


(197)












SU9516 platform
6
(13.6%)
1
(2.3%)
3
(6.8%)
12***
(27.3%)
15
(34%)


Analogs (44)





*Neither of the β-Gal stabilizing compounds gave a >25% in α7+/LacZ MB or MT assays


**1 β-gal stabilizer also increased MT >25% over DMSO


***1 β-gal stabilizer also increased MB >25% over DMSO






Top Hits from the Screen


Next the top 6 compounds for activation of ITGA7 in myotubes, based on maximum response (Table 18) were selected and the screen was performed with a larger number of concentrations in order to achieve more accurate dose-response curves (FIGS. 7A-7F). From these dose-response curves, GraphPad Prism nonlinear-regression analysis was used to determine the EC50 in myotubes (Table 18). The maximum predicted increase was calculated as the maximum increase generated in the α7+/LacZ multiplied by 2 due to the single allelic copy of the LacZ reporter (FIGS. 7A-7F).









TABLE 18







Summary of the top 6 compounds found to be effective in myotubes.

















Online







Online
Screen
EC50


Compound

Known
Screen
Fold-
α7+/LacZ
MT FOLD


Name
MW
effects
Score
change
MT
INCREASE





SU9516
241.3
Cdk2


6.0 × 10−6 M
2.4




inhibitor


MLS000532969
236.3
NA
44
1.7
2.2 × 10−6 M
2.0


MLS003126425
399.4
NA
43
1.9
7.0 × 10−6 M
2.0


MLS001060533
483.3
NA
42
1.9
4.7 × 10−6 M
1.5


MLS000683232
257.2
NA


5.3 × 10−6 M
1.7


MLS000683234
237.3
NA
46
2.6
2.7 × 10−5 M
1.7









SU9516 Increases α7 Integrin Protein in C2C12 Myotubes

In order to confirm the on-target activity of SU9516, C2C12 myotubes were treated with either DMSO control or 12 μM SU9516 for 48 hours. Western blot analysis of the myotube protein extracts were then performed for α7B Integrin and normalized to GAPDH (FIGS. 8A and 8B). SU9516 treated myotubes displayed an increase of approximately 1.6-fold in α7 Integrin protein levels compared to DMSO treated controls (FIGS. 8A and 8B). These results confirm the myogenic cell-based assay had successful identified novel small molecules that would target an increase in α7 integrin in skeletal muscle.


Duchenne muscular dystrophy is a fatal neuromuscular disease for which there is currently no cure and limited treatment options. Studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD. Loss of the α7 integrin in dystrophin deficient mdx mice results in more severe muscle disease, while transgenic or viral mediated expression of the α7 integrin can rescue mouse models of DMD. These results support the hypothesis that the α7β1 integrin is major modifier of disease progression in DMD. In support of the idea that drug-based modulation of α7β1 integrin in skeletal muscle may serve as a therapeutic avenue for DMD, it has been shown that prednisone, the current front line therapy for DMD, acts to increase laminin-α2 and α7β1 integrin in the muscles of mdx mice, GRMD dog and DMD patient cells. Together these preclinical studies indicate the α7 integrin is a “druggable” target for the treatment of DMD.


In this study a muscle cell-based assay was used to identify small molecules that promote an increase in α7 integrin in myogenic cells. A screen of FDA-approved and novel compound libraries identified several chemical platforms which may be promising for the treatment of DMD. From the FDA-approved libraries it was identified that the Fe-chelating compounds Ciciopirox and Deferoxamine as α7β1 integrin enhancers. In addition 2,2-dipyridyl which is within this same compound family was tested using the muscle cell-based assay and was confirmed to also increase α7 integrin levels. Since Ciclopirox, deferoxamine and 2,2-dipyridyl have been shown to increase stability of the transcription factor hypoxia inducible factor-1 (HIF-1) by preventing its breakdown, bioinformatic analysis was performed on the α7 integrin promoter. Analysis of a 2.8 kb fragment of the proximal α7 integrin promoter sequence using MATINSPECTOR (Genomatix) revealed the presence of a HIF-1 binding site and flanking sequences that promote HIF-1 binding. These results indicate Ciclopirox, Deferoxamine and 2,2-dipyridyl act to increase α7 integrin gene expression by inhibiting proteosomal breakdown of HIF-1 in muscle cells resulting in increased cellular levels of HIF-1 protein in skeletal muscle. Interestingly, increased HIF-1 levels are associated with increased angiogenesis and therefore not only would these drugs increase membrane stability through increased α7 integrin gene expression, but they may also increase muscle vascularization, improving blood flow and reducing the ischemia associated with dystrophic muscle.


To identify small novel molecular probes that increase the α7 integrin in skeletal muscle, the MLSMR at the National Chemical and Genomics Center (NCGC) at NIH was screened. Using the muscle cell-based assay 380,000 compounds in the Molecular Library and Small Molecule Repository (MLSMR) were screened using dose-response quantitative high through-put screening (qHTS). From the primary screen, 1,500 hit compounds were identified as actives. Analysis of these compounds using the hierarchical cluster approach revealed 321 clusters in which 17 clusters contained more than 10 compounds and 210 singletons. From these studies 500 compounds were cherry-picked for further testing which included confirmation of activity in the primary screening assay and secondary assays which included β-galactosidase reporter stabilizer, myostatin and cell-toxicity assays. In addition primary HTS active compounds were tested to determine if they increased β-galactosidase reporter in myoblasts and myotubes. From these counter-screens seven novel hit compounds were selected for further analysis to determine whether they increase the α7 integrin in a dose-dependent fashion. Analysis in mouse and DMD human muscle cells revealed that all seven increased both α7 integrin transcript and protein levels. These compounds gave a maximal increase in α7 integrin protein in human DMD muscle cells which would be in the therapeutic range as predicted from transgenic mouse studies.


Studies of the seven novel α7 integrin enhancing compounds showed that two molecular platforms are related, while the other compounds are unrelated to each other. One compound, SU9156 which was used as a positive control in the screen is currently in clinical trials as a potential anti-cancer therapeutic. SU9516 (3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1, 3-dihydro-indol-2-one) is a 3-substituted-indolinone compound that binds to cdk2 and selectively inhibits its catalytic kinase activity. X-ray crystallography studies showed that the small molecule compound inhibited cdk2/cyclin A through competitive inhibition of ATP. In human leukemic cells, SU9516 caused the pronounced down-regulation of the anti-apoptotic protein McI-1 through transcriptional repression, increased proteasomal degradation, inhibition of RNA Po1 II CTD phosphorylation and oxidative damage. Thus, SU9516 has demonstrated its potential as a viable pharmacological drug for the development of anti-neoplastic therapeutics and has reached clinical trials for the same. The molecular mechanism by which SU9516 enhances integrin is currently unknown. SU9516 has also been reported to inhibit glycogen synthase kinase 3β (GSK-3β), which is involved in normal cell death. Levels of inactive p-S9-GSK3β are reduced and total GSK3β is elevated in the muscles of patients with myotonic dystrophy type 1 (DM1). Inhibition of GSK3β in both DM1 cell culture and mouse models reduced muscle weakness and myotonia in DM1 mice. Hence, compounds normalizing GSK3β activity might be beneficial for improving muscle function in patients with DM1.


Using a novel muscle cell-based assay an exhaustive screen of small molecule compound libraries has been conducted and identified several FDA-approved and novel molecules that increase α7 integrin in skeletal muscle. These small molecules can serve as molecular probes to dissect the signaling pathways that regulate levels of the α7β1 integrin in skeletal muscle. In addition these molecules may serve as platforms to develop novel therapeutics that target an increase in α7 integrin for the treatment of muscular dystrophy.


Example 4

This example describes methods for identifying and analyzing suitable analog compounds useful for the methods and assays disclosed herein. In one embodiment, Stryka-969 was identified as a top “hit” from of the over 400,000 compounds screened using the assay disclosed herein due to its efficacy, large range of potency, and its lack of cellular toxicity (REF paper in prep). The chemical structure of Stryka-969 along with the dose-response curve generated in ITGA7+/LacZ myotubes is illustrated FIG. 7B. While Stryka-969 also functions equally well in myoblasts, the therapeutic target for enhanced α7 integrin protein are differentiated myofibers and thus this example focuses primarily on the Stryka-969 analogs function in ITGA7+/LacZ myotubes. The maximum increase of ˜2-fold should translate into a 3-fold change in ITGA7 transcription due to the heterogenetic nature of the assay and single reporter. This increase is well within the therapeutic range previously reported for dystrophic mice.



FIG. 10 illustrates results obtained from some analogs of Stryka-969. These compounds were scored based on their structural similarity to Stryka-969 using the Tanimoto coefficient which measures the similarity between two. These compounds were then used to treat ITGA7+/LacZ myotubes at eight different concentrations in order to produce dose-response curves (FIG. 10; n=3/concentration). The dose-response results suggest that the amide group may help increase α7 Integrin. This group is maintained in MLS000061149, which displays similar activity in the Burkin assay to the parent compound Stryka-969 (FIG. 10). The other compounds were less similar to Stryka-969 and displayed little to no relevant response (FIG. 10). Together, these studies indicate that SAR analysis will further elucidate the functional groups capable of maximizing the α7 integrin enhancing activity of this compound platform.


To assess the on-target effects of Stryka-969, cultured telomerase-expressing Human DMD myotubes were used. After a minimum of 10 days of differentiation, myotubes were treated with either DMSO or varying concentrations of Stryka-969. After 48 hours the media was aspirated and the myotubes were washed in PBS and then scraped into ice-cold RIPA buffer with protease inhibitors. After protein concentration was determined by BCA, standard. SDS-PAGE and western blotting procedures for α7B Integrin and α-Tubulin were followed. A maximum increase of ˜1.7-fold over DMSO levels with a treatment of 20 μM Stryka-969 (FIG. 11) was obtained. This shows that Stryka-969 is an α7 integrin enhancing compound with strong therapeutic potential for the treatment of DMD patients.


Pre-clinical assessment of therapeutic small molecules, including ADMET studies, can help to prioritize small molecules with the highest therapeutic value. A step to ensuring that small molecules will be therapeutically on-target in patients is to use cultured Human patient cells for assessment. This ensures that given biologic availability has a conserved mechanism of action between species and the treatment should effectively produce the desired effect in the specific tissue.


Briefly, either 1 mg or 5 mg of each analog are suspended in DMSO at 10 μM concentration. Working plates are made to give a minimum of 8-point dose response per compound. Drugs are added to ITGA7+/LacZ myotubes for 48 hours, at which time the FDG assay is performed as previously described. Using such analogs of Stryka-969 that achieve a minimum of 1.5-fold increase in the Burkin Lab ITGA7+/LacZ based assay, 6 cm plates with cultured Human DMD myotubes are treated with a range of concentrations based on the dose-response discussed above (n=3/concentration). After 48 hours the myotubes are washed and lysed for western blot analysis. Several proteins, including α7B and β1A Integrins, Laminin-β2, and utrophin levels, are assessed using standard western blotting techniques. If compounds fail to produce a minimum of 1.5-fold α7 integrin increase they may be eliminated from further studies. The behavior of Stryka-969 on the cultured Human DMD myotubes discussed herein indicates that analogs of this compound may lead to increased α7 integrin in the Human cells. Western blots using a broad range of concentrations can be used to avoid missing an optimal dose for a small molecule, which could occur if the EC100 concentration from the initial assay for use in the human DMD myotubes is solely relied upon.


The activity of other α7 integrin compounds also can be determined using β-galactosidase cleavage of the non-fluorescent compound fluorescein di-β-D-galactopyranoside (FDG) to fluorescein in both myoblasts and myotubes. For myoblast assays using analog compounds, 1×104 cells are plated on 96 well black well culture plates and cells grown for 24 hours. The parent and analog compounds are added and the FDG bioassay is performed 48 hours after the addition of compounds. For myotube assays. 2.5×105 myoblasts on 96 well black cell culture plates are grown for 24 hours. Cell differentiation medium is added daily and after 7 days of differentiation, and compounds are added to myotubes and incubated for 48 hours. β-galactosidase levels are then be quantified using the FDG assay. These assays re performed in triplicate for each compound screened. For each assay the parent compound and analogs are added to cells with a dose range from 0.5-20 μM in DMSO and a DMSO only control is added for each assay. The dose curve class and. EC50 for each compound is calculated, plotted and compared to the profile of the parent hit molecule. Analog molecules that show activity (both positive and negative) in myoblasts and myotubes inform the SAR for each molecular platform series. In parallel microsome and plasma stability, permeability and solubility of confirmed lead compounds able to increase α7 integrin in myotubes is also evaluated to help determine the best templates for further pharmacokinetic evaluation. Lead compounds are selected for further studies based on the most potent activity in myotubes (the target tissue) and optimal physical and structural drug-like properties in accordance with Lipinski's Rules.


Positive analogs are then assessed to determine whether they increase α7 integrin transcript and protein using mouse C2C12 and human DMD myogenic cells. For myotube analysis, C2C12 and DMD myoblasts are differentiated in DMEM supplemented with 2% horse serum and 50U/ml of penicillin/streptomycin. Compounds or DMSO are added to C2C12 and DMD myoblasts and myotubes at EC100 dose calculated from the FDG screen above. All studies are done in triplicate, and RNA is extracted using TriZol reagent. RNA is reverse transcribed to cDNA using a Superscript II kit (Invitrogen). Primer sequences are used that specifically amplify mouse or human transcripts using SYBR Green technology and quantitative RT-PCR, and reactions carried out in an ABI Prism 7000 Sequence Detection System. The CT value and a standard curve from a dilution series of cDNA from non-treated cells is calculated by the accompanying ABI Prism 7000 SDS software. Transcripts are normalized to 18S rRNA transcript and reported as fold change from control cells. Experiments are performed in triplicate and statistical significance (p<0.05) determined using ANOVA.


Immunoblot analysis is used to assess the protein level expression in treated and control cells. C2C12 and human DMD myoblasts and myotubes are cultured in triplicate experiments and treated with lead compounds or DMSO as described above. Cells are harvested using a cell scraper and extracted proteins. Extracted protein is quantified by a Bradford assay. Equal amounts of total protein are separated on SDS-PAGE gels at 40 mA for 1 hour and protein is transferred to nitrocellulose membranes. Blots are incubated with Ponceau S to confirm equal protein loading. Rabbit anti-α7 integrin antibodies, A2-345 and B2-347, at 1:500 with will be used to detect mouse and human α7A and α7B integrin respectively. Bands are scanned with an Odyssey Infrared Imaging System. Blots are re-probed with an anti-GAPDH to normalize band intensities for protein loading. A LiCoR Odyssey scanner and software is used to quantify band intensities and statistical significance (p<0.05) is determined using ANOVA.


The pharmacokinetic properties of the compounds having desirable drug-like structures are analyzed for in vivo activity. Three 8-week-old C57Bl/6 mice by will be treated by intraperiontoneal injection (i.p) with either the control solute or the compounds under investigation at the EC100 determined in the examples discussed above. At 0 mins (before administration) and then 30 mins, 1 hr, 2 hrs, 4 hrs, 8 hrs, 12 hrs and 24 hrs after compound administration, 50 μL of blood is collected by retro-orbital bleeds and serum isolated by centrifugation. After administration, animals re observed and signs of toxicity including weight loss, convulsions, uncoordinated movement, torpidity, temperature changes, dispenia or death. In a separate series of pharmacokinetic experiments to assess muscle tissue levels of analogs, three 8-week-old C57Bl/6 mice are treated control solute or the compounds at the same time points listed above. At each time point mice are humanely euthanized by CO2 inhalation and the gastroenemius. TA, heart, intestine and diaphragm muscles dissected for analysis. The muscle and intestine is extracted to determine the levels of each lead analog within the tissue.


Purified compounds serve as a control to identify the small molecule signature after LC-MS/MS analysis. Pharmacokinetic profiles include: serum half-life (t1/2), Volume of distribution (Vd), drug concentration in serum (C0 or CSS), Elimination rate constant (ke), Clearance (CL), Bioavailability (f), peak serum concentration (Cmax) and time to reach Cmax (tmax). A lead compound with a suitable pharmacokinetic profile is then selected for further studies. Formulation is developed with the objective of developing a lead α7 integrin enhancing compound that can be orally administered. For experimental rigor, samples will be coded before analysis so those collecting data are blinded to the treatment group.


The pharmacokinetic profile for the lead compound identified above and the calculated optimal dose are used to determine if the on-target in vivo activity of the drug increases α7 integrin into the therapeutic range as determined by transgenic mouse studies. Off-target activity of the lead compound also will be examined. Three week-old mdx mice are administered with a suitable lead compound as determined using the above examples, or a vehicle by oral gavage daily for 14 days using dosing determined in pharmacokinetic studies. C57Bl/10 mice are included as wild-type controls. A minimum of 22 male mdx mice per experimental and control group are used as determined by Power analysis (Power=0.8, α=0.05 and r=0.5). Mice are weighed weekly and behavioral changes recorded. Mice are euthanized at 5 weeks-of-age and Tibialis anterior (TA), gastroenemius, diaphragm and cardiac muscles harvested. Expression of α7 and β1 integrin is quantified by qRT-PCR and western blots as discussed herein. For off-target activity, transcript levels of integrin α3, α5, α6 and extracellular matrix genes are quantified. Primers are used that specifically amplify these mouse transcripts using SYBR Green technology and quantitative RT-PCR reactions performed in an ABI Prism 7000 Sequence Detection System. The CT value for each is calculated using ABI Prism 7000 SDS software and transcripts normalized to 18S rRNA and reported as fold change from control tissue. Experiments are performed in triplicate and statistical significance (p<0.05) determined using ANOVA. Transcripts that change >2-fold are confirmed by western blot analysis.


To determine if the lead compound shows therapeutic benefit, a preliminary muscle histology study also is performed. Before harvesting tissue, mice are injected with Evans Blue dye (EBD) and histology performed for EBD uptake, percentage of myofibers with centrally located nuclei, inflammation, myofiber cross-sectional area and fibrosis as previously described. For experimental rigor, samples are coded before analysis so those collecting data will blinded to the treatment group. Data analyzed by ANOVA and a p-value<0.05 will be considered statistically significant.


These examples can be used to determine the PK/PD of a lead α7 integrin enhancing compound. The lead compound with favorable PK/PD is then assessed for on-target efficacy and off-target activity.


Statements of the Disclosure


Paragraph 1. A method for treating a subject with muscular dystrophy, comprising administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one of or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy.


Paragraph 2. The method disclosed in the preceding paragraph, wherein the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1A (MDC 1A), merosin deficient congenital muscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).


Paragraph 3. The method disclosed in any one of the preceding paragraphs wherein the muscular dystrophy is DMD, MDC1A or FCMD.


Paragraph 4. The method disclosed in any one of the preceding paragraphs, wherein the muscular dystrophy is DMD.


Paragraph 5. The method disclosed in any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.


Paragraph 6. The method disclosed in the preceding paragraph, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.


Paragraph 7. The method disclosed in paragraph 6, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog, a different compound selected from Formulas 1-16 and/or Tables 1-16 and 18.


Paragraph 8. The method disclosed in any one of the preceding paragraphs, further comprising selecting a subject with muscular dystrophy.


Paragraph 9. The method disclosed in the preceding paragraphs, wherein selecting a subject with muscular dystrophy comprises diagnosing the subject with muscular dystrophy prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.


Paragraph 10. A method of enhancing muscle regeneration, repair, or maintenance in a subject, comprising:


administering an effective amount of an α7β1 integrin modulatory agent to the subject in need of muscle regeneration, repair, or maintenance, wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one of or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.


Paragraph 11. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein the α7β1 modulatory agent is administered prior to the subject experiencing muscle damage or disease.


Paragraph 12. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the method is a method of enhancing muscle maintenance in a subject.


Paragraph 13. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.


Paragraph 14. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered to a subject at risk of acquiring a muscle disease or damage.


Paragraph 15. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, further comprising selecting a subject in need of enhancing muscle regeneration, repair, or maintenance.


Paragraph 16. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.


Paragraph 17. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired production of a component of α7β1 integrin prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.


Paragraph 18. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.


Paragraph 19, The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.


Paragraph 20. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog, or a different compound selected from Formulas 1-16 and/or Tables 1-16 and 18.


Paragraph 21. A method of prospectively preventing or reducing muscle injury or damage in a subject, comprising, administering an effective amount of an α7β1 integrin modulatory agent to the subject wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby prospectively preventing or reducing muscle injury or damage in the subject.


Paragraph 22. The method of the preceding paragraph, wherein the subject is at risk of developing a muscle injury or damage.


Paragraph 23. The method of prospectively preventing or reducing muscle injury or damage in a subject in any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.


Paragraph 24. The method of prospectively preventing or reducing muscle injury or damage in a subject of the preceding paragraph, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.


Paragraph 25. The method of prospectively preventing or reducing muscle injury or damage in a subject of the preceding paragraph, wherein the additional α7β1 integrin modulatory agent is laminin-111, laminin-111 fragment, valproic acid, or a valproic acid analog, or a different compound selected from Formulas 1-16 and/or Tables 1-16 and 18.


Paragraph 26. A method of enhancing α7β1 integrin expression, comprising contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression.


Paragraph 27. The method of the preceding paragraph, wherein the cell is a muscle cell.


Paragraph 28. The method of enhancing α7β1 integrin expression of any of the preceding paragraphs, wherein the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.


In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims
  • 1. A method for treating a subject with muscular dystrophy, comprising administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is MLS000534926-01 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy.
  • 2. The method of claim 1, wherein the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1A (MDC1A), merosin deficient congenital muscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).
  • 3. The method of claim 2, wherein the muscular dystrophy is DMD, MDC1A or FCMD.
  • 4. The method of claim 2, wherein the muscular dystrophy is DMD.
  • 5. The method of claim 1, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.
  • 6. The method of claim 5, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.
  • 7. The method of claim 6, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.
  • 8. The method of claim 1, further comprising selecting a subject with muscular dystrophy.
  • 9. The method of claim 8, wherein selecting a subject with muscular dystrophy comprises diagnosing the subject with muscular dystrophy prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.
  • 10. A method of enhancing muscle regeneration, repair, or maintenance in a subject, comprising: administering an effective amount of an α7β1 integrin modulatory agent to the subject in need of muscle regeneration, repair, or maintenance, wherein the α7β1 integrin modulatory agent is MLS000534926-01, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.
  • 11. The method of claim 10, wherein the α7β1 modulatory agent is administered prior to the subject experiencing muscle damage or disease.
  • 12. The method of claim 10, wherein the method is a method of enhancing muscle maintenance in a subject.
  • 13. The method of claim 12, wherein the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.
  • 14. The method of claim 13, wherein the α7β1 integrin modulatory agent is administered to a subject at risk of acquiring a muscle disease or damage.
  • 15. The method of claim 10, further comprising selecting a subject in need of enhancing muscle regeneration, repair, or maintenance.
  • 16. The method of claim 15, wherein selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.
  • 17. The method of claim 15, wherein selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired production of a component of α7β1 integrin prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.
  • 18. The method of claim 10, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.
  • 19. The method of claim 18, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.
  • 20. The method of claim 19, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.
CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No. 15/961,408, which is a continuation application of U.S. patent application Ser. No. 15/619,256, now U.S. Pat. 9,980,943, which is a divisional application of U.S. patent application Ser. No. 14/776,898, now U.S. Pat. 9,707,210, which is the U.S. National Stage of International Application No. PCT/US2014/029085, filed Mar. 14, 2014, which was published in English under PCI Article 21(2), which in turn claims the benefit of U.S. Provisional Patent Application No. 61/798,479, filed on Mar. 15, 2013, each of which is herein incorporated by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Numbers R43 AR060030, R21 NS058429-01, and R21 AR060769 awarded by the National Institutes of Health. The government has certain rights in the invention.

Provisional Applications (1)
Number Date Country
61798479 Mar 2013 US
Divisions (1)
Number Date Country
Parent 14776898 Sep 2015 US
Child 15619256 US
Continuations (2)
Number Date Country
Parent 15961408 Apr 2018 US
Child 16236244 US
Parent 15619256 Jun 2017 US
Child 15961408 US