Compounds for treating and preventing viral infections and methods of use thereof

Abstract

Compounds and pharmaceutical compositions are provided. Methods are also provided for utilizing the compounds in the treatment or prevention of flavivirus infections, including Zika Virus or Dengue Virus infections.

Description

70729-02COMPOUNDS FOR TREATING AND PREVENTING VIRAL INFECTIONS AND METHODS OF USE THEREOFPRIORITY

[0001] This patent application is related to and claims the priority benefit of U.S. Provisional Patent Application No. 63 / 679,232 filed August 5, 2024. The content of the foregoing application is hereby incorporated by reference in its entirety into this disclosure.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. AI095366 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure generally relates to small molecules, compositions of matter, materials and methods for treating a viral infection in a subject, specifically addressing Zika virus (ZIKV) or dengue virus seroty pe 2 (DENV2) infections.BACKGROUND

[0004] This section introduces aspects that may help facilitate a better understanding of the disclosure. These statements are to be read in this context and are not to be understood as admissions regarding prior art.

[0005] The reemergence of several flavivirus species each year results in roughly 400 million infections worldwide and represents a significant public health concern. In particular, the repeated outbreaks of Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) demonstrate the need for the development of effective treatment options. While a vaccine is available against JEV and, presently, there are several vaccine candidates that show promise against DENV, no vaccine or specific antiviral compound is available for the prevention or treatment of ZIKV infection.

[0006] ZIKV, a mosquito-borne flavivirus first identified in 1947, has emerged as a global public health concern, particularly following large-scale outbreaks in the Americas during 2015-2016. Although most infections are asymptomatic or mild, ZIKV is associated with severe neurological outcomes, including microcephaly in neonates and Guillain-Barre syndrome in adults. Giri et al., Intrinsically disordered side of the Zika virus proteome, Frontiers in Cellular & Infection Microbiology 6: 144 (2016); Nicholls et al., Structure-guided paradigm shifts in flavivirus70729-02 assembly and maturation mechanisms, Advances in Virus Research 108: 1-28 (2020). Despite extensive global spread, now documented in over 85 countries, and sustained transmission in regions such as Brazil and India, no approved vaccine or targeted antiviral therapy is currently available. Gestuveo et al.. Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection, Nature Communications 12(1): 2766 (2021). ZIKV remains on the World Health Organization’s list of priority pathogens.

[0007] ZIKV is an enveloped particle containing a positive-sense, single-stranded ribonucleic acid (RNA) genome (~1 Ikb) that is translated as a single polypeptide across the endoplasmic reticulum (ER) membrane upon viral infection of a host cell. Nicholls et al. (2020), supra. The polyprotein encoded by the genome is co- and post-translationally cleaved into three structural proteins - Capsid (C), Envelope (E), and pre-Membrane / Membrane (prM / M) - and seven non- structural proteins (NS1, NS2A, NS2B, NS3, NS4A,NS4B, andNS5). Among these, the C protein plays a central role in virion assembly by binding to viral RNA and initiating nucleocapsid formation at the ER membrane. Morando et al., Dynamics of Zika virus capsid protein in solution: the properties and exposure of the hydrophobic cleft are controlled by the a-helix 1 sequence. Biochemistry 58(20): 2488-2498 (2019). The C protein also engages in host-virus interactions, modulating immune responses and cellular metabolism. Shang et al., Crystal structure of the capsid protein from zika virus, J Molecular Biology 430(7): 948-962 (2018). Structural studies have revealed a dimeric conformation consisting of five a-helices (al- a5), a pre-al loop, and disordered terminals, which are essential for its function. Tan et al., Capsid protein structure in Zika virus reveals the flavivirus assembly process, Nature Communications 11(1): 895 (2020); Shang et al., (2018), supra Li et al., Structural insight into the Zika virus capsid encapsulating the viral genome, Cell Research 28(4): 497-499 (2018). The pre-al loop region is responsible for interactions with lipid membranes, while the a4 region is posited to interact with the viral RNA genome. Kumar et al., Drugs to limit Zika vims infection and implication for maternal-fetal health, Frontiers in Virology 2: 928599 (2022). The fifth helix (a5) is believed to remain in the ER membrane after polyprotein processing where it is degraded.

[0008] While several small molecules have shown in vitro anti-ZIKV activity, none are known to directly target the C protein-RNA interaction essential for nucleocapsid assembly. Accordingly, there remains a need for therapeutic agents capable of disrupting C protein function to inhibit the replication of ZIKV and. potentially, other flavivirus species.70729-02BRIEF SUMMARY

[0009] The present invention generally relates to compounds for use in treating or preventing a flavivirus infection in a subject. In certain embodiments, a compound is provided that comprises the following structure:or is a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein:X is a hydroxyl group ( — OH), hydrogen (H), or NH2; andR1 is (i) a six-membered carbocyclic group, or (ii) a fused bicyclic carbocyclic group comprising two six-membered rings, wherein each of (i) or (ii) is optionally substituted. R1 can be the six-membered carbocyclic group of (i) and substituted with F, Cl, a hydroxyl group, or a methoxy. R1 can be the fused bicyclic carbocyclic group of (ii) and substituted with F and a hydroxyl group. In certain embodiments, X is O', NH+, or H+(such as where the compound is a radical). In certain embodiments, X is a hydroxyl group. In certain embodiments, X is NH2.

[0010] The compound can comprise the structure of:or be a pharmaceutically acceptable salt, solvate, or hydrate thereof.70729-02

[0011] The compound can comprise the structure of:(Compound C), or be a pharmaceutically acceptable salt, solvate, or hydrate of either of the foregoing. In certain embodiments, the compound is:(Compound C), or is a pharmaceutically acceptable salt, solvate, or hydrate thereof.70729-02

[0012] The compound can comprise the structure of:(Compound B'), or be a pharmaceutically acceptable salt, solvate, or hydrate of either of the foregoing.

[0013] The compound can comprise the structure of:70729-02(Compound A"), or be a pharmaceutically acceptable salt, solvate, or hydrate of any of the foregoing structures.

[0014] Pharmaceutical compositions are also provided. In certain embodiments, the pharmaceutical compositions comprise a therapeutically effective amount of: (a) any compound hereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and (b) a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can be formulated for administration via a route selected from the group consisting of parenteral, oral, and intranasal. The pharmaceutical composition can be formulated for administration intramuscularly, intravenously, subcutaneously, or intranasally.

[0015] In certain embodiments, a radical is provided. The radical can be of any of the compounds described herein.

[0016] Still further, methods for treating or preventing a viral infection in a subject are provided. Such a method can comprise administering to the subject a therapeutically effective amount of: (a) any compound hereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; or (b) any pharmaceutical composition hereof. The viral infection can be a flavivirus infection and, optionally, a Zika viral (ZIKV) or a dengue viral (DENV) infection.

[0017] In certain embodiments, the method comprises administering a co-therapy to the subject. The co-therapy can comprise: (a) a medicament selected from the group consisting of an immunomodulator, an anti-inflammatory compound, an antibiotic, an antifungal compound, and an antimicrobial compound; (b) an intravenous drip; or (c) both (a) and (b). The co-therapy can be administered to the subject simultaneously or sequentially with the compound or pharmaceutical composition. In certain embodiments comprising administration of a co-therapy, administering is parenteral, oral, or intranasal. In certain embodiments comprising administration of a co-therapy, the administering is intravenous, intramuscular, or subcutaneous.

[0018] A compound hereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or a pharmaceutical composition hereof is also provided for use in treating or preventing a flavivirus infection in a subject. As noted above, the flavivirus can be ZIKV. The flavivirus can be DENV.70729-02The compound or pharmaceutical composition can be formulated for administration via a route selected from the group consisting of parenteral, oral, and intranasal.

[0019] Processes for the manufacture of a medicament for the treatment or prevention of a flavivirus infection in a subject are also provided. In certain embodiments, the process comprises formulating a compound hereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, with one or more pharmaceutically acceptable carriers or excipients.

[0020] Methods of inhibiting ZIKV or DENV replication are also provided. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of: (a) any compound hereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; or (b) any pharmaceutical composition hereof. In certain embodiments, the therapeutically effective amount is from about 6.25 pM to about 12.5 pM.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying figures provide detailed visual representations and data analyses as follows:

[0022] FIG. 1A illustrates a three-dimensional model depicting the interaction between a Capsid (C) protein and a ribonucleic acid (RNA) molecule, generated using the HDOCK server. Dashed lines indicate non-covalent interactions between the RNA molecule and the side chains (labeled SC) of the C protein (labeled C).

[0023] FIG. IB illustrates an inverted-pyramid schematic summarizing the compound selection workflow, encompassing both in-silico screening and the in vitro testing as described herein.

[0024] FIG. 1C displays three-dimensional binding poses of ten selected compounds with a Zika Virus (ZIKV) C protein. FIG. ID presents two-dimensional interaction maps of these compounds with a ZIKV C protein, where arrowed and dashed lines indicate interaction between ligand and C protein residues.

[0025] FIG. 2A presents graphical data depicting the cytotoxicity profiles of uninfected Vero cells treated with each of the ten indicated compounds / inhibitors at various concentrations, or a positive control Ribavirin, each measured at 48 hours post-infection (hpi). The compounds were dissolved in 2% fetal bovine serum (FBS) supplemented with Dulbecco’s Modified Eagle Medium (DMEM) to achieve the desired concentrations.

[0026] FIG. 2B presents graphical data depicting the cytotoxicity profiles of virus-infected cells (multiplicity of infection (Mol) = 1.0) treated with each of the five indicated inhibitors or a positive control Ribavirin, measured at 48 hpi.70729-02

[0027] FIG. 3 presents graphical data depicting the cytotoxicity profiles of virus-infected cells (Mol = 3.0) treated with each of the five indicated inhibitors or a positive control Ribavirin, measured at 48 hpi (subparts a-d) and 96 hpi (subparts e-h).

[0028] FIG. 4A is a representative image of plaque reduction assays for untreated, Ribavirin- treated. and compound D-treated ZIKV.

[0029] FIG. 4B presents graphical data depicting a titer analysis of Ribavirin-treated ZIKV plaque reduction assays.

[0030] FIG. 4C presents graphical data from a titer analysis from compound D-treated ZIKV plaque reduction assays.

[0031] FIGS. 4D and 4E present data from compound D-treated ZIKV supernatants, analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (FIG. 4D) and enzyme- linked immunosorbent assay (ELISA) (FIG. 4E), respectively.

[0032] FIG. 4F presents a Western blot image from untreated and compound D-treated ZIKV- infected cell lysates.

[0033] FIG. 4G shows results from virus plaque assays on Vero cells after no treatment (farthest left; control), and treatment with different concentrations of Ribarvirin, prepared in phosphate- buffered saline supplemented with calcium and magnesium ions (PBS++) (middle and right images).

[0034] FIG. 4H shows results from virus plaque assays on Vero cells after no treatment (farthest left; control), and treatment with different concentrations of compound D, prepared in PBS++.

[0035] FIG. 41 shows results from virus plaque assays (time-of-compound-addition assay) on Vero cells after no treatment (control) and treatment with compound D, with images taken at 2- hour intervals and collected at 48 hpi.

[0036] FIG. 5 shows results from an immunofluorescence assay (IF A) performed on cells infected with virus at Mol = 1.0, analyzed at 48 hours hpi following treatment with varying concentrations of compound D. The untreated virus control is designated as DMSO. Sample names or concentrations are listed vertically along the left margin, and the corresponding fluorescent targets are labeled horizontally along the top.

[0037] FIG. 6 includes multiple subparts: (a) and (b) show cryo-transmission electron microscopy (cryo-TEM) micrographs of purified ZIKV, either untreated (a) or treated with the and compound D at a concentration of 5 iiM (b). Subpart (c) presents an SDS-PAGE analysis, and (d) presents a Western blot analysis, both of purified supernatant and purified virus samples obtained from each of the three concentrated bands within the discontinuous density gradients for both treatment conditions. The E (Envelope) protein was detected in both the bands of untreated virus (upper and middle), while no E protein was detected in any treated ZIKV band.70729-02

[0038] FIGS. 7A-7E present data from entry-inhibition assays demonstrating reduced viral infection with increasing concentrations of compound D. FIG. 7A shows graphical data from a cytotoxicity' assay indicating cell viability7across increasing compound D concentrations. FIG. 7B presents a Western blot analysis of lysates from compound D- treated, virus-infected cells, showing reduced levels of the viral E protein. FIGS. 7C and 7D present plaque assay results indicating a reduction in viral load upon treatment with compound D. FIG. 7E shows images from a plaque assay on Vero cells to assess viral entry' inhibition, with images taken after no treatment (farthest left; control), and treatment with different concentrations of compound D (made in PBS++) as follows: 3. 125 pM. 6.25 pM, and 12.5 pM (from left to right).

[0039] FIG. 8 shows results from an IFA performed on cells infected with ZIKV virus at a Mol = 1.0 following preincubated treatment with varying concentrations of compound D. The untreated virus control is designated as DMSO. Sample names or concentrations are listed vertically along the left margin of the figure, and the corresponding fluorescent targets are labeled horizontally along the top. The data demonstrate a reduced viral load, consistent with inhibition of viral entry'.

[0040] FIGS. 9A-9E present results of binding evaluations conducted using fluorescence spectroscopy, together with calculations of dissociation constant (Kd) for compound D. FIGS. 9A and 9B show binding data for ZIKV NS2B-NS3 protease, and FIGS. 9C and 9D show binding data for ZIKV envelope (E) protein. FIG. 9E presents Dengue virus plaque assay titers after treatment with different concentrations of compound D. Statistical significance 'as evaluated using a one-w ay analysis of variance (ANOVA). Significant differences were observed at 12 pM (p = 0.0072), 25 pM (p < 0.0001), and 50 pM (p < 0.0001).

[0041] FIG. 10 presents graphical data depicting the cytotoxicity profiles of ZIKV -infected cells (Mol = 1.0) following treatment with either compound D or compound C, measured at 48 hpi. WT depicts a positive control that was not treated with either compound.DETAILED DESCRIPTION

[0042] For the purposes of promoting an understanding of the principles of the present disclosure, references will now' be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

[0043] The present disclosure is based, at least in part, on the discovery of small-molecule compounds that competitively bind to a region of the capsid (C) protein, thereby disrupting virion assembly as evidenced by treated viral particles exhibiting distorted morphology7. The compounds can target and disrupt the ability of C protein to interact with viral RNA (an important step of70729-02 virion assembly) and significantly reduce ZIK.V and DENV infection following treatment at relatively low concentrations (e.g., less than about 6.25 pM). Unexpectedly, the compounds can also inhibit viral entry at concentrations of about 12.5 pM.

[0044] In certain embodiments, the compound comprises the following structure:or is a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein:X is a hydroxyl group ( — OH), hydrogen (H), or NH2; andR1 is a six-membered carbocyclic group or a fused bicyclic carbocyclic group comprising two six-membered rings, each optionally substituted. R1 can be substituted with fluorine (F). R1 can be substituted with chlorine (Cl). R1 can be substituted with a hydroxyl group. R1 can be substituted with a methoxy ( — OCH3). In certain embodiments where R1 is a fused bicyclic carboxylic group, the first ring bears one or more substituents selected from F and hydroxyl.

[0045] In certain embodiments, X is H. In certain embodiments, X is O', NH+, or H+(such as where the compound is a radical). In certain embodiments, X is a hydroxyl group. In certain embodiments, X is NH2. In certain embodiments, R1 isa point of attachment.

[0046] In certain embodiments, the compound comprises the following structure:(Compound A),70729-02(Compound A"), or is a pharmaceutically acceptable salt, solvate, or hydrate of any of the foregoing. In certain embodiments, the compound is:70729-02(Compound A"), or is a pharmaceutically acceptable salt, solvate, or hydrate of any of the foregoing.

[0047] In certain embodiments, the compound comprises the following structure:(Compound B'), or is a pharmaceutically acceptable salt, solvate, or hydrate of either of the foregoing. In certain embodiments, the compound is:70729-02(Compound B), or is a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the compound is:(Compound B'), or is a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0048] In certain embodiments, the compound comprises the following structure:70729-02(Compound C), or is a pharmaceutically acceptable salt, solvate, or hydrate of either of the foregoing. In certain embodiments, the compound is:(Compound C), or is a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the compound is:(Compound C), or is a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0049] In certain embodiments, the compound comprises the lol I owing structure:70729-02or is a pharmaceutically acceptable salt, solvate, or hydrate thereof. In certain embodiments, the compound is:or is a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0050] In certain embodiments, the compound comprises one of the following structures: ),70729-0270729-02

[0051] The compounds contain one or more chiral centers or can otherwise be capable of existing as multiple stereoisomers. Accordingly, various embodiments of the compound can include pure stereoisomers, as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. The compound can be capable of existing as geometric isomers, such as pure geometric isomers or mixtures of geometric isomers.

[0052] Radicals of any of the foregoing compounds are also provided. As used herein, a “radical” of a disclosed compound refers to any reactive species derived from the compound by removal of one or more atoms or groups so to provide at least one open valence site available for bonding e.g., covalent bonding). Such radicals include, without limitation, neutral, anionic, or cationic species, and can be generated at any atom of the parent structure by homolytic or heterolytic bond cleavage, dehydrogenation, or loss of a substituent. Unless otherwise indicated, the term encompasses all positional isomers of such radicals, regardless of charge, stereochemistry', or substitution pattern, and includes radicals generated in situ or prepared as isolated intermediates for subsequent derivatization, conjugation, or other chemical transformation.

[0053] The compound (or radicals thereof) can be synthesized in accordance with methods known in the art. Various methods of synthesis are exemplified in the Examples.

[0054] Salts

[0055] The compounds (and radicals) hereof can be presented as a pharmaceutically acceptable salt. A “pharmaceutically acceptable salt” of a compound (or radical) refers to those salts whose counter ions can be used in pharmaceuticals. Such salts include (i) acid addition salts, which can be obtained by reaction of the free base of the parent compound with inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid,70729-02 and the like, or with organic acids, such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethane sulfonic acid, / ;-tol uene sulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, malonic acid, and the like, and (ii) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methyl glucamine, and the like. Pharmaceutically acceptable salts are well-known to those skilled in the art, and any such pharmaceutically acceptable salt is contemplated herein.

[0056] In various embodiments, suitable basic salts are formed from bases which fonn non-toxic salts. Illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases can also be formed, e.g., hemisulphate and hemicalcium salts.

[0057] Pharmaceutically acceptable salts can be synthesized from the parent compound (or radical thereof) which contains a basic or acidic moiety by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference.

[0058] The compound, radical, or pharmaceutically acceptable salt or hydrate thereof, can exist in unsolvated or solvated forms, including hydrated forms. Solvated forms can be equivalent in activity to unsolvated forms. As used herein, the structural formulae encompass not only all pharmaceutically acceptable salts of the compounds and radicals, but also any hydrates and / or solvates thereof. The term “solvate” refers to a compound, or a salt thereof, that contains a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is referred to as a “hydrate.”

[0059] Certain functional groups, such as the hydroxy, amino, and like, can form complexes and / or coordination conjugates with water and / or various solvents. Accordingly, the formulae are to be understood to include and represent those various hydrates and / or solvates. Non-hydrates and / or non-solvates of the compounds are also included.

[0060] Pharmaceutical Compositions

[0061] In view of the above, also provided is a composition (e.g., a pharmaceutical composition) for the treatment of a flavivirus infection (e.g, a ZIKV or DENV infection) comprising at least70729-02 compound (or radical) hereof and a pharmaceutically acceptable carrier or excipient. “Pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as, but not limited to, a buffering agent, a preserving agent, an anesthetic agent, a solubilizing agent, an isotonic agent, a wetting agent, and a stabilizer. The term also encompasses any of the agents approved by a regulatory agency, such as the U.S. Food and Drug Administration, or listed in the U.S. Pharmacopeia for use in animals (e.g., mammals, such as humans). The carrier can be a phosphate-buffered saline solution, water, or an emulsion such as an oil / water or water / oil emulsion.

[0062] Also provided is a pharmaceutical composition comprising any of the compounds (or radicals) hereof (or a pharmaceutically acceptable salt, solvate or hydrate thereof) and a pharmaceutically acceptable carrier or excipient, for use in the treatment of a flavivirus infection (e.g., aZIKV or DENV infection).

[0063] The compounds and radicals can be fonnulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration. For example, the pharmaceutical composition can be formulated for and administered via oral or parenteral, intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular, inhalation and / or subcutaneous routes. Indeed, in at least one embodiment, a compound and / or composition as described herein can be administered directly into the blood stream, into muscle, or into a nasal cavity.

[0064] For example, in at least one embodiment, the present compounds can be systemically administered (orally, for example) in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the compositions and preparations can vary and can be between about 1 to about 99% weight of the active ingredient(s) and a binder, excipients, a disintegrating agent, a lubricant, and / or a sweetening agent (as are known in the art). The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0065] The compounds, radicals, and pharmaceutical compositions hereof can be fonnulated as parenteral formulations. Parenteral formulations are typically aqueous solutions, which can contain carriers or excipients such as salts, carbohydrates, and buffering agents (preferably at a pH of from 3 to 9), but they can be more suitably formulated as a sterile, non-aqueous solution or as a dried from to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or sterile saline. Preparation under sterile conditions, by lyophilization to produce a sterile,70729-02 lyophilized powder for a parenteral formulation, can be accomplished using methods well-known in the art. The solubility of the compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, for parenteral formulation can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

[0066] The compounds / compositions can also be administered via infusion or injection (e.g., using needle (including microneedle) injectors and / or needle-free injectors). Solutions of the composition can be aqueous, optionally mixed with a nontoxic surfactant and / or can contain carriers or excipients such as salts, carbohydrates and buffering agents (preferably at a pH of from 3 to 9), but, for some applications, they can be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or phosphate-buffered saline (PBS). For example, dispersions can be prepared in glycerol, liquid PEGs, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can further contain a preservative to prevent the growth of microorganisms.

[0067] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredients that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example and without limitation, water, ethanol, a poly ol (e.g., glycerol, propylene glycol, liquid PEG(s), and the like), vegetable oils, nontoxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, the proper fluidity can be maintained by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The action of microorganisms can be prevented by the addition of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certain cases, it will be desirable to include one or more isotonic agents such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the incorporation of agents formulated to delay absorption, for example, aluminum monostearate and gelatin.

[0068] Sterile injectable solutions can be prepared by incorporating the compound(s) and / or composition in the required amount of the appropriate solvent with one or more of the other ingredients set forth above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparations70729-02 are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0069] The amount of the compound (or pharmaceutically acceptable salt, solvate, or hydrate thereof) to be administered to a subject can vary’ significantly, depending on the type of viral infection being treated, the stage of infection, the route of administration, and tissue distribution. As used herein, the terms ‘'therapeutically effective,” “therapeutically effective dose,” “therapeutically effective amount,” “prophylactically effective amount,” or “prophylactically effective dose” mean (unless specifically stated otherwise) a quantity of a compound which, when administered either one time or over the course of a treatment cycle affects the health, wellbeing or mortality of a subject (e.g.. and without limitation, inhibits ZIK.V or DENV replication, and / or delays the onset of and / or reduces the severity of one or more of the symptoms associated with a viral infection). Useful dosages of the compounds can be determined by comparing their in vitro activity, and the in vivo activity in animal models. Methods of the extrapolation of effective dosages in mice and other animals to human subjects are known in the art. Indeed, the dosage of the compound can vary significantly depending on the condition of the host subject, the viral infection being treated, how advanced the pathology7is, the route of administration of the compound and tissue distribution, and the possibility of co-usage of other therapeutic treatments (such as additional drugs in combination therapies such as, for example antibiotics, antiinflammatory' compounds, immunomodulators, etc.). The amount of the composition required for use in treatment (e.g., the therapeutically or prophylactically effective amount or dose) can vary' not only with the particular application, but also with the salt selected (if applicable) and the characteristics of the subject (such as, for example, age, condition, sex, the subject’s body mass, tolerance to drugs), and will ultimately be at the discretion of the attendant physician, clinician, or other healthcare provider.

[0070] The amount to be administered to a subject can range, for example, from about 0.05 mg to about 30 mg, about 0.05 mg to about 25 mg, about 0.05 mg to about 20 mg, about 0.05 mg to about 15 mg. about 0.05 mg to about 10 mg. about 0.05 mg to about 9 mg, about 0.05 mg to about 8 mg, about 0.05 mg to about 7 mg, about 0.05 mg to about 6 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 4 mg, about 0.05 mg to about 3 mg, about 0.05 mg to about 2 mg, about 0.05 mg to about 1 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about 0.01 mg to about 20 mg, about 0.3 mg to about 10 mg, about 0. 1 mg to about 20 mg, or about 0.8 mg to about 3 mg. The ordinarily skilled artisan will readily appreciate that the dose can vary w ithin the various ranges provided above based on the factors pointed out above and can be at the treating physician’s discretion.70729-02

[0071] Therapeutically effective or prophylactically effective amounts or doses can range, for example, from about 0.05 mg / kg of patient body weight to about 30.0 mg / kg of patient body weight, or from about 0.01 mg / kg of patient body weight to about 5.0 mg / kg of patient body weight, including but not limited to 0.01 mg / kg, 0.02 mg / kg. 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg. 0.2 mg / kg. 0.3 mg / kg. 0.4 mg / kg. 0.5 mg / kg. 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg, all of which are kg of patient body weight. The total therapeutically or prophylactically effective amount of the bispecific adaptor can be administered in single or divided doses and can, at the practitioner's discretion, fall outside of the typical range given herein.

[0072] In another embodiment, the compound can be administered in a therapeutically or prophylactically effective amount of from about 0.5 g / m2to about 500 mg / m2, from about 0.5 g / m2to about 300 mg / m2, or from about 100 g / m2to about 200 mg / m2. In other embodiments, the amounts can be from about 0.5 mg / m2to about 500 mg / m2, from about 0.5 mg / m2to about 300 mg / m2, from about 0.5 mg / m2to about 200 mg / m2, from about 0.5 mg / m2to about 100 mg / m2, from about 0.5 mg / m2to about 50 mg / m2, from about 0.5 mg / m2to about 600 mg / m2, from about 0.5 mg / m2to about 6.0 mg / m2, from about 0.5 mg / m2to about 4.0 mg / m2, or from about 0.5 mg / m2to about 2.0 mg / m2. The total amount can be administered in single or divided doses and can, at the physician's discretion, fall outside of the typical range given herein. These amounts are based on meters of body surface area. All ranges specified in this paragraph are inclusive of the stated end points and include all 0.5 g / m2increments encompassed in each specified range.

[0073] In other embodiments, the amount of the compound (or pharmaceutically acceptable salt, solvate, or hydrate thereol) to be administered to a subject can range, for example, from about 50 nmol / kg to about 3.000 nmol / kg of subj ect body weight, about 50 nmol / kg to about 2,000 nmol / kg, about 50 nmol / kg to about 1,000 nmol / kg, about 50 nmol / kg to about 900 nmol / kg, about 50 nmol / kg to about 800 nmol / kg, about 50 nmol / kg to about 700 nmol / kg, about 50 nmol / kg to about 600 nmol / kg, about 50 nmol / kg to about 500 nmol / kg, about 50 nmol / kg to about 400 nmol / kg, about 50 nmol / kg to about 300 nmol / kg, about 50 nmol / kg to about 200 nmol / kg. about 50 nmol / kg to about 100 nmol / kg, about 100 nmol / kg to about 300 nmol / kg, about 100 nmol / kg to about 500 nmol / kg, about 100 nmol / kg to about 1,000 nmol / kg, or about 100 nmol / kg to about 2,000 nmol / kg of subject body weight. In other embodiments, the dose can be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg. about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 600 nmol / kg, about 700 nmol / kg, about 800 nmol / kg, about 900 nmol / kg, about 1,000 nmol / kg, about 2,000 nmol / kg, or about 3,000 nmol / kg of subject body weight. In other embodiments, between about 20 pg / kg to about 3 mg / kg of subject body weight can be administered. The amount can be between about70729-020.2 mg / kg to about 0.4 mg / kg of subject body weight or about 50 pg / kg subject body weight. All ranges specified in this paragraph are inclusive of the stated end points and include all 1 nmol / kg or 10 pg / kg increments, as applicable, encompassed in each specified range.

[0074] Uses and Methods

[0075] Further provided is a method of treating and / or preventing a viral infection in a subject (e.g., a ZIKV or DENV infection). The method comprises administering to the subject therapeutically effective amounts of a compound hereof (or pharmaceutically acceptable salt, solvate, or hydrate thereof) or a pharmaceutical composition hereof. In certain embodiments, for example, the method comprises administering to the subject a therapeutically effective amount of compound D, compound C, or compound C. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising compound D, compound C, or compound C, and a pharmaceutically acceptable carrier or excipient.

[0076] The terms “treat,” "treating." "treated." and "Treatment” refer to therapeutic treatment. Such treatment can have a prophylactic effect. A viral infection can be considered “treated,” for example and without limitation, when the symptoms or signs of the infection are ameliorated, such as by a reduction in viral load, a decrease in the severity or duration of clinical symptoms, complete or partial elimination of detectable virus, inhibition of viral replication or spread, stabilization of the infection (e.g., prevention of progression to more severe disease), prevention or reduction of virus-associated complications, or any other effect on the viral infection that a physician or other healthcare provider would consider to constitute therapeutic or prophylactic treatment.

[0077] The term “subject,” as used herein, means an animal, such as a mammal, and in particular a human. In veterinary applications, the subject can be a laboratory, an agricultural, a domestic, or a wild animal. Examples of such animals include, but are not limited to, a rodent, a rabbit, a monkey, a chimpanzee, a dog, a cat, a cow-, a horse, a pig, a sheep, a goat, a bear, a panda, a lion, a tiger, a leopard, an elephant, a zebra, a giraffe, a gorilla, a dolphin, or a whale.

[0078] The viral infection can be caused by any flavivirus. In certain embodiments, the flavivirus is ZIKV or DENV (including any serotype thereof), or any variant, strain, isolate, or recombinant thereof. In certain embodiments, the flavivirus is ZIKV or DENV.

[0079] The compound (or pharmaceutically acceptable salt or hydrate thereof) or pharmaceutical composition comprising same can be administered to the subject using any suitable method known in the art. The compound (or pharmaceutically acceptable salt or hydrate thereof) or pharmaceutical composition can be administered as a monotherapy, for example.70729-02

[0080] The terms “administer,” “administering,” “administered,” and “administration” refer to methods of introducing the compound (or a pharmaceutically acceptable salt, solvate, or hydrate thereof) or a pharmaceutical composition comprising the compound (or a pharmaceutically acceptable salt, solvate, or hydrate thereof) into a subject. Examples of suitable routes of administration include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, and transdermal. The compounds and pharmaceutical compositions can be administered directly into the blood stream, into muscle, or into a nasal cavity, for example. Suitable routes for parenteral administration include, but are not limited to, intravenous, intra-arterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular, and subcutaneous. Use can be made of needle injectors, including microneedles, needle-free injectors, and infusions. The compounds and pharmaceutical compositions can be administered in unit dosage forms and / or formulations containing conventional non-toxic pharmaceutically acceptable carriers or excipients (or vehicles or adjuvants).

[0081] In certain embodiments, the compounds hereof (or pharmacally acceptable salt, solvate, or hydrate thereof) or the pharmaceutical compositions hereof can be administered in combination with a co-therapy, such as an immunomodulator, an anti-inflammatory or symptomcontrol medication (e.g., corticosteroids, antipyretics, and / or NS AfDs), antibiotics (e.g., broadspectrum antibiotics), antifungals, antimicrobials, and / or supportive / organ-protective therapies (e.g., intravenous fluids (with or without electrolytes), and / or antiemetics).

[0082] Where multiple therapeutics and / or therapies are co-administered, dosages may be adjusted accordingly, as is recognized in the pertinent art. “Co-administration” and combination therapy are not limited to simultaneous administration but also include sequential administration, in either order, by the same or different routes, and treatment regimens in which a compound is administered at least once during a course of treatment that involves administering another therapy to a subj ect.

[0083] The methods of treating or preventing a viral infection hereof can comprise administering any of the compounds to the subject and administering any of the aforementioned co-therapies to the subj ect.

[0084] In certain embodiments, a method for inhibiting flavi virus infection of a subject. The method comprises administering to the subject a therapeutically effective amount of: (i) any compound (or pharmaceutically acceptable salt, solvate, or hydrate thereof); or (ii) any pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compound or pharmaceutical composition are administered systemically. In certain embodiments, the compound or pharmaceutical composition are administered parenterally, including but not limited to intravenously or intramuscularly. In certain70729-02 embodiments, the compound or pharmaceutical composition are administered via inhalation and / or intranasally. In certain embodiments, the compound or pharmaceutical composition are administered orally. The flavivirus can be any flavivirus. In certain embodiments, the flavivirus is ZIKV. In certain embodiments, the flavivirus is DENV.

[0085] Also provided are methods for inhibiting flavivirus replication. The flavivirus can be ZIKV. The flavivirus can be DENV. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of: (i) any compound (or pharmaceutically acceptable salt, solvate, or hydrate thereof); or (ii) any pharmaceutical composition comprising the same and a pharmaceutically acceptable earner or excipient.

[0086] In certain embodiments, a use of a compound or radical hereof (such as, for example, a therapeutically effective amount of compound D, compound C or compound C), a pharmaceutically acceptable salt, hydrate, or solvate of the compound, or a composition in the manufacture of a medicament for the treatment of a viral infection in a subj ect is provided. The compound can be any compound hereof. The medicament can be for use as a monotherapy, or for use as a co-therapy in combination with administration of any of the aforementioned co-therapies.

[0087] General

[0088] Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular embodiments described. Other implementations may be possible.

[0089] While the compounds and pharmaceutical compositions are illustrated and described in detail in the foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only certain embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.

[0090] It is intended that the scope of the present compounds, compositions, methods, and uses are defined by the following claims. However, this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. Those skilled in the art will understand that various alternatives to the embodiments described herein can be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.

[0091] Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section.

[0092] All publications, patents, patent application publications, journal articles, textbooks, and other publications referred to in this document are indicative of the level of skill of those in the70729-02 art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. If there is inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0093] Various techniques and mechanisms will sometimes describe a connection or link between two components. Words such as attached, linked, coupled, connected, and similar tenns with their inflectional morphemes are used interchangeably, unless the difference is noted or made otherwise clear from the context. These words and expressions do not necessarily signify direct connections but include connections through mediate components. It should be noted that a connection between two components does not necessarily mean a direct, unimpeded connection, as a variety of other components may reside between the two components of note. Consequently, a connection does not necessarily mean a direct, unimpeded connection unless otherwise noted.

[0094] Certain Definitions

[0095] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

[0096] The term '‘about” or “approximately” means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean a range of up to 20%, preferably up to 10%. more preferably up to 5%, and more preferably still up to 1% of a given value. By way of further example, "about” or “approximately” can mean within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term “about” means within an acceptable error range for the particular value, such as ± 1-20%, preferably ± 1-10% and more preferably ±1-5%.

[0097] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those limits are also included.70729-02

[0098] A phrase referring to “at least one of’ a list of items refers to any combination of those items, including single members. As an example, “at least one of a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0099] The terms “a.” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.

[0100] The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology’ employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.

[0101] The terms and expressions employed are used as terms of description and not of limitation. Where certain terms are defined and are otherwise described or discussed elsewhere in the “Detailed Description.” all such definitions, descriptions, and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. Furthermore, while subheadings may be used in the “Detailed Description,” such use is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one subheading is intended to constitute a disclosure under each and every' other subheading.

[0102] It is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein. Such modifications and variations are considered within the scope of the invention as claimed herein.EXAMPLES

[0103] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.Example 1: Identification of Capsid-Binding Compounds

[0104] Although prior structural studies, such as Ma et al., Solution structure of dengue virus capsid protein reveals another fold, Proceedings of Nat 'I Academy Sciences USA 101(10): 3414- 3419 (2004), have proposed general orientations of flaviviral capsid (C) proteins in complex with viral RNA, the specific amino acid residues mediating these interactions remain undefined. To address this gap and to elucidate potential RNA-binding residues within a monomeric ZIKV C70729-02 protein, computation approaches were employed. Specifically, in silico molecular docking techniques were used to simulate and predict residue-level interactions between the monomeric C protein and viral RNA sequences. These methodologies enabled the identification of candidate binding interfaces likely to contribute to RNA recognition and nucleocapsid assembly.

[0105] In particular, the protein-RNA interaction modeling was conducted using the HDOCK server, an established hybrid docking algorithm publicly available through the Huang Laboratory, School of Physics, Huazhong University of Science & Technology. The three-dimensional structural coordinates of the ZIKV C protein and the viral RNA were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB-PDB). Specifically, the C protein structure was retrieved under accession number 5YGH, and the RNA structure under accession number 5TPY. These experimentally derived structural models served as input for the docking simulations to predict energetically favorable binding interactions between the C protein and RNA.

[0106] 5 residues were identified that interact with RNA via salt-bridge, including Arg55, Lys82, Lys85, Arg93, and Asn96, which could play important roles in the interaction between C protein and viral RNA. The receptor grid was generated based on these residues for compound screening (FIG. 1A)

[0107] Using the identified residues as a spatial foundation for binding requirements, a broadscale in silico screening was performed. A total of 18,123 small-molecule compounds were selected for virtual screening from three curated libraries within the Life Chemicals Inc. database. The libraries used for this study included: (1) the Antiviral Screening Compound 2D Similarity Library, (2) the Antiviral Combined Ligand and Structure-Based Library, and (3) the RNA- Focused Library. Following compilation of these libraries, molecular docking was performed using the Glide module of the Schrodinger software suite, applying a hierarchical three-tiered screening protocol. The screening was conducted sequentially using: (i) High-Throughput Virtual Screening (HTVS), (ii) Standard Precision (SP) docking, and (iii) Extra Precision (XP) docking. For each compound that progressed to the XP stage, at least ten binding poses were generated and evaluated to assess binding orientation and interaction likelihood. The overall screening and selection workflow is illustrated in FIG. IB.

[0108] The Glide module within the Schrodinger drug discovery' platform was employed to perform virtual screening of the three compound libraries, along with a known control compound, against the ZIKV C protein, as described above. See, e.g, Sharma et al.. Small molecule inhibitors possibly targeting the rearrangement of Zika virus envelope protein, Antiviral Research 182: 104876 (2020); Kumar et al., Discovery7and characterization of small molecule inhibitors of Zika virus replication. bioRxiv, 2022.12.15.520558 (2022). The screening protocol incorporated the70729-02 previously outlined three-tiered approach - HTVS, SP, and XP. Compounds that progressed through the final XP (Extra Precision) stage were prioritized based on Glide docking scores and pose quality.

[0109] To further refine candidate selection, binding poses were subjected to Molecular Mechanics Generalized Bom Surface Area (MM-GBSA) free energy calculations using the Prime module within the Schrodinger suite. Final compound ranking was determined using a combination of Glide score and MM-GBSA binding free energy, thereby enhancing confidence in predicted ligand-protein interaction strength.

[0110] After the virtual and manual screening based on docking score, binding energies, and interacting residues, 10 compounds were selected for in vitro testing as the final candidates. The three-dimensional binding poses and two-dimensional interaction profiles of each of the selected 10 compounds are detailed in FIGS. 1C and ID. All compounds interacted with C protein through at least 2 common residues, supporting an interaction hotspot during C protein-RNA docking. Table 1 provides a summary of the calculated interaction scores and predicted binding residues for the ten compounds selected, as determined using the Glide module of the Schrodinger platform.

[0111] Table 1.70729-02

[0112] In addition, Table 1 includes corresponding CC50 values derived from zft vitro cytotoxicity assays described in Example 2 below. These CC50 values were calculated using GraphPad Prism software based on a standard curve generated in accordance with established assay protocols.

[0113] As a reference control, the screening parameters for Ribavirin, an FDA-approved broadspectrum antiviral agent, were also evaluated. Ribavirin served as a positive control for ZIKV inhibition in vitro. Docking simulations performed using the Glide module indicated that Ribavirin exhibited both a lower docking score and less favorable binding free energy (as calculated by MM-GBSA) against the ZIKV C protein compared to the 10 experimental compounds identified in this study. These results support the superior binding affinity and predicted activity of the disclosed candidate compounds relative to Ribavirin.Example 2: Compound Rescue of Cells After Viral Infection

[0114] Prior to screening the finalized group of ten small molecules against ZIKV infection within cells, the ten compounds were first examined for cytotoxic effect using Vero cells in the absence of virus for 48 hours. The ten compound selected from virtual screening (Example 1) were purchased from Life Chemicals, Inc., and the control drug Ribavirin was purchased from Sigma- Aldrich. All compounds were dissolved in dimethyl sulfoxide (DMSO), while Ribavirin was dissolved in cell-culture grade Mili-Q water.

[0115] The cytotoxicity of the selected compounds was evaluated using a standard MTT -based colorimetric assay. All assays disclosed herein (including this Example 1) were conducted using Vero cells (African green monkey kidney epithelial cells) obtained from the American Type Culture Collection (ATCC). 10,000 cells / well in 96-well plates were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2X Penicillin-Streptomycin (PenStrep) antibiotic solution. Cell cultures were maintained overnight at 37 °C in a humidified incubator with 5% CO2. The next day, prior to compound treatment, cells were washed once with IX phosphate-buffered saline (PBS) to remove residual serum components, and then infected with ZIKV (Mol 1.0 and 3.0) for 1 hour at 37 °C in 2% FBS-supplemented DMEM media. All inhibitors were diluted in the same media used for infection. After infection, the inoculum was removed, cells were again washed with PBS, and the media was replaced with the various concentrations of inhibitor mixed media. At 48 hours postinfection (hpi), the media was removed and MTT (0.5 mg / ml) mixed media was added to cells for 3.5 hours. Following incubation, the resulting formazan crystals were solubilized using DMSO.70729-02Absorbance measurements were recorded using a SpectraMax iD5 spectrophotometer (Molecular Devices, San Jose, CA) at wavelengths of 590 nm and 630 nm. The differential absorbance (590 nm - 630 nm) was calculated to correct for background signal and used to quantify cell viability . Experimental values were normalized against untreated control wells containing only vehicle (DMSO) and plotted to determine relative cytotoxicity.

[0116] In comparison to the untreated cells, most of the ten lead compounds were non-cytotoxic to cells at concentrations up to 200 pM, while three of the compounds were found to be highly cytotoxic to cells at a concentration of 25 pM. All compound cytotoxicity results, including CC50 values, are shown in Table 1 and FIG. 2A. Based on this initial in vitro screen, a total of five compounds were selected for further investigation - compound D, compound F, compound E, compound M, and compound I.

[0117] The five initial lead compounds that exhibited no significant cytotoxicity in Vero cells were subsequently evaluated for their ability to confer protection against ZIKV -induced cytopathic effects. ZIKV (French Polynesian / MR766 strain) was used for all virus culture and infection assays described herein, including the procedures outlined in this Example 2.

[0118] For infection, 50,000 Vero cells / well in 24-well plates were seeded as described above. Thereafter, the cells were exposed to ZIKV at a multiplicity of infection (Mol) of 1.0 in DMEM media containing 2% FBS and incubated for one hour at 37 °C to allow for viral adsorption. Following infection, cells were washed with PBS to remove unbound virus and then treated with each test compound in mixed media. Compounds were applied at a series of two-fold serial dilutions, with concentration ranges determined based on each compound's previously calculated CC50 value. The desired concentrations of compounds were achieved by diluting them in 2% FBS and antibiotics supplemented in DMEM media.

[0119] As with all viral infection studies described herein, supernatant containing ZIKV was harvested at 72 hours hpi, clarified by centrifuged at 3000 rpm for 5 minutes to remove cell debris, and stored for downstream assays. The antiviral efficacy of each compound was assessed by measuring cell viability relative to untreated infected controls at defined time points postinfection.

[0120] As shown in FIG. 2B, after 48 hpi, compounds D, E, and F displayed significant inhibition of virus and rescued cell viability (-80-90%) at concentrations <100 pM in comparison to virus- infected only cells (-60% cell viability). The positive control Ribavirin inhibited ZIKV infection at much higher concentrations (>100 pM) compared to the experimental compounds. Unlike the other initial lead compounds, compound M failed to rescue cell viability after ZIKV infection and instead displayed a combinatory effect of increasing cell death in the presence of virus.70729-02

[0121] To further assess the anti-viral efficacy of the remaining compounds D, E, and F under conditions of increased viral load and prolonged exposure, Vero cells were infected with ZIKV at a higher Mol 3.0 (pursuant to the previously described protocol). Following a one-hour adsorption period at 37 °C, cells were washed with PBS (as described above) and treated with a single dose of each compound at varying concentrations. Treated cultures were then incubated for extended durations of 48 and 96 hpi.

[0122] Cell viability at 48 hpi following treatment with compound D (<100 pM) was greater than Ribavirin-treated cells at concentrations up to 200 pM (FIGS. 3A and 3B). Similarly, compound E also generated a significant increase in cell viability of virus-infected cells compared to Ribavirin (FIG. 3D). However, compound F could not rescue the cells at higher virus multiplicity (FIG. 3C). At a longer incubation of 96 hpi, both Ribavirin and compound E lost the capacity to rescue cells from infection (FIGS. 3E and 3H). Conversely, compound D showed remarkable rescue of cell viability even after a prolonged incubation period, indicating excellent inhibitory potential against ZIKV. Therefore, compound D was selected for further testing against ZIKV using more rigorous experimental techniques to confirm its qualitative and quantitative characteristics.Example 3: Inhibition of ZIKV Proliferation 7w Vitro

[0123] To study the mechanism of inhibition that compound D provided, plaque assays were performed using collected untreated and compound treated supernatants. Specifically, IxlO5Vero cells / well were seeded into 12- well plates as described above. For viral quantification, infectious supernatants harvested from the ZIKV -infected cultures were subjected to 10-fold serial dilutions in PBS supplemented with calcium and magnesium ions (Ca2+ / Mg2+) and 0.5% FBS. Diluted viral samples were incubated for 1 hour at room temperature. Thereafter, the inoculum was applied to the cell monolayers and allowed to adsorb. Following adsorption, the inoculum was removed, and cells were washed with PBS. An agar overlay medium was then applied to restrict viral diffusion. After a 4-day incubation period, plaques were visualized by staining with 0.01 % neutral red solution, followed by overnight incubation at 30 °C. Plaques were counted 24 hours later to determine viral titer.

[0124] A statistically significant reduction in viral titer was observed in cultures treated with the test compound D as compared to untreated infected controls (FIGS. 4A-4I). Specifically, the difference in plaque-forming units (PFU) between treated and untreated samples was significant, with a p-value of less than 0.001 (**p < 0.001), as determined by appropriate statistical analysis. These results confirm the antiviral efficacy of compound D under the tested conditions. Indeed, compound D reduced the ZIKV titer by roughly 2-logs at a concentration of 3. 124 pM, and nearly70729-024-logs at a concentration of 6.25pM (FIGS. 4A, 4C, 4G, and 4H). For comparison, the positive control Ribavirin reduced viral titer by 2-logs at a concentration of 100 pM (FIGS. 4A and 4B).

[0125] The ability of compound D to inhibit ZIKV infection at such a low concentration was further tested using a time-of-addition assay. The inhibitor (compound D) concentration was set at 5 pM, based on prior concentration-dependent inhibition results obtained from plaque assays. Vero cells were seeded and infected with ZIKV at a Mol of 1.0, as described above. Following a one-hour infection period and subsequent washing with PBS, the compound D was administered at various time points post-infection (2, 4, 6, 8, 10, 12, and 24 hpi). Additional conditions included pre-treatment with compound D 2-hours prior to infection (-2 hpi), co-treatment at the time of infection (0 hpi), and treatment after the time of infection. At 48 hpi, supernatants were harvested, clarified by centrifugation to remove cell debris, and stored at - 80 °C for downstream analysis, including testing for infectious particle production using plaque assays (protocols described above). Intriguingly, no plaques were produced at any time point of infection (FIG. 41), supporting that compound D may prevent the entry of the virus into the cells at only 5 pM concentration. Furthermore, this data supports that the compound sufficiently prevented viral replication and proliferation when added to infected cells, even as late as 24 hpi.

[0126] To quantify the effect of inhibitor treatment on viral replication, total ZIKV RNA was extracted from the collected supernatants corresponding to each treatment condition. Quantitative reverse transcription PCR (qRT-PCR) was then performed to determine the relative viral RNA load by measuring the number of ZIKV genome copies present in each sample. The resulting data allowed for comparative analysis of viral replication levels between treated and untreated conditions across all time points.

[0127] Specifically, viral RNA from harvested infectious supernatant was purified using a Viral RNA purification kit (Qiagen N.V., Hilden, Germany) and stored at -20 °C. The purified RNA was then quantified via qRT-PCR using a SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Thenno Fisher Scientific Inc., Waltham. MA) and previously published primers targeting the coding region for E protein. See for primers, Kumar et al., Experiments and simulation on ZIKV NS2B-NS3 protease reveal its complex folding. Virology 556: 110-123 (2021). The qRT-PCR assays were performed in biological triplicate using 96-well plates on an Applied Biosystems QuantStudio 6 Pro Real-Time PCR System. Quantification of viral RNA was achieved using a standard curve generated from purified and serially diluted ZIKV-cDNA plasmids of known copy number. This approach enabled accurate determination of viral genome copy number in each sample and facilitated direct comparison of viral load across treatment conditions.

[0128] As show n in FIG. 4D, treatment with the compound at a concentration of 6.25 pM resulted in a statistically significant reduction in viral genome copies in the supernatant, as compared to70729-02 the untreated control (p < 0.0001). Specifically, viral RNA levels were reduced by approximately one logarithmic order of magnitude, indicating robust inhibition of viral replication under these conditions. The antiviral effect was even more pronounced at a compound D concentration of 12.5 p.M, which resulted in a reduction in viral genome copies by at least two logarithmic orders of magnitude (> 2 logs) relative to the untreated control. This dose-dependent inhibition further supports the efficacy of compound D in suppressing ZIKV replication at therapeutically relevant concentrations.

[0129] Enzyme-linked immunosorbent assays (ELIS As) targeting the ZIKV envelope (E) protein were performed on both treated and untreated culture supernatants to evaluate whether E protein levels correlated with viral genome quantities (as measured by qRT-PCR) and infectious virus titers. These assays served to assess the extent to which inhibition of viral replication was accompanied by a reduction in structural protein release, providing an additional measure of antiviral efficacy.

[0130] Briefly, sandwich ELISA assays were conducted to measure the concentration of ZIKV E protein in culture supernatants following infection and compound challenge. High binding ELISA plates (Millipore Sigma, Burlington, MA) were first coated with capture (ZV-67) antibodies diluted in carbonate bicarbonate buffer (Sigma-Aldrich Corporation, St. Louis. MO) the night before. After washing with PBS containing 0.5% Tween 20 (PBS-T). the coated plates were then incubated with blocking buffer (PBST + 5% skim milk) for 1 hour at 37 °C to minimize nonspecific binding. Following additional PBS-T washes, untreated and inhibitor-treated samples (serving as antigen sources) were two-fold serially diluted in blocking buffer and added to the ELISA plates. Plates were incubated at 37 °C for 1 hour to permit antigen-antibody binding, followed by repeated washing with PBS-T.

[0131] Biotinylated ZV-67 antibody, diluted in blocking buffer, was then added to each well as the primary detection antibody and incubated under the same conditions. After further PBS-T washes, horseradish peroxidase (HRP)-conjugated streptavidin diluted in blocking buffer was added as the “secondary” detection reagent. Following incubation and washing, tetramethylbenzidine (TMB) substrate solution was added to each well to initiate colorimetric development. A blue color indicated the presence of ZIKV E protein. The reaction was allowed to proceed for 10 minutes in the dark and was terminated by the addition of 1 N hydrochloric acid (HC1). Plates were incubated for an additional 5 minutes, and absorbance was measured at 450 nm using a SpectraMax iD5 spectrophotometer (Molecular Devices, LLC, San Jose, CA) to quantify E protein levels.

[0132] As illustrated in FIG. 4E, incubation of ZIKV infected cells with compound D at either 12.5 (iM or 25 (iM maintained saturation levels of E protein in the cell supernatant 48 hpi. Notably,70729-02 however, the E protein signal in the compound D-treated samples became undetectable at lower dilutions compared to the untreated control, indicating that the initial concentration of E protein in the treated supernatants was substantially reduced. These results further support that compound treatment led to a marked decrease in viral protein production and release.

[0133] Western blotting was also performed to determine whether cells infected with ZIKV could produce E protein after 72 hours of incubation with various concentrations of inhibitor (compound D). Briefly, the cell lysates were isolated at 48 hpi from untreated and inhibitor-treated virus- infected cells. After collecting the culture supernatants, the remaining adherent cells were washed twice with PBS and harvested into pre-chilled microcentrifuge tubes. The cell suspensions were centrifuged at 8,000 rpm for 5 minutes at 4 °C to pellet the cells, and the resulting supernatants were discarded. The cell pellets were then lysed by incubation with radioimmunoprecipitation assay (RIP A) buffer for 5 minutes, wi th mixing performed by gentle pipetting to ensure complete resuspension. The lysates were subsequently incubated on ice for 15 minutes, with intermittent vortexing to enhance protein extraction. To remove insoluble debns, the lysates were centrifuged at 14,000 rpm for 20 minutes at 4 °C, and the clarified supernatants were collected in another prechilled microcentrifuge tube and stored at -80 °C.

[0134] Total protein concentrations of the cell lysates were determined using the Bradford assay, with absorbance measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA). Equal amounts of protein (100 pg) from untreated and inhibitor-treated cell lysates, corresponding culture supernatants, and purified virus samples were prepared and separated on 10% SDS-poly acry lamide gels. Electrophoresis was performed at a constant voltage of 120 V for 1 hour and 20 minutes. Following electrophoresis, the gels were either transferred to nitrocellulose membranes for immunoblotting or stained directly using Coomassie Brilliant Blue stain. For blotting, membranes were incubated overnight at 4 °C in blocking buffer composed of 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) to prevent non-specific antibody binding.

[0135] The next day, the membranes were washed with TBST and then rocked gently for 1 hour at room temperature with mouse primary antibody (ZV-67 - 1 :200) against E protein of ZIKV and mouse GAPDH antibody (1:10,000), both diluted in blocking buffer. After primary antibody incubation and subsequent TBST washes, membranes were incubated with species-specific antimouse secondary antibodies for 1 hour at room temperature with gentle rocking. Following additional washes to remove unbound secondary' antibodies, the blots were imaged using the Odyssey® infrared imaging system (LI-COR Biosciences, Lincoln, NE), enabling quantitative detection of target proteins.70729-02

[0136] As shown in FIG. 4F, the presence of E protein is only detected at lower concentrations of inhibitor treatment while higher concentrations of inhibitor eliminate the production of E protein. Such results suggest that compound D may also function as a viral genome replication inhibitor in addition to preventing the assembly of flavivirus immature particles.

[0137] The results obtained from the Western blot analysis were corroborated by immunofluorescence assay (IF A) targeting the ZIKV E protein. The IFA provided spatial and qualitative confirmation of E protein expression within infected cells and served as an independent validation of the protein-level differences observed between untreated and inhibitor-treated samples.

[0138] Briefly, for immunofluorescence analysis, 25,000 Vero cells per well were seeded into 24- well plates and infected with ZIKV at a Mol of 1.0, followed by treatment with varying concentrations of the test inhibitor (compound D), as described above. At 48 hpi, supernatants were collected for downstream analysis and the adherent cells were fixed with 3.75% formaldehyde in PBS for 15 minutes at 37 °C. Following fixation, cells were washed with PBS and permeabilized using 0.1% Triton X-100 in PBS for 10 minutes at 37 °C.

[0139] Cells were then incubated with blocking buffer (1% bovine serum albumin in TBST) for 1 hour at 37 °C to minimize non-specific binding. After washing, the cells were incubated overnight at 4 °C with the primary antibody ZV-67 (diluted 1:200 in blocking buffer), which is specific for the ZIKV E protein. After further PBS washes, cells were incubated with a tetramethylrhodamine (TRITC)-conjugated secondary antibody (diluted 1 :500 in blocking buffer) for 1 hour at 37 °C. Nuclei were counterstained with DAPI, followed by final PBS washes. Fluorescent images were captured using a Cytation 7 imaging reader (BioTek Instruments, Inc., Winooski, VT).

[0140] Since treating ZIKV with compound D resulted in a ~ 4-log reduction in titer at a concentration of 6.25 pM (as compared to untreated), a concentration 5pM was utilized to generate virus for morphological visualization under cryogenic transmission electron microscopy (cryo-TEM).

[0141] Specifically, cryo-TEM was employed to visualize morphological differences between untreated and compound D-treated ZIKV particles. For sample preparation, glow-discharged lacey carbon grids (400-mesh copper grids with carbon film; Ted Pella, Inc., Redding, CA) were used. A 2.5 pL aliquot of each purified virus sample was applied to the grids, followed by blotting with Whatman filter paper for 2.5 seconds to remove excess liquid. The grids were then plunge- frozen in liquid ethane cooled by surrounding liquid nitrogen using a Cryo-Pl unger 3 (CP3) freezing system (Gatan, Inc., Pleasanton, CA). Cryo-frozen grids were imaged using a Talos70729-02F200C transmission electron microscope (Thermo Fisher Scientific Inc., Waltham, MA) operating at 200 kV and equipped with a Ceta 16M CMOS detector.

[0142] As shown in FIG. 6A, the untreated virus particles were smooth and round, illustrating the wild type (WT) morphology of mature infectious ZIKV particles. However, the treated virus particles displayed a distorted morphology and seem to generate protein aggregates roughly half the size of WT particles FIG. 6B. This data supports the inability of ZIKV to proliferate when treated with the test compound, instead producing distorted protein aggregates.

[0143] SDS-PAGE and western blot analysis in FIGS. 6C and 6D confinn the presence of E protein in purified untreated samples but not in the treated virus samples, supporting that long term incubation of infected cells with only 5pM of compound D is sufficient to either prevent the release of viral particles capable of being purified or aggregate the particles / E proteins to prevent infection and purification.Example 4: Compound D as an Entry Inhibitor

[0144] Due to the significant inhibition of ZIKV observed with exposure to relatively low concentrations of compound D (as compared to Ribavirin), the potential of compound D to also function as an entry' inhibitor directly was assessed.

[0145] To allow time for the compound to bind to the glycoproteins, compound D was incubated with ZIKV (Mol 1.0) at desired concentrations for 30 minutes at room temperature. After incubation, the mixture was used to infect cells for 1 hour at room temperature. A solution of fresh 2% FBS media and antibiotics was then added to the cells after a single PBS wash and the infection was allowed to progress for 48 hours. At 48 hpi. the supernatant was harvested, aliquoted. and stored at -80 °C for subsequent downstream assays.

[0146] Results of MTT-based colorimetric assays demonstrated that pre-treatment of ZIKV wdth compound D prior to infection rescued cell viability compared to an untreated viral infection. In particular, ZIKV infection alone resulted in approximately 60% cell death, as measured by reduced metabolic activity in infected cells. However, treatment with the test compound D significantly improved cell viability, with a pronounced protective effect observed at concentrations as low was 3.125 pM (FIG. 7A). At this concentration, cell viability was substantially restored, indicating the test compound’s ability to inhibit viral entry or early-stage infection processes.

[0147] To further investigate compound D’s potential to inhibit viral entry. Western blot analysis was performed on cellular lysates collected post-infection (using the protocols previously described herein). The results revealed a concentration-dependent reduction in the expression of the ZIKV E protein. Specifically, increasing concentrations of compound D were inversely70729-02 correlated with E protein levels, supporting the conclusion that the compound interferes wi th early stages of the viral life cycle, including entry and / or initial replication events (FIG. 7B).

[0148] Similar results were obtained through direct plaque assays (performed using the previously described protocols), during which cells were incubated with compound D-preincubated virus for 1-hour. washed to remove any unbound virus, and then covered with an agarose overlay to generate plaques. As seen in FIGS. 7C and 7D, nearly complete inhibition of viral entry was seen at 12.5pM, with substantial decreases in titer also observed at 3.125 pM and 6.25 pM (see also FIG. 7E). This implies that compound D had multiple protein targets that affected both entry and replication, in addition to altering viral assembly via C protein-RNA disruptions.

[0149] Furthermore, IF As using cells infected with ZIKV that had been preincubated with compound D also complemented the plaque assay data (FIG. 8). Altogether, compound D presents a highly robust ability' to prevent ZIKV entry, in addition to reducing viral replication and infectious particle release, at an economically low concentration.Example 5: Compound D Displays Promiscuous Binding Across Both ZIKV Proteins and Flavivirus Species

[0150] To identify the specific viral protein target(s) affected by compound D, binding interactions were evaluated using purified structural and non-structural ZIKV proteins. In particular, changes in intrinsic fluorescence of tryptophan residues within purified ZIKV E protein and NS2B-NS3 protease were monitored upon exposure to increasing concentrations of test compound D. This fluorescence-based assay was used to assess conformational or microenvironmental changes indicative of direct binding between the compound and the viral proteins.

[0151] Primarily, to purify the viral proteins, cells were seeded in 8x T-175 flasks and then infected at > 90% confluency with 0.6 Mol of ZIKV for 1 hour at room temperature. Fresh 2% FBS supplemented DMEM media was added to the cells of 4 flasks while 5 pM of compound D mixed 2% FBS supplemented DMEM media was added to the other 4 flasks. The flasks were incubated at 37 °C with 5% CO2, and supernatant w as harvested at 72 hpi.

[0152] The supernatant was first clarified via centrifugation at 5000 rpm for 30 minutes in 50 ml falcon tubes to remove cellular debris. Then, the supernatant was centrifuged in Ti-50.2 polycarbonate tubes with a 24% sucrose cushion underlay at 32,000 rpm and 4 °C for 2 hours. The sucrose cushion process was repeated once more to remove any residual cellular debris. The resulting pellet was resuspended in 20 mM Tris, 120 mM NaCl, and 1 mM EDTA, pH 8.0 (NTE buffer) overnight and then added to the top of a discontinuous K-tartrate gradient (35-10%, in 5% intervals) in an Ultra-Clear ultracentrifuge tube (Beckman Coulter, Inc., Brea, CA). All visible70729-02 bands for both untreated and treated samples were collected by puncturing a 20-gauge needle through the side wall of the tube and extracting the band via 3 ml syringe. The collected samples were then buffer exchanged to remove K-tartrate and glycerol and concentrated using 100 kDa cutoff Concentrators (Ami con). The concentrated samples were then checked for presence of viral proteins through SDS-PAGE and Western blots.

[0153] NS2B-NS3 protein was purified using the method described by Kumar et al. Kumar et al. (2021), supra. Briefly, E protein was cloned into pET-21(d) expression vector. E. coli (DE3). BL21 cells were transformed, incubated to the specified ODeoo and the protein was induced using 1 mM isopropyl (3-D-l -thiogalactopyranoside (IPTG). The cells were again incubated for another 4 hours before pelleting. The pellet was resuspended in a resuspension buffer consisting of 50 mM Tris-Cl, 300 mM NaCl, 40 mM imidazole, 5% glycerol, pEI 8, and phenylmethylsoulfonyl fluoride (PMSF). Cells were lysed through sonication and centrifuged for 30 minutes at 13,000 rpm. The pellet was washed twice with a wash buffer containing 25 mM Tris-Cl, 2 M urea, 1% Triton X- 100, pH 8. The soft pellet was resuspended in solubilization buffer: 25 mM Tris-Cl, 300 mM NaCl, 8 M urea, pH 8 and centrifuged for 1 hour at 16,000 rpm. The supernatant was diluted using a solubilization buffer and protein was refolded dropwise in resuspension buffer at 4 °C. The refolded protein was then purified using Ni-NTA affinity' chromatography using an elution buffer: 50 mM Tris-Cl, 300 mM NaCl. 1 M imidazole, 5% glycerol, pH 8. Protein was aliquoted and stored at -80 °C before exchanging into 20 mM Tris-Cl, 150 mM NaCl, 5% Glycerol, pH 8 buffer. The identity of envelope protein w as confirmed using SDS PAGE.

[0154] The intrinsic fluorescence of try ptophan residues of envelope and NS2B-NS3 protease were then used to assess the binding of the compound with the proteins. For envelope and protease proteins, buffer consisting of 50 mM Tris-Cl (pH 8) and 20 mM sodium phosphate pH 7.4 was used, respectively. The compound D w as titrated in a solution containing the protein in increasing concentrations from 0.5 nM to 10 pM. The fluorescence spectra were recorded at room temperature using spectrophotometer (Horiba Scientific Fluorolog, model no. 1073) in a 1 mm pathlength quartz cuvette. The tryptophan residues were excited at 295 nm and emission spectra were measured from 310-450 nm wavelength with slit widths kept at 3 nm. To remove any inner- filter effect of the compound, the corresponding absorption spectra w ere also recorded from 250- 450 nm and the fluorescence data was corrected using the equation: Fcor =FobsxantilogWhere FCOr and Fobs represents the corrected and observed fluorescence of the tryptophan residue. Aexand Aemare the excitation and emission absorbance, respectively.70729-02

[0155] As can be seen in FIGS. 9A and 9C, the compound quenched the tryptophan fluorescence of both ZIKV proteins, indicating a high degree of binding. Further, the dissociation constants (Kd) calculated by plotting normalized / .max (E protein: nm; NS2B-NS3 protease: nm) values versus compound D concentrations are shown in FIGS. 9B and 9D. The Ka values were calculated as 19.26 nM (R2 = 0.88) and 8.5 nM (R2 = 0.90) for envelope-F3210-0047 and NS2B-NS3 protease-F3210-0047 complexes, respectively. Accordingly, compound D bound to E protein and NS2B-NS3 protease, and displayed a degree of interaction promiscuity. Having multiple binding partners as a small molecule inhibitor could be beneficial as antiviral option, since a single point mutation in the binding site of one protein during viral genome replication would be unlikely to render the inhibitor obsolete.

[0156] Compound D was also shown to inhibit the proliferation of another member of the Flavivirus genera, namely Dengue virus serotype 2 (DENV2). Using a similar approach to ZIKV inhibition testing, cells were infected with DENV2 (Mol 1.0) for 1 hour at room temperature, washed to remove unbound virus, and then incubated in compound D-containing media for 72 hours. The collected supernatant was then used in plaque assays (according to the protocols described herein) to determine viral titer.

[0157] As shown in FIG. 9E, compound D demonstrated potent antiviral efficacy against DENV2. At a concentration of 25 pM, compound D reduced viral titer by approximately three logarithmic orders of magnitude, and at 50pM, it completely eliminated detectable infectious virus. These results confirm the compound’s strong inhibitory activity' and its potential to fully inhibit not only ZIKV proliferation via binding to multiple protein targets at different points in the viral life cycle, but also DENV2. Indeed, while lower than the binding affinities between compound D and ZIKV C protein, the in-silico binding affinity' between compound D and DENV2 C protein was also found to be energetically favorable. Taken together, such findings support compound D as a prime candidate for further testing as a general, cross-species flavivirus inhibitor.Example 6: Comparative Antiviral Efficacy of Compound D and Analog

[0158] To compare the antiviral efficacy of a reference compound (compound D, known to be effective as shown above) with an analog (compound C, an iteration of compound D), ZIKV- infected Vero cells were treated with each compound in vitro at various dosage concentrations and their relative ability to reduce viral infection was measured (FIG. 10). ZIKV -infected, but untreated cells w ere used for a control (WT).

[0159] The inhibitory' potency of compound D and compound C was also evaluated in an infected-cell assay. Dose-response curves were generated and fitted to determine the absolute IC5070729-02 values. Compound D exhibited an absolute IC50 of approximately 4.94 * 1 O'7M (494 nM), whereas compound C demonstrated an absolute IC50 of approximately 7.21 x 10'7M (721 nM), indicating that the compound D was approximately 1.5-fold more potent than compound C.

[0160] Curve-fit analysis further revealed that compound D achieved near-complete inhibition at high concentrations, with a steep inhibition profile and an excellent fit (R2= 0.9962). In contrast, compound C achieved slightly less complete inhibition, exhibited greater variability in the steep portion of the curve, and had a less stable Hill slope, reflected in a broader confidence interval and reduced fit quality (R2= 0.9652).

[0161] These data support that both compounds exhibited sub-micromolar potency against the virus, while compound D demonstrated greater potency, more complete inhibition, and a more reliable dose-response profile.

Claims

70729-02CLAIMS1. A compound comprising the following structure:or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein:X is a hydroxyl group ( — OH), hydrogen (H), or NH2; andR1 is (i) a six-membered carbocyclic group, or (ii) a fused bicyclic carbocyclic group comprising two six-membered rings, wherein each of (i) or (ii) is optionally substituted.

2. The compound of claim 1, wherein R1 is the six-membered carbocyclic group of (i) and substituted with F, Cl, a hydroxyl group, or a methoxy, or R1 the fused bicyclic carbocyclic group of (ii) and substituted with F and a hydroxyl group.

3. The compound of claim 1 or claim 2, wherein X is a hydroxyl group.

4. The compound of claim 1 comprising the structure of:or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

5. The compound of claim 1, comprising the structure of:70729-02(Compound C), or a pharmaceutically acceptable salt, solvate, or hydrate of either of the foregoing.

6. The compound of claim 1, comprising the structure of:(Compound B) or70729-02(Compound B'), or a pharmaceutically acceptable salt, solvate, or hydrate of either of the foregoing.

7. The compound of claim 1, comprising the structure of:(Compound A"), or a pharmaceutically acceptable salt, solvate, or hydrate of any of the foregoing.70729-028. A pharmaceutical composition comprising a therapeutically effective amount of:(a) the compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and (b) a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition of claim 8 formulated for administration via a route selected from the group consisting of parenteral, oral, and intranasal.

10. A radical of the compound of any one of claims 1-7.

11. A method for treating or preventing a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of:(a) the compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; or(b) the pharmaceutical composition of claim 8 or claim 9.

12. The method of claim 11, wherein the viral infection is a flavivirus infection and, optionally, a Zika virus (ZIKV) or a dengue virus (DENV) infection.

13. The method of claim 11, further comprising administering a co-therapy to the subject, wherein the co-therapy comprises:(a) a medicament selected from the group consisting of an immunomodulator, an antiinflammatory’ compound, an antibiotic, an antifungal compound, and an antimicrobial compound;(b) an intravenous drip; or(c) both (a) and (b).

14. The method of claim 13, wherein the co-therapy is administered to the subject simultaneously or sequentially with the compound or pharmaceutical composition.

15. The method of claim 11 or claim 12, wherein administering is parenteral, oral, or intranasal.

16. The method of claim 1 1 or claim 12, wherein administering is intravenous, intramuscular, or subcutaneous.70729-0217. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or the pharmaceutical composition of claim 8 or claim 9, for use in treating or preventing a flavivirus infection in a subject.

18. The compound or pharmaceutical composition of claim 17, wherein the flavivirus is ZIKV or DENV.

19. The compound or pharmaceutical composition of claim 17, formulated for administration via a route selected from the group consisting of parenteral, oral, and intranasal.

20. A process for the manufacture of a medicament for the treatment or prevention of a flavivirus infection in a subject, the process comprising formulating the compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, with one or more pharmaceutically acceptable carriers or excipients.

21. A method for inhibiting Zika virus (ZIKV) or dengue virus (DENV) replication, the method comprising administering to the subject a therapeutically effective amount of:(a) the compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; or(b) the pharmaceutical composition of claim 8 or claim 9.

22. The method of claim 21, wherein the therapeutically effective amount is from about 6.25 pM to about 12.5 pM.

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