Patent Application
20230304024
The contents of the electronic sequence listing (STAN-1783_SEQ_LIST.xml; Size: 15,606 bytes; and Date of Creation: Jun. 1, 2023) is herein incorporated by reference in its entirety.
Methods of controlled regulation of gene expression have been increasingly important in a wide range of areas, including, but not limited, to gene therapy, synthetic biology, plant management, environmental clean-up, bacterial and microbial management and synthetic genetic circuits. Control of gene expression holds vast potential at revolutionizing therapeutics, animal models, and biotechnological processes and is useful to integrate multiple input signals for cell-based therapy and animal model development. Despite rapid advances in recent years, precise control of gene expression remains a challenge due to unpredictability stemming from unintended interactions between biological components, such as transcription factors, etc. A fundamental goal in cellular engineering is to predictably and efficiently express genes at a desired level and under precise control. Such genetically engineered cells hold great promise for advancing therapeutics, diagnostics, animal models, and biotechnological processes.
To date, a variety of different gene modulation technologies for modulating gene expression in a cell have been developed. Such gene modulation technologies include RNA interference, DNA editing and expression, and chemical compounds that suppress, enhance, or modify gene expression. These can be in the form of RNA, DNA, or protein, and can be introduced into cells in culture through direct application to media, lipofection, electroporation, or viral transduction.
However, because of the wide applicability of gene modulation to both research and therapeutic applications, there is a continued interest in the development of new ways to modulate transcription of a target gene in a cell, specifically to modulate expression of genes without genetic modification.
Methods of modulating transcription of a target gene in a cell, without genetic modification, where the methods may be in vitro or in vivo, are provided. Aspects of the methods employ a transcription factor-chemical inducer of proximity (TF-CIP) system to modulate, e.g., enhance or reduce, transcription of a target gene in a cell. Embodiments of the methods include providing in a cell a chemical inducer of proximity (CIP) which links a first endogenous anchor transcription factor that binds to a promoter of the target gene and a second endogenous transcription modulating factor (e.g., a transcription factor or transcription repressor), wherein CIP mediated linkage of the anchor transcription factor and transcription modulating factor modulates transcription of the target gene in the cell without a need for genetic modification. Also provided are compositions that find use in practicing methods of the invention.
Thus, the reporter system is multiplexed and useful for defining apoptotic responses in many different cell types. The FOXO3A reporter consists of an array of FOXO3A binding sites taken from different pro-apoptotic genes including TP53, PUMA, BIM and others. These genes have the ability to kill cells when simply overexpressed. Ten base pairs on either side of the actual human FOXO3A binding site are included to take advantage of the fact that transcriptional specificity is due to the concerted binding of proteins in an enhanceosome (PMID9510247, 18206362). Thus, the reporter system is multiplexed and useful for defining apoptotic responses in many different cell types.
Linkers can be chosen and constructed from these and other published components to provide solubility to the TF-CIP compounds.
Methods of modulating transcription of a target gene in a cell (which may be in vitro or in vivo) are provided. Aspects of the methods employ a transcription factor-chemical inducer of proximity (TF-CIP) to modulate, e.g., enhance or reduce, transcription of a target gene in a cell (e.g., as illustrated in
Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. § 112, are not to be construed as necessarily limited in any way by the construction of “means” or “steps” limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. § 112 are to be accorded full statutory equivalents under 35 U.S.C. § 112.
As summarized above, aspects of the invention include methods of modulating transcription of a target gene in a cell. The methods may be viewed as inducible methods of modulating transcription of a target gene. As the methods are inducible, the modulation of transcription of the target gene is not constitutive, but instead occurs in response to an applied stimulus, e.g., the provision of a CIP, such as described in greater detail below. As the methods are methods of inducibly modulating transcription of a target gene, they are methods of changing transcription of a target gene in some manner, e.g., enhancing transcription of a target gene or reducing transcription of a target gene. The magnitude of change in transcription (relative to a suitable control, e.g., an identical system but for the absence of a CIP), may vary, where in some instances the magnitude of the change, e.g., enhancement or reduction, is 2-fold or greater, such 5-fold or greater, e.g., 10-fold or greater.
As summarized above, aspects of the invention include methods of modulating transcription of a target gene. The term gene refers to a genomic region that encodes a functional RNA, including non-coding RNAs, microRNAs, enhancer RNAs or RNAs that may be translated into a protein product. The term gene is used in its conventional sense to refer to a region or domain of a chromosome that includes not only a coding sequence, e.g., in the form of exons separated by introns, but also regulatory sequences, e.g., enhancers/silencers, promoters, terminators, non-coding RNAs, micro RNAs etc.
The specific target gene that is the focus of a given method may vary. In some instances, the target gene is a gene whose expression is to be enhanced, such as a pro-apoptotic gene (e.g., PUMA (BBC3), BIM (BCL2L11), BID, BAX, BAK, BOK, BAD, HRK, BIK BMF, and NOXA(PMAIP1), or a gene whose activity is inhibited such as the anti-apoptotic gene BCL6, a beneficially therapeutic gene (e.g., a rate-limiting enzyme (such as TPH2), a haploinsufficient gene (such as ARID1B), etc. In some instances, the target gene is an over-expressed gene whose expression is to be reduced, e.g., an oncogene (such as MYC), a trisomy gene (such as a chromosome 21 gene), or an amplified gene etc.
The above categories of genes are merely exemplary of the types of genes that may be target genes of the subject methods. Additional examples of target genes include, but are not limited to: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FOR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, Upases, lipoxygenases, lyso/ymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases); chemokines (e.g. CXCR4, CCR5), the RNA component of telomerase, vascular endothelial growth factor (VEGF), VEGF receptor, tumor necrosis factors nuclear factor kappa B, transcription factors, cell adhesion molecules, Insulin-like growth factor, transforming growth factor beta family members, cell surface receptors, RNA binding proteins (e.g. small nucleolar RNAs, RNA transport factors), translation factors, telomerase reverse transcriptase); and the like.
As reviewed above, embodiments of the methods employ a Chemical Inducer of Proximity (CIP). A CIP is a compound that induces proximity of a first endogenous anchor transcription factor that binds to a promoter of the target gene and a second endogenous transcription modulating factor under intracellular conditions. As the CIPs of the invention induce proximity of at least one endogenous transcription factor with another endogenous transcription modulating factor (e.g., a transcription factor or transcription repressor), the CIPs of the invention may be referred to as Transcription Factor-Chemical Inducers of Proximity (TF-CIP) and are generally illustrated in
Any convenient compound that functions as a CIP may be employed. A wide variety of compounds, including both naturally occurring and synthetic substances, can be used as CIPs. Applicable and readily observable or measurable criteria for selecting a CIP include: (A) the ligand is physiologically acceptable (i.e., lacks undue toxicity towards the cell or animal for which it is to be used); (B) it has a reasonable therapeutic dosage range; (C) it can cross the cellular and other membranes, as necessary, and (D) binds to the target domains of the endogenous anchor transcription factor and the endogenous transcription modulating factor. As such, a desirable criterion is that the compound is relatively physiologically inert, but for its CIP activity. In some instances, the ligands will be non-peptide and non-nucleic acid. Of interest in some applications are compounds that can be taken orally (e.g., compounds that are stable in the gastrointestinal system and can be absorbed into the vascular system).
CIP compounds of interest include small molecules and are non-toxic. By small molecule is meant a molecule having a molecular weight of 5000 g/mole (Dalton) or less, such as 2500 g/mole (Dalton) or less, including 1000 g/mole (Dalton) or less, e.g., 500 g/mole (Dalton) or less. In some instances, the CIP employed in embodiments of the invention has a molecular weight ranging from 250 to 1500 g/mole, such as 300 to 1200 g/mole. By non-toxic is meant that the inducers exhibit substantially no, if any, toxicity at concentrations of 1 g or more/kg body weight, such as 2.5 g or more /kg body weight, including 5g or more/kg body weight.
CIP compounds employed in embodiments of the invention include a first ligand that specifically binds to the anchor transcription factor covalently linked to a second ligand that specifically binds to the transcription modulating factor. In other words, the CIP compounds include a linker component, which may be a bond or a linking moiety, which links a first ligand that specifically binds to the anchor transcription factor covalently and a second ligand that specifically binds to the transcription modulating factor. The terms “specific binding,” “specifically bind,” and the like, refer to the ability of the first and second ligands to preferentially bind directly to their corresponding anchor and transcription modulator factors relative to other molecules or moieties in the cell. In certain embodiments, the affinity between a given ligand and its corresponding factor when they are specifically bound to each other in a binding complex is characterized by a KD (dissociation constant) of 10−5 M or less, 10−6 M or less, 10−7 M or less, 10−8 M or less, 10−9 M or less, 10−10 M or less, 10−11 M or less, 10−12 M or less, 10−13 M or less, 10−14 M or less, or 10−15 M or less (it is noted that these values can apply to other specific binding pair interactions mentioned elsewhere in this description, in certain embodiments).
The nature of the first and second ligands, as well as the linker components, of the CIP compounds may vary. In any given CIP compound, the first and second ligands will be chosen based on the nature of their corresponding anchor and transcription modulating factors, where examples of such and their corresponding ligands are provided below. Specificity of activity with respect to a particular cell type may be provided through selection of the first and second ligands of the CIP, which can be configured to recruit anchor transcription factors and transcription modulatory factors in a manner that provides for desired cell or conditional specificity. For example, CIPs can be engineered to induce proximity of anchor transcription factors and transcription modulatory factors that are primarily present in a target cell of interest, such that the CIP exhibits highly selective activity for that cell. The selectivity of a given CIP may be described by the following formula:
(selectively of expression of anchor transcription factor)×(selectively of expression of the transcription modulating factor)×(genomic specificity of anchor transcription factor)=selectivity of induced activity
Chemical linkage of BCL6 inhibitors, such as B13812(PMID32275432), to estrogen compounds that then bind and induce proximity to the cell death (pro-apoptotic) promoters, such as those for TP53, PUMA and BIM, convert the inhibitor of cell death to an activator of cell death in cells having high concentrations of estrogen receptors, such as breast cancer cells. Examples of the TF-CIPs synthesized by the above protocol and designed to hijack BCL6's repressive activity and convert it to an activator of cell death are shown in
Also shown is an example of a structure using a similar strategy employing an androgen analogue linked to a BCL6 inhibitor based on the previously described molecule B13812(PMID32275432). The later type of molecule may find use in treating prostatic cancer where the AR gene is amplified or overexpressed.
These reporter systems and direct measures of cancer cell killing were used to assess the TF-CIP molecules made by rational design that are illustrated in
The therapeutic effectiveness of ER-TF-CIPs can be improved in several ways. First, the estrogen analogue can be chosen from many published estrogen analogues, including those that have a higher affinity for the estrogen receptor than estrodiol (DOI: 10.1002/cbic.200200499; PMID:12794859; PMID:15300835; PMID:2064992 PMID; 2362442; PMID:3702438). The latter would have certain advantages in treating women who are not post menopausal and have high levels of estrogen that could compete with the ER-TFCIP. The therapeutic effectiveness of an ER-TFCIP can be improved by use of different linkers which will be discussed and illustrated in more detail later in the application. Different linkers could position the Estrogen Receptor more effectively to the BCL6 protein, or could give the ER-TF-CIP superior pharmacologic features. The therapeutic effectiveness of an ER-TFCIP can be improved by use of different BCL6 ligands, some of which are illustrated in the following publications: (PMID:32275432; PMID:30335946; PMID:28930682; PMID:27482887).
The studies described in the section above teach one how to build and evaluate a TF-CIP made from existing components. In cases where existing components or the ligands for the anchor and regulatory transcription factors are not available, the following methods to detect and evaluate these novel ligands may be employed.
A protocol for selecting a ligand to be used in a TC-CIP for an anchor transcription factor is provided using the protocol illustrated in
An additional way that a totally novel TF-CIP can be detected in a library of small molecules involves the use of the reporters shown in
In some instances, the first and second ligands of the CIPs are small molecules, which in some instances each have a molecular weight ranging from 50 Daltons to 1000 Daltons, such as to 400 to 800 Daltons. The chemical structures of the first and second ligands may vary widely, where the first and second ligands may be chosen to provide for the desired specific binding to the target anchor transcription or transcription modulatory factors. The first and second ligands may be selected so as to have little, if any, impact on the activity of the endogenous factor, e.g., anchor transcription factor or transcription modulating factor, to which they are configured to bind.
For a given target gene, anchor transcription factors and ligands therefore that may be employed in a TF-CIP may be identified using any convenient protocol. In some instances, a protocol as described in
As described above, the first and second ligands of the CIPs may be bound to each other by a bond, or via a linking moiety, i.e., linker. When employed, any convenient linker may be employed to link the first and second ligands to each other. Linkers of interest are linkers that provide for a stable association of the first and second ligands in a manner such that the first and second ligands are capable of specifically binding to their respective endogenous factors in the cell. As the linker provides for stably associating the first and second ligands with each other, the first and second ligands do not dissociate from each other under cellular conditions, e.g., conditions at the surface of a cell, conditions inside of a cell, etc. Linkers may be provided for stable association of the first and second ligands using any convenient binding, such as covalent or non-covalent binding, where in some instances the linker component is covalently bound to both the first and second ligands.
Where a CIP includes a linker covalently bound to the first and second ligands, any convenient protocol for forming a covalent bond between the linker and each of the ligands may be employed, where linking protocols of interest include, but not limited to, addition reactions, elimination reactions, substitution reactions, pericyclic reactions, photochemical reactions, redox reactions, radical reactions, reactions through a carbene intermediate, metathesis reaction, among other types of bond-forming reactions. In some embodiments, the linkers employ reactive linking chemistry such as where reactive linker pairs (e.g., as provided by moieties on the ligands and linkers) include, but are not limited to: maleimide/thiol; thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol; N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester), or pentafluorophenyl-ester (PFP ester)/amine; bissuccinimidylester/amine; imidoesters/amines; hydrazine or amine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl or amine; carbohydrate—periodate/hydrazine or amine; diazirine/aryl azide chemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide; carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol and amine/BMPH (N-[β-Maleimidopropionic acid]hydrazide.TFA)/thiol; azide/triarylphosphine; nitrone/cyclooctyne; azide/tetrazine and formylbenzamide/hydrazino-nicotinamide. In certain embodiments, a linker employs a cycloaddition reaction, such as a [1+2]-cycloaddition, a [2+2]-cycloaddition, a [3+2]-cycloaddition, a [2+4]-cycloaddition, a [4+6]-cycloaddition, or cheletropic reactions, including linkers that undergo a 1,3-dipolar cycloaddition (e.g., azide- alkyne Huisgen cycloaddition), a Diels-Alder reaction, an inverse electron demand Diels Alder cycloaddition, an ene reaction or a [2+2] photochemical cycloaddition reaction. In some embodiments, the linker may include an alkyl chain, an alkoxy chain, an alkenyl chain or a alkynyl chain, where the number of carbon atoms in the chain may vary, ranging in some instances from 2 to 25, such as 5 to 20, where one or more carbon atoms are replaced with NH or CH3—N as reactive functionalities for covalent bonding.
In some instances, the linker is selected from a group comprising the following, where n refers to the total number of carbon or carbon-substituent atoms which may be present, sub-counted by k, m, and/or p:
The structures of linker molecules and non-inclusive selected examples are shown in
As summarized above, the CIP employed in embodiments of the invention includes a first ligand that specifically binds to an endogenous anchor transcription factor that binds to a promoter of a target gene, e.g., as illustrated in
Anchor transcription factors of the invention are those transcription factors that bind to a transcription factor-binding site or response element of the target gene of the cell and modulate, e.g., enhance or repress, transcription thereof. As the anchor transcription factors are endogenous, they originate from the cell and are not heterologous to the cell. As such, an anchor transcription factor of a cell in which a method of invention is carried out is one that is encoded by a chromosomal gene, where the chromosomal gene is not heterologous to the cell, i.e., the gene has not been introduced into the chromosome of the cell, e.g., by a vector, such as a viral vector or has been genetically modified in anyway.
The endogenous anchor transcription factor may vary widely depending on the nature of the particular target gene and type of modulation, e.g., transcription enhancement or reduction, desired. Examples of anchor transcription factors that may be employed in embodiments of the invention include, but are not limited to: general transcription factors that are involved in the formation of a preinitiation complex, e.g., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH; and upstream transcription factors, e.g., proteins that bind somewhere upstream of the initiation site to stimulate or repress transcription. Endogenous anchor transcription factors employed in given embodiments of the invention may be any of the transcription factors described in Lambert et al., “The Human Transcription Factors,” Cell (February 8, 2018) 172: 650-665 as well as those listed in the supplementary materials therefore and also at http://humantfs.ccbr.utoronto.ca/. Specific anchor transcription factors finding use in exemplary applications of the invention are reviewed in greater detail below.
As summarized above, the CIP employed in embodiments of the invention includes a second ligand that specifically binds to an endogenous transcription modulatory factor, e.g., as illustrated in
As summarized above, aspects of methods of invention include providing a CIP in a cell in which transcription of a target gene is to be modulated. The cell that is provided with the CIP compound may vary depending on the specific application being performed. Cells of interest include eukaryotic cells, e.g., animal cells, where specific types of animal cells include, but are not limited to yeast, insect, worm or mammalian cells. Various mammalian cells may be used, including, by way of example, equine, bovine, ovine, canine, feline, murine, non- human primate and human cells. Among the various species, various types of cells may be used, such as hematopoietic, neural, glial, mesenchymal, cutaneous, mucosal, stromal, muscle (including smooth muscle cells), spleen, reticulo- endothelial, epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary, fibroblast, and other cell types. Hematopoietic cells of interest include any of the nucleated cells which may be involved with the erythroid, lymphoid or myelomonocytic lineages, as well as myoblasts and fibroblasts. Also, of interest are stem and progenitor cells, such as hematopoietic, neural, stromal, muscle, hepatic, pulmonary, gastrointestinal and mesenchymal stem cells, such as ES cells, epi-ES cells and induced pluripotent stem cells (iPS cells). As summarized above, the cells that are provided with the CIP compounds contain at least the endogenous anchor transcription factor and endogenous transcription modulatory factor. As such, the cells are cells that naturally include the anchor transcription factor and transcription modulatory factor and have not been engineered to include these factors. As desired, the cells may be in vitro or in vivo. In some instances, the cell in which transcription of the target gene is to be modulated is part of a multicellular organism.
Aspects of the invention include providing the CIP in the cell, e.g., as described above, in a manner sufficient to induce proximity of the anchor transcription factor and transcription modulatory factor, e.g., as described above. Any convenient protocol for providing the CIP in the cell may be employed. The particular protocol that is employed may vary, e.g., depending on whether the target cell is in vitro or in vivo. In certain instances, the CIP is provided in the cell by contacting the cell with the CIP. For in vitro protocols, contact of the CIP compound with the target cell may be achieved using any convenient protocol. For example, target cells may be maintained in a suitable culture medium, and the CIP compound introduced into the culture medium as described specifically in the figures.
For in vivo protocols, any convenient administration protocol may be employed. Depending upon the binding affinity of the CIP compound, the response desired, the manner of administration, the half-life, the number of cells present, various protocols may be employed. Thus, the CIP can be incorporated into a variety of formulations, e.g., pharmaceutically acceptable vehicles (also referred to herein as pharmaceutical delivery vehicles or carriers), for therapeutic administration. More particularly, the CIP of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments (e.g., skin creams), solutions, suppositories, injections, inhalants and aerosols. As such, administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. In pharmaceutical dosage forms, the CIPs may be administered alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following examples are illustrative and not limiting.
For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
In those embodiments where an effective amount of an active agent is administered to a living subject, the amount or dosage is effective when administered for a suitable period of time, such as one week or longer, including two weeks or longer, such as 3 weeks or longer, 4 weeks or longer, 8 weeks or longer, etc., so as to evidence a desired therapeutic effect. For example, an effective dose is the dose that, when administered for a suitable period of time, such as at least about one week, and maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer, will results in a desired therapeutic effect. In some instances, an effective amount or dose of active agent will not only slow or halt the progression of the disease condition but will also induce the reversal of the condition, i.e., will cause an improvement one or more symptoms of the condition. For example, in some instances, an effective amount is the amount that when administered for a suitable period of time, usually at least about one week, and maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks, or longer will improve one or more symptoms of a subject suffering from a disease condition, where the magnitude of improvement (e.g., as measured using a suitable protocol with relevant control) may vary, for example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in some instances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more. In certain embodiments, the methods include removing the CIP from the cell at some point after provision of the CIP. Removal of the CIP from the cell may be accomplished using any convenient protocol, e.g., by removing the CIP from the medium in which the cell is present, by ceasing administration of the CIP to the animal comprising the cell, by contacting the cell with an inhibitor of the CIP induced proximity, by contacting the cells with a molecule that displaces the CIP and binds to only one of the endogenous anchor transcription or transcription modulatory factors, etc. One specific type of inhibitor of the action of the TF-CP would be a one-sided molecule consisting of the ligand for either the anchor or the hijacked transcription factor without the linker or other moiety.
As summarized above, aspects of the invention further include methods of inducibly modulating transcription of a target gene. Such methods include providing a chemical inducer of proximity (CIP) in a cell (e.g., a eukaryotic cell) containing an endogenous anchor transcription factor and endogenous transcription modulatory factor, e.g., as described above, under conditions sufficient to modulate transcription of the target gene. The CIP and cell may be as described above. The transcription modulation may vary. In some instances, the modulating includes enhancing transcription of the gene, e.g., where the gene is beneficial with respect to the disease condition, e.g., by enhancing a desired activity in the cell, such increasing expression of a proapoptotic gene where death of the cell is desired, increasing expression of a therapeutically beneficial gene where increased amounts of the product of such gene are beneficial with respect to a given disease condition, etc. In such instances, the magnitude of enhancement may vary, where examples include from substantially no to some expression, and in some instances the magnitude may be 2-fold or greater, such a 5-fold or greater, including 10-fold or greater. In some instances, the modulating includes reducing transcription of the target gene, e.g., where the gene is harmful, e.g., c-myc or a triplet expansion gene, e.g., such as Huntington, etc. In such instances, the magnitude of reduction may vary, where examples include from some expression to substantially none, if any, expression, and in some instances the magnitude of reduction may be 2-fold or greater, such a 5-fold or greater, including 10-fold or greater.
In some instances, the cell is a cell of a subject suffering from a disease condition, i.e., a cell obtained from such a subject or a cell that is part of such a subject. Disease conditions from which the subject may be suffering may vary, where examples of such disease conditions include, but are not limited to: neoplastic disease conditions, e.g., cancers; neurological conditions, immune disorders, gastrointestinal diseases, cardiovascular diseases and the like.
The subject methods find use in the treatment of a variety of different conditions in which the modulation of target gene transcription in a host is desired. By treatment is meant that at least an amelioration of one or more of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
Where the methods are methods of treating a subject for a condition, the methods may further include assessing that the subject has the given condition, e.g., so as to confirm that a given CIP is suitable for use in treating the subject for the condition. Any convenient diagnostic protocol appropriate for a given condition may be employed, where the choice of such protocol will necessarily depend on the specific condition to be treated.
A variety of subjects are treatable according to the subject methods. In some instances, the subjects are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subjects are humans.
The following sections provide further details regarding illustrative embodiments of the methods of the invention.
Embodiments of the invention include methods of enhancing transcription of a pro-apoptotic gene in a cell, e.g., as illustrated in
The methods may result in enhancing transcription of a variety of different proapoptotic genes. Proapoptotic genes are genes the expression products of which promote or cause apoptosis, i.e., programmed cell death that occurs in multicellular organisms, which may be characterized by a variety of cell changes, such as blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay, and death. Specific proapoptotic genes of interest for transcription that may be enhanced in embodiments of the invention include, but are not limited to: PUMA (BBC3), BIM (BCL2L11), BID, BAX, BAK, BOK, BAD, HRK, BIK, BMF, and NOXA, and the like. Their relative ability to kill breast cancer cells when simply overexpressed are shown in Table 1. These measurements are useful in picking transcription factors and ligands for targeting the TF-CIP to a specific effective pro-apoptotic gene, such as BMF and HRK.
Pro-apoptotic genes are of particular interest because they are expressed at levels that balance the anti-apoptotic genes, allowing the cell to survive by virtue of this balanced steady state.
Aspects of the methods of these embodiments include providing in the cell, e.g., via a protocol as described above, a chemical inducer of proximity (CIP) which links a first endogenous anchor transcription factor that binds to a promoter of the proapoptotic gene and a second endogenous oncogenic transcription factor, wherein CIP mediated linkage of anchor and oncogenic transcription factors enhances transcription of the proapoptotic gene in the cell. In some instances, CIPs employed in these embodiments are generally as described above and include a first ligand that specifically binds to the anchor transcription factor and a second ligand that specifically binds to the oncogenic transcription factor, where these first and second ligands are joined by a bond suitable linker, e.g., as described above.
A variety of different anchor transcription factors may be employed in methods of these embodiments.
In TF-CIPs of these embodiments, any convenient ligand for these anchor transcription factors may be employed, where suitable ligands include small molecule ligands that are capable of specifically binding to the target anchor transcription factor without any relevant negative impact on the anchor transcription factor's ability to bind to target DNA binding site. The molecular weight of these ligands may vary, and in some instances ranges from 50 Daltons to 1200 Daltons such as 200 to 500 Daltons. A general method for identifying suitable ligands for use in such TF-CIPs is provided in
In addition to the anchor transcription factor ligand, the CIPs employed in these embodiments also include a ligand for an oncogenic transcription factor. This embodiment is of particular significance in treatment of cancer, where the TF-CIP causes the cancer cell to kill itself with its own driver. Oncogenic transcription factors are transcription factors whose activity contributes to a neoplastic, e.g., cancerous, disease condition. The oncogenic transcription factor may vary, e.g., depending on the particular nature of the disease condition being treated, where examples of oncogenic transcription factors include, but are not limited to: hormonal receptors (e.g., estrogen, androgen and progesterone receptors and the like), oncogene drivers (e.g., MYC, MLL fusion proteins, ETS fusion proteins, SS18-SSX fusion proteins and the like), translocated fusion oncogenes and proteins that regulate cell cycle entry (e.g., E2F family members and the like), etc.
Ligands for exemplary cancer drivers or modulators are illustrated in
In some instances, the oncogenic transcription factor is a hormonal receptor. Hormonal receptors that may be employed as the oncogenic transcription factor in embodiments of the invention include, but are not limited to: estrogen receptor (ER), e.g., such as those provided in
In some instances, the oncogenic transcription factor is BAF53a (a.k.a. ACTL6a). BAF53a is a subunit of the BAF or mSWI/SNF chromatin regulatory complex (PMID: 9845365), which opposes polycomb mediated repression over the genome (PMID: 27941796) and plays prominent roles in activating developmentally repressed genes (PMID: 20110991). In these cancers, BAF53a drives both initiation and proliferation and is expressed highly and specifically. In a specific embodiment of this invention TF-CIPs can be applied in Squamous Cell Cancer (SCC). Thus, using the overexpressed BAF53a to kill the SCC is a specific treatment for SCC. Using the systematic approach defined in
The first and second ligands of the CIPs employed in embodiments of the above methods may be linked to each other by any convenient linker. As reviewed above, linkers of interest are linkers that provide for a stable association of the first and second ligands in a manner such that the first and second ligands are capable of specifically binding to their respective endogenous factors in the cell. As the linker provides for stably associating the first and second ligands with each other, the first and second ligands do not dissociate from each other under cellular conditions, e.g., conditions at the surface of a cell, conditions inside of a cell, etc. Linkers may be provided for stable association of the first and second ligands using any convenient binding, such as covalent or non-covalent binding, where in some instances the linker component is covalently bound to both the first and second ligands. In some embodiments, the linker may be an alkyl chain, an alkoxy chain, an alkyenyl chain or a alkynyl chain, where the number of carbon atoms in the chain may vary, ranging in some instances from 2 to 25, such as 5 to 20, where one or more carbon atoms are replaced with NH or CH3—N to provide covalent bonding to the first and second ligands. Linkers may be bound to the first and second ligands at positions that do not negatively impact the ability of the ligands to bind to their respective endogenous factors. In some embodiments there will be no discrete linker, but rather a linking component that is a molecule which induces proximity of the targeted transcription factor to the anchoring transcription factor on the promoter of the target gene. Examples of such linking components include molecular glues, in which the two different sides of the singular molecule will each bind a separate transcription factor, e.g., as illustrated in
Methods of enhancing transcription of pro-apoptotic genes finds use in, for example, treatment of oncogenic transcription factor mediated neoplastic disease conditions, e.g., cancer. Cancers which may be treated using embodiments of the invention include oncogenic receptor (e.g., hormonal receptor, such as estrogen, progesterone and androgen receptor) mediated cancers, oncogenic driver (e.g., myc) mediated cancers, translocated fusion oncogene (e.g., those shown in
Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.), Sezary Syndrome, Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell Carcinoma, Nonmelanoma, etc.), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer (e.g., Endometrial, etc.), Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenström Macroglobulinemia, Wilms Tumor, and the like. Cancers that may be treated further include, epithelial cancers, such as carcinomas, such as acinar carcinoma , acinic cell carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenocortical carcinoma, alveolar carcinoma, ameloblastic carcinoma, apocrine carcinoma, basal cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, cholangiocellular carcinoma, chorionic carcinoma, clear cell carcinoma, colloid carcinoma, cribriform carcinoma, ductal carcinoma in situ, embryonal carcinoma, carcinoma en cuirasse, endometrioid carcinoma, epidermoid carcinoma, carcinoma ex mixed tumor, carcinoma ex pleomorphic adenoma, follicular carcinoma of thyroid gland, hepatocellular carcinoma, carcinoma in situ, intraductal carcinoma, Hürthle cell carcinoma, inflammatory carcinoma of the breast, large cell carcinoma, invasive lobular carcinoma, lobular carcinoma, lobular carcinoma in situ (LCIS), medullary carcinoma, meningeal carcinoma, Merkel cell carcinoma, mucinous carcinoma, mucoepidermoid carcinoma, nasopharyngeal carcinoma, non-small cell carcinoma , non-small cell lung carcinoma (NSCLC), oat cell carcinoma, papillary carcinoma, renal cell carcinoma, scirrhous carcinoma, sebaceous carcinoma, carcinoma simplex, signet-ring cell carcinoma, small cell carcinoma , small cell lung carcinoma, spindle cell carcinoma, squamous cell carcinoma, terminal duct carcinoma, transitional cell carcinoma, tubular carcinoma, verrucous carcinoma, and the like.
Embodiments of the methods may include assessing whether a subject suffering from a neoplastic disease has a particular type of cancer. For example, where a subject has breast cancer, the methods may include assessing whether the breast cancer is ER and/or PR positive, and then employing an appropriate TFCIP to treat the particular cancer. For example, if the breast cancer is ER positive, a CIP having a ligand that binds to ERα may be employed. Another example is prostatic cancer driven by the translocated ETS family members to a gene that drives high level expression of the ETS fusion protein. Here the ETS fusion protein can be hijacked by, for example, an ERG binding ligand, e.g., selected as described in
Embodiments of the invention include methods of reducing transcription of a specific gene whose overexpression contributes to a pathologic process. Examples include, but are not limited to, oncogenes that are pathogenic due to amplication of their DNA (for example BAF53a in squamous cell carcinoma, genes that are overexpressed as a result of trisomy (for example Down Syndrome), genes that are overexpressed for a variety of reasons and that lead to a disease state (for example Tumor Necrosis Factor in arthritis); and alleles containing triplet repeats. The general approach is illustrated in
In addition to the anchor transcription factor ligand, the CIPs employed in these embodiments may also include a ligand for a transcription modulating factor that reduces transcription of the target gene, e.g., oncogene, in the cell. Transcription modulatory factors that reduce transcription of the oncogene in the cell when complexed with the anchor transcription factor via the CIP may vary, and include transcriptional repressors. Examples of transcriptional repressors include, but are not limited to, heterochromatin protein 1 (HP1) repressor proteins, KRAB repressor proteins, H3K9 methyltransferases, histone deacetylases etc. Any convenient ligands for these transcription modulatory factors may be employed, where suitable ligands include small molecule ligands that are capable of specifically binding to the target transcriptional modulatory factor without any relevant negative impact on the factor's ability to reduce transcription of the target oncogene when complexed with the anchor transcription factor by a CIP. The molecular weight of these ligands may vary, and in some instances ranges from 75 to 1000 such as 200 to 400 Daltons. Examples of such ligands include both agonists and antagonists. Suitable ligands for the anchor transcription factor may be chosen using any convenient protocol, such as in silico screening protocols, and the like, such as described below.
The first and second ligands of the CIPs employed in embodiments of the above methods may be linked to each other by any convenient linker component. As reviewed above, linker components of interest such as those described above provide for a stable association of the first and second ligands in a manner such that the first and second ligands are capable of specifically binding to their respective endogenous factors in the cell. As the linker component provides for stably associating the first and second ligands with each other, the first and second ligands do not dissociate from each other under cellular conditions, e.g., conditions at the surface of a cell, conditions inside of a cell, etc. Linker components may be provided for stable association of the first and second ligands using any convenient binding, such as covalent or non-covalent binding, where in some instances the linker component is covalently bound to both the first and second ligands. In some embodiments, the linker may be an alkyl chain, an alkoxy chain, an alkyenyl chain or a alkynyl chain, where the number of carbon atoms in the chain may vary, ranging in some instances from 2 to 25, such as 5 to 20, where one or more carbon atoms are replaced with NH or CHs-N. Linkers may be bound to the first and second ligands at positions that do not negatively impact the ability of the ligands to bind to their respective endogenous factors. In other cases, the first and second ligand are contained within one two-sided molecule or molecular glue. Examples of molecular glues include FK506, rapamycin, and cyclosporin A, etc.
Methods of reducing transcription of oncogenes finds use in, for example, treatment of oncogene mediated neoplastic disease conditions, e.g., cancer, where examples of such cancers include, but are not limited to, those described above.
Embodiments of the invention also include reducing transcription of mutant extended nucleotide repeat (NR) containing alleles. For example, where NR containing genes are mono allelic, embodiments of the methods may include suppressing expression of the NR containing monoallele by employing an anchor transcription factor that binds to the allele containing the NR. In such embodiments, the target gene is a gene that includes a mutant extended NR, such as a TNR, where the mutant extended nucleotide repeat domain is not present in normal versions of the gene. By mutant extended nucleotide repeat (NR) is meant a domain (i.e., region) of the gene that includes multiple adjacent repeats of units of 2 or more nucleotides, where a given repeating unit of nucleotides may vary in length, ranging in some instances from 2 to 10 nucleotides, such as 3 to 6 nucleotides, where examples of repeat unit lengths include units of 2 nucleotides (e.g., where the mutant extended nucleotide repeat is a dinucleotide repeat), 3 nucleotides (e.g., where the mutant extended nucleotide repeat is a trinucleotide repeat), 4 nucleotides (e.g., where the mutant extended nucleotide repeat is a tetranucleotide repeat), 5 nucleotides (e.g., where the mutant extended nucleotide repeat is a pentanucleotide repeat) or 6 nucleotides (e.g., where the mutant extended nucleotide repeat is a hexanucleotide repeat). Within a given domain, the domain may be homogeneous or heterogeneous with respect to the nature of the repeat units that make up the domain. For example, a given domain may be made up of a single type of repeat unit, i.e., al the repeat units of the domain share the same (i.e., identical) sequence of nucleotides, such that it is a homogenous mutant NR domain, Alternatively, a given domain may be made up of two or more different types of repeat units, i.e., repeat units that have differing sequences, such that it is a heterogeneous mutant NR domain. The mutant extended nucleotide repeat domain may be present in a coding or non-coding region of the target gene. In some instances, the extended nucleotide repeat domain is present in a coding region of the target gene. In some instances, the extended nucleotide repeat domain is present in a non-coding region of the target gene. The length and particular sequence of the mutant extended nucleotide repeat may vary.
In some instances, the mutant extended nucleotide repeat is a mutant extended trinucleotide repeat. By mutant extended trinucleotide repeat is meant a domain (i.e., region) of the gene that includes multiple adjacent repeats of the same three nucleotides, where the length and particular sequence of the mutant extended trinucleotide repeat may vary and the mutant extended trinucleotide repeat domain is not present in normal versions of the gene. The extended trinucleotide repeat domain may be present in a coding or non-coding region of the target gene. In some instances, the extended trinucleotide repeat domain is present in a coding region of the target gene. In some instances, the extended trinucleotide repeat domain is present in a non-coding region of the target gene. In embodiments, the mutant repeat domain is present in a non-coding region of the target gene, such as the CTG expansion located in the 3′ untranslated region of the dystrophia myotonica-protein kinase gene, which leads to Myotonic dystrophy (DM), In some instances, the mutant repeat domain is present in a coding region of the target gene, such that in some instances its presence in the target gene results in a corresponding domain or region (e.g., polyQ domain) in a product encoded by the gene. In some instances of the method, the mutant extended TNN domain is a CTG repeat domain. In certain cases, the mutant extended trinucleotide repeat domain includes 26 or more CTG repeats (e.g,, 30 or more, 35 or more, etc.).
The mutant extended trinucleotide repeat may vary in terms of nucleotide composition and length. Specific trinucleotides of interest include, but are not limited to: CAG, CTG, CGG, CCC, GAA, and the like. In some instances, the mutant extended trinucleotide repeat domain is a CAG repeat domain. The particular length of the repeat domain (e.g., CAG repeat domain) may vary with the respect to the specific target gene so long as it results in deleterious activity, and in some instances is 25 repeats or longer, such as 26 repeats or longer. 30 repeats or longer, including 35 repeats or longer, 40 repeats or longer, 50 repeats or longer or even 60 repeats or longer. Specific target genes and expressed proteins of interest, diseases associated therewith and the specific length of repeat sequences of extended CAG repeats of interest, include (but are not limited to) those provided in the table, below.
The pathogenic repeat lengths shown are approximate and represent the most common range of pathogenic repeat lengths. The lower of the two numbers shown for each pathogenic repeat length indicates the length at which pathogenic effects of the expansion begin to occur, Although both cellular copies of autosomal genes responsible for NR diseases may contain NR domains, commonly one copy of the targeted gene is mutated to have an expanded NR segment, whereas the other copy (i.e., allele) contains a unexpanded
Embodiments of the invention include methods of enhancing transcription of a therapeutically beneficial gene in a cell, e.g., as illustrated in general in
The methods may result in enhancing transcription of a variety of different therapeutically beneficial genes that may or may not be haploinsufficient genes. Therapeutically beneficial genes are genes the expression products of which are beneficial with respect to a given disease condition. Therapeutically beneficial genes may be genes in which an increase in the amount of expression product thereof results in the improvement in one or more symptoms of a disease condition associated with a low amount, e.g., an amount below that of a normal control, of expression product of that gene. As illustrated in
Aspects of the methods of these embodiments include providing in the cell, e.g., via a protocol as described above, a chemical inducer of proximity (CIP) which links a first endogenous anchor transcription factor that binds to a promoter of the therapeutically beneficial gene and a second endogenous transcription modulatory factor, e.g., transcription factor, wherein CIP mediated linkage of anchor and transcription modulatory factors enhances transcription of the therapeutically beneficial gene in the cell. In some instances, CIPs employed in these embodiments are generally as described above and include a first ligand that specifically binds to the anchor transcription factor and a second ligand that specifically binds to the transcription modulatory factor, where these first and second ligands are joined by a bond or suitable linker, e.g., as described above.
A variety of different anchor transcription factors may be employed in methods of these embodiments, where anchor transcription factors may readily be chosen based on the specific therapeutically beneficial gene and transcription factors therefor. For example, where the beneficial therapeutic gene is TPH2, any transcription factor therefore may be employed as the anchor transcription factor, where examples of such transcription factors include, but are not limited to: FEV, EN1, GATA2, GATA3, LMX1 B, POU3F2, INSM1, ESR2, CTCF, NR3C1, and REST, etc. The selection of pairs of transcription factors selectively expressed in serotonergic cells of the dorsal raphe is illustrated in
A parallel strategy may be used to increase dopamine synthesis by stimulating production of the rate-limiting enzyme, tyrosine hydroxlase, in the substantia nigra in early Parkinson's disease, where dopaminergic cells have not been entirely lost, as illustrated in
As a general strategy to encode cell specificity into a TF-CIP, transcription factor pairs are targeted that are selectively co-expressed in the cell type of interest as illustrated in
In another example, where the beneficial therapeutic gene is ARID1 B, the anchor transcription factor could be chosen using the systematic process described in
In addition to the anchor transcription factor ligand, the CIPs employed in these embodiments also include a ligand for a transcription modulatory factor, e.g., which in some instances is a second endogenous transcription factor expressed in tissue type harboring the target cell, e.g., the dorsal raphe (for enhancement of TPH2 for serotonin production) or the human brain (for ARID1B). The transcription modulatory factor may vary, e.g., depending on the particular nature of the disease condition being treated and the cell in which transcription modulation is desired. For example, where the target beneficial therapeutic gene is TPH2, transcription modulatory factors that may be employed include, but are not limited to FEV, BRD4, P300/CBP, PAXS, POU6F2, KLF5, SOX14, POU3F3, SATB2, nBAF etc. In another example, where the target beneficial therapeutic gene is ARID1B, transcription modulatory factors that may be employed include, but are not limited to TBR1 and others chosen to optimize cell type specificity using the quantitative proteome map of the human body (PMID 32916130) and the procedures described in
Any convenient ligands for these transcription factors may be employed, where suitable ligands include small molecule ligands that are capable of specifically binding to the target transcription factor without any relevant negative impact on the transcription factor's ability to enhance transcription of the target beneficial therapeutic gene when complexed with the anchor transcription factor by a CIP, i.e., the transcription-activating activity of the transcription factor. A systematic process for selecting the ligand is illustrated in
Methods of these embodiments find use in treating any disease condition for which increased transcription of a beneficial therapeutic gene is desired. Examples of such disease conditions include, but are not limited to: disease conditions associated abnormal expression of rate-limiting enzymes, e.g., TPH2 in disease conditions characterized by altered brain serotonin such as depression, anxiety, panic disorder, obsessive compulsive disorder, attention deficit hyperactivity disorder, sleep and circadian rhythm disorders, irritable bowel syndrome, PMS/hormone dysfunction, fibromyalgia, obesity, alcoholism, aggression, hyperserotonemia, social disorders, autism spectrum disorder, language disorders, etc. (PMIDs, 30552318, 32883965, 22826343, 22698760, 14675805). In addition, haploinsufficiency disease conditions, e.g., ARID1B causing intellectual disability, autism spectrum disorders, etc.; CHD7 gene causing CHARGE syndrome; RUNX2 gene causing Cleidocranial dysostosis; ADAMTS2, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, PLOD1, FKBP14, TNXB, COL12A1, B4GALT7, B3GALT6, CHST14, DSE, C1R, C1S, SLC39A13, ZNF469, PRDM5 causing Ehlers-Danlos syndromes; TNFAIP3 causing Haploinsufficiency of A20, FBN1 causing Marfan syndrome; SHANKS causing 22813 deletion syndrome, SCN1A causing Dravet syndrome, etc. A list of curated genes that produce human disease when the dosage is reduced by 50% and may be treated by embodiments of the invention is available from the Clinical Genome Resource at: https://search.clinicalgenome.org/kb/curations.
Aspects of the present disclosure further include combination therapies. In certain embodiments, the subject method includes administering a therapeutically effective amount of one or more additional active agents in combination with a CIP of the invention. By combination therapy is meant that a CIP can be used in a combination with another therapeutic agent to treat a single disease or condition. Alternatively, a second TF-IP could be used to overcome drug-induced resistance to a first CIP or to boost the apoptotic activity of the first CIP. This could be accomplished by using a ligand to another anchoring transcription factor that binds to one or more of the apoptotic gene promoter/enhancers. In some embodiments, a CIP compound of the present disclosure is administered concurrently with the administration of another therapeutic agent, which can be administered as a component of a composition including the compound of the present disclosure or as a component of a different composition. In certain embodiments, a composition including a compound of the present disclosure is administered prior or after administration of another therapeutic agent. The subject compounds can be administered in combination with other therapeutic agents in a variety of therapeutic applications. Therapeutic applications of interest for combination therapy include those applications those described above.
As reviewed above, in some instances the CIPs are employed for treating neoplastic conditions. As such, the CIPs of such embodiments can be used jointly with any agent useful in the treatment of a neoplastic condition, such as anti-cancer agents and anti-tumor agents. Agents of interest which can be used jointly with the subject CIS compounds in such instances include, but are not limited to, Cancer chemotherapeutic agents, Agents that act to reduce cellular proliferation, Antimetabolite agents, Microtubule affecting agents, Hormone modulators and steroids, natural products and biological response modifiers, e.g., as described in greater detail below.
Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used. Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. Pat. No. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623);
duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD). Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide. Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine. Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like. Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine. Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like. Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g. aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation. Therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation. Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc. Taxanes are suitable for use. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis). Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose). Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxol derivative described in U.S. Pat. No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701. Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.
In some instances, the CIPs of the invention are employed in combination with immunotherapy agents. Examples of immunotherapy include anti-PD-1/PD-L1 immunotherapies, such as anti-PD-1/PD-L1 therapeutic antagonists, where such antagonists include but are not limited to e.g., OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), Tecentriq™ (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242 and the like. Nivolumab (OPDIVO®) is a humanized IgG4 anti-PD-1 monoclonal antibody used to treat cancer. Pembrolizumab (KEYTRUDA®), formerly known as MK-3475, lambrolizumab, etc., is a humanized antibody used in cancer immunotherapy targeting the PD-1 receptor. Atezolizumab (Tecentriq™) is a fully humanized, engineered monoclonal antibody of IgG1 isotype against the PD-L1 protein. Durvalumab (MedImmune) is a therapeutic monoclonal antibody that targets PD-L1. Avelumab (also known as MSB00107180; Merck KGaA, Darmstadt, Germany & Pfizer) is a fully human monoclonal PD-L1 antibody of isotype IgG1. BMS-936559 (also known as MDX-1105; Bristol-Myers Squibb) is a blocking antibody that has been shown to bind to PD-L1 and prevent its binding to PD-1 (see e.g., U.S. NIH Clinical Trial No. NCT00729664). CA-170 (Curis, Inc.) is a small molecule PD-L1 antagonist. BMS-202, BMS-8, BMS-37, BMS-242 are small molecule PD-1/PD-L1 complex antagonists that bind PD-1 (see e.g., Kaz et al., (2016) Oncotarget 7(21); the disclosure of which is incorporated herein by reference in its entirety). Anti-PD-L1 antagonists, including e.g., antibodies, useful in the methods described herein include but are not limited to e.g., those described in U.S. Pat. Nos. 7,722,868; 7,794,710; 7,892,540; 7,943,743; 8,168,179; 8,217,149; 8,354,509; 8,383,796; 8,460,927; 8,552,154; 8,741,295; 8,747,833; 8,779,108; 8,952,136; 8,981,063; 9,045,545; 9,102,725; 9,109,034; 9,175,082; 9,212,224; 9,273,135 and 9,402,888; the disclosures of which are incorporated herein by reference in their entirety. Anti-PD-1 antagonists, including e.g., antibodies, useful in the methods described herein include but are not limited to e.g., those described in U.S. Pat. Nos. 6,808,710; 7,029,674; 7,101,550; 7,488,802; 7,521,051; 8,008,449; 8,088,905; 8,168,757; 8,460,886; 8,709,416; 8,951,518; 8,952,136; 8,993,731; 9,067,998; 9,084,776; 9,102,725; 9,102,727; 9,102,728; 9,109,034; 9,181,342; 9,205,148; 9,217,034; 9,220,776; 9,308,253; 9,358,289; 9,387,247 and 9,402,899; the disclosures of which are incorporated herein by reference in their entirety.
Also provided are pharmaceutical preparations of the CIP compounds. The CIP compounds can be incorporated into a variety of formulations for administration to a subject. More particularly, the CIP compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The formulations may be designed for administration via a number of different routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, intravenous, etc., administration. In pharmaceutical dosage forms, the compounds may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxy-propylmethycellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. The compounds of this invention and their pharmaceutically acceptable salts which are active on topical administration can be formulated as transdermal compositions or transdermal delivery devices (“patches”). Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive. Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated by reference in its entirety. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
Optionally, the pharmaceutical composition may contain other pharmaceutically acceptable components, such a buffers, surfactants, antioxidants, viscosity modifying agents, preservatives and the like. Each of these components is well-known in the art. See, for example, U.S. Pat. No. 5,985,310, the disclosure of which is herein incorporated by reference. Other components suitable for use in the formulations of the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). In an embodiment, the aqueous cyclodextrin solution further comprise dextrose, e.g., about 5% dextrose.
Dosage levels of the order of from about 0.01 mg to about 140 mg/kg of body weight per day are useful in representative embodiments, or alternatively about 0.5 mg to about 7 g per patient per day. For example, inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. As such, unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular peptidomimetic compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
Also provided are kits and systems that find use in practicing embodiments of the methods, such as those described as described above. The term “system” as employed herein refers to a collection of two or more different active agents, present in a single composition or in disparate compositions, that are brought together for the purpose of practicing the subject methods. The term “kit” refers to a packaged active agent or agents. For example, kits and systems for practicing the subject methods may include one or more pharmaceutical formulations. As such, in certain embodiments the kits may include a single pharmaceutical composition, present as one or more unit dosages, where the composition may include one or more expression/activity inhibitor compounds. In yet other embodiments, the kits may include two or more separate pharmaceutical compositions, each containing a different active compound.
In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, portable flash drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
The following examples are offered by way of illustration and not by way of limitation.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Breast cancer is responsible for over 40,000 deaths per year in the US. About 1 in 8 women will develop breast cancer and of these one in 39 will die of their cancer. If surgery is not effective in curing the cancer a number of treatments are used that include hormonal therapy, HER2 monoclonal antibodies and immunotherapy. Each of these is making a contribution to the effective treatment of breast cancer, yet many of the drugs used such as anthracyclines and others are non-specific in their antitumor actions and hence have broad toxicity that can be lethal. Therefore, the field has sought more specific routes for treatment. Here is described a new way of producing more specific death of the cancer cells relative to that of other cells and hence better treatments with fewer side effects.
We have devised a general way of hijacking cancer-drivers to activate cell death pathways thereby killing any cancer cell that has a driving mutation. In the specific case of breast cancer, we hijack the estrogen receptor (ER) and/or the progesterone receptor (PR) to activate PUMA, BIM and other cell death genes. To do this we have invented small molecule chemical inducers of proximity (CIP) that have estrogen-like compounds linked at the C17 or C7 position to a ligand for a transcription factor(s) that normally binds to the regulatory regions of cell death genes, such as PUMA, BAX, BIM and BID and NOXA genes. Thus, estrogen, which normally drives the tumor instead activates a set of genes which results in programmed cell death. By this means the breast cancer pathways that drive proliferation are hijacked to specifically kill the cancer cell.
A large number of breast cancers are ER positive and PR positive. These hormonally responsive cancers are often treated using antagonists to these receptors and thereby reduce or slow the growth of the cancer. However, cancers are almost never successfully treated by agents that simply reduce the rate of growth of the cancer, because they simply recur when the treatment is stopped. Hence diverting the cancer's driving proliferative force to kill cancer cells is useful. We have invented a series of molecules (CIP) which chemically link estrogen-like molecules at C17 to a small linker and then to a ligand for transcription factors that control genes that induced apoptosis or programmed cell death such as the PUMA, BIM, BAX, BID and NOXA genes. These genes function downstream of p53 to initiate cell death when DNA damage occurs. In this way these genes suppress the formation of cancer and this is one of the major effectors of p53's tumor suppressor pathway. Many breast cancers have p53 inactivated and hence are unable to respond to DNA damage signals that might eliminate a cancer at its inception. In the following described embodiment, the transcription factor FOXO3a has been chosen, which regulates the PUMA, BIM and the BAX genes. The means for defining the anchor transcription factors are illustrated in
A specific example of a TF-CIP that may be employed in such embodiments is estradiol linked to a small molecule binder of a transcription factor that activates the programmed cell death genes. Other embodiments of the invention use progesterone, any of several proapoptotic genes and any one of the following breast selective transcription factors: 1) FOXO3A, 2) PPARgamma, 3) HIF1A, 4) E2F1; 5) RUNX1 and MAZ, which bind to the PUMA as well as other the promoters for other killer genes, such as BIM. Normally estrogen will bind to the estrogen receptor and induce proliferation. In this embodiment of the present invention, a small molecule linking the estrogen receptor to a transcription factor (TF) which binds and coordinately activates killer genes is employed as a new therapy for ER positive breast cancer.
One of the many advantages of this approach of hijacking a cancer driver pathway to activate a cell death pathway is that it allows one to exploit the combinatorial specificity of the transcription factors to specifically activate the killer genes as well as the specificity of the cancer driver itself. This is illustrated generally in
(selectively of expression of TF-A)×(selectively of expression of the cancer driver)×(genomic specificity of TF-A)=selectivity of cell killing
There are many drugs and treatments used for treating human breast cancer including hormonal therapy, HER2 monoclonal antibodies and immunotherapy, each of which has a degree of specificity for the tumor. There are also a large number of drugs that are used in nearly all metastatic breast cancers that have little or no specificity for the cancer cell. Many of these merely stop the growth of the cancer cell as well as other cells.
The advantage of our approach is that it harnesses the cancer's own specific driving mechanism to activate a transcription factor that kills ER-positive breast cancer cells. An additional advantage of our approach is that it may be tailored to be very specific for the cancer cell. For example, if the estrogen receptor is expressed at a selective level of 6.5:1 in breast cancer cells and the transcription factor that bound the cell death gene were expressed at selective level of 10:1 the breast cancer cells would be killed with a 65-fold specificity thereby establishing an effective therapeutic window. The therapeutic window may be predicted as follows:
To identify transcription factors that could activate the expression of cell death genes we began by defining the cell death gene that was most effective at killing breast cancer cell lines. We examined each of the cell death genes shown in Table 1 using doxycycline inducible expression and found that several were effective, rapidly killing over 50% of the cells. To identify transcription factor pairs that may be used to selectively activate these genes only in target cells we used the procedures outlined In
We then sought to identify transcription factors (TFs) with binding motifs within ±1 kb region around BIM TSS. This region has chromatin accessible to TFs binding as revealed by analysis of ATAC-seq data of two randomly selected patients from BRCA (Breast Invasive Carcinoma) cohort of TCGA (The Cancer Genome Atlas). Our method of transcription factor identification is described step-by-step in
We analyzed expression of the selected 337 TFs in 5 cancer cell lines using publicly available RNA-seq data sets. The list of TFs exhibiting consistently high expression across cancer cell lines include YBX1, ATF4, HIF1A, MAZ, NFE2L2, TGIF1, SMAD2, TFDP1, MYC, DDIT3, STAT3, PNRC2, HES1, FOXO3A, RUNX1 and PPARG. Among these TFs, RUNX1, HIF1A, PAX9 and PPARG are highly specific to breast tissue (according to the Human Protein Atlas). HIF1A is master transcriptional regulator of adaptive response to hypoxia and plays important role in tumor angiogenesis and in hypoxia induced cell death.
Our method of developing ligands for anchoring TF's is described in a step-by-step approach in
The TF-CIP, Estrogen-like ER binder-Linker(n)-FOXO3a binder illustrated below is synthesized as follows. The first component of our invention, which consist of three parts (A-L-B hijackers) is an estrogen-like molecule including, but not limited to, those shown in
The second component the CIP, which consists of three parts (A-L-B hijackers) is a linker (L) of n carbon atoms designed to bridge the distance between the ER and the transcription factor (FOXO3a in this specific example). A suitable linker such as those described above is employed.
The third component of the CIP, which consists of three parts (A-L-B hijackers), is a small molecule that binds to the TF regulating the expression of the cell death genes: PUMA, BAX, BID and BIM. In
Molecules of the general structure A-L-B are tested for their ability to cause programmed cell death in estrogen-dependent cell lines such as MCF7, and MCF10 breast cancer cell lines. Although several hundred combinations of FOXO3A ligands (
With the TF-CIPs illustrated in
We anticipate that resistance could arise for several reasons. For example, the binding site for the anchor TF could be mutated or the TF mutated to no longer serve as an anchor. In this case we use the method provided in
Another example of using TF-CIPs as a way to cope with this possible complication, would involve constructing a TF-CIP to recruit a protein possessing a highly acidic domain to the PUMA TSS. Acidic domains are known for their ability to activate transcription. To identify acidic proteins highly specific to breast tumors, we used the following criteria: (1) Isoelectric point less than 5; (2) The protein is either nuclear or cytosolic (In later case it should have less than 500 amino acids to allow efficient transport to the nucleus); and (3) The average protein expression in tumor exceeds expression at normal tissue at least at 4 times. For this comparison we used gene expression databases consisting of 1085 samples from BRCA TCGA patients and 291 samples from healthy individuals. We found 28 proteins satisfying all 3 criteria: CENPF, CTXN1, DBNDD1, EPN3, ESRP1, FOXA1, GPRCSA, HN1, IF16, KRT8, LMNB1, MCM4, MUC1, PKIB, PRC1, PRR15, PYCR1, RACGAP1, S100P, SDC1, SLC9A3R1, SPP1, STARD10, TFF1, TNNT1, TPD52, UBE2S, ZWINT.
For the treatment of ER negative cancers, the progesterone receptor is targeted using an analogous group of molecules based on progesterone analogues that hijack killer genes.
A solution of 1-(5-chloro-4-((8-methoxy-1-methyl-3-(2-(methylamino)-2-oxoethoxy)-2-oxo-1,2-dihydroquinolin-6-yl)amino)pyrimidin-2-yl)piperidine-4- carboxylic acid (10 mg, 0.02 mmol, according to lit.1) t-Boc-N-amido-PEG3-amine and (30 mg, 0.1 mmol), HATU (21 mg, 0.05 mmol) and DIPEA (50 uL, 0.4 mmol) in DMF (0.25 mL) was stirred at room temperature for 1 h. The crude reaction was purified by HPLC to afford compound Int-1 (16 mg, 95%). MS obsd. [(M+H)+]:805.9
A solution of Int-1 (16 mg, 0.02 mmol) was dissolved in DCM (1 mL) and added TFA 0.2 mL and stirred at room temperature for 0.5 h. The crude reaction was purified by HPLC to afford compound 1, (10 mg, 50%) as white solid. MS obsd. [(M+H)+]:705.8. 1H NMR (500 MHz, DMSO) δ 8.88; (s, 1H), 8.08; (s, 1H), 7.99; (q, J=4.6 Hz, 1H), 7.89; (t, J=5.7 Hz, 1H), 7.78; (s, 3H), 7.58-7.51; (m, 2H), 7.01; (s, 1H), 4.56; (s, 2H), 4.51; (dt, J=13.2, 3.4 Hz, 2H), 3.80-3.97; (m, 6H), 3.61-3.51; (m, 9H), 3.40; (t, J=6.2 Hz, 2H), 3.20; (q, J=6.0 Hz, 2H), 3.03-2.86; (m, 4H), 2.66; (d, J=4.7 Hz, 3H), 2.42; (tt, J=11.6, 3.9 Hz, 1H), 1.71; (dd, J=13.4, 3.6 Hz, 2H), 1.50; (qd, J=12.4, 4.2 Hz, 2H).
A solution of 2-((((13S,E)-3-hydroxy-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-17H-cyclopenta[a]phenanthren-17-ylidene)amino)oxy)acetic acid (20 mg, 0.06 mmol, according to lit.1) t-Boc-N-amido-PEG3-amine and (17 mg, 0.06 mmol), HATU (33 mg, 0.09 mmol) and DIPEA (33 uL, 0.2 mmol) in DMF (0.5 mL) was stirred at room temperature for 1 h. The crude reaction was purified by flash chromatography to afford coupling product Int-2 (20 mg, 70%) as white solid
Int-3 (60 mg, 0.1 mmol) was dissolved in 1 mL DCM and subjected to 0.2 mL TFA at room temperature for 0.5 h. The crude reaction was purified by HPLC to afford compound Int-3 (30 mg, 60%). MS obsd. [(M+H)+]: 518.7
A solution of Int-3 (30 mg, 0.06 mmol), and 1-(5-chloro-4-((8-methoxy-1-methyl-3-(2-(methylamino)-2-oxoethoxy)-2-oxo-1,2-dihydroquinolin-6-yl)amino)pyrimidin-2-yl)piperidine-4-carboxylic acid (30 mg, 0.06 mmol, prepared according to WO 2018/108704 A1), HATU (38 mg, 0.1 mmol) and DIPEA (50 uL, 0.3 mmol) in DMF (1 mL) was stirred at room temperature for 1 h. The crude reaction was purified by HPLC to afford compound 2, (10 mg, 50%) as white solid. MS obsd. [(M+H)+]:1031.1. 1H NMR (500 MHz, DMSO) δ 9.07; (s, 1H), 9.03; (s, 2H), 8.15-8.08; (m, 1H), 7.94; (q, J=4.1 Hz, 1H), 7.86; (t, J=5.6 Hz, 1H), 7.52; (d, J=1.5 Hz, 2H), 7.41; (q, J=5.7 Hz, 1H), 7.07-6.99; (m, 2H), 6.51; (dq, J=8.2, 2.4 Hz, 1H), 6.44; (q, J=2.5 Hz, 1H), 4.56; (s, 2H), 4.47; (d, J=13.0 Hz, 2H), 4.34; (d, J=2.6 Hz, 2H), 4.02; (s, 1H), 3.91-3.84; (m, 5H), 3.45-3.60; (m, 8H), 3.43; (t, J=6.0 Hz, 2H), 3.39; (ddd, J=7.5, 4.7, 1.8 Hz, 2H), 3.31-3.24; (m, 2H), 3.19; (q, J=5.9 Hz, 2H), 2.97; (m, 3H), 2.74; (m, 1H), 2.65; (dd, J=4.6, 1.2 Hz, 3H), 2.54; (d, J=8.5 Hz, 2H), 2.28; (td, J=8.5, 4.1 Hz, 1H), 2.15; (q, J=4.4 Hz, 1H), 1.91-1.80; (m, 3H), 1.76-1.60; (m, 3H), 1.57-1.45; (m, 2H), 1.20-1.36; (m, 5H), 0.91-0.85; (m, 3H).
A solution of 3-(2-(2-(1-(5 -chloro-4-((8-methoxy-1-methyl-3-(2-(methylamino)-2-oxoethoxy)-2-oxo-1,2-dihydroquinolin-6-yl)amino)pyrimidin-2-yl)piperidine-4-carboxamido)ethoxy)ethoxy)propanoic acid (7 mg, 0.009 mmol), DHT (6 mg, 0.018 mmol), EDCI (6 mg, 0.04 mmol), DMAP (5 mg, 0.018), HOAt (5 mg, 0.009 mmol) in 0.25 mL DMF was stirred at room temperature for 1 h. The crude reaction was purified by HPLC to afford compound 3 (2 mg, 30%) as white solid. MS obsd. [(M+H)+]:963.1. 1H NMR (500 MHz, DMSO) δ 10.84; (s, 1H), 8.82; (s, 1H), 8.06; (s, 1H), 7.98; (s, 1H), 7.84; (q, J=8.3 Hz, 2H), 7.45-7.60; (m, 2H), 7.00; (s, 1H), 4.55; (s, 2H), 4.53 — 4.45; (m, 3H), 3.87; (d, J=6.7 Hz, 4H), 3.61; (t, J=6.1 Hz, 4H), 3.52-3.47; (m, 9H), 3.24-3.15; (m, 5H), 3.00; (s, 2H), 2.91; (t, J=13.0 Hz, 4H), 2.78; (s, 3H), 2.78-2.75; (m, 4H), 2.66; (d, J=4.7 Hz, 3H), 2.53; (s, 6H), 2.43-1.99; (6H, m), 1.77-0.73; (22H, m).
Compound 4 was prepared according to procedure of scheme 1.
MS obsd. [(M+H)+]: 699.8. 1H NMR (500 MHz, DMSO) δ 8.76; (s, 1H), 8.00 (s, 1H), 7.92; (q, J=4.5 Hz, 1H), 7.70; (t, J=5.7 Hz, 1H), 7.59; (s, 1H), 7.51-7.44; (m, 2H), 6.93; (s, 1H), 4.50-4.41; (m, 4H), 3.80; (m, 5H), 2.94; (d, J=6.7 Hz, 2H), 2.80-2.90; (m, 2H), 2.60-2.69; (m, 3H), 2.58; (d, J=4.7 Hz, 3H), 2.35-2.26; (m, 2H), 1.62; (dd, J=13.4, 3.7 Hz, 2H), 1.43-1.17; (m, 20H).
Compound 5 was prepared according to procedure of scheme 2.
MS obsd. [(M+H)+]: 1075.2. 1H NMR (500 MHz, DMSO) δ 8.93; (s, 1H), 8.81; (s, 1H), 8.00; (s, 1H), 7.89; (d, J=2.3 Hz, 1H), 7.79; (t, J=5.7 Hz, 1H), 7.46; (s, 2H), 7.33; (q, J=5.2 Hz, 1H), 6.90-6.95; (m, 2H), 6.43; (dd, J=8.4, 2.7 Hz, 1H), 6.36; (d, J=2.6 Hz, 1H), 4.48; (s, 2H), 4.42; (dd, J=10.5, 7.0 Hz, 2H), 4.26; (s, 2H), 3.79; (d, J=6.7 Hz, 4H), 3.40-3.50; (m, 12H) 3.20; (m, 2H), 3.11 (q, J=5.9 Hz, 3H), 2.83 (td, J=12.9, 2.8 Hz, 2H), 2.73-2.62; (m, 2H), 2.58; (d, J=4.7 Hz, 3H), 2.53-2.44; (m, 1H), 2.34; (m, 2H), 2.20-2.02; (m, 13H), 1.83-1.70; (m, 3H), 1.67-1.60; (m, 2H), 1.42-1.27; (m, 2H), 0.80; (s, 3H).
Compound 6 was prepared according to procedure of scheme 2.
MS obsd. [(M+H)+] 987.1: 1H NMR (500 MHz, DMSO) δ 8.93; (s, 1H), 8.80; (s, 1H), 8.00; (s, 1H), 7.89; (d, J=5.3 Hz, 2H), 7.78; (t, J=5.6 Hz, 2H), 7.46; (s, 2H), 7.34; (q, J=9.6 Hz, 2H), 7.00-6.91; (m, 3H), 6.42; (d, J=8.3 Hz, 2H), 6.37; (d, J=7.7 Hz, 2H), 4.45; (d, J=29.5 Hz, 5H), 4.26; (s, 2H), 3.79; (d, J=6.7 Hz, 7H), 3.33; (dt, J=16.2, 5.8 Hz, 11H), 3.19; (q, J=5.9 Hz, 5H), 3.11; (q, J=6.0 Hz, 4H), 2.83; (t, J=12.4 Hz, 4H), 2.73-2.61; (m, 4H), 2.58; (d, J=4.7 Hz, 4H), 2.48; (d, J=9.6 Hz, 4H), 2.38-2.29; (m, 3H), 2.20; (d, J=13.2 Hz, 2H), 2.06; (t, J=11.8 Hz, 2H), 1.82-1.73; (m, 4H), 1.63-1.24; (m, 15H), 1.24-1.15; (m, 4H), 0.79; (s, 3H).
Compound 7 was prepared according to procedure of scheme 2.
MS obsd. [(M+H)+]: 1025.3.1H NMR (500 MHz, DMSO) δ 8.95; (br, 2H), 8.76; (s, 1H), 7.99; (s, 1H), 7.92; (s, 1H), 7.68; (t, J=5.8 Hz, 1H), 7.47; (d, J=2.1; Hz, 1H), 7.30; (t, J=5.9 Hz, 1H), 6.98-6.91; (m, 1H), 6.43; (dd, J=8.5, 2.6 Hz, 1H), 6.37; (d, J=2.6 Hz, 1H), 4.49-4.41; (m, 2H), 4.23; (s, 1H), 3.79; (d, J=5.2 Hz, 3H), 3.09-2.95; (m, 4H), 2.92; (d, J=4.2 Hz, 3H), 2.86-2.78; (m, 5H), 2.06; (s, 1H), 1.78; (t, J=14.9 Hz, 3H), 1.62-1.42; (m, 7H), 1.14; (d, J=7.5 Hz, 7H), 0.80; (s, 3H).
Compound 8 was prepared according to procedure of scheme 2.
MS obsd. [(M+H)+]: 955.1. 1H NMR (500 MHz, DMSO) δ 8.93; (s, 1H), 8.79; (s, 1H), 8.00; (s, 1H), 7.92; (d, J=5.0 Hz, 1H), 7.69; (t, J=5.6 Hz, 2H), 7.46; (q, J=2.3 Hz, 2H), 7.33; (t, J=6.0 Hz, 2H), 6.95; (d, J=10.7 Hz, 3H), 6.42; (dd, J=8.4, 2.6 Hz, 2H), 6.36; (d, J=2.7 Hz, 2H), 4.47; (s, 2H), 4.44; (d, J=12.6 Hz, 2H), 4.23; (s, 2H), 3.79; (d, J=5.4 Hz, 6H), 3.03; (p, J=6.8 Hz, 4H), 2.93; (q, J=6.5 Hz, 3H), 2.85-2.76; (m, 4H), 2.66; (dd, J=11.1, 6.1 Hz, 3H), 2.64-2.56; (m, 5H), 2.50-2.45; (m, 3H), 2.33-2.24; (m, 3H), 2.23-2.16; (m, 2H), 2.05; (t, J=11.2 Hz, 2H), 1.81-1.70; (m, 5H), 1.65-1.58; (m, 3H), 1.47-1.35; (m, 5H), 1.35-1.13; (m, 17H), 0.79; (s, 3H).
A solution of 2-((((13S,E)-3-hydroxy-13-methyl-6,7,8,9,11,12,13,14,15,16-decahydro-17H-cyclopenta[a]phenanthren-17-ylidene)amino)oxy)acetic acid (15 mg, 0.06 mmol, according to lit.1 N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1-(5-chloro-4-((8-methoxy-1-methyl-3-(2-(methylamino)-2-oxoethoxy)-2-oxo-1,2- dihydroquinolin-6-yl)amino)pyrimidin-2-yl)-N-methylpiperidine-4-carboxamide (15 mg, 0.02 mmol, according to scheme 1), HATU (20 mg, 0.05 mmol) and DIPEA (50 uL, 0.3 mmol) in DMF (0.25 mL) was stirred at room temperature for 1 h. The crude reaction was purified by HPLC to yield 9 (2 mg, 15%) as white solid. MS obsd. [(M+H)+]: 1045.2. 1H NMR (500 MHz, DMSO) δ 8.94; (s, 2H), 8.76; (s, 1H), 7.99; (d, J=4.6 Hz, 1H), 7.89; (d, J=6.2 Hz, 1H), 7.50-7.43; (m, 2H), 7.31; (q, J=6.3 Hz, 1H), 7.00-6.91; (m, 3H), 6.43; (td, J=8.2, 2.5 Hz, 2H), 6.37; (dd, J=8.5, 2.5 Hz, 2H), 4.55; (s, 1H), 4.47; (d, J=1.8 Hz, 2H), 4.26; (d, J=2.2 Hz, 2H), 3.79; (dd, J=5.7, 2.1 Hz, 5H), 3.47; (s, 2H), 3.46-3.36; (m, 8H), 3.35; (s, 11H), 3.23-3.14; (m, 1H), 2.99; (d, J=17.6 Hz, 2H), 2.93-2.79; (m, 4H), 2.74; (d, J=3.1 Hz, 2H), 2-2.61; (m, 1H), 2.65; (s, 3H), 2.58; (d, J=4.7 Hz, 3H), 2.38; (dd, J=18.3, 9.5 Hz, 1H), 2.21; (t, J=13.5 Hz, 2H), 2.08; (d, J=11.9 Hz, 2H), 1.80; (td, J=11.2, 3.4 Hz, 2H), 1.76; (s, 4H), 1.59; (d, J=10.5 Hz, 2H), 1.45-1.37; (m, 4H), 1.35-1.27; (m, 3H), 1.27; (s, 9H), 1.25; (d, J=12.0 Hz, 1H), 1.19; (dd, J=13.2, 6.2 Hz, 2H), 0.80; (s, 3H).
Methods for regulating cellular processes within distinct populations of neurons are needed to elucidate relationships between molecular mechanisms, circuits, and behavior; and to develop cell type- or circuit-selective treatments for neurological disorders. We provide here a novel, non-genetic, small molecule-based method—transcription factor-chemically induced proximity (TF-CIP)—that harnesses the cell type- and circuit-specificity of endogenous transcription factors to regulate gene expression in subsets of neurons. TF-CIP utilizes a bifunctional small molecule to draw together an “anchor” transcription factor, which naturally binds to a target gene, and a “hijacked” transcription factor, which enhances or represses transcription of the target gene. Cell type specificity is determined by the intersection of expression for each transcription factor. The TF-CIP approach can be adapted for any organism and because transcription factors are well conserved, it is reasonable to expect that the same TF-CIP small molecule can be used to modulate neuronal processes across animal species. We use a TF-CIP to modulate the expression of the rate-limiting enzyme for serotonin synthesis, TPH2, as a means to tune serotonin neurotransmission from subsets of serotonergic neurons (
As an example, we detail the construction and testing of TF-CIPs in cells and animals to modulate the expression of TPH2, the rate limiting enzyme for brain serotonin synthesis. Briefly, the approach involves using the steps illustrated in
ARID1B (BAF250b) mutations are the most common cause of de novo intellectual disability and a frequent cause of autism spectrum disorder (PMID: 30349098). ARID1B is dosage-sensitive and loss of function mutations in one allele can cause intellectual disability, autism spectrum disorder, and/or Coffin-Siris syndrome. Hence, increasing ARID1B expression by 2-fold would be therapeutic. We have shown that we can do this in human induced pluripotent stem cells (iPS) taken from Coffin-Siris patients with a loss of function mutation in one allele and another normal allele. IPS cells were differentiated into neural progenitors by recruiting a transcriptional activator to the promoter of the ARID1B gene resulting in normal expression of ARID1 B (
To develop a TF-CIP to increase ARID1B expression by two-fold we found that ARID1B is controlled by another autism transcription factor, TBR1, which binds to the ARID1B regulatory regions and controls transcription of this gene (PMID: 27325115). Using the steps provided in
Designing small molecule pharmaceutics with cell-specific activity is a central challenge in medicine. The relative lack of cell-specific medicines is one reason why many therapeutic drugs have unwanted and sometimes life-threatening side-effects. A special feature of CIP molecules that can engender them with cell specific-activity is that they preferentially bind to both target proteins at the same time. For example, the CIP molecule rapamycin binds with 20,000 times higher affinity to both of its target proteins, FRB and FKBP than to FRB alone (PMID: 15796538). Because of this, CIP molecules are unlikely to function in cells that only express one of the target proteins. Thus, CIP molecules intrinsically encode cell specificity from the intersection of expression of their two target proteins.
In this example, we describe a method to identify transcription factor targets for cell-specific CIPs using single-cell gene expression data and statistical overlap and correlation analyses (see
To identify co-expressed transcription factors in the target cells, two orthogonal approaches are used: (1) Pearson correlation and (2) statistical overlap analysis. A Pearson correlation matrix is generated representing the expression relationship between pairs of transcription factors across individual target cells. Transcription factor pairs that show positive (r>0.5) and significant (P<0.05) correlation are included in a list called Coexpressed_Targe_TFs. To perform overlap analysis, lists of cells that express a given transcription factor are generated and then analyzed with the geneOverlap package in R (Shen L, Sinai ISoMaM (2021). GeneOverlap: Test and visualize gene overlaps. R package version 1.30.0, http://shenlab-sinai.github.io/shenlab-sinai/) to generate matrices depicting the Odds Ratio (measures strength of overlap) and Jaccard Index (measures similarity between two lists) for every transcription factor pair. Transcription factor pairs that show significant (P<0.05) overlap in their expression are also added to the Target_TFs list.
Next, transcription factors pairs that show co-expression in non-target cells are excluded. For this analysis, single-cell RNA expression data from across human tissues is used (e.g., publicly available Single-Cell Atlas from the Human Protein Atlas). If applicable, single cell expression data from the target cell type is excluded from this analysis. From this subdataset of non-target cells, single-cell expression data for each of the transcription factors that are expressed in the target cells are extracted and analyzed by Pearson correlation and overlap analysis, as above. Transcription factor pairs that are not significantly correlated and which do not overlap in their expression in non-target cells are added to a list Nontarget_TFs. The lists of Target_TFs and Nontarget_TFs are intersected, resulting in a reduced list of cell-specific transcription factor pairs. Additional parameters by which transcription factor pairs may be excluded are: a) expression level of the transcription factors below a set threshold, b) the percent of target cells expressing the transcription factor pair, and c) whether a binding motif for either transcription factor is present in regulatory regions near the target gene. Following this method, we identified 14 pairs of transcription factors that show selective co-expression in dopaminergic neurons of the human substantia nigra: RORA:SOX6, RORA:AEBP2, AEBP:LCORL, PBX1:SETBP1, PBX1:PIAS1, PBX1:ZEB1, FOXP2:ZEB1, FOXP2:KLF12, MYT1L:ZNF91, ZFHX3:ZNF91, ZFHX3:ZNF420, ZNF91:ZNF420, AFF3:THRB, and TAX1BP1:TCF25. These transcription factors may serve as targets for cell-specific CIPs to enhance dopamine synthesis in patients with early-stage Parkinson's disease, as described in an earlier embodiment.
In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase “means for” or the exact phrase “step for” is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. § 112(6) is not invoked.
This application is a continuation application of PCT Application Serial No. PCT/US2021/058231 filed on Nov. 5, 2021; which application claims priority to the filing date of U.S. Provisional Patent Application Ser. No. 63/110,575, filed Nov. 6, 2020; the disclosures of which applications are incorporated herein by reference.
This invention was made with Government support under contract CA163915 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63110575 | Nov 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/058231 | Nov 2021 | US |
| Child | 18143492 | US |
