INTRODUCTION

[001] COVID-19 is a global health crisis caused by the novel coronavirus SARS-CoV-2. In severe cases, SARS-CoV-2 infection causes respiratory failure and death. The recent pandemic spread of SARS-CoV-2 infections has resulted in more than 95 million COVID-19 cases and more than 2 million deaths (as of 1/20/2021).

[002] A SARS-CoV-2 variant, SARS-CoV-2 VUI 202012/01, or B.l.1.7, has been identified in the United Kingdom, United States, France, Spain, Denmark and Australia and Japan. This variant has caused more than 50% of CO VID-19 cases from October 5 and December 13, 2020, in the United Kingdom, primarily in individuals less than age 60. An observed increase in the virus reproduction number (Ro), the number of people that one infected person can infect, from 1.1 to 1.5 suggests that UK variant B.l.1.7 is significantly more contagious than the original Wuhan strain (Wuhan-Hu-1). Previous studies revealed the importance of a specific mutation in increasing transmissibility of SARS-CoV-2: an aspartate to glycine mutation at position 614 (D614G) of the spike glycoprotein (Korber B et al. "Tracking changes in SARS-CoV-2 spike: Evidence that D614G increases infectivity of the COVID-19 virus." Cell. 2020; 182(4):812-827). D614G was found in 10% of global sequences by March 1, 2020, and 78% of global sequences by May 29 ^th . The D614G mutation caused higher infectious titers of spike-pseudotyped virus, indicating a greater replicative efficiency and transmissibility.

[003] SARS-CoV-2 drug discovery efforts to date have focused on traditional targets involved with the virus life cycle including the Spike protein RBD, polymerase and helicase. There is a strong need to discover drugs that counteract functional transmission effects mediated by position 614 of the Spike protein. The continued persistence of this highly infectious SARS-CoV- 2 variant represents an important problem that could potentially be controlled with therapeutic solutions.

SUMMARY

[004] Described are methods to rapidly target structural elements that distinguish viral variants and identify compounds having antiviral activity against those variants. The methods comprise identifying viral variations, such as new mutations, associated with functional mechanisms U'.g., altered infectivity or disease severity), generating structural models of the variants, and using molecular docking to screen chemical compound libraries for binding to the variants using the models. Once compounds are identified in silico using molecular docking, they are screened in vitro to evaluate binding and antiviral activity. In some embodiments, the virus is SARS-CoV-2 and the viral variant is SARS-CoV-2 D614G, which has increased infectivity relative to the parent SARS-CoV-2 virus. In some embodiments, the compounds are small molecules less than 500 molecular weight (daltons). The methods can be used to rabidly identify antiviral therapeutics to combat new virus variants. The methods can be used to identify compounds that specifically bind to mutant proteins in the viral variants.

[005] Described are methods of identifying compounds that bind the SARS-CoV-2 spike glycoprotein D614G variant. The methods comprise modeling the SARS-CoV-2 spike protein based on a cryoEM structure, mapping the location of the D614 in the model, substituting glycine for the aspartate in the model, and using in silico high-throughput molecular docking to analyze binding of compounds to the D614G site. In mapping the D614G mutation, a structural pocket was identified that was generated by the aspartate to glycine mutation. In some embodiments, the structural pocket formed at position 614 of the spike protein in a SARS-CoV-2 D14G variant is used as the basis for selecting drug candidates using the high-throughput molecular docking. The compounds can be from any list or library of known or theoretical compounds. In some embodiments, the spike protein is modeled from the cryoEM structure PDB ID: 6M17 and/or PDB ID: 6VSB. In some embodiments, the compounds are small molecules less than 500 molecular weight (daltons). The methods can be used to rabidly identify antiviral therapeutics that target SARS-CoV-2 D614G.

[006] We have identified a pocket in a SARS-CoV-2 D614G spike protein created by the D614G mutation. In some embodiments, this pocket is used in molecular docking analyses to identify compound that specifically bind to the SARS-CoV2 D614G virus.

[007] Using the described methods, compounds were identified that bind to and/or occupy the pocket formed when the aspartic acid at position 614 of the SARS-CoV-2 spike protein is mutated to glycine. Compounds that bind to and/or occupy the D614G pocket are expected to inhibit virus infection by blocking crucial transmissibility mechanisms.

[008] Described are small molecule drugs that are useful as antiviral therapeutics in preventing and/or treating infection caused by a SARS-CoV-2 variant having a mutation of an aspartate to a glycine at position 614 of the spike protein (D614G mutation) (SARS-CoV-2 D614G). The small molecule antiviral drugs include FDA approved drugs and drugs not currently FDA approved. The SARS-CoV-2 D614G strain can be, but is not limited to SARS-CoV-2 VUI 202012/01 and B.1.1.7. An infection caused by a SARS-CoV-2 D614G can be COVID-19. In some embodiments, the identified drugs can be used to inhibit SARS-CoV-2 D614G replication and/or infection. Exemplary small molecule drugs suitable for preventing or treating SARS-CoV-2 D614G infection are listed in Table 2.

[009] Described are small molecule drugs that specifically bind SARS-CoV-2 D614G variants. The described compounds bind to SARS-CoV-2 isolates having an aspartate to glycine mutation at position 614 of the spike protein with higher affinity than the compounds bind SARS- CoV-2 isolates having an aspartate at position 614 of the spike protein when measured under similar conditions. In some embodiments, the described small molecules bind in a pocket created by the aspartate to glycine mutation at position 614 of the spike protein. The small molecule antiviral drugs include FDA approved drugs and drugs not currently FDA approved. The SARS- CoV-2 isolate can be, but is not limited to SARS-CoV-2 VUI 202012/01 and B. l.1.7. Examplary small molecule drugs that bind to SARS-CoV-2 isolates having an aspartate to glycine mutation at position 614 of the spike protein with higher affinity than the compounds bind SARS-CoV-2 (without the D614G mutation) are listed in Table 2.

[010] The described small molecule drugs may alter interaction between the SI subunit of one spike protein in a trimer and the S2 subunit of a neighboring spike protein in the trimer. Although an understanding of mechanism is not required for practice, it is believed that some or all of the small molecule drugs may act by binding to the D614G spike protein and inhibit cell fusion.

[OH] The identified small molecules can be used to prevent or treat SARS-CoV-2 infection in a subject. In some embodiments, the described small molecules are administered to a subject at risk of infection by SARS-CoV-2 and/or SARS-CoV-2 D614G. In some embodiments, the described small molecules are administered to a subject that has tested positive for SARS-CoV-2 and/or SARS-CoV-2 D614G. In some embodiments, the described small molecules are administered to a subject that has been exposed to SARS-CoV-2 and/or SARS-CoV-2 D614G. In some embodiments, the described small molecules are administered to a subject suspected of having been exposed to SARS-CoV-2 and/or SARS-CoV-2 D614G. In some embodiments, the described small molecules are administered to a subject at risk of being exposed to SARS-CoV-2 and/or SARS-CoV-2 D614G. In some embodiments, the described small molecules are administered to a subject suffering from or diagnosed with COVID- 19 or SARS-CoV-2 and/or SARS-CoV-2 D614G infection. In some embodiments, the described small molecules are administered to a subject to treat acute lung injury in a subject suffering from SARS-CoV-2 and/or SARS-CoV-2 D614G infection. [012] In some embodiments, a small molecule drug identified as being useful in the prevention and/or treatment of SARS-CoV-2 D614G infection is an FDA approved drug.

[013] In some embodiments, the compounds identified in the molecular docking analyses of SARS-CoV-2 D614G can be combined with existing medications such as, but not limited to, antiviral medications, remdesivir, anti-inflammatory medications, and steroids.

[014] In some embodiments, the small molecule drug is selected from the drugs in Table 2.

BRIEF DESCRIPTION OF THE FIGURES

[015] FIG. 1. Drawing illustrating the location of mutated positions that distinguish the United Kingdom variant SARS-CoV-2 VUI 202012/01 (SARS-CoV-2 UK) from Wuhan-Hu- 1 (GENBANK accession number MN908947). NTD = N-terminal domain; RBD = receptor binding domain; FP = fusion peptide; HR1 = heptad repeat 1 ; HR2 = heptad repeat 2; TM = transmembrane anchor; IC = intracellular tail.

[016] FIG. 2. Ribbon diagram model of SARS-CoV-2 UK variant spike glycoprotein based on the structure of the trimeric protomer (PDB 6VSB). Sites that differ SARS-CoV-2 UK variant and Wuhan-Hu-1 are shown as with spheres. One chain of the spike trimer is shown with the RBD in the up conformation.

[017] FIG. 3. Ribbon diagram model of the SARS-CoV-2 UK variant spike protein trimer complexed to ACE2. ACE2 is shown in above the trimer. The modeled interaction between ACE2 and the RBD was based on the 2019-nCoV RBD/ACE2 complex (PDB 6M17).

[018] FIG. 4. Illustration of the structural modeling of the SARS-CoV-2 D614G variant prefusion spike protein trimer. The spike protein Receptor Binding Domain in the up conformation is shown interacting with ACE2 (top). Position 614 is shown as red in the box at the interface between the SI subunit and the S2 subunit of the neighboring molecule in the trimer.

[019] FIG. 5. Diagram illustrating molecular modeling ofN501 of Wuhan-Hu-1 and Y501 of SARS-CoV-2 UK relative to ACE2. The N501Ymutation in SARS-CoV-2 UK variant increases affinity for ACE2 by altering intermolecular interactions. Asn501 (N501) in the spike glycoprotein from Wuhan-Hu- 1 forms a H bond (dashes) with Tyr41 in ACE2 (upper panel). Tyr501 (Y501) in SARS-CoV-2 UK forms an aromatic stack with Tyr41 and a H bond with Lys353 of ACE2 (lower panel).

[020] FIG. 6. Diagrams illustrating molecular modeling of mutations in SARS-CoV-2 UK that hinder intermolecular interactions between subunits of the spike glycoprotein. Ala570 in the spike glycoprotein from Wuhan-Hu-1 forms an intermolecular van der Waals contact (dashes) with the main chain amide of Lys964 from the neighboring chain (upper left panel). Asp570 in the SARS-CoV-2 UK variant is expected to form a repulsive interaction (dashes) because of a potential clash with the main chain of the neighboring chain (lower left panel). Asp614 in the spike glycoprotein from Wuhan-Hu- 1 forms an interm olecular H bond with Thr859 a neighboring chain (upper middle panel). Gly614 in the SARS-CoV-2 UK variant results in a loss of a H bond with Thr859 at the interface between two spike protein subunits of the trimeric protomer forming a potentially druggable structural pocket (lower middle panel). Ser982 in the spike glycoprotein from Wuhan-Hu-1 forms an intermolecular H bond with Thr547 of a neighboring chain (upper right panel). Ala982 in the spike protein of the UK variant prevents H bonding with Thr547 of a neighboring chain (lower right panel).

[021] FIG 7. Illustration showing selection of FDA approved drugs that bind SARS-CoV-2 D614G. In the top panel, aspartic acid (D) at position 614 (arrow) is at the intermolecular interface between SI and S2 (PDB 6VSB). In the bottom panel, the model is shown with spike protein variant position 614 glycine (G). 1,207 FDA approved drugs were positioned in the D614G pocket by molecular docking. Sulfoxone is shown in the D614G pocket (stick model, bottom panel). Sulfoxone is predicted to bind with an estimated AG value of -24.4 kcal/mol by AutoDock Vina. [022] FIG. 8. Diagram illustrating molecular modeling of sulfoxone in the G614 pocket of SARS-CoV-2 UK. Sulfoxone was predicted to bind the interface between subunits of the spike protomer at position 614 with an estimated AG value of -24.4 kcal/mol.

[023] FIG. 9. Graph illustrating replication kinetics of the USA-WA1/2020 SARS-CoV-2 isolate on ACE2 -transfected A549 cells. Replication kinetics were evaluated in a 6-well plate and viral burden was determined by plaque assay on Vero E6 cells.

[024] FIG. 10. Graph illustrating antiviral activity of remdesivir against USA-WA1/2020 strain of SARS-CoV-2 on ACE2-A549 cells. Viral burden was measured by plaque assay on Vero E6 cells. Each data point represents the geometric mean of three independent samples and error bars correspond to one standard deviation. The dashed line signifies the plaque assay limit of detection.

DETAILED DESCRIPTION

[025] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “an oligomer” includes a plurality of oligomers and the like. The conjunction "or" is to be interpreted in the inclusive sense, i.e., as equivalent to "and/or," unless the inclusive sense would be unreasonable in the context.

[026] In general, the term "about" indicates insubstantial variation in a quantity of a component of a composition not having any significant effect on the activity or stability of the composition. When the specification discloses a specific value for a parameter, the specification should be understood as alternatively disclosing the parameter at "about" that value. All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as "not including the endpoints"; thus, for example, "within 10-15" includes the values 10 and 15. Also, the use of "comprise," "comprises, " "comprising,” "contain," "contains," "containing," "include," "includes," and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. To the extent that any material incorporated by reference is inconsistent with the express content of this disclosure, the express content controls.

[027] Unless specifically noted, embodiments in the specification that recite "comprising" various components are also contemplated as "consisting of' or "consisting essentially of' the recited components. Embodiments in the specification that recite "consisting essentially of' various components are also contemplated as "consisting of'. "Consisting essentially of' means that additional component(s), composition(s) or method step(s) that do not materially change the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods.

[028] A "SARS-CoV-2related betacoronavirus" is a virus that is considered highly similar to or phylogenetically similar to 2003 SARS-CoV or 2019 SARS-CoV-2. A SARS-CoV-related betacoronaviruses can be a betacoronavirus in Lineage B, subgenus Sarbecovirus, or Lineage D, subgenus Nobecovirus. Betacoronaviruses in Lineage A, subgenus Embecovirus (including common human coronaviruses OC43 and HKU1) and Lineage C, subgenus Merbecovirus (including Middle East respiratory syndrome coronavirus) are not considered SARS-CoV-related betacoronaviruses.

[029] A "SARS-CoV-2 spike protein" (also termed "spike protein," "S protein," or S glycoprotein") is a glycoprotein on mature SARS-CoV-2. The SARS-CoV-2 spike protein forms a trimer, with three receptor-binding SI heads on top of a trimeric membrane fusion S2 stalk (Shang J et al. "Structural basis of receptor recognition by SARS-CoV-2." Nature. 2020; 581(7807):221-224). SARS-CoV-2 SI subunit contains a receptor-binding domain (RBD) that specifically recognizes angiotensin-converting enzyme 2 (ACE2) as a receptor to gain entry in host cells. The SARS-CoV-2 spike glycoprotein is activated by proteolysis at the S1/S2 boundary (furin cleavage site), after which SI dissociates from S2 and undergoes a structural change to mediate fusion between the virus and host cell membranes. The fusion peptide sequence in S2, and Heptad Repeat sequences 1 and 2 in S2, are essential elements for membrane fusion.

[030] "SARS-CoV-2 D614G" is a SARS-CoV-2 virus variant having an aspartate (aspartic acid) to glycine mutation are position 614 of the spike protein amino acid sequence. A SARS-CoV- 2 D614G virus may have one or more additional mutations relative to the original SARS-CoV-2 Wuhan strain (Wuhan-Hu- 1, GENBANK accession number MN908947). Examples of SARS- CoV-2 D614G include, but are not limited to, SARS-CoV-2 VUI 202012/01 (also called SARS- CoV-2 UK). The D614G mutation has been suggested to increase viral infectivity and enhance replication in human lung epithelial cells, therefore supporting an increase in viral fitness.

[031] A “homologous” sequence (e.g., nucleic acid sequence or amino acid sequence) refers to a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (z.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences.

[032] Peptide variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. An amino acid sequence modification can be a substitution, insertion, or deletion. Insertions include amino and/or carboxyl terminal additions as well as intrasequence insertions of single or multiple amino acid residues. Deletions include the removal of one or more amino acid residues from the peptide sequence. Substitutions include substitution of an amino acid residue at a given position in the amino acid sequence with a different amino acid. Insertions, deletions, and substitutions can occur at a single position or multiple positions. Insertions, deletions, and substitutions can occur at adjacent positions and/or non- adjacent positions. In some embodiments the one or more of the substitutions is a conservative amino acid substitution. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final S-protein polypeptide. For a peptide differing by 0, 1, 2, or 3 amino acids from a reference sequence, the peptide can have substitutions, insertions, or deletions of 0, 1, 2, or 3 amino acids in any combination or order.

[033] A "derivative" or "structural analog" of a first compound is a compound that has a three dimensional structure that is similar to at least a part of that the first compound. In some embodiments, a derivative or structural analog is a compound that is derived from, or imagined to derive from, another compound such as by substitution of one atom or group with another atom or group. In some embodiments, derivatives or structural analogs are compounds that at least theoretically can be formed from a common precursor compound.

[034] An “active ingredient” is any component of a drug product intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body of humans or other animals. Active ingredients include those components of the product that may undergo chemical change during the manufacture of the drug product and be present in the drug product in a modified form intended to furnish the specified activity or effect. A dosage form for a pharmaceutical contains the active pharmaceutical ingredient, which is the drug substance itself, and excipients, which are the ingredients of the tablet, or the liquid in which the active agent is suspended, or other material that is pharmaceutically inert. During formulation development, the excipients can be selected so that the active ingredient can reach the target site in the body at the desired rate and extent.

[035] A “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount (dose) of a described active pharmaceutical ingredient or pharmaceutical composition to produce the intended pharmacological, therapeutic, or preventive result. An "effective amount" can also refer to the amount of, for example an excipient, in a pharmaceutical composition that is sufficient to achieve the desired property of the composition. An effective amount can be administered in one or more administrations, applications, or dosages. [036] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of active pharmaceutical ingredient and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration. [037] The terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease or condition in a subject. Treating generally refers to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term treatment can include: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. Treating can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with coronavirus infection that or those in which infection is to be prevented. Treating can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the inflammation without preventing viral replication.

[038] The recently solved cryoEM structure of the SARS-CoV-2 spike protein was used to provide a basis for a novel molecular docking approach in an attempt to identify compounds that alter SARS-CoV-2 D614G infectivity and/or replication. By modeling the spike protein and the effect of an aspartate to glycine substitution at position 614, it was found that the D614G mutation in the SARS-CoV-2 spike protein creates a pocket (D614G pocket).

[039] Using molecular docking analysis, candidate drugs were identified based upon their predicted interactions with the SARS-CoV-2 D614G binding site, the D614G pocket. In some embodiments, small molecules less than or equal to 500 MW are screened by in silica molecular docking for binding in or to the D614G pocket. In some embodiments, molecular docking is used to identify small molecules (<500 MW) that bind to SARS-CoV-2 D614G with higher affinity than SARS-CoV-2 having an aspartate at position 614 of the spike protein. In some embodiments, molecular docking is used to identify small molecules (<500 MW) having a negative AG for binding to the D614G pocket of SARS-CoV-2 D614G. In some embodiments, molecular docking is used to identify small molecules (<500 MW) having a negative AG of -30 or less, -29 or less, -28 or less, -27 or less, -26 or less, -25 or less, -24 or less, -23 or less, -22 or less, -21 or less, -20 or less, -19 or less, -18 or less, -17 or less, -16 or less, or -15 or less, for binding to the D614G pocket of SARS-CoV-2 D614G.

[040] In silico molecular docking indicates that the compounds listed in Table 2 have the potential to bind specifically to SARS-CoV-2 D614G and inhibit or reduce SARS-CoV-2 D614G cell binding, cell fusion, infectivity and/or replication.

[041] In some embodiments, the small molecules identified by the molecular docking analyses bind to the D614G pocket and create allosteric changes that impair cell binding, cell fusion, infectivity and/or replication of SARS-CoV-2 D614G.

[042] In some embodiments, the small molecules identified by the molecular docking analyses that bind to the D614G pocket of a SARS-CoV-2 D614G virus are selected from the group consisting of: Adenosine, Adenosine monophosphate, Adenosine diphosphate, Adenosine triphosphate, Adenosine triphosphate disodium, Probenecid, gamma-Glutamylcysteinylglycine, Fludarabine Phosphate, Vidarabine Phosphate, Carbutamide, Etebenecid, Salmeterol, Sodium sulfoxone, 2,2-dichloro-N-[l,3-dihydroxy-l-(4-nitrophenyl)propan-2-yl]a cetamide, Sulfadoxine, 5-Methoxysulfadiazine, Olsalazine, Sulfamethoxypyridazine, Quinethazon, [5-(6-Aminopurin-9- yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate;[5-(2,4-dioxopyrimidin-l-yl)-3,4- dihydroxyoxolan-2-yl]methyl dihydrogen phosphate, Biotin, Mucocis, Ethaverine, Tartrate, Dicumarol, D-Tartaric Acid, Sulfamethizole, Aminopterin, 5-(2-Oxo-hexahydro-thieno[3,4- d]imidazol-6-yl)-pentanoic acid, Ketanserin, lofendylate, Sulfamerazine, Methallenestril, Copper oleate, Tartaric Acid, DL-Amethopterin, 4-amino-N-[(5Z)-3,4-dimethyl-2,5-dihydro-l,2-oxazol- 5-ylidene]benzene-l-sulfonamide, Tolbutamide, Fusaric Acid, arsenic(3+);2,3- bis(sulfanyl)butanedioic acid, 1 Ibeta-Prostaglandin El, Chlorthalidone, Methotrexate, Protirelin, Mecillinam, Glisoxepide, Methantheline bromide, Polygris, Mephobarbital, Pranoprofen, Mesoridazine besylate, Glybuzole, Papaverine, Melatonin, 4-Butyl-3-propyl-lH-quinolin-2- one;2,4,6-trinitrophenol, Nialamide, Azaribine, Succinate, Mephentoin, Dantrolene, Clidanac, Oxyfedrine hydrochloride, Nadolol, Succimer, lodoquinol, Proglumide, and Metharbital or a salt or a derivative or structural analog thereof.

[043] In some embodiments, the small molecules of Table 2 can be administered to a subject to decrease SARS-CoV-2 D614G disease burden. In some embodiments, the small molecules or Table 2 can be administered to a subject to decrease SARS-CoV-2 D614G viral transmission. In some embodiments, the small molecules or Table 2 can be administered to a subject to inhibit SARS-CoV-2 D614G infection, decrease the likelihood of infection, decrease the severity of infection, and/or decrease the severity or duration of infection. In some embodiments, the small molecules or Table 2 can be administered to a subject to decrease the severity or duration of one or more symptoms associated with SARS-CoV-2 D614G infection. In some embodiments, the small molecules or Table 2 can be administered to a subject to inhibit SARS-CoV-2 D614G entry into host cells. In some embodiments, the small molecules or Table 2 can be administered to a subject to reduce disease burden caused by SARS-CoV-2 D14G infection.

[044] In some embodiments, the small molecules of Table 2 can be used to inhibit SARS- CoV-2 D614G entry into ACE2-expressing host cells, such as, but not limited to, human airway epithelia cells. In some embodiments, the small molecules of Table 2 can be used to inhibit SARS- CoV-2 D614G entry into human airway epithelia cells.

[045] Described are methods of decreasing disease burden, decreasing viral transmission, preventing infection, decreasing the likelihood of infection, decreasing the severity of infection, decreasing the severity or duration of infection and/or decreasing the severity or duration of one or more symptoms associated with SARS-CoV-2 D614G infection. The methods comprise administering one or more of the small molecules of Table 2 to a subject that is infected with a SARS-CoV-2 D614G, suspected of being infected with a SARS-CoV-2 D614G, or at risk of being infected with a SARS-CoV-2 D614G.

[046] In some embodiments, the methods comprise administering one or more of the described small molecules of Table 2 to a subject.

[047] In some embodiments, one or more of the described small molecules is administered to a subject at their recognized dosage levels. In some embodiments, the described small molecules are administered according to their recognized administration route. Any of the small molecules of Table 2 can be provided as a liquid formulation, as an aerosol, as a tablet, as a coated tablet, as a chewable table, as a powder, or as a capsule or a combination thereof. The small molecule can be administered orally, by inhalation, or parenterally, or a combination thereof. Parenteral administration can be, but is not limited to, intramuscular administration and intravenous administration. In some embodiments, the small molecule is administered orally. In some embodiments, the small molecule is administered orally in water or phosphate buffered saline at pH 7.4. In some embodiments, the small molecule is administered parenterally. In some embodiments, the drug is administered by nasal administration. In some embodiments, the drug is administered as a nasal spray. In some embodiments, the small molecule is administered 1, 2, 3, 4, 5, or 6 times per day.

[048] Compounds identified in the molecular docking analysis, e.g., the compounds listed in Table 2, can be administered to a subject to reduce the duration of SARS-CoV-2 D614G infection, reduce the severity of SARS-CoV-2 D614G infection, reduce the severity of one or more SARS- CoV-2 D614G infection-related symptoms, reduce the likelihood of SARS-CoV-2 D614G infection, reduce the likelihood of one or more SARS-CoV-2 D614G infection-related symptoms, reduce SARS-CoV-2 D614G replication, reduce the likelihood of developing a SARS-CoV-2 D614G -related illness, or reduce the likelihood of developing COVID-19.

[049] In some embodiments, the compounds identified in the molecular docking analysis are administered to a subject at risk of infection by SARS-CoV-2 D614G, a subject that has tested positive for SARS-CoV-2 D614G, a subject that has been exposed to SARS-CoV-2 D614G, a subject suspected of having been exposed to SARS-CoV-2 D614G, a subject at risk of being exposed to SARS-CoV-2 D614G, a subject suffering from or diagnosed with COVID-19, or a subject suffering from acute lung injury due to SARS-CoV-2 D614G infection.

[050] In some embodiments, the compounds are formulated with one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers), thereby forming a pharmaceutical composition or medicament suitable for in vivo delivery to a subject, such as a human. The phrase “pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

[051] A pharmaceutical composition or medicament includes a pharmacologically effective amount of the active compound and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[052] Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. [053] The pharmaceutical compositions can contain other additional components commonly found in pharmaceutical compositions. Such additional components can include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.).

[054] A carrier can be, but is not limited to, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. A carrier may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. A carrier may also contain isotonic agents, such as sugars, polyalcohols, sodium chloride, and the like into the compositions.

[055] Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the subject from a pharmacological/toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a subject. In some embodiments, a pharmaceutically acceptable compound is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[056] In some embodiments, the pharmaceutical compositions further comprise one or more additional active ingredients. The additional active ingredient can be, but is not limited to, an additional antiviral therapeutic, a pain reliver, or a nasal decongestant. In some embodiments, the additional active ingredient comprises an additional antiviral therapeutic. In some embodiments, the additional active ingredient comprises a pain reliever. The pain reliever can be, but is not limited to, acetaminophen, NSAID, ibuprofen, or naproxen. In some embodiments, the pain reliever is acetaminophen. The amount of acetaminophen in the formulation can be about 325 to about 1000 mg. In some embodiments, the additional active ingredient comprises a nasal decongestant. The nasal decongestant can be, but is not limited to, phenylephrine and pseudoephedrine.

[057] The pharmaceutical composition can be in a liquid formulation, a tablet, a coated tablet, a chewable tablet, a powder (e.g., a lyophilized powder), or a capsule. The pharmaceutical composition can be administered orally, by inhalation, (e.g., nasally) or parenterally. Parenteral administration can be, but is not limited to, intramuscular administration and intravenous administration. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered parenterally.

[058] It is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

EXAMPLES

Example 1. Sequences of UK variant B.l.1. 7 (SARS-CoV-2 D614G) and reference strain Wuhan- Hu-1.

[059] The Wuhan-Hu-1 sequence was used as a wild-type reference (GENBANK accession number MN908947). A Threat Assessment Brief, December 20, 2020, was used to define mutations that distinguish the UK variant B.l.1.7 (e.g, SARS-CoV-2 D614G) (European Center for Disease Prevention and Control).

Example 2. Modeling mutations in the UK variant B.1.1. 7 spike glycoprotein

[060] The cry oEM structure of the SARS-CoV-2 (Wuhan-Hu- 1) RBD/ACE2-B0AT 1 complex (Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank PDB ID: 6M17, FIG. 3) (YanR et al. "Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2." Science. 2020; 367(6485): 1444-1448) was used as the basis for modeling N501Y. The prefusion SARS-CoV-2 (Wuhan-Hu- 1) spike glycoprotein with a single receptor-binding domain up (RCSG Protein Data Bank PDB ID: 6VSB, FIG. 2) (Wrapp D et al. "Cryo-EM structure ofthe 2019-nCoV spike in the prefusion conformation." Science. 2020; 367(6483): 1260-1263) was used as the basis for modeling D570A, D614G, T716I, S982A and D1118H. Side chains were mutated in COOT (Emsley P et al. "Features and development of coot." Acta Crystallogr D Biol Crystallogr. 2010 66(4):486-501) using rotamers that represent a local energy minimum of torsional angles.

[061] The majority of substitutions in the spike protein that distinguish UK variant B.l.1.7 were determined to be located at sites of intermolecular interaction (4 out of 7 mutations). One mutation (N501Y) enhanced the affinity of the spike protein with ACE2 (Starr TN, Greaney AJ, Hilton SK, et al. Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding. Cell. 2020; 182(5): 1295-1310. e20). N501Y was modeled based on the cryoEM structure of the Wuhan-Hu- 1 spike protein/ ACE2 complex (Yan R et al. "Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. " Science. 2020; 367(6485): 1444-1448) (FIG. 3, PDB 6M17), indicating that the gain in affinity likely results from aromatic interactions (TC stacking) between Tyr501 and Tyr41 of ACE2 (FIG. 5).

[062] The modeling analyses indicated that three mutations in the spike protein that distinguish UK variant B.1.1.7 are located at interfaces between subunits of the trimeric protomer. Unlike the mutation at position 501, which increased affinity for ACE2, the 3 substitutions at spike trimer interfaces likely reduce intermolecular binding affinity. The mutations likely influence stability/lability in a manner that enhances dynamic virus processes that include spike protein cleavage, structural rearrangement and host cell fusion mechanisms.

[063] Intermolecular interactions between individual chains of the SARS-CoV-2 spike glycoprotein were observed at positions A570, D614, and S982 in the original Wuhan strain (Wuhan-Hu-1) (FIG. 6).

[064] The A570D substitution in the UK variant B.1.1.7 variant introduces a side chain unable to form van der Waals or H bond interactions with the neighboring chain because of steric clash with the backbone amide of K964 (FIG. 6).

[065] Using the recently solved cryoEM structure of the prefusion SARS-CoV-2 Spike glycoprotein with a single RBD in the up conformation (Wrapp, Wang et al 2020) (PDB code 6VSB, FIG. 2) and the cryoEM structure of the SARS-CoV-2 RBD/ACE2-B0AT1 complex (PDB 6M17) (Yan, Zhang et al. 2020), the location of position 614 in relation to the ACE2 binding site in the RBD was mapped. An atomic model of the SARS-CoV-2 D614G variant (FIG. 4) was generated by mutating the side chain using Crystallographic Object-Oriented Toolkit (COOT) (Emsley et al. "Features and development of Coot." Acta Crystallographica. Section D, Biological crystallography, 66(4):486-501 (2010)) and energy minimization (Adams et al. "PHENIX: a comprehensive Python-based system for macromolecular structure solution." Acta Crystallographica. Section D, Biological crystallography, 66(2):213-221 (2010)). D614 in the spike protein of Wuhan-Hu-1 was determined to form an intermolecular H bond with Thr859 in the neighboring chain of the trimer (FIG. 6, FIG. 7). Modeling of the D614G substitution in the UK variant B.l.1.7 indicated this mutation removes side chain atoms at this position (thus preventing intermolecular H bonding potential) and results in the formation of a structural pocket at the interface between 614G in SI and 859T in S2 of the neighboring chain of the Spike glycoprotein trimer ((FIG. 6, FIG. 7, and FIG. 8). The D614G mutation in the spike protein has been observed in the majority of SARS-CoV-2 sequences since June 2020 (Korber B et al. "Tracking changes in SARS-CoV-2 spike: Evidence that D614G increases infectivity of the COVID-19 virus." Cell. 2020; 182(4): 812-827), including the UK variant B.1.1.7 (Kirby T. New variant of SARS-CoV-2 in UK causes surge of CO VID-19. Lancet Respir Med. 2021. doi: S2213- 2600(21):5-9).

[066] S982 was determined to be located in the S2 subunit of the Wuhan-Hu- 1 spike glycoprotein, forming a H bond with T547 in the neighboring chain of the spike protein trimer (FIG. 6). The S982A in UK variant B.l.1.7 lacks intermol ecular H bonding potential between spike protein subunits at this site (FIG. 6).

[067] Collectively, these data suggest that: 1) the UK variant B.l.1.7 exhibits a change that enhances affinity for the coronavirus receptor ACE2 (N501Y), and 2) mutations may enhance dynamic virus fusion mechanisms by reducing intermolecular stability of spike protein trimer (A570D, D614G, S982A). Changes in intermolecular contacts are expected to improve dynamic mechanisms involved in spike protein cleavage, structural rearrangement and host cell membrane fusion. Emerging mutations such as D614G can serve as the basis for drug discovery efforts to target specific highly transmissible variants such as UK variant B.l.1.7.

[068] P681H represents a potentially important difference between Wuhan-Hu- 1 sequence and UK variant B.1.1.7. Position 681 is located adjacent to the RRAR proprotein convertase motif considered a hallmark of high pathogenesis (PRRAR in Wuhan-Hu-1 (Shang J et al. "Structural basis of receptor recognition by SARS-CoV-2." Nature. 2020; 581(7807):221-224), HRRAR in B. l.1.7). This site is cleaved by furin and other proteases to separate the SI and S2 subunits of the spike protein, which undergo structural rearrangement and fusion with host cell membranes mediated by heptad repeat domains in S2. Since S1/S2 cleavage occurs in endosomes with acid pH, a protonated histidine at position 681 of the UK variant B.l.1.7 has the potential to influence the rate of spike protein cleavage and subsequent mechanisms to gain cell entry.

[069] To address the question of whether or not mutations that distinguish the UK variant B. l.1.7 influence T and B cell responses, we analyzed solvent accessibility of positions that differ with Wuhan-Hu-1. Deletion of positions 69-70 and 144-145 represents a loss of T and B cell epitopes, although antibodies directed against these sites are not expected to exhibit neutralizing activity that prevents ACE2 binding. The N501Y mutation present in UK variant B.l.1.7 has the potential to influence T and B cell epitopes, although N and Y are semiconserved neutral polar amino acids. The non-conservative A570D, D614G, P681H differences have the potential to influence HLA binding, but seem unlikely to represent significant antibody binding targets for neutralizing antibodies (primarily directed at the interface between the RDB of the spike protein and ACE2). The D1118H difference is non-conservative, however the side chain is not solvent exposed in the Wuhan-Hu-1 spike protein or UK variant B.1.1.7 model indicating that this position is unlikely to represent an epitope for neutralizing antibody binding. T716I and S982A represent conservative differences with the potential to cause minor alteration of T and B cell epitopes. Although neutralizing antibodies and T cell responses participate in productive immune responses against SARS-CoV-2, evidence suggests that dominant functional antiviral responses are driven by CD4 ⁺ Thi T cells and cytotoxic CD8 ⁺ T cells (Grifoni A et al. "Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID- 19 disease and unexposed individuals." Cell. 2020;181(7):1489-1501). Since T cells recognize peptides in the context of multiple HLA molecules encoded by highly polymorphic genes (HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP), differences between peptides derived from the Wuhan-Hu- 1 spike protein or UK variant B.1.1.7 are not expected to dramatically influence the overall function of polyclonal T cell responsiveness to infection. However, surveillance of neutralizing antibody responses and T responses to peptides derived from the UK variant B.1.1.7 in COVID- 19 patients will be required to understand functional effects on immune recognition.

[070] A summary of the findings from the modeling for each of the 9 sites that differ between sequences of SARS-CoV-2 spike glycoproteins from the original Wuhan strain (Wuhan-Hu-1) and UK variant B.1.1.7 are shown in Table 1.

Table 1. SARS-CoV-2 UK variant B.1.1.7 mutations - location and likely effects determined from molecular modeling.

Example 3. Molecular docking analysis: in silica screen of small molecules (MW <500) for binding the SARS-CoV-2 Spike glycoprotein D614G variant (SARS-CoV-2 D614G)

[071] The highly infectious characteristic of the SARS-CoV-2 surface glycoprotein D614G variant suggests that this position is of importance in virus transmissibility. The location of this position in the Spike glycoprotein is distant from the RBD, in the SI subunit, where D614 forms intermolecular contacts with a neighboring S2 subunit in the prefusion trimeric form of the Spike protein (PDB 6VSB). Since the D614G variation was associated with enhanced infectivity, drugs that bind the SARS-CoV-2 surface glycoprotein at position 614 are expected inhibit virus transmissibility and/or infectivity.

[072] A model of the spike protein trimer of the UK variant B.1.1.7 described above was used as the basis for molecular docking analyses. We used molecular docking (DOCK6 (Brozell et al. "Evaluation of DOCK 6 as a pose generation and database enrichment tool. J. Computer-Aided Molecular Design, 26(6) 749-773 (2012)) and/or Glide (Schrodinger; Friesner, Banks et al 2004) to position libraries of compounds into the structural pocket specific to the SARS-CoV-2 D614G variant, the interface site formed between position D614G and T859 of neighboring chains in the spike protein trimer Candidate compounds were obtained from two libraries: 1) 1,207 FDA approved drugs, and 2) 139,735 small molecules in the Plated Set repository at the NTH National Cancer Center Developmental Therapeutics Program (NCI DTP) (Monga, Sausville 2002). FDA approved drugs with off-target activity against SARS-CoV-2 D614G may be rapidly translated to the clinic. The larger NCI DTP library includes a significantly more diverse collection of small molecules.

[073] Each drug was positioned in 500-1000 different orientations and scored based on polar (e.g., H bond) and non-polar e.g., van der Waals) interactions. The highest scoring drugs, based on estimated AG in kcal/mol, were identified as antiviral candidates against the highly infectious SARS-CoV-2 D614G variant. The results are shown in Table 2.

[074] The steps for molecular docking approach are: 1) define atomic coordinates of SARS- CoV-2 D614G structural model, 2) select atoms located at targeted site, 3) generate molecular surface, 4) define atom charge, 5) format structural coordinates of small molecule libraries, 6) generate spheres that represent potential ligand atoms at D614G site, 7) generate a scoring grid based on polar and non-polar interactions, and 8) identify compounds predicted to bind to the D614G pocket using molecular docking analysis. The compounds identified in the molecular docking analyses are ranked. The output is ranked in 3-dimensional mol2 format.

[075] To prepare the site for docking, all water molecules were removed. The molecular surface of the structure was explored using sets of spheres to describe potential binding pockets at the SARS-CoV-2 D614G variant interface between the equivalent of chains A and B of PDB 6VSB. The sites selected for molecular docking were defined using the SPHGEN program in DOCK (Kolb P et al. 'Docking and chemoinformatic screens for new ligands and targets." Curr Opin Biotechnol . 2009; 20(4):429-436; and Shoichet BK et al. "Lead discovery using molecular docking. Curr Opin Chem Biol. 2002; 6(4): 439-446), which generates a grid of points that reflect the shape of the selected site, then filtered through CLUSTER. The CLUSTER program groups the selected spheres to define the points that were used by DOCK6.7 (UCSF) to match potential ligand atoms with spheres. Intermolecular AMBER energy scoring (van der Waals plus columbic), contact scoring, and bump filtering were implemented in the DOCK program algorithm. Atomic coordinates for 1,207 FDA approved small were positioned in a structural pocket located at position G614 of chain A, and position T859 in chain B. Each drug was docked in 500-1,000 different orientations and scored on the basis of predicted polar (hydrogen bond) and nonpolar (van der Waals) interactions. The most favorable orientation and scores (contact and electrostatic) were calculated. PyMOL (Schrodinger) was used to generate molecular graphic images.

[076] The highest scoring compounds, those with the lowest AG values, based on predicted H bond and van der Waals contacts, are further tested for direct antiviral activity against SARS- CoV-2 D614G.

[077] Molecular docking output grid scores are interpreted by AG values calculated from DOCK6.7, AutoDock Vina, and/or Glide Small molecules predicted to bind with the highest affinity correspond to the lowest AG molecular docking output values. For example, probenecid, with a molecular docking score of AG -28.3 kcal/mol from DOCK6.7 (UCSF), is predicted to bind SARS-CoV-2 D614G with higher affinity compared to sulfoxone, with a molecular docking score of AG -24.4 kcal/mol. [078] Ten drugs were identified in an initial screen that were estimated to bind the UK variant B.1.1.7 at the D614G site with AG values less than -24.0 kcal/mol, including the anti-leprosy drug sulfoxone, AG -24.4 kcal/mol (FIG. 7 and FIG. 8).

[079] We generated a novel atomic model and applied a rapid high-throughput screen to identify drugs selected to bind an emerging viral variant. Using this same strategy, targetable positions in other emerging viral variants can be rapidly modeled and antiviral agents that specifically target the variants quickly identified.

Table 2. Compounds predicted to bind to SAR-CoV-2 D614G

The compounds listed in Table 2 are understood to include salts and/or derivatives or structural analogs thereof.

Example 4. Measurement of antiviral effects of selected small molecules on SARS-CoV-2 D614G.

[080] Predictions from the molecular docking studies are experimentally validated using an infectious SARS-CoV-2 cell-based drug susceptibility assay. The drug susceptibility assays will determine the antiviral activity of these small molecules by measuring the ability of the agent to inhibit the production of infectious virus over time in a human lung cell line.

[081] The compounds identified in the molecular docking analyses are expected to bind in the D614G pocket and alter spike protein interactions or activity. It is expected that pretreatment and/or co-treatment will have a beneficial effect.

[082] The antiviral activity of the selected compounds is tested in virus propagated in Vero E6 cells. Antiviral activity is tested in ACE2-A549 human lung cells to evaluate replication kinetics of wild-type SARS-CoV-2 (USA-WA1/2020 strain) and SARS-CoV-2 D614G (such as the Germany/BAVPATl/2020 isolate (Phan 2020)) (FIG. 9). Virus is inoculated onto ACE2-A549 cells at a multiplicity of infection (MOI) equivalent to 0.03. Wild-type virus was observed to replicate rapidly through a 4 day experiment reaching peak viral titers of 6.1 logic PFU/mL. Slower replication kinetics may give the drug an unfair advantage and faster kinetics and may underestimate drug effectiveness. Thus, we chose an MOI of 0.03 for our plate evaluations.

Toxicity of each agent in the cell lines is analyzed using the commercially available WST-1 assay. Toxicity screens are conducted in parallel with drug assays to determine if any observable antiviral activity is due to cytotoxicity. The EC50/95 and CC50/95 values are determined by estimating sigmoid-Emax models to describe the viral burden data and WST-1 data via maximum likelihood estimation. Compounds that inhibit the production of infectious virus by >2-logw plaque forming units (PFU)/ml at concentrations that are physiologically achievable in humans (based on known pharmacokinetic data for repurposed agents) are further evaluated.

[083] As a control, the antiviral potential of remdesivir was assessed against the USA- WA1/2020 strain of SARS-CoV-2 on ACE2-A549 cells at an MOI of 0.03. Antiviral activity of the compounds identified in the molecular docking analyses are tested in a similar manner. Clinically relevant doses of drugs are used when applicable. Duration of pretreatment is dependent upon the properties of the drug, including potential catalysis and stability. Briefly, ACE2-A549 cells were seeded into 6-well plates 24 h prior to the drug susceptibility assay. After the 24 h incubation period, confluent cell monolayers were infected with SARS-CoV-2 at the above mentioned MOI and virus was allowed to adsorb for 1 h. Unbound virus was then removed from cell monolayers by washing twice with warm saline. Medium containing various concentrations of remdesivir was added to different wells. One well served as a no-treatment control and did not receive drug. Cell supernatants were sampled daily, clarified by high-speed centrifugation, and frozen at -80°C until the end of the study. Infectious viral burden was measured in all samples simultaneously by plaque assay on Vero E6 cells. The effective concentration 50 (EC50) value was determined over the course of the study by calculating the area under the viral burden-time curve for each regimen. An inhibitory sigmoid-Emax model was fit to the area under the curve estimates to yield the EC50 value. The results are shown in FIG. 10. Remdesivir was effective at suppressing infectious virus production yielding an EC50 value of 0.4 pM. This EC50 value is clinically achievable in humans, supporting the feasibility of this agent as a treatment strategy for COVID-19. The study is repeated to test antiviral activity against SARS-CoV-2 D614G. Compounds identified as inhibiting viral infectivity or replication in this assay are further analyzed for efficacy in vivo. [084] Compounds identified in the molecular docking analyses are also tested against live SARS-CoV-2 D614 strain infectivity of one or more of: cell culture ACE-2 expressing mammalian cells, primary airway epithelia cells, animal models, or humans.

Example 5. Combination therapy.

[085] Compounds identified as having antiviral activity against SARS-CoV-2 D614G are combined with other antivirals and tested as described above to identify combinations with enhances antiviral effectiveness.