Patent Application
20240116879
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the etiological agent of the COVID-19 respiratory disease, the largest scale respiratory virus pandemic the world has witnessed since the 1918 Spanish flu. Coronaviruses (CoVs) generally cause mild flu-like symptoms in humans but have caused two smaller scale pandemics in the last two decades: SARS-CoV (2003) and MERS (2012) (Costa et al., Arch Virol 165, 1517-1526 (2020)). Recent phylogenetic mapping traced all human coronaviruses to animal origins (Cui et al., Nat Rev Microbiol 17, 181-192 (2019)). While the middle zoonotic carrier of the virus between the animal of origin and humans seem to vary between CoVs, the chronological surfacing of human CoV pandemics seems to follow a dangerous trend of increasing lethality of each pandemic, thereby underscoring the need for a better understanding and targeting of the current and future CoV etiologic agents.
After more than a year since the first cases of SARS-CoV-2 human infection, this virus is expected to remain a global threat until vaccines are available and adopted worldwide. Furthermore, despite the increase in number of approved vaccines their implementation has been hindered by the scarcity of doses available worldwide. On top of production rate, pharmaceutical companies are currently facing challenges with the recently reported SARS-CoV-2 variants, against which not all vaccines have proven sustained efficacy, highlighting the need for a synergistic antiviral-based approach (Greaney et al., Cell Host & Microbe 29, 463-476.e466 (2021)). While recent treatments have been approved for use within hospital settings, there are no known FDA approved cures for the infection (Release, FDA news (2020)).
The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development.
The present disclosure is based, in part, on results of analysis of the CoV RNA genome that revealed many conserved RNA structures in the 5′-end region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following conservation analysis of this region, a small molecule library based on the amiloride scaffold was screened against a less virulent human coronavirus, OC43, to identify lead ligands. Next, antiviral activity was replicated in SARS-CoV-2 via a rapid RT-q-PCR based assay and authentic viral infections. Reporter screens confirmed the importance of RNA structures in the 5′ end of the viral genome. Finally, NMR chemical shift perturbations studies of the first six stem loops of the 5′-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain internal or bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. This combination of methods has revealed the first small molecules to target RNA structures at the 5′-end of the CoV genome. These molecules serve as chemical probes to further understand coronavirus RNA biology, and the combination of methods allows rapid identification of future RNA-targeted antivirals.
Accordingly, one aspect of the present disclosure provides a compound of Formula (I), a pharmaceutical acceptable salt, a hydrate, a prodrug, an ester, or a derivative thereof:
wherein R1 and R2 are independently H, C1-C8 alkyl, substituted C1-C8 alkyl, C1-C8 alkenyl, substituted C1-C8 alkenyl, C3-C6 substituted cycloalkyl, a C3-C6 unsubstituted cycloalkyl, aryl, or substituted aryl;
R3 and R4 are independently H, C1-C8 alkyl, substituted C1-C8 alkyl, C1-C8 alkenyl, substituted C1-C8 alkenyl, C3-C6 substituted cycloalkyl, a C3-C6 unsubstituted cycloalkyl, aryl, substituted aryl, or R3 and R4 may be taken together to form a C3-C6 substituted cycloalkyl, a C3-C6 unsubstituted cycloalkyl, C3-C6 substituted cycloalkenyl, a C3-C6 unsubstituted cycloalkenyl, a substituted heterocycle, a unsubstituted heterocycle, or a C3-C24 heteroaromatic;
R5 is C1-C8 substituted alkyl, C1-C8 unsubstituted alkyl, C3-C6 substituted cycloalkyl, C3-C6 unsubstituted cycloalkyl, a substituted heterocycle, an unsubstituted heterocycle, C3-C24 aromatic, C3-C24 heteroaromatic,
and
R6, R7, R8, R9, R10, R11, R12, R13, and R14 independently are H, Cl, Br, F, CF3, C1-C6 substituted alkyl, C1-C6 unsubstituted substituted alkyl, OCH3, NH2, NHCH3, N(CH3)2, CN, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; and X is O, S, or NH.
In some embodiments, R1 and R2 may be independently H, C1-C4 alkyl, C1-C6 substituted alkyl, C1-C4 alkenyl, C3-C5 substituted cycloalkyl, a C3-C5 unsubstituted cycloalkyl, aryl, or substituted aryl; R3 and R4 may be independently H, C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C3-C5 substituted cycloalkyl, a C3-C5 unsubstituted cycloalkyl, aryl, substituted aryl, or R3 and R4 may be taken together to form a C3-C5 substituted cycloalkyl, a C3-C5 unsubstituted cycloalkyl, C4-C14 heteroaromatic, a substituted heterocycle, or an unsubstituted heterocycle; R5 may be C1-C4 substituted alkyl, C1-C4 unsubstituted alkyl, C3-C5 substituted cycloalkyl, C3-C5 unsubstituted cycloalkyl, C4-C14 aromatic, C4-C14 heteroaromatic, a substituted heterocycle, an unsubstituted heterocycle,
R6, R7, R8, R9, R10, R11, R12, R13, and R14 may be independently H, Cl, Br, F, OCH3, NH2, NHCH3, N(CH3)2, or phenyl; and X may be O.
In some embodiments, R1, R2, R3, and R4 may be independently H, CH3, CH3CH2,, CH(CH3)2, C(CH3)3, or phenyl; R5 may be
R6, R7, R8, R9, R10, R11, R12, R13, and R14 independently may be H, CH3, F, CF3, or phenyl; and X may be O.
In some embodiments, R1, R2, R3, and R4 may be independently H or CH3; R5 is,
R9, R10, R11, R12, R13, and R14 independently may be H or phenyl; and X may be O.
In some embodiments, the compound may be DMA-132, wherein R1 and R2 are H; R3, and R4 are CH3;
R5 is
and R10, R11, R12, R13, and R14 are H as shown in the compound of Formula (II):
In some embodiments, the compound may be DMA-135, wherein R1 and R2 are H; R3, and R4 are CH3:
R5 is
and R9 is phenyl as shown in the compound of Formula (III):
In some embodiments, the compound may be DMA-155, wherein R1 and R2 are H; R3, and R4 are CH3;
R5 is
and R10, R11, R13, and R14 are H; R12 is phenyl as shown in the compound of Formula (IV):
Another aspect of the present disclosure provides a pharmaceutical composition comprising, consisting of, or consisting essentially of any of the compounds as described above and herein and a pharmaceutically acceptable carrier and/or excipient.
Another aspect of the present disclosure provides a method of inhibiting viral replication in a cell. This method may comprise administering to the cell an effective amount of any of the compounds or an effective amount of the pharmaceutical composition as described above and herein.
Another aspect of the present disclosure provides a method of preventing or treating a viral infection in a subject. This method may comprise administering to the subject an effective amount of any of the compounds or an effective amount of the pharmaceutical composition as described above and herein.
In some embodiments, the virus may comprise a coronavirus. In some embodiments, the coronavirus may be selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus), SARS-CoV (beta coronavirus), and SARS-CoV-2, or a combination thereof. In certain embodiments, the coronavirus may comprise SARS-CoV-2.
Still another aspect of the present disclosure provides a method of preventing or treating COVID-19 in a subject. This method may comprise administering to the subject an effective amount of any of the compounds or an effective amount of the pharmaceutical composition as described above and herein.
In some embodiments, for any of the methods described above and herein, the compound or the pharmaceutical composition may be administered in conjunction with another antiviral or anti-inflammatory agent. In certain embodiments, the compound or the pharmaceutical composition and another antiviral or anti-inflammatory agent may be combined in the same formulation or pill.
Still another aspect of the present disclosure provides use of any of the compounds or the pharmaceutical composition described above and herein as an antiviral agent.
In some embodiments, the antiviral agent targets coronavirus RNA. By way of non-limiting example, the coronavirus may comprise 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus), SARS-CoV (beta coronavirus), SARS-CoV-2, or a combination thereof. In certain embodiments, the coronavirus may comprise SARS-CoV-2.
Yet another aspect of the present disclosure provides use of any of the compounds or the pharmaceutical composition described above and herein as an antiviral agent for preventing or treating COVID-19.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The accompanying figures and examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying figures and examples relating to one or more embodiments, in which.
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
As used herein, the term “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”
Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
As used herein, “treatment,” “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition. As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
As used herein, the term “administering” an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.
The term “biological sample” as used herein includes, but is not limited to, a sample containing tissues, cells, and/or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and tears. In one embodiment, the biological sample is a biopsy (such as a tumor biopsy). A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician).
The term “disease” as used herein includes, but is not limited to, any abnormal condition and/or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as cancer, cancer metastasis, and the like.
“Contacting” as used herein, e.g., as in “contacting a sample” refers to contacting a sample directly or indirectly in vitro, ex vivo, or in vivo (i.e. within a subject as defined herein). Contacting a sample may include addition of a compound to a sample (e.g., a sample comprising cells infected with a viral infection), or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture.
As used herein, the term “viral infection” refers to those conditions characterized by the presence of a virus in the body (of a subject). The subject may show symptoms (e.g., fever, chills, stuffy/runny nose, etc.) or be asymptomatic. The methods and formulations provided herein are also useful in the prevention of a viral infection and may be used as a prophylactic for a subject at risk of developing a viral infection (e.g., a medical professional). The viral infection may be caused by any kind of virus. In some embodiments, the virus is capable of infecting a human subject. In some embodiments the virus is capable of infecting the mucosal/nasal/lungs/respiratory tract. Suitable examples include influenza, coronaviruses, rhinoviruses, respiratory syncytial virus, and the like. In some embodiments, the viral infection may be caused by a coronavirus. In some embodiments, the coronavirus may be selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus), SARS-CoV (beta coronavirus), and SARS-CoV-2, or a combination thereof. By way of non-limiting example, the coronavirus may comprise SARS-CoV-2.
As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient).
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
One aspect of the present disclosure provides a compound of Formula (I), a pharmaceutically acceptable salt, a hydrate, a prodrug, an ester, or a derivative thereof:
wherein R1 and R2 are independently H, C1-C8 alkyl, substituted C1-C8 alkyl, C1-C8 alkenyl, substituted C1-C8 alkenyl, C3-C6 substituted cycloalkyl, a C3-C6 unsubstituted cycloalkyl, aryl, or substituted aryl;
R3 and R4 are independently H, C1-C8 alkyl, substituted C1-C8 alkyl, C1-C8 alkenyl, substituted C1-C8 alkenyl, C3-C6 substituted cycloalkyl, a C3-C6 unsubstituted cycloalkyl, aryl, substituted aryl, or R3 and R4 may be taken together to form a C3-C6 substituted cycloalkyl, a C3-C6 unsubstituted cycloalkyl, C3-C6 substituted cycloalkenyl, a C3-C6 unsubstituted cycloalkenyl, a substituted heterocycle, a unsubstituted heterocycle, or a C3-C24 heteroaromatic;
R5 is C1-C8 substituted alkyl, C1-C8 unsubstituted alkyl, C3-C6 substituted cycloalkyl, C3-C6 unsubstituted cycloalkyl, a substituted heterocycle, an unsubstituted heterocycle, C3-C24 aromatic, C3-C24 heteroaromatic,
and
R6, R7, R8, R9, R10, R11, R12, R13, and R14 independently are H, Cl, Br, F, CF3, C1-C6 substituted alkyl, C1-C6 unsubstituted substituted alkyl, OCH3, NH2, NHCH3, N(CH3)2, CN, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; and X is O, S, or NH.
In general, R1 and R2 may be independently H, C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C3-C5 substituted cycloalkyl, a C3-C5 unsubstituted cycloalkyl, aryl, or substituted aryl. R3 and R4 may be independently H, C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 alkenyl, C3-C5 substituted cycloalkyl, a C3-C5 unsubstituted cycloalkyl, aryl, substituted aryl, or R3 and R4 may be taken together to form a C3-C5 substituted cycloalkyl, a C3-C5 unsubstituted cycloalkyl, C4-C14 heteroaromatic, a substituted heterocycle, or an unsubstituted heterocycle. In some embodiment, R1, R2, R3, and R4 may be independently H, CH3, CH3CH2, CH(CH3)2, C(CH3)3, or phenyl. In certain embodiments, R1, R2, R3, and R4 may be independently H or CH3. Particularly, R1 and R2 may be H; R3, and R4 may be CH3.
In general, R5 may be C1-C4 substituted alkyl, C1-C4 unsubstituted alkyl, C3-C5 substituted cycloalkyl, C3-C5 unsubstituted cycloalkyl, C4-C14 aromatic, C4-C14 heteroaromatic, a substituted heterocycle, an unsubstituted heterocycle,
In certain embodiments, R5 may be
R5 may be
Still particularly, R5 may be
In general, R6, R7, R8, R9, R10, R11, R12, R13, and R14 may be independently H, Cl, Br, F, OCH3, NH2, NHCH3, N(CH3)2, or phenyl. In some embodiments, R6, R7, R8, R9, R10, R11, R12, R13, and R14 may be independently H, CH3, F, CF3, or phenyl. In certain embodiments, R9, R1, R11, R12, R13, and R14 may independently be H or phenyl. Particularly, R10, R11, R12, R13, and R14 may be H. Particularly, R9 may be phenyl. Particularly, R10, R11, R13, and R14 may be H and R12 may be phenyl.
Generally, X may be O.
In some embodiments, the compound may be DMA-132, wherein R1 and R2 are H; R3, and R4 are CH3, R5 is
and R10, R11, R12, R13, and R14 are H as shown in the compound of Formula (II):
In some embodiments, the compound may be DMA-135, wherein R1 and R2 are H; R3, and R4 are CH3, R5 is
and R9 is phenyl as shown in the compound of Formula (III):
In some embodiments, the compound may be DMA-155, wherein R1 and R2 are H; R3, and R4 are CH3, R5 is
R10, R11, R13, and R14 are H; R12 is phenyl as shown in the compound of Formula (IV):
B. Pharmaceutical Compositions
In another aspect, the present disclosure provides compositions comprising one or more of the compounds as described above and herein and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.
When used to treat or prevent a disease, such as a viral infection, the compounds described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents (e.g., therapeutic agents) useful for treating such diseases and/or the symptoms associated with such diseases. Such agents may include, but are not limited to, antiviral agents, NSAIDS, and the like. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.
Pharmaceutical compositions comprising the compound(s) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.
The compounds may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.
Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.
For topical administration, the compound(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.
Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.
For nasal administration or administration by inhalation or insufflation, the compound(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
For ocular administration, the compound(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.
For prolonged delivery, the compound(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The compound(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).
Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.
The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more-unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The compound(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.
The amount of compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compound(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.
Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.
Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and/or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and/or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.
The compounds provided herein have many uses. Hence, one aspect of the present disclosure provides a method inhibiting viral replication in a cell and/or subject. This method may comprise administering to the cell and/or subject an effective amount of any of the compounds or the pharmaceutical composition described above and herein such that the viral replication is inhibited in the cell and/or subject
Another aspect of the present disclosure provides a method of preventing or treating a viral infection in a subject. This method may comprise administering to the subject an effective amount of any of the compounds or the pharmaceutical composition described above and herein such that the viral infection is prevented or treated in the subject.
In some embodiments, the virus may comprise a coronavirus. In some embodiments, the coronavirus may be selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus), SARS-CoV (beta coronavirus), SARS-CoV-2, and combinations thereof. In certain embodiments, the coronavirus may comprise SARS-CoV-2.
Another aspect of the present disclosure provides a method of preventing or treating COVID-19 in a subject. This method may comprise administering to the subject an effective amount of any of the compounds or the pharmaceutical composition described above and herein such that COVID-19 is prevented or treated in the subject.
Still another aspect of the present disclosure provides use of any of the compounds or the pharmaceutical composition described above and herein as an antiviral agent. In some embodiments, any of the compounds or the pharmaceutical composition described above and herein may be used in conjunction with another antiviral or anti-inflammatory agent. In certain embodiments, the agents may be combined in the same formulation or pill.
In some embodiments, the antiviral agent targets coronavirus RNA. By way of non-limiting example, the coronavirus may comprise 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (beta coronavirus), SARS-CoV (beta coronavirus), SARS-CoV-2, or a combination thereof. In certain embodiments, the coronavirus may comprise SARS-CoV-2.
In some embodiments, for any of the methods described above and herein, the compound or the pharmaceutical composition may be administered in conjunction with another antiviral or anti-inflammatory agent. In certain embodiments, the compound or the pharmaceutical composition and another antiviral or anti-inflammatory agent may be combined in the same formulation or pill.
Yet another aspect of the present disclosure provides use of any of the compounds or the pharmaceutical composition described above and herein as an antiviral agent for preventing or treating COVID-19. In some embodiments, any of the compounds or the pharmaceutical composition described above and herein may be used in conjunction with another antiviral or anti-inflammatory agent. In certain embodiments, the agents may be combined in the same formulation or pill.
The following Examples are provided by way of illustration and not by way of limitation.
Analysis of Sequence Conservation: Representative betacoronavirus sequences were selected according to official taxonomy as represented by the International Committee for the Taxonomy of Viruses (Lefkowitz et al., Nucleic acids research 46, D708-D717 (2018)). Multiple sequence alignments of coronavirus sequences were constructed using BLAST and MAAFT as implemented in GLUE (Altschul et al., Nucleic acids research 25, 3389-3402 (1997); Katoh and Standley, Mol Biol Evol 30, 772-780 (2013); Singer et al., BMC Bioinformatics 19, 532-532 (2018)). Alignments were manually inspected and adjusted using Se-Al. Position coverage and percentage identity were calculated and visualized using JalView (Waterhouse et al., Bioinformatics (Oxford, England) 25, 1189-1191 (2009)).
Cells and Virus: Vero E6 (African green monkey kidney; ATCC CRL-1586) cells were cultured in MEM medium supplemented with 10% FBS (ThermoFisher Scientific) and maintained at 33° C. Cells were infected with human coronavirus OC43 (ATCC VR-1558) at indicated MOI (multiplicity of infection) and incubated 1 hr at 33° C. for adsorption. Unbound virus was removed, and cells were refed fresh medium with various concentrations of DMAs. Media from infected cells were harvested 24 hrs post-infection, and virus titers were determined by plaque assay on Vero E6 cells. SARS-CoV-2 (USA-WA1/2020 strain; BEI Resources) was propagated and titered on VeroE6 cells, with sequence confirmation of a P2 stock to confirm stability of the viral genome.
Effects of SARS-CoV-2 5′- and 3′-End Sequence Elements on Luciferase Reporter Activity: The reporter plasmid pCoV-2-5′UTR-FLuc-3′UTR contains the SARS-CoV-2 5′UTR and adjacent coding sequences in ORFla fused in-frame with the firefly luciferase open reading frame, followed by the SARS-CoV-2 3′UTR. For reporter plasmid pCoV-2-5′UTR-FLuc, the Cov-2 3′UTR as replaced with vector-encoded sequence. The plasmids were kindly provided by Dr. Shin-Ru Shih (Chang-Gung University, Taiwan). Plasmid pRL, the Renilla luciferase control reporter vector, was purchased from Promega. CoV-2-5′UTR-FLuc-3′UTR, Cov-2-5′UTR-FLuc, and RLuc RNAs were in vitro synthesized from these plasmid templates using the MEGAscript T7 Transcription Kit (ThermoFisher Scientific).
Vero E6 cells were seeded in 24-well plates. Two hundred (200) ng of reporter RNA, 5 μl of SuperFect (Qiagen), and 400 μl of MEM with 10% FBS were combined and added to one well of cells. Cells were incubated at 33° C. for 4 hrs and media were changed, and various concentrations of DMAs were added. Two days after transfection, reporter luciferase activities were determined by measuring Renilla luciferase (RLuc) and firefly luciferase (FLuc) activities using the Dual Luciferase Reporter Assay System (Promega).
Cytotoxicity Assays: Various concentrations of DMAs were added to Vero E6 cells in culture. The cells were incubated at 33° C. for 96 hrs. Cell viability was determined by MTT assay and measured at 570 nm according to the manufacturer's instructions (EMD Millipore). All experiments were performed in triplicate. The concentration of DMAs required to reduce cell viability to 50% of the control cells was expressed as CC50.
SARS-CoV-2 Antiviral Assays: Analysis of SARS-CoV-2 growth in cell culture supernatants was performed using a simplified RT-qPCR assay as explained before (Mugisha et al., mSphere 5, e00658-20 (2020)). In brief, Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.1 i.u./cell in 96-well plates, virus inoculum was removed 1 hour post-adsorption and replaced with media containing serial dilutions of the DMA compounds. 5 μL of cell culture media containing released virions were collected at 24 hpi and processed as detailed in previous studies. Viral RNA levels were quantitated by RT-qPCR using primers specific to SARS-CoV-2 N gene and a standard curve derived from in vitro synthesized RNA encoding N.
In addition, antiviral activity was tested against SARS-CoV-2 using a TCID50 assay, VeroE6 cells were seeded at 1×105 per well in a 24-well plate at 37° C. for 24 hours. Cells and samples were then transferred to a Biosafety Level 3 facility. Stocks of SARS-CoV-2 were diluted in DMEM/2% FBS for a solution of 20,000 pfu/mL. Growth media was aspirated from 24-well plates and replaced with 495 μL of DMEM/2% FBS containing 20,000 pfu/mL SARS-CoV-2 for an M.O.I. of 0.1. 5 μL of DMSO or compound were then immediately diluted into each well for final concentrations of 50 μM and 10 μM. Plates were incubated at 37° C. for 72 hours. Media was then harvested, centrifuged at RT for 10 min at 1,500×g, then used for TCID50 assay. Serial dilutions of supernatant from the treated cells were added to VeroE6 cells in 96 well plates and cells are monitored for cytopathic effect (CPE). Viral titer was calculated from the numbers of positive wells using a modified Reed and Muench method.
Synthesis and Purification of RNA Stem Loop Constructs Present at 5′-UTR: SL1 through 6 of the 5′-end were in vitro transcribed using a standard protocol, from synthetic DNA templates from Integrated DNA Technologies (Coralville, IA) (Milligan and Uhlenbeck, Methods Enzymol 180, 51-62 (1989); Milligan et al., Nucleic Acids Research 15, 8783-8798 (1987)). The 3-6 ml reactions involved the use of purified recombinant T7 RNA polymerase expressed in BL21 (DE3) cells. Depending on the labeling scheme of each RNA, double labeled, 13C and 15N, rNTPs and unlabeled rNTPs were utilized in the reaction. Nucleotide labeling of the SLs was based on the abundance of nucleotides in bulges and loops. The labeling pattern for the 1H-13C HSQC experiments was as follows: SL1 C(13C, 15N)-labeled and A(13C, 15N)-labeled, SL2 U(13C, 15N)-labeled, SL3 AU(13C, 15N)-labeled, SL4 A(13C,15N)-labeled, SL5a AU(13C, 15N)-labeled, SL5b AU(13C, 15N)-labeled, and SL6 AC(13C, 15N)-labeled. For the 1H-1H NOESY experiments, the labeling scheme was SL6 UG(2H) AC(2H3′-5″)-, CUG(2H) A(2H3′-5″)-, and ACUG(2H)-selectively labeled constructs, along with a CG(2H), AU(2H3′-5″-)-selectively labeled SL1 construct.
Next, the SLs were purified on denaturing gels, ranging from 8 to 16%, and extracted using electroelution. After desalting with a Millipore Amicon Ultra-4 centrifugal filter, the RNAs were annealed by heating for 2 min at 95° C. and flash-cooled on ice. The samples were thoroughly washed and concentrated down with a 100% D2O buffer of 25 mM K2HPO4, 50 mM KCl at pH 6.2. Using the NanoDrop™ 2000 software (Thermo Fisher), the theoretical extinction coefficients of the SLs were calculated in order to determine RNA concentrations. Samples for NMR titrations contained 100 μM of RNA in D2O buffer with a final volume of 200 μL.
NMR Profiling of DMA Interactions with 5′-End Structures: A 900 MHz spectrometer was used to record all NMR data. The 1H-13C HSQC titrations were recorded with 100 μM of selectively labeled SL1 through 6 in a 100% D2O buffer of 25 mM K2HPO4, 50mM KCl at pH 6.2 with a 200 μL sample volume. Titrations of the DMA molecules into the different SLs were collected at a molar ratio of 5:1, DMA to RNA, at a temperature of either 298, 303, or 308K. For each construct, temperature optimization experiments were done in order to determine the optimum temperature to conduct the titrations with. Also, based on the optimized temperatures, 1H-1H NOESY spectra (tm=250 ms) of selectively labeled SL6 samples and their DMA-155 complexes were collected with a 200 μM sample concentration in 25 mM K2HPO4, 50 mM KCl pH 6.2 in 100% D2O at a 200 μL sample volume. At similar conditions, 1H-1H NOESY spectra (tm=250 ms) of selectively labeled SL1 were collected in the absence and presence of DMA-135 and 155 at a 5:1 molar ratio. The NMR spectra were processed using NMRPipe/NMRDraw and analyzed with NMRView J or Sparky (Delaglio et al., Journal of Biomolecular NMR 6, 277-293 (1995); Johnson and Blevins, Journal of Biomolecular NMR 4, 603-614 (1994); Lee et al., Bioinformatics 31, 1325-1327 (2014)).
Virtual Ligand Screening against SARS-CoV-2 5′-End RNA Structures:
FARFAR model generation: FARFAR is part of the Rosetta 3 software package can be obtained for free academic usage (https://www.rosettacommons.org/software/license-and-download). Each SL was generated through the following protocol.
rna_helix.py is a python wrapper for the Rosetta executable rna_helix. rna_helix.py is available in $ROSETTA/tools/rna_tools/bin, where $ROSETTA is the Rosetta installation path. Below are the commands to generate SL1.
rna_helix.py-seq acc ggu-resnum 8-10 17-19-o helix_2.pdb
For each helix in a SL other than the nucleotides that flank bulges and loops we prebuild as idealized A-form helices with the above commands. Modeling helical residues as idealized A-form significantly reduces computational time allowing for more models to be built.
FARFAR modeling is performed through the rna denovo executable.
rna_denovo -nstruct 1000 -s helix_1.pdb helix_2.pdb -fasta input.fasta-secstruct_file input.secstruct -minimize_rna true -out.file: silent farfarout
where -nstruct is the max number of models requested, -fasta is the path of a fasta file containing the RNA sequence, -secstruct is the path of a file (input. secstruct) containing the RNA secondary structure in dot-bracket notation, -minimize rna true minimizes the RNA after fragment assembly, -s specifies the path to the pdb files that contain static structures of our helices, and -out:file:silent specifies the output file path to store all generated models. Each SL was run on 100 cores for 24 hours, the number of generated models is reported in table FARFAR (Table 1).
To reduce the number of models to dock against, fixed-width clustering using the rna_cluster executable was performed.
rna_cluster -in:file:silent farfar.out -nstruct 15 -cluster:radius RADIUS
Where -in:file:silent is a silent file of all models for a given SL, -nstruct is the max number of clusters requested, and -cluster:radius is max distance in heavy-atom RMSD between members of the same cluster.
Small Molecule Docking: Virtual docking simulations were performed using ICM (Abagyan et al., Journal of Computational Chemistry 15, 488-506 (1994)) (Molsoft LLC. La Jolla, CA), employing the SARS-CoV-2 5′-UTR structures obtained from FARFAR modeling and clustering. RNA structural elements' binding pockets were defined using ICM Pocket Finder module and all the small molecules protonation states were adjusted to pH=7.0 using ChemAxon© (www.chemaxon.com). RNA ensembles were then combined into conformational stacks using ICM's “impose conformations.” Then, to create “flexible receptors” to dock against that would reflect all of the conformations of each structure, ICM's “create 4D grid” function was used for each docking project. Each of the structures was then docked against a library of 55 DMA molecules. The DMA library was saved in .sdf file format, which was indexed for VLS using ICM-Pro. The virtual screening simulation was implemented with a conformational search and optimization with a limit of 10 conformers per molecule. The thoroughness was left at level 10.
Indicator Displacement Assay (IDA): A serial dilution of the seven SLs RNA (SL1, SL2, SL3, SL4, SL5a, SL5b, and SL6) was performed in Tris buffer (50 mM Tris-HCl, 50 mM KCl, at pH 7.4) in a 96 well plate in triplicate. 8 μL of each dilution were transferred to a 384 well plate followed by 8 uL of a 500 nM solution of RiboGreen™ dye (Invitrogen) in the same buffer. The plates were excited at 487 nm (8 nm slit) and emission was recorded at 525 nm (8 nm slit, focal height 11.3 mm) using a CLARIOstar plate reader (BMG labtech). The affinity of the dye for the RNA construct was determined by fitting the raw fluorescence in in GraphPad Prism version 8.3.1 for Macintosh (GraphPad Software, La Jolla California USA, (www.graphpad.com)) by fitting to the [Agonist] vs. response—variable slope model that using Equation (1).
Y=Bottom+(X{circumflex over ( )}HillSlope)*(Top—Bottom)/(X{circumflex over ( )}HillSlope+EC50{circumflex over ( )}HillSlope)−Equation (1)
Where Y is normalized % change in fluorescence intensity, X is RNA concentration, Bottom is lowest fluorescence % change and Top is highest fluorescence % change. Affinity of the dye for the RNA construct was used as the ideal RNA concentration for small molecule titrations.
The two most promising small molecule leads (DMA-135 and -155) were then screened against SL1, SL4, SL5a, SL5b, and SL6 but not against the short stem loops SL2 and SL3 for which the RiboGreen™ dye showed weak affinity.
A serial dilution of DMA-135 and -155 (0, 0.5, 1.0, 2.5, 5, 10.0, 12.5, 15, 20, 25, 40, 50, 75, 100, 225, 400, 450 μM) was performed in phosphate buffer Tris buffer (50 mM Tris-HCl, 50 mM KCl, at pH 7.4) in a 96 well plate in triplicate. 8 μL of each dilution were transferred to a 384 well plate, followed by 8 μL of a solution of Stem loop RNA and 0.5 μM of RiboGreen™ (Invitrogen). The 384 well plates were at 4000 rpm for 1 min, and incubated in the dark for 30 min. The plates were excited at 487 nm (8 nm slit) and emission was read at 525 nm (8 nm slit, focal height 11.3 nm) using a CLARIOstar plate reader (BMG Labtech). Percent fluorescence indicator displacement (%FID) was calculated by subtracting and, subsequently, dividing by the blank wells with RNA-dye complex and no small molecule as shown in Equation (2).
Where F0 is the fluorescence of the blank well with RNA+dye and no small molecule and F is the fluorescence of the well with all three components (RNA+dye+small molecule).
Each technical triplicate was averaged, and the resulting FID values were averaged between three independent experiments. The binding curve and EC50 value was obtained by using a non-linear fit curve agonist vs. response with variable slope at four parameters (GraphPad Prism Software version for Macintosh 8.3.1, La Jolla, California, USA (www.graphpadprism.com)) as shown in Equation (1). Reported values are averages of three independent experiments ±standard deviation.
Current candidates for treatment have limited approval for emergency use in severe COVID-19 cases. Remdesivir, for example, is an RNA-dependent RNA polymerase inhibitor (RdRp) initially developed during the Ebola outbreak and revisited at the start of the pandemic (Shannon et al., Antiviral Res 178, 104793-104793 (2020)). The compassionate use of the candidate antiviral across many countries reported mixed results, with overall faster recovery time from the virus but no difference in mortality rates (Singh et al., Diabetes Metab Syndr 14, 641-648 (2020)). While more randomized trials are needed for a final verdict on the efficacy of Remdesivir in critical patients, its stereospecific multi-step synthetic process highlights the need for new, scalable and more efficacious antivirals. Baricitinib has been recently approved for emergency use for COVID-19 treatment in conjunction with Remdesivir. Also known as Olumiant, this small molecule was approved in 2018 as treatment for moderate to severe rheumatoid arthritis (Genovese et al., The Lancet Rheumatology 2, e347-e357 (2020)). It is proposed that the anti-inflammatory effects of the drug help in decreasing inflammatory cascades associated with COVID-19. While promising, Baricitinib has yet to receive approval as a stand-alone treatment and, so far, has been shown to improve recovery time by one day when compared to Remdesivir alone treatment (Release, FDA news (2020)).
The recent emergence of multiple coronaviruses pandemics clearly indicates that SARS-CoV-2 most likely will not be the last CoV pandemic (Costa et al., Arch Virol 165, 1517-1526 (2020)). The current limited tools and lack of cures underscore the need for a new approach in developing antivirals that would not only provide novel routes to combat the current pandemic but also provide invaluable information on targetable structures that can aid in the prevention of and fight against future CoV outbreaks. Indeed, small molecules are uniquely poised to achieve this goal as their design and development provides both a better understanding of CoV biology and identify druggable targets that could aid in the development of pan-coronavirus antivirals.
Several steps in the coronavirus replication cycle offer potential therapeutic targets for viral inhibition (
CoV antivirals to date have been developed to target viral proteins, including to prevent endocytosis, assembly of viral protein for export, and condensation of viral genome for packaging (Tiwari et al., Drug Discov Today 25, 1535-1544 (2020)). While this protein-centric approach has proven successful in a few cases, the sequence and structural conservation of RNA structural motifs pose an attractive complementary target for small molecule antiviral development, a strategy that has shown promise against a plethora of viruses (Nagy and Pogany, Nature Reviews Microbiology 10, 137-149 (2012); Hermann, Wiley Interdiscip Rev RNA 7, 726-743 (2016)). Specifically, international efforts that allowed for identification and tracking of SARS-CoV-2 variants highlighted the large number of mutations accumulated in protein-coding regions (Fang et al., Nucleic Acids Research 49, D706-D714 (2020)). At the same time, recent global cataloguing of mutations in the untranslated regions (UTRs) revealed a significantly lower rate of mutation, highlighting the potential of UTRs as drug targets (Mukherjee and Goswami, PLOS ONE 15, e0237559 (2020)). Recently published data on genome-wide secondary structure of the virus obtained by in vitro and in vivo SHAPE (selective 2′ hydroxyl acylation analyzed by primer extension) and DMS (dimethyl sulfate) probing of SARS-CoV-2 infected Vero E6 cells, as well as NMR characterization, recapitulates the computationally predicted stem loops (SL) at the 5′ region of the genome (
Drug-like small molecules offer the ability to develop chemical probes that reveal function and to design bioavailable clinical candidates for treatment. While RNA targeting has lagged behind protein targeting, recent successes in both the laboratory and the clinic support its potential role. The first US FDA-approved small molecule targeting RNA other than the ribosome was approved for treatment of spinal muscular atrophy in August of 2020 (O'Keefe. (FDA Press Announcements 2020). Effective small molecule targeting in the laboratory has also been observed for a plethora of disease relevant RNAs, including viral RNAs. Specifically, small molecules targeting structures within the 5′-UTR region have shown antiviral activity for a number of positive-sense RNA viruses such as HCV, FMDV, and EV71 (Hermann, in RNA Therapeutics, A. L. Garner, Ed. (Springer International Publishing, Cham, 2018), pp. 111-134; Davila-Calderon et al., Nature Communications 11, 4775 (2020); Lozano et al., RNA Biol 12, 555-568 (2015)). Recent studies have begun evaluating the potential of small molecules against the frameshifting elements of SARS-CoV-2 RNA in the coding region. These recent reports include evaluation of known SARS-CoV pseudoknot binders as well as development of a small molecule binder to the attenuator hairpin preceding the pseudoknot (Kelly et al., J Biol Chem 295, 10741-10748 (2020); Bhatt et al., Science 372, 1306-1313 (2021); Sreeramulu et al., Angew Chem Int Ed Engl, 60, 19191-19200 (2021); Haniff et al., ACS Central Science 6, 1713-1721 (2020)). Coupling of the latter to the known ribonuclease targeting chimera (RIBOTAC) technology results in recruitment of cellular ribonucleases leading to viral genomic RNA degradation. In addition, a fragment-based screen against structures in the 5′-UTR of SARS-CoV-2 was successful in identifying potential ligands (Sreeramulu et al., Angew Chem Int Ed Engl, 60, 19191-19200 (2021)). While the antiviral activity of these molecules has not been published, these preliminary results, combined with known RNA-targeted antivirals for other positive sense RNA viruses, stand as proof-of concept of the targetability of SARS-CoV-2 RNA motifs.
Successful efforts to target viral 5′-UTR structures have often leveraged the synthetic tuning of a known RNA binding scaffold (Hermann, in RNA Therapeutics, A. L. Garner, Ed. (Springer International Publishing, Cham, 2018), pp. 111-134; Davila-Calderon et al., Nature Communications 11, 4775 (2020); Lefebvre et al., Biochemical and Biophysical Research Communications 455, 378-381 (2014)). This approach was employed to develop RNA-targeted antivirals based on dimethylamiloride (DMA) that target the internal ribosomal entry site (IRES) region in the 5′-UTR of EV71. The DMA scaffold had been previously reported to be poised for tuning for specific RNA constructs via a facile three-step synthesis (Davila-Calderon et al., Nature Communications 11, 4775 (2020)). Further investigation and functionalization of the scaffold resulted in a bioactive antiviral analogue that formed a repressive ternary complex with IRES stem loop II RNA and the human AUF1 protein, ultimately inhibiting translation and compromising viral replication. The successes in DMA exploration highlight the scaffold's potential for tuning as well as broad applicability as an antiviral scaffold.
The present disclosure reports DMA analogues that show promising antiviral properties by reducing SARS-CoV-2 viral titer in a dose-dependent manner in infected cells. In addition, dual luciferase reporter assays confirmed the antiviral activity of the small molecules to be dependent on the 5′-UTR and proximal region of the SARS-CoV-2 genome. Investigation of possible conserved RNA binding sites of the lead small molecules revealed putative bulge-like binding sites in stem loops 1, 4 and 5a, located in the 5′-UTR of the SARS-CoV-2 genome, as well as in the adjacent stem loop 6 located in ORFla, supporting both the targetability of 5′-region stem loops. Taken together, these results establish conserved CoV RNA structures as antiviral targets and reveal lead molecules with promising antiviral properties.
As the functional significance of the 5′-end stem loops (SLs) is still being elucidated, sequence conservation was examined in the 5′-end region across the Betacoronavirus genus as a preliminary approach to assessing the suitability of the known structures in this region as therapeutic targets. Sequence conservation within the contexts of folded RNA domains would imply selective pressures to exert biological function. A multiple sequence alignment spanning the 5′-UTR and the adjacent region including representatives of all five betacoronavirus subgenera was constructed (
Targeting of the 5′-end stem loop structures with the DMA scaffold was pursued, which has been previously reported as an RNA binding scaffold that can be successfully optimized for selectivity for distinct RNA elements (Davila-Calderon et al. , Nature Communications 11, 4775 (2020)). To quickly assess potential CoV antiviral activity, human OC43 betacoronavirus was used due to its lower virulence and thus suitability for use in standard cell culture facilities (Chen et al., Signal Transduction and Targeted Therapy 5, 89 (2020)). Vero E6 cells were infected with human coronavirus OC43 at an MOI=1. After a one-hour adsorption, a panel of twenty-three DMAs at 50 μM or 100 μM were added to the cells and incubated for 24 hours at 33° C. Virus titers were determined by plaque formation on Vero E6 cells. The results of small molecule screening against OC43 with Vero E6 cells are shown in
To determine the antiviral activity and potency of the lead small molecules against SARS-CoV-2, a simplified qRT-PCR assay was utilized to monitor SARS-CoV-2 viral RNA levels in supernatants of infected Vero E6 cells (Mugisha et al., mSphere 5, e00658-20 (2020)). Similar to Remdesivir, DMA-135 and DMA-155 led to a dose dependent 10-30-fold decrease in cell-free viral RNA levels within 24 hours of infection with an approximate IC50 of 10 and 16 μM, respectively (
Antiviral activity of the three most active DMA leads (DMA-132, -135, and -155) was confirmed using Vero E6 cells infected with wild-type SARS-CoV-2. Small molecule treatment was performed at 10 μM and 50 μM, respectively. DMA-132 and -135 showed dose-dependent reduction in viral titer compared to DMSO, as measured by median tissue culture infectious dose (TCID50) assay, without measurable effect on cellular viability as measured by ATP content (
DMA-132, -135, and -155 were tested for longer term (96 hour) toxicity in Vero E6 cells. Small molecules do not significantly reduce cell viability <10 μM, supporting a potential therapeutic window, though more extensive cytotoxicity studies are warranted. In particular,
To assess the effect of DMAs on reporter gene expression directed by SARS-CoV-2 sequence elements, a reporter plasmid, pCoV2-5′UTR-FLuc-3′UTR was used as template for in vitro synthesis of CoV-2-5′UTR-FLuc-3′UTR RNA (
Towards understanding potential mechanisms by which the DMAs inhibit SARS-CoV-2 replication, single-point 13C-1H TROSY HSQC titrations of DMA-132, -135 and -155 into stem loops 1-6 (SL1-6) of the 5′-region were carried out. This region of the 5′-UTR to NMR profile was selected following the reasoning that it contains SLs with assigned functions that can be prepared at sizes amenable to facile NMR screening, shows a degree of selective pressure to maintain sequence and structure, and contains several non-canonical RNA elements that were hypothesized as good targets for the DMAs. An advantage of using 13C-1H TROSY HSQC titrations to profile small molecule-RNA interactions is that the extent of NMR signal perturbation provides a convenient proxy on the degree of binding specificity, even in the absence of chemical shift assignments. The designs of the individual SLs were based on the reasoning that DMAs likely bind the 5′-region stem loops at non-canonical structural elements such as bulge, internal or apical loops. Therefore, isolated SL domains were in vitro transcribed using 13C/15N rNTPs that would maximize NMR signal detection for the non-canonical elements over the base paired regions (
Notably, SL1 and SL6 experienced the most significant NMR signal perturbations upon addition of excess DMAs (
For SL6, addition of excess DMA-155 induced a combination of CSPs and LB to a greater extent than DMA-132 or DMA-135. To further assess the nature of these signal perturbations on the SL6 structure, a 1H-1H NOESY (tm=250 ms) spectrum was recorded with UG(2H), AC(2H3′-5″)-selectively labeled SL6 constructs, which was chosen based on the abundance of A and C residues in the bulge (
Moreover, the affinity of the three leads for the stem loop constructs was investigated via in vitro indicator displacement assays (IDA) (Davila-Calderon et al., Nature Communications 11, 4775 (2020); Wicks, Methods 167, 3-14 (2019); Patwardhan, et al., Org Biomol Chem 17, 1778-1786 (2019)). DMA-155 was revealed to be a strong binder with highest affinity for SL6, in agreement with NOE studies (
To generate potential 3D models of each SL, Fragment Assembly of RNA with Full-Atom Refinement (FARFAR) was used. FARFAR was chosen to generate preliminary models as it has consistently been demonstrated to be the most accurate RNA 3D prediction algorithm (Miao et al., Rna 23, 655-672 (2017); Rangan and Zheludev, RNA, 26, 937-959 (2020)). Between 5,000 and 100,000 models were generated for each SL and then ˜15 representative clusters were generated.
The lowest energy conformation from each cluster was used to generate an ensemble for each SL that was submitted to ICM pocket finder to find and characterize possible binding pockets (see, Table 1).
Notably, SL2, which showed no change in 18C-1H HSQC NMR chemical shifts upon small molecule addition, did not have any identifiable binding pocket. SLs 1, 3, and 5b presented binding pockets with low to intermediate scores in terms of volume, area, hydrophobicity, burriedness and druggability score (DLID) parameters often used to describe a binding pocket's fitness. Notably, SL3 and 5b presented minor chemical shift perturbations upon small molecule addition. 13C-1H TROSY HSQC NMR experiments showed significant changes upon small molecule binding to SL1, 5a, and SL6 (
Previously published 55 member DMA library was then docked against the clustered FARFAR-generated stem loop structures (
Discussion: Screening of synthetic RNA-focused libraries in recent years has provided the field with many RNA-binding bioactive small molecules and some of the highest hit rates among small molecule screening approaches (Morgan et al., Nucleic Acids Research 46, 8025-8037 (2018)). Amiloride, a known RNA-binding scaffold, has been synthetically tuned for a range of RNA secondary structures and recently yielded a novel antiviral lead for the treatment of enterovirus 71. In this case, amiloride inhibited viral translation by binding the viral 5′-UTR and modulating RNA:host protein interactions. SARS-CoV-2 also contains a highly conserved 5′-end that is reported to play a crucial role in viral replication and hijacking of host cell translational machinery. The presence of multiple bulge or internal loops, the secondary structural elements that amilorides have been reported to bind most effectively, makes the 5′-UTR and the adjacent SL6 ideal therapeutically relevant targets for small molecule probing. An initial DMA focused library screen against OC43-infected Vero E6 cells allowed for the identification of three lead compounds, namely DMA-132, -135, and -155 that significantly reduced viral titer. Initial structure-activity relationships could also be resolved, highlighting the critical substitution of the dimethylamine group at the C5 position and rigid aromatic substituents at the C6 position. These molecules were also found to be active against SARS-CoV-2 in both screening-format qRT-PCR and infectious viral titer assays. Luciferase assays revealed the presence of the 5′-UTR and proximal region as necessary and sufficient for translation inhibition upon small molecule treatment. Markedly, all in vitro assays identified DMA-155 as the strongest SARS-CoV2 inhibitor, with the largest reduction of luciferase signal and highest decrease in viral titer at 10 μM.
NMR profiling of leads DMA-132, -135 and -155 against each of the major 5′-UTR and adjacent stem loop domains revealed that the DMAs bind preferentially to SLs that contain large internal or bulge loops. Interestingly, SL1's NMR data revealed CSPs and LB with DMA-135, which signifies its binding onto a specific surface with a disruption in stacking interactions, unlike DMA-155. Notably, SL6, which contains a moderately conserved and weakly paired ˜16-nt bulge loop, showed one of the most significant CSPs when titrated with DMAs. Specifically, NOE experiments revealed that DMA-155 binds specifically to SL6, a finding that was corroborated by in vitro indicator displacement assays (IDA), which identified SL6 as the preferred target for DMA-155. Interestingly, the 5′-side of the SL6 bulge loop has been proposed to interact with the SARS-CoV-2 Nucleocapsid (N) protein under phase separation conditions and may also impact genome packaging (Iserman et al., bioRxiv, 2020.2006.2011.147199 (2020)). The modulation of the N protein:SL6 interaction is to be investigated as a potential antiviral mechanism in future work.
Finally, in silico analysis corroborated the experimental trend observed with NMR experiments, identifying some of the stem loops with predicted bulges (SL1, SL4, SL5a, SL6) as those with binding pockets with highest druggability. The small molecules that showed highest antiviral activity, namely DMA-132, -135, and -155 scored highest in the stem loops that reported significant chemical shift perturbations upon small molecule binding. Additionally, refined docking of the three small molecule leads against motif-specific clusters revealed SL1 to be the preferred target for DMA-135 and SL6 as the favored target for DMA-155, in line with NMR and IDA results.
In conclusion, the present disclosure identified drug-like small molecules that reduced SARS-CoV-2 replication and were the first antivirals to target the conserved RNA stem loops in the 5′-end region of SARS-CoV-2. Work is underway to further characterize the mode of action of these ligands, particularly putative impacts on RNA:protein interactions and specific steps in the viral replication cycle. Indeed, these leads are uniquely poised to further elucidate the relationship between in vitro preferential binding and small molecule mediated SL-specific alterations of virus:host interactions. These small molecules offer the opportunity to understand the contribution of individual 5′-end stem loops to viral proliferation in a system where mutational studies are difficult due to genome size. Once characterized, it is expected that these amiloride-based ligands to serve as chemical biology tools to help understand CoV RNA molecular biology, such as N-dependent genome packaging and other cellular stages of the viral RNA replication process. Importantly, an efficient framework has been established to identify novel RNA-targeted CoV antivirals that will serve not only the SARS-CoV-2 pandemic but future coronavirus pandemics.
One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein is presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
This PCT application claims benefit of priority to U.S. Provisional Patent Application No. 63/120,912, filed Dec. 3, 2020, the contents of which is hereby incorporated by reference in its entirety.
This invention was made with Government support under Federal Grant Nos. R35 GM124785 and R01 GM126833 awarded by the National Institutes of Health (NIH). The Federal Government has certain rights to this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/61774 | 12/3/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63120912 | Dec 2020 | US |
