WO2022169932A1 - Novel treatments for coronavirus and sars-cov-2 infections - Google Patents

Abstract

Described are host-targeted anti-viral therapeutics that target host proteins, pathways, or functions, and inhibit coronavirus infection, cell entry, or replication or reduce viral-induced toxicity. Also described are method of using the host-targeted anti-viral therapeutics to treat or prevent coronavirus infection or disease.

Description

Novel Methods of Treatment for SARS-CoV-2 Infections

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/145,763, filed February 4, 2021, which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT.

[2] This invention was made with government support under grant number TR001427, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[3] The Sequence Listing written in file 573151_T18424_SeqListing.txt is 1 kilobyte in size, was created January 27, 2022, and is hereby incorporated by reference

INTRODUCTION

[4] 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. SARS-CoV- 2 gains access to airway cells through binding to the angiotensin converting enzyme 2 (ACE2).

[5] Viruses require host factors at every step in their life cycle. Both viral factors and host factors can be targeted for antiviral drug design. Host factors represent druggable targets (z.e., host-targeting antivirals) with the potential for broad-spectrum activity against multiple viruses within a given virus family and even across virus families.

[6] There remains a need for antiviral therapies for use in vulnerable immunocompromised individuals, breakthrough infections, in regions where vaccine access is limited, and in the event that antigenically distinct SARS-CoV-2 variants emerge

SUMMARY

[7] Described are pharmaceutical compositions and formulations comprising one or more of amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, or an RNA function inhibitor targeting the XRN1 gene for use in the prevention and/or treatment of a coronavirus infection. Methods of using the pharmaceutical compositions to treat a subject infected by a coronavirus, suspected of being infected by a coronavirus, or at risk of being in infected by a coronavirus are described. In some embodiments, the compounds, pharmaceutical compositions, and methods are used to treat other viral infections, included viruses causing respiratory symptoms.

[8] Described are host-targeted anti-viral therapeutics that target host proteins, pathways, or functions, and inhibit coronavirus infection, cell entry, or replication or reduce viral-induced toxicity. The host-targeted anti-viral therapeutics include, but are not limited to, amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an inhibitory RNA agent targeting the CCZ1 gene, an inhibitory RNA agent targeting the EDC4 gene, or an inhibitory RNA agent targeting the XRN1 gene. The host- targeted anti-viral therapeutics can be used to treat a subject infected by a coronavirus, suspected of being infected by a coronavirus, or at risk of being in infected by a coronavirus The coronavirus can be, but is not limited to, a human cold-causing coronavirus, a betacoronavirus, a SARS-CoV-related betacoronavirus, a SARS-CoV, a SARS-CoV-2, or a MERS-CoV. An infection caused by a coronavirus can be, but is not limited to, common cold, SARS, MERS, and COVID- 19. An infection caused by a SARS- CoV-related betacoronavirus can be, but is not limited to, COVID- 19.

[9] The identified host-targeted anti-viral therapeutics can be used to prevent or treat coronavirus infection in a subject. In some embodiments, the host- targeted anti-viral therapeutics are administered to a subject at risk of infection by coronavirus. In some embodiments, the host- targeted anti-viral therapeutics are administered to a subject that has tested positive for coronavirus. In some embodiments, the host-targeted anti-viral therapeutics are administered to a subject that has been exposed to coronavirus. In some embodiments, the host-targeted anti-viral therapeutics are administered to a subject suspected of having been exposed to coronavirus. In some embodiments, the host-targeted anti-viral therapeutics are administered to a subject at risk of being exposed to coronavirus. In some embodiments, the host-targeted anti-viral therapeutics are administered to a subject suffering from or diagnosed with a coronavirus infection. In some embodiments, the host- targeted anti-viral therapeutics are administered to a subject to reduce one or more symptoms associated with a coronavirus infection. In some embodiments, the host-targeted anti-viral therapeutics are administered to a subject to reduce morbidity associated with coronavirus infection.

[10] The coronavirus can be a member of the subfamily Orthocoronavirinae. A Orthocoronavirinae can be, but is not limited to, an alphacoronavirus, a betacoronavirus, a gammacoronavirus, or a deltacoronavirus. Alphacoronaviruses include, but are not limited to, human coronavirus 229E and human coronavirus NL63. Betacoronavirus include, but are not limited to, SARS-CoV, a MERS-CoV, a SARS-CoV-2, human coronavirus OC43, and KHU4.

[U] The identified host-targeted anti-viral therapeutics can be used to prevent or treat SARS-CoV-2-related betacoronavirus infection in a subject. In some embodiments, the identified host-targeted anti-viral therapeutics are administered to a subject at risk of infection by a SARS-CoV-related betacoronavirus. In some embodiments, the identified host- targeted anti-viral therapeutics are administered to a subject that has tested positive for a SARS-CoV-related betacoronavirus. In some embodiments, the identified host- targeted anti-viral therapeutics are administered to a subject that has been exposed to a SARS-CoV-related betacoronavirus. In some embodiments, identified host-targeted antiviral therapeutics are administered to a subject suspected of having been exposed to a SARS-CoV-related betacoronavirus. In some embodiments, the identified host-targeted anti-viral therapeutics are administered to a subject at risk of being exposed to a SARS- CoV-related betacoronavirus. In some embodiments, the identified host-targeted anti-viral therapeutics are administered to a subject suffering from or diagnosed with a SARS-CoV- related betacoronavirus illness. In some embodiments, the host- targeted anti-viral therapeutics are administered to a subject to reduce one or more symptoms associated with a SARS-CoV-related betacoronavirus infection. In some embodiments, the host-targeted anti-viral therapeutics are administered to a subject to reduce morbidity associated with SARS-CoV-related betacoronavirus infection

[12] In some embodiments, the SARS-CoV-related betacoronavirus is SARS-CoV or SARS-CoV-2. An infection caused by a SARS-CoV-related betacoronavirus can be, but is not limited to, COVID-19. In some embodiments, the described compositions and formulations can be used to inhibit SARS-CoV-related betacoronaviruses replication and/or infection.

[13] The described host-targeted anti-viral therapeutics can be formulated with one or more additional pharmaceutically active ingredients. The described host-targeted anti-viral therapeutics can be formulated with one or more adjuvants, carriers, excipients, or a combination thereof.

[14] The compositions and formulations can be formulated for oral administration, parenteral administration, IV administration, injection, or inhalation (e.g., nasal delivery). The compositions and formulations can be formulated or manufactured as a solid, a powder (e.g., a lyophilized powder), a tablet (e.g., a pill), a capsule, or a liquid. In some embodiments, the compositions and formulations can be formulated for nasal delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

[15] FIG. 1 A. Diagram illustrating Vero E6 screen.

[16] FIG. IB. Diagram illustrating HEK293T-hACE2 screens.

[17] FIG. 2A-B. Diagrams illustrating MAGeCK analysis of genes the promote SARS- CoV-2 infection (A) or OC43 infection (B) in Vero E6 cells.

[18] FIG. 3A-B. MAGeCK analysis of SARS-CoV-2 infection of HEK293T-hACE2 cells at the initial infection (A) and at MOI 0.01 reinfection (B)

[19] FIG. 3C. MAGeCK analysis of SARS-CoV-2 infection of HEK293T-hACE2 cells at MOI 0.1 reinfection.

[20] FIG. 3D-E. MAGeCK analysis of SARS-CoV-2 infection of HEK293T-hACE2 at MOI 0.3, with sgRNAs sequenced from resistant clones in the initial infection (E) and reinfection (F).

[21] FIG. 4A-B. MAGeCK analysis for OC43 infection of HEK293T-hACE2 cells at initial infection (A) and at MOI 0.01 reinfection (B).

[22] FIG. 4C. MAGeCK analysis for OC43 infection of HEK293T-hACE2 cells at MOI 0.1 reinfection.

[23] FIG. 5A-B. (A) Western blot with antibodies directed to CTSL, CCZ1, and EDC4 illustrating gene knockdown in cells transduced with a gene-specific shRNA or empty vector control (EV). Actin expression served as a loading control. (B) Graph illustrating viral genome copy numbers in shRNA-expressing cells compared to the EV control (n = 3 experiments). Error bars denote standard errors of mean and P values were determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).

[24] FIG. 5C. Western blot illustrating EDC4 and XRN1 levels in SAEC-hACE2 cells transduced with a gene-specific sgRNA or in wild- type cells (WT). Actin expression served as a loading control.

[25] FIG. 5D. Graphs illustrating viral titers at various times in SAEC-hACE2 WT, EDC4ko and XRNlko cells infected with SARS-CoV-2 or OC43 and MOI 0.01. Error bars denote standard errors of mean and P values were determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). [26] FIG. 6A. Bar graph illustrating drug inhibition of SARS-CoV-2-induced cytotoxicity in Vero E6 cells following MOI 0.3 infection. Error bars are the mean and standard deviation of 6 biological replicates from two technical replicates

[27] FIG. 6B. Bar graph illustrating SARS-CoV-2 genome replication was measured by RT-qPCR at 2 dpi of Vero E6 cells at MOI 0.01. One-way ANOVA was used to compare inhibitor-treated toxicity values to virus-alone controls. Error bars are the mean and standard deviation of 3 biological replicates.

[28] FIG. 6C-D. Graphs illustrating EC50 (ability of inhibitors to reduce SARS-CoV-2 cytotoxicity) and CC50 (toxicity of inhibitors alone) curves in Vero E6 cells for ABE and AMX. The selectivity indices are also shown. Non-linear regression of the data points was used to determine the EC50 and CC50 values. Error bars indicate standard deviation for all panels (n = 3 biological replicates).

[29] FIG. 6E-F. Graphs illustrating EC50 (ability of inhibitors to reduce SARS-CoV-2 cytotoxicity) and CC50 (toxicity of inhibitors alone) curves in Vero E6 cells for AZ1 and HAR. The selectivity indices are also shown. Non-linear regression of the data points was used to determine the EC50 and CC50 values. Error bars indicate standard deviation for all panels (n = 3 biological replicates).

[30] FIG. 6G-H. Graphs illustrating EC50 (ability of inhibitors to reduce SARS-CoV-2 cytotoxicity) and CC50 (toxicity of inhibitors alone) curves in Vero E6 cells for NIN and PMZ. The selectivity indices are also shown. Non-linear regression of the data points was used to determine the EC50 and CC50 values. Error bars indicate standard deviation for all panels (n = 3 biological replicates).

[31] FIG. 61. Graphs illustrating EC50 (ability of inhibitors to reduce SARS-CoV-2 cytotoxicity) and CC50 (toxicity of inhibitors alone) curves in Vero E6 cells for UC2. The selectivity indices are also shown. Non-linear regression of the data points was used to determine the EC50 and CC50 values. Error bars indicate standard deviation for all panels (n = 3 biological replicates).

[32] FIG. 7A-B. Bar graphs illustrating TCID50 of the indicated compound on inhibition of SARS-CoV-2 (A) and OC43 (B) in SAEC-hACE2 cells compared to DMSO control.

[33] FIG. 7C-H. Graphs illustrating phospholipidosis as measured in SAEC-hACE2 cells across several drug concentrations after 3 days of treatment and normalized to 5 DM of amiodarone (AMD) as a positive lipidosis control. Vertical dashed coincide with inhibitor concentrations used in FIG. 7A-B. Error bars denote standard errors of mean and P values were determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[34] FIG. 7I-N. Graphs illustrating cytotoxicity as measured by LDH release. Vertical dashed coincide with inhibitor concentrations used in FIG. 7A-B. Error bars denote standard errors of mean and P values were determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[35] FIG. 8A. Graphs illustrating EC50 determination for amlexanox and archazolid in inhibiting SARS-CoV-2 injection in human SAEC-ACE2 cells.

[36] FIG. 8B. Graph illustrating effect of AMX and ARCA on TCID50 for viral infection in human SAEC-ACE2 cells

[37] FIG. 8C. Graph illustrating relative viral replication in SEAC ACE2 cells treated with ARC.

[38] FIG. 8D. Graph illustrating effect of various drugs on TCID50 for HCoV in human SAEC-ACE2 cells.

[39] FIG. 9. Graphs illustrating effect of AMX in decreasing SARS-CoV-2 viral load in lung of infected mice.

[40] FIG. 10A. Graphs illustrating cytotoxicity of SARS-CoV-1 in the presence of ABE, AMX, AZ1, BRT, CFZ, and CPZ (black bars - no virus, grey bars - SARS-CoV-2 (MOI 0.2).

[41] FIG. 10B. Graphs illustrating cytotoxicity of SARS-CoV-1 in the presence of HAR, INDY, NIN, OPB, and PMX (black bars - no virus, grey bars - SARS-CoV-2 (MOI 0.2).

[42] FIG. 11. Graphs illustrating IC50 and EC50 determination by cytotoxicity measurement.

[43] FIG. 12. Graphs illustrating SARS-CoV-2 genome equivalents in Vero E6 cells treated with the indicated inhibitors (ABE = 38.5 pM, AMX = 502.9 pM, AZ1 = 47.3 pM, HAR = 70.7 pM, NIN = 7.4 pM, OPB = 28.7 pM, PMZ = 87.9 pM).

[44] FIG. 13 A. Graphs illustrating % cytotoxicity induced by SARS-CoV-2 in cells treated with the indicated combinations. EC50 for HAR + no NIN = 7.16 pg/mL, EC50 for HAR + 2.5 pg/mL NIN = 4.97 pg/mL. EC50 for NIN + no ABE = 1.230 pg/mL, EC50 for 10 pg/mL ABE = 0.1227 pg/mL. (HAR and NON target different networks (cellular pathways), ABE and NIN target the same networks).

[45] FIG. 13B. Graph illustrating % cytotoxicity induced by SARS-CoV-2 in cells treated with the indicated combination. EC50 for AZ 1 + no PMZ = 5.15 pg/mL, EC50 for AZ1 + 15 pg/mL PMZ = 5.998 pg/mL. (AZ1 and PMZ target different networks). [46] FIG. 14A-B. Graphs illustrating EC50 determination of azelastine and diphenhydramine in inhibiting SARS-CoV-2 replication.

[47] FIG. 14C. Graphs illustrating EC50 determination of hydroxyzine in inhibiting SARS-CoV-2 replication.

[48] FIG. 15. Graphs illustrating exemplary synergistic activity between a host-targeted anti-viral therapeutic and diphenhydramine in inhibiting SARS-CoV-2 injectivity and/or replication.

DETAILED DESCRIPTION

[49] 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 “a drug” includes a plurality of drugs 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.

[50] In general, the term “about” indicates 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” or “from 10 to 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.

[51] 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.

[52] Coronaviruses, subfamily Orthocoronavirinae, are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Mild illnesses in humans include some cases of the common cold. More lethal varieties can cause SARS, MERS and COVID- 19. Coronaviruses include, alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. Alphacoronaviruses include human coronavirus 229E and human coronavirus NL63. Betacoronaviruses include, lineage A betacoronaviruses (subgenus Embecovirus), lineage B betacoronaviruses (subgenus Sarbecovirus). lineage C betacoronaviruses (subgenus Merbecovirus), and lineage D betacoronaviruses (subgenus Nobecovirus)' . Lineage B betacoronaviruses include, SARS-CoV, MERS-CoV, SARS- CoV-2, human coronavirus OC43, and KHU4.

[53] A “SARS-CoV-related 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.

[54] An RNA function inhibitor comprises any polynucleotide or nucleic acid analog containing a sequence whose presence or expression in a cell causes the degradation of or inhibits the function or translation of a specific cellular RNA, usually an mRNA, in a sequence-specific manner. Inhibition of RNA can thus effectively inhibit expression of a gene from which the RNA is transcribed. RNA function inhibitors are selected from the group comprising: siRNA, microRNA, interfering RNA or RNAi, dsRNA, RNA Polymerase II transcribed DNAs encoding siRNA or antisense oligonucleotides, RNA Polymerase III transcribed DNAs encoding siRNA or antisense oligonucleotides, ribozymes, antisense oligonucleotides, and antisense nucleic acid, which may be RNA, DNA, or artificial nucleic acid. SiRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 19-25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that direct destruction or translational repression of their mRNA targets. Antisense oligonucleotides comprise sequence that is complimentary to an mRNA. Antisense oligonucleotides include, but are not limited to: morpholinos, 2'-O-mcthyl polynucleotides, DNA, RNA and the like. RNA polymerase III transcribed DNAs contain promoters selected from the list comprising: U6 promoters, Hl promoters, and tRNA promoters. RNA polymerase II promoters include Ul, U2, U4, and U5 promoters, snRNA promoters, microRNA promoters, and mRNA promoters. These DNAs can be delivered to a cell wherein the DNA is transcribed to produce small hairpin siRNAs, separate sense and anti-sense strand linear siRNAs, or RNAs that can function as antisense RNA or ribozymes.

[55] An RNA function inhibitor may be polymerized in vitro, recombinant RNA, contain chimeric sequences, or derivatives of these groups. The RNA function inhibitor may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid may be single, double, triple, or quadruple stranded.

[56] An RNA interference (RNAi) polynucleotide is a molecule capable of inducing RNA interference through interaction with the RNA interference pathway machinery of mammalian cells to degrade or inhibit translation of messenger RNA (mRNA) transcripts of a transgene a sequence specific manner. RNAi polynucleotides may be selected from the group comprising: siRNA, microRNA, double-strand RNA (dsRNA), short hairpin RNA (shRNA), and expression cassettes encoding RNA capable of inducing RNA interference. siRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 21-25 base pairs and having a nucleotide sequence identical (perfectly complementary) or nearly identical (partially complementary) to a coding sequence in an expressed target gene or RNA within the cell. An siRNA may have overhangs, such as dinucleotide 3’ overhangs.

[57] An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. An siRNA molecule of the invention comprises a sense region and an antisense region. In one embodiment, the siRNA of the conjugate is assembled from two oligonucleotide fragments wherein one fragment comprises the nucleotide sequence of the antisense strand of the siRNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siRNA molecule. In another embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. [58] MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nucleotides long that direct destruction or translational repression of their mRNA targets. For miRNAs, the complex binds to target sites usually located in the 3' UTR of mRNAs that typically share only partial homology with the miRNA. A "seed region"— a stretch of about seven (7) consecutive nucleotides on the 5' end of the miRNA that forms perfect base pairing with its target— plays a key role in miRNA specificity. Binding of the RISC/miRNA complex to the mRNA can lead to either the repression of protein translation or cleavage and degradation of the mRNA. Recent data indicate that mRNA cleavage happens preferentially if there is perfect homology along the whole length of the miRNA and its target instead of showing perfect base -pairing only in the seed region (Pillai et al, 2007).

[59] An antisense oligonucleotide (ASOs) comprises an oligonucleotide having a nucleobase sequence that is complementary to a target nucleic acid or region or segment thereof. In certain embodiments, an antisense oligonucleotide is specifically hybridizable to a target nucleic acid or region or segment thereof. ASOs can comprise nucleobase sequence and optionally one or more additional features, such as a conjugate group or terminal group. ASOs may be single-stranded and double-stranded compounds. ASOs include, but are not limited to, oligonucleotides, ribozymes, and morpholinos (peptide- conjugated phosphorodiamidate oligonucleotides (PPMOs) or simply phosphorodiamidate oligonucleotides (PMOs)). Antisense nucleic acids act by hybridization of an antisense nuclei acid to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound to the target. Antisense nucleic acid can inhibit gene expression by reducing the levels of target RNA in a cell or by inhibiting translation, splicing, or activity of an RNA in a cell.

[60] 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 (i.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.

[61] The term “complementarity” refers to the ability of a polynucleotide to form hydrogen bond(s) (hybridize) with another polynucleotide sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of bases, in a contiguous strand, in a first nucleic acid sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e, g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). Percent complementarity is calculated in a similar manner to percent identify.

[62] 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.

[63] 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.

[64] 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. [65] 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.

[66] CRISPRko and CRISPRa screening was used to identify host targets (receptors or other proteins) whose functions modulate coronavirus infection of cells or replication in infected cells. Host-targeted anti-viral therapeutics were then identified that target the identified host targets and exhibit activity against coronavirus infectivity or replication.

[67] Described herein are compositions and formulations comprising host-targeted antiviral therapeutics. The described compositions and formulations can be used in methods for therapeutic treatment and/or prevention of symptoms and diseases associated of coronavirus infection. Such methods comprise administration of the compositions and formulations as described herein to a subject, e.g., a human or animal subject.

[68] The coronavirus can be a virus in the subfamily Orthocoronavirinae. Coronaviruses include, alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. Alphacoronaviruses include, but are not limited to, human coronavirus 229E and human coronavirus NL63. Betacoronaviruses include, but are not limited to, lineage A betacoronaviruses (subgenus Embecovirus), lineage B betacoronaviruses (subgenus Sarbecovirus), lineage C betacoronaviruses (subgenus Merbecovirus), and lineage D betacoronaviruses (subgenus Nobecovirus). Lineage B betacoronaviruses include, but are not limited to, SARS-CoV, MERS-CoV, a SARS-CoV-2, SARS-CoV- related betacoronaviruses, human coronavirus OC43, and KHU4.

[69] The described compositions and formulations can be used to prevent or treat coronavirus infection in a subject. In some embodiments, the described compositions and formulations are administered to a subject at risk of infection by coronavirus. In some embodiments, the described compositions and formulations are administered to a subject that has tested positive for a coronavirus. In some embodiments, the described compositions and formulations are administered to a subject that has been exposed to coronavirus. In some embodiments, the described compositions and formulations are administered to a subject suspected of having been exposed to coronavirus. In some embodiments, the described compositions and formulations are administered to a subject at risk of being exposed to coronavirus. In some embodiments, the described compositions and formulations are administered to a subject suffering from or diagnosed with coronavirus infection. In some embodiments, the described compositions and formulations are administered to a subject to treat one or more symptoms associated with a coronavirus infection. In some embodiments, the described compositions and formulations are administered to a subject to treat or prevent one or more symptoms associated with a SARS- CoV-2 infection. Coronavirus infections include, but are not limited to, colds caused by coronavirus, SARS, MERS, and COVID-19.

[70] In some embodiments, the described compositions and formulations can be administered to a subject to decrease coronavirus disease burden. In some embodiments, the described compositions and formulations can be administered to a subject to decrease coronavirus viral transmission. In some embodiments, the described compositions and formulations can be administered to a subject to inhibit coronavirus infection, decrease the likelihood of infection, decrease the severity of infection, and/or decrease the duration of infection. In some embodiments, the described compositions and formulations can be administered to a subject infected with coronavirus to decrease viral load in the subject. In some embodiments, the described compositions and formulations can be administered to a subject to inhibit coronavirus entry into host cells. In some embodiments, the described compositions and formulations can be administered to a subject to reduce the likelihood the subject will require hospitalization due to coronavirus infection. [71] Described are methods of decreasing viral load, decreasing disease burden, decreasing viral transmission, preventing infection, decreasing the likelihood of infection, decreasing the severity of infection, and/or decreasing duration of coronavirus infection. The methods comprise administering one or more of the described host-targeted anti-viral therapeutics to a subject that is infected with a coronavirus, suspected of being infected with a coronavirus, or at risk of being infected with a coronavirus. In some embodiments, the methods comprise administering a pharmaceutical composition comprising an effective dose of one or more of the described host-targeted anti-viral therapeutics to a subject that is infected with a coronavirus, suspected of being infected with a coronavirus, or at risk of being infected with a coronavirus.

[72] Host-targeted anti-viral therapeutics include, amlexanox, USP25/28 inhibitor AZ 1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

[73] Amlexanox (AMX; CAS No. 68302-57-8) is an anti-inflammatory antiallergic immunomodulator and inhibits TANK binding kinase 1 (TBK1) and its adaptor protein TBK-binding protein 1 (TBKBP1). Amlexanox has been used to treat recurrent aphthous ulcers and inflammatory conditions.

[74] In some embodiments, an effective amount of Amlexanox is about 25 to about 150 mg per day. In some embodiments, an effective amount of Amlexanox is about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 125 mg, or about 150 mg. In some embodiments, an effective amount of Amlexanox is about 25 to about 50 mg, about 25 mg, about 30 mg, about 40 mg, or about 50 mg 1-3 times per day.

[75] USP25/28 inhibitor AZ1 (AZ1, CAS No. 2165322-94-9) is noncompetitive dual inhibitor of the ubiquitin-specific protease (USP) 25/28.

[76] Harmine (HAR; CAS No. 442-51-3) is known to be an inhibitor of the DYRK1A enzyme pathway and reversibly inhibits monoamine oxidase A (MAO-A), but not Monoamine oxidase B (MAO-B).

[77] In some embodiments, an effective amount of harmine is about 0.01 to about 3 mg/kg per day.

[78] Nintedanib (NIN; CAS No. 656247-17-5) competitively inhibits both nonreceptor tyrosine kinases (nRTKs) and receptor tyrosine kinases (RTKs). Nintedanib has used for the treatment of idiopathic pulmonary fibrosis and non-small-cell lung cancer.

[79] In some embodiments, an effective amount of nintedanib is about 100 to about 300 mg per day. In some embodiments, an effective amount of nintedanib is about 100, about 125, about 150 mg, about 175 mg, about 200 mg, about 225, mg, about 250 mg, about 275 mg, or about 300 mg. In some embodiments, an effective amount of nintedanib is about 100 mg about 125 mg, or about 150 mg twice per day.

[80] inhibitor.

[81] Promethazine (PMZ; CAS No. 60-87-7) is an antihistamine and antipsychotic. Promethazine can suppress clathrin function in cells through signaling receptor inhibition. Promethazine is a first-generation antihistamine and antipsychotic that has been used to treat allergies, insomnia, and nausea.

[82] In some embodiments, an effective amount of promethazine is about 5 to about 100 mg, about 5 to about 80 mg, or about 6.25 to about 50 mg per day. In some embodiments, an effective amount of promethazine is about 6.25 mg, about 12.5 mg, about 25 mg, about 20 mg 1-4 times per day. In some embodiments, an effective amount of promethazine is about 6.25 to about 12.5 mg three times per day. In some embodiments, an effective amount of promethazine is up to 25 mg every 4-6 hours.

[83] Archazolid A, (ARCA, PubChem CID No. 16680454) is a vacuolar-type ATPase inhibitor.

[84] Olaparib (OPB; CAS No. 763113-22-0) is poly ADP ribose polymerase (PARP) inhibitor.

[85] In some embodiments, an effective amount of olaparib is about 100 to about 800 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg. In some embodiments, an effective amount of olaparib is about 100 to about 400 mg twice per day. In some embodiments, an effective amount of olaparib is about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg twice per day.

[86] Abemaciclib (ABE; CAS No. 1231929-97-7) is a CDK inhibitor selective for CDK4 and CDK6.

[87] In some embodiments, an effective amount of abemaciclib is about 50 to about 400 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg. In some embodiments, an effective amount of abemaciclib is about 50 about 200 mg orally 2 times a day. In some embodiments, an effective amount of abemaciclib is about 50 mg, about 100 mg; about 150 mg; about 200 mg twice per day.

[88] In some embodiments, an RNA function inhibitor targeting the CCZ1 gene comprises an antisense oligonucleotide or an siRNA that inhibits expression of the CCZ1 gene by at least 20%, at least 30%, or at least 50%.

[89] In some embodiments, an RNA function inhibitor targeting the EDC4 gene comprises an antisense oligonucleotide or an siRNA that inhibits expression of the EDC4 gene by at least 20%, at least 30%, or at least 50%.

[90] In some embodiments, an RNA function inhibitor targeting the XRN1 gene comprises an antisense oligonucleotide or an siRNA that inhibits expression of the XRN1 gene by at least 20%, at least 30%, or at least 50%.

[91] In some embodiments, the described host- targeted anti-viral therapeutics are administered to a subject at dosage levels recognized or recommended for treating other conditions.

[92] In some embodiments, the described host-targeted anti-viral therapeutics are administered according to their recognized administration routes.

[93] In some embodiments, a host-targeted anti-viral therapeutic is 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.

[94] A pharmaceutical composition or medicament includes a pharmacologically effective amount of host-targeted anti-viral therapeutic 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.

[95] Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming 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.

[96] 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 antiinflammatory agents (e.g., antihistamine, diphenhydramine, etc.).

[97] 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.

[98] 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.

[99] In some embodiments, any two or more of the amlexanox, USP25/28 inhibitor AZ1 , harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN 1 gene are combined for treatment and/or prevention of coronavirus infection.

[100] 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 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.

[101] In some embodiments, the additional active ingredient comprises an additional antiviral therapeutic. In some embodiments, any one or more of amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, or an RNA function inhibitor targeting the XRN 1 gene is combined with a second antiviral therapeutic for treatment and/or prevention of coronavirus infection. In some embodiments, the additional antiviral therapeutic is selected from the group consisting of: azelastine, diphenhydramine and hydroxyzine.

[102] The compositions and formulations can be formulated for oral administration, parenteral administration, IV administration, or injection. The host- targeted anti-viral therapeutics can be provided as a liquid formulation, as a tablet, as a coated tablet, as a chewable tablet, as a powder (e.g., a lyophilized powder), or as a capsule, he host-targeted anti-viral therapeutics 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 host-targeted anti-viral therapeutics is administered orally. In some embodiments, the host-targeted antiviral therapeutics is administered by inhalation. In some embodiments, the host-targeted anti-viral therapeutics is administered parenterally.

[103] The described compositions and formulations can be used in methods for therapeutic treatment and/or prevention of symptoms and diseases associated with coronavirus infection. Such methods comprise administration of the compositions and formulations as described herein to a subject, e.g., a human or animal subject.

[104] In some embodiments, the described pharmaceutical compositions are used for treating or managing clinical presentations associated with coronavirus infection. In some embodiments, a therapeutically or prophylactically effective amount of a host-targeted antiviral therapeutic is administered to a subject in need of such treatment, prevention or management. In some embodiments, administration of host-targeted anti-viral therapeutic can be used to decrease the number, severity, and/or frequency of symptoms associated with coronavirus infection in a subject.

[105] The described pharmaceutical compositions can be used to treat at least one symptom associated with coronavirus infection in a subject. In some embodiments, the subject is administered a therapeutically effective amount of one or more host- targeted antiviral therapeutics, thereby treating the symptom. In some embodiments, the subject is administered a prophylactically effective amount of one or more host-targeted anti-viral therapeutics thereby preventing infection by a coronavirus or preventing development of one or more symptoms associated with coronavirus infection.

[106] Symptoms associated with coronavirus infection can be, but are not limited to, an inflammatory response, a cytokine storm, an infLammasome-associate response, an IL- 1 [1- associated response, an NLRP3 -associated response, lung fibrosis, pulmonary fibrosis, ground glass opacities, pulmonary fibrosis, acute respiratory distress syndrome (ARDS), pneumonia, and combinations thereof.

[107] Described are methods of reducing the duration of coronavirus infection, reducing the severity of coronavirus infection, reducing the likelihood of coronavirus infection, reducing the number of coronavirus particles, or reducing the likelihood of developing a coronavirus infection symptom in a subject, the method comprising administering to the subject any of the described combinations or formulations comprising one or more host- targeted anti-viral therapeutics.

[108] In some embodiments, the described pharmaceutical compositions are administered to a subject at risk of infection by a coronavirus, a subject that has tested positive for a coronavirus, a subject that has been exposed to a coronavirus, a subject suspected of having been exposed to a coronavirus, a subject at risk of being exposed to a coronavirus, or a subject suffering from or diagnosed with a coronavirus infection.

[109] Described are methods of reducing the duration of SARS-CoV-related betacoronavirus infection, reducing the severity of SARS-CoV-related betacoronavirus infection, reducing the likelihood of SARS-CoV-related betacoronavirus infection, reducing SARS-CoV-related betacoronavirus replication, or reducing the likelihood of developing a SARS-CoV-related betacoronavirus-related illness in a subject, the methods comprising administering to the subject any of the described combinations or formulations of diphenhydramine and lactoferrin (e.g., any of the described pharmaceutical compositions). The SARS-CoV-related betacoronavirus can be, but is not limited to, SARS-CoV-2. The methods comprise administration of a therapeutically effective amount of the combinations, formulations, or pharmaceutical compositions as described herein to a subject, e.g., a human or animal subject in need of such treatment.

[110] In some embodiments, methods or treating SARS-CoV-2 infection, or COVID-19, are described, the methods comprising administering to a subject at risk of infection by SARS-CoV-2, diagnosed with infection by SARS-CoV-2, or diagnosed with COVID- 19 one or more effective doses of amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and/or an RNA function inhibitor targeting the XRN1 gene.

[111] Listing of embodiments:

[112] 1. A method of treating a subject suffering from infection by or susceptible to infection by a coronavirus comprising administering to a subject a therapeutically effective amount of one or more host-targeted anti-viral therapeutics selected from the group consisting of: amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

[113] 2. The method of embodiment 1, wherein the coronavirus is selected from the group consisting of: a betacoronavirus, a SARS-CoV-related betacoronavirus, a SARS, SARS-CoV-2, and a MERS, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, and KHU4.

[114] 3. A method of treating a subject suffering from a coronavirus-related illness comprising administering to the subject a therapeutically effective amount of one or more host-targeted anti-viral therapeutics selected from the group consisting of: amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

[115] 4. The method of embodiment 3, wherein the coronavirus-related illness is selected from the group consisting of: a betacoronavirus, a SARS-CoV-related betacoronavirus, a SARS, SARS-CoV-2, and a MERS, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, and KHU4.

[116] 5. A method of preventing infection by a coronavirus comprising administering to a subject a therapeutically effective amount of one or more host- targeted anti-viral therapeutics selected from the group consisting of: amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

[117] 6. The method of embodiment 5, wherein the coronavirus is selected from the group consisting of: a betacoronavirus, a SARS-CoV-related betacoronavirus, a SARS, SARS-CoV-2, and a MERS, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, and KHU4.

[118] 7. The method of any one of embodiments 1-6, wherein the RNA function inhibitor comprises an antisense oligonucleotide, a peptide-conjugated phosphorodiamidate, an siRNA, or a shRNA.

[119] 8. The method of any one of embodiments 1-6, wherein the method further comprises administering one or more additional active ingredients.

[120] 9. The method of embodiment 4, wherein the additional active ingredients are selected from the group consisting of: azelastine, diphenhydramine, and hydroxyzine.

[121] 10. The method of any one of embodiments 1-4 and 4-8, wherein the subject has tested positive for a coronavirus, has been exposed to a coronavirus, is suspected of having been exposed to a coronavirus, is at risk of being exposed to a coronavirus, is suffering from or diagnosed with a coronavirus-related illness, or is suffering from acute lung injury due to a coronavirus-related illness.

[122] 11. The method of any one of embodiments 2 and 4-7, wherein the coronavirus- related illness is a common cold, SARS, MERS, or COVID- 19.

[123] 12. The method of embodiment 1, wherein treating a subject suffering from coronavirus infection comprises decrease coronavirus disease burden, decreasing viral load, decreasing viral transmission, decreasing the severity of infection, and/or decreasing the duration of infection, decreasing coronavirus entry into host cells, or decreasing viral replication.

[124] 13. The method of embodiment 3, wherein treating a subject suffering from a coronavirus-related illness comprises: decreasing the severity of a symptom or decreasing the duration of a symptom.

[125] 14. The method of embodiment 5, wherein preventing infection by a coronavirus comprises inhibiting viral entry into host cells.

[126] 15. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: amlexanox. [127] 16. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: USP25/28 inhibitor AZ1.

[128] 17. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: harmine.

[129] 18. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: nintedanib.

[130] 19. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: UC2288.

[131] 20. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: promethazine.

[132] 21. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: archazolid.

[133] 22. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: abemaciclib.

[134] 23. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: an RNA function inhibitor targeting the CCZ1 gene.

[135] 24. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: an RNA function inhibitor targeting the EDC4 gene.

[136] 25. The method of any one of embodiments 1-14, wherein the one or more host- targeted anti-viral therapeutics comprises: and an RNA function inhibitor targeting the XRN1 gene.

[137] 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. Study design

[138] The objectives of this study were to identify host factors that promote HCoV replication and to determine whether these host factors can be targeted with commercially available drugs to block viral infection in vitro.

[139] To achieve this goal, we performed genome-wide CRISPR knockout screens in Vero E6 cells using a newly generated Vervet sgRNA library and in HEK293T-hACE2 cells using the commercially available human Brunello sgRNA library (FIG. 1 A). Vero E6 cells transduced with the Vervet or C. sabaeus sgRNA library were infected with SARS- CoV-2 or OC43 at MOI 0.01, resistant (surviving) cells were expanded and re-infected at MOI 0.1. Genomic DNA was extracted from multiple replicates of control cells, the initial infections, and re-infections for the purpose of sgRNA sequencing.

[140] Two screens were performed in HEK293T-hACE2 cells transduced with the Brunello sgRNA library (FIG. IB). In the first screen, cells were infected with SARS-CoV- 2 or OC43 at MOI 0.01 and resistant cells were re-infected with either MOI 0.01 or MOI 0.1 of the corresponding virus. In the second screen, cells with infected with SARS-CoV- 2 at MOI 0.3 and re-infected at MOI 0.03. Genomic DNA was extracted from multiple replicates of control cells, the initial infections, and re-infections for the purpose of sgRNA sequencing. Genes targeted by enriched sgRNAs were compared between replicates, infection conditions, HCoVs, cell lines, and previously published screens to identify common and unique host factors as well as putative pan-HCoV host factors. Genes of interest were selected for validation and targeted for knockdown using shRNAs followed by infection with HCoVs. Commercially available inhibitors to other important genes identified in our study were evaluated for their capacity to prevent virus-induced toxicity and viral replication in vitro. ECso and IC50 values for efficacious compounds were determined.

[141] A. Virus stock generation and viral titer determination. SARS-CoV-2 strain UF-1 (GenBank accession number MT295464.1) was originally isolated from a COVID-19 patient at the University of Florida Health Shands Hospital via nasal swab. SARS-CoV-2 and OC43 were propagated in Vero E6 cells (ATCC) grown in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals) and Pen-Strep (100 U/ml penicillin, 100 pg/ml streptomycin; Gibco) at 37°C and 5% CO2. Virus stocks were prepared by infecting Vero E6 cells at MOI 0.01, centrifuging culture supernatants collected at 3 dpi for 5 mins at 1000xg, and filtering through a 0.44 pm PVDF filter (Millipore) followed by a 0.22 pm PVDF filter (Restek). The virus stocks were aliquoted and stored at -80°C. Virus stocks or supernatants from infected SAEC-hACE2 cells were titered using a standard TCID50 assay. In brief, Vero E6 cells were seeded at 2x 10⁴ cells per well in a 96-well plate (Corning) and allowed to attach overnight. Virus stocks or supernatants from infected cells were serially diluted onto cells, with a total of 8 replicates per dilution. Monolayers were visualized in the BSL3 using an EVOS XL Core microscope (Thermo Fisher Scientific) and scored positive or negative for cytopathic effect (CPE) at 7 dpi.

[142] B. Viral genome copy number enumeration. For SARS-CoV-2, supernatants and cells were harvested into AVL buffer from the QIAamp Viral RNA Kit (Qiagen) and RNA was purified according to the manufacturer’s recommendations. The samples underwent reverse transcription and cDNA synthesis using the iTaq Universal SYBR Green One-Step Kit (BioRad) and primers targeting the nucleocapsid (N) gene of SARS-CoV-2 (NproteinF-GCCTCTTCTCGTTCCTCATCAC (SEQ ID NO: 1),

NproteinR-AGCAGCATCACCGCCATTG (SEQ ID NO: 2)). qPCR was carried out on a Bio-Rad CFX96 and viral genome copy numbers were extrapolated using CT values from a standard curve generated using a control plasmid containing the N protein gene (Integrated DNA Technologies). For OC43, RNA from infected cells was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations and amplified using the Applied Biosystems AgPath-ID One-Step RT-PCR Kit (Thermo Fisher Scientific) and primers and probe targeting the N gene of OC43. GAPDH levels were determined for each sample for normalization purposes using previously described primers. All samples were run in triplicate for each primer pair and normalized viral genome copy numbers were calculated using the comparative cycle threshold method.

[143] C. Genome-wide CRISPR sgRNA screens. The human CRISPR Brunello library (Addgene 73178) was amplified following a previously published protocol. We constructed a Vervet domain-targeted sgRNA library since one was not commercially available. For both libraries, lentiviruses were produced in HEK293T cells by co-transfection of library plasmids together with the packaging plasmid psPAX2 (Addgene 12260) and envelope plasmid pMD2.G (Addgene 12259). CRISPR screens were carried out in two cell lines: AGM Vero E6 cells were transduced with the newly generated Vervet sgRNA library and human HEK293T-hACE2 cells (Genecopoeia) were transduced with the Brunello sgRNA library. For each screen, 1.2><10⁸ cells were transduced with lentivirus-packaged sgRNA library at MOI 0.3 in the presence of 8 pg/ml polybrene (Sigma) to achieve ~500-fold overrepresentation of each sgRNA. After 48 h, 0.6 pg/ml puromycin (Gibco) was added to eliminate non-transduced cells and cultures were expanded in Matrigel-coated (Coming) T300 flasks. Control replicates were collected at this time to determine input library composition and additional replicates were infected with SARS-CoV-2 or OC43 at the indicated MOIs. Cells surviving initial infections were collected when they had expanded to confluency. A portion of each replicate was stored in DNA/RNA Shield (Zymo Research) at -80°C for genomic DNA extraction and the remaining cells were reseeded and reinfected at the indicated MOIs. Cells surviving reinfections were also harvested at confluency for genomic DNA extraction.

Genomic DNA was extracted from each sample (detailed extraction methods are described in supplemental methods). The sgRNA regions were then amplified and indexed for Illumina sequencing using a one-step PCR method and primers specific to the LentiCRISPRv2 -based Vervet and Brunello libraries. Brunello and Vervet DNA samples were amplified in ten 100 pl reactions using the NEBNext High-Fidelity 2X Master Mix Kit (New England Biolabs), 0.5 pM of forward and reverse primers and 10 pg of DNA template per reaction with the following program: initial denaturation at 98°C for 3 min,

24 cycles of denaturation at 98°C for 10 s, annealing 60°C for 15 s and extension 72°C for

25 s, and final extension at 72°C for 2 min. 256 bp amplicons were quantified on a 2% agarose gel stained with SYBR Safe (Invitrogen), using the Gel Doc quantification software (Bio-Rad). Amplicons were first pooled in an equimolar fashion and then the pools were gel-extracted using the PureLink Quick Gel Extraction Kit (Thermo Fisher Scientific). The sequencing was carried out using a NovaSeq 6000 sequencer (Illumina). The Brunello amplicons were sequenced using the S4 2x150 cycles Kit (Illumina) while the Vervet- AGM amplicons were sequenced with the SP 1x100 cycles Kit (Illumina).

[144] D. Computational analysis. The FASTX-Toolkit was used to demultiplex raw FASTQ data which were further processed to generate reads containing only the unique 20 bp sgRNA sequences. The sgRNA sequences from the library were assembled into a Burrows-Wheeler index using the Bowtie build-index function and reads were aligned to the index. The efficiency of alignment was checked and the number of uniquely aligned reads for each library sequence was calculated to create a table of raw counts. Ranking of genes corresponding to perturbations that are enriched in infected cultures was performed using a robust ranking aggregation (a-RRA) algorithm implemented in the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) tool through the test module. Tables with raw counts corresponding to each sgRNA in reference (initial pool) and selected (virus-infected) samples were used as an input for MAGeCK test. Gene-level ranking was based on FDRs and candidates with FDRs < 0.25 were considered as significant hits. Ranking of genes corresponding to positively selected and negatively selected perturbations was performed using a robust ranking aggregation (a-RRA) algorithm implemented in MAGeCK through the test module. Tables with raw counts corresponding to each sgRNA in reference (initial pool) and selected (exposed to virus) samples were used as an input for MAGeCK test. Gene-level ranking was based on false discovery rate (FDR) and candidates with FDR < 0.25 were considered as significant hits. We submitted FastQ files to Gene expression omnibus (GSE177545) and all CRISPR screen data to BioGRID ORCS database.

[145] E. Validation of host factors in promoting viral infections. To validate selected host factors for their capacity to promote HCoV infection in vitro, wc transduced HEK293T- hACE2 cells with lentivirus-packaged shRNAs targeting CTSL, CCZ1, or EDC4 (TRC Human shRNA Library collection) or the empty vector pLKO.1. In addition, we generated hACE-expressing SAEC cells by transduction with pLENTI_hACE2_HygR plasmidpackaged lentiviruses (Addgene 155296) and selection of transduced cells with hygromycin (300 pg/ml). Next, SAEC-hACE2 cells were transduced with lentivirus- packaged sgRNAs targeting EDC4 and XRN1, designed with PAVOOC and synthesized by the Molecular Cloning Facility (Department of Biological Science, Florida State University). Transduced cells were expanded under puromycin selection (0.8 ug/ml) for at least 7 days to generate stable knockdown or knockout cell lines. For knockout SAEC- hACE2 cells, monoclonal colonies were isolated from pools of cells and tested individually. To confirm gene knockdown and knockout, cell lysates prepared from knockdown/knockout and control cells were tested by western blotting with antibodies directed to CTSL (Invitrogen, BMS1032), CCZ1 (Santa Cruz Biotechnology, sc-514290), EDC4 (Cell Signaling Technology, 2548S), XRN1 (Cell Signaling Technology, 70205S), and actin as a loading control (Sigma- Aldrich, MAB1501R). Once knockdown or knockout was confirmed, cells were infected with SARS-CoV-2 or OC43 at MOI 0.01 and RNA was extracted at 2 dpi for viral genome copy number enumeration, or cell supernatants were tested at various timepoints for quantification of viral infectious particles by TCID50 as described above. Cell viability of SAEC-hACE2 knockout cells was compared to control cells using the CellTiterGlo 2.0 Cell Viability Assay (Promega) according to manufacturer’s instructions. Knockout cells displayed no gross reduction in viability. [146] F. Identification and testing inhibitors of host factors from CRISPR screens. Online databases and published literature were used to find small molecule inhibitors targeting a subset of top-scoring genes in the CRISPR screens. Amlexanox (AMX) was purchased from InvivoGen. Abemaciclib (ABE), AZ1, olaparib (OPB), and nintedanib (NIN) were purchased from Selleckchem. Harmine (HAR), INDY, chlorpromazine (CPZ), promethazine (PMZ), UC2288 (UC2), and CID 1067700 (CID) were purchased from Millipore Sigma. Drugs were diluted according to the manufacturers’ recommendations and single-use aliquots were frozen at -80°C until the time of assay. Drugs were diluted down to 2x concentrations and mixed with 2x concentrations of virus to generate lx concentrations, then added to the monolayers. Cells were infected at a MOI of 0.2 as determined by preliminary experiments to generate an ideal dynamic range of the colorimetric CYTOTOX96® Non-Radioactive Cytotoxicity Assay (Promega). Infections progressed for 72 h at which time supernatant from treatments and controls were processed for LDH release according to the manufacturer’s recommendations. Absorbance at 450 nm was read using an accuSkan FC microplate reader (Fisher Scientific) with Skanlt software (Fisher Scientific). Absorbance values were background subtracted and transformed to percent of virus-infected controls. These percentages were compared to the values obtained from virus infected-cell cytotoxicity values by one-way ANOVA using GraphPad Prism version 9. Assays were carried out in biological duplicate and in three independent experiments. The concentration of drug alone that resulted in 50% of maximum toxicity (cytotoxic concentration 50; CC50) and the concentration of drug that inhibited 50% of the vehicle treated SARS-CoV-2 induced cytotoxicity (effective concentration 50; EC50) were determined by serially diluting small molecule inhibitors during SARS-CoV-2 infection of Vero E6 cells. EC50 and CC50 values were calculated by transforming inhibitor concentrations to log then using the non-linear fit with variable slope function (GraphPad Prism version 9) to determine best fit variables using the percent of maximum SARS-CoV- 2 induced cytotoxicity measurements at each drug concentration performed in technical duplicate. Candidate inhibitors were assayed for their ability to prevent OC43 and SARS- CoV-2 replication in SAEC-hACE2 cells by TCID50. SAEC-hACE2 cells were infected in triplicate at an MOI of 0.01 in the presence of the indicated inhibitors for 3 days. TCID50 were determined by diluting the supernatant of each replicate across octuplet columns of Vero E6 cells. Five days later TCID50 was read. The data are from two independent repetitions. Phospholipidosis was determined in SAEC-hACE2 cells with the HCS LipidTOX Red Neutral Lipid Stain according to the manufacturer’s instructions using a Biotek Cytation 3 plate imager. Cytotoxicity in SAEC-hACE2 cells was determined as above for Vero E6 cells.

[147] G. Statistical analysis. For the genome-scale CRISPR analysis, the embedded statistical tools in the MAGeCK/VISPR pipelines were used. Further details are provided in the supplemental materials. All other statistical analyses were carried out using GraphPad Prism 9.0. To compare the mean normalized viral genome copy number values in targeted shRNA knockdown experiments (FIGs. 5 A-D), P values were determined using one-way ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001), with error bars representing standard errors of mean (n = 3 experiments). For testing inhibitory activity of small molecules on SARS-CoV-2 infection of Vero E6 cells (FIG. 6), a one-way ANOVA test was used for comparison of toxicity values for inhibitor-treated infected cells and infected- only control cells (no treatment), with error bars denoting standard deviation for all panels (n = 3 experiments). Non-linear regression of data points was used to determine the ECso and IC50 values for indicated compounds.

Example 2. Genome-wide CRISPR screens in Vero E6 cells identify host factors required for HCoV infection.

[148] To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43.

[149] Due to the highly cytopathic nature of HCoV infection in the Vero E6 cells derived from African Green Monkey (AGM; Chlorocebus sabaeus), \\x carried out genome-wide screens using a custom Vervet CRISPR knockout library. Vero E6 cells were transduced with the Vervet CRISPR library and infected with SARS-CoV-2 or OC43 at a multiplicity of infection (MOI) 0.01. We observed -60% visible cytopathic effect (CPE) for SARS- CoV-2 and -85% CPE for OC43. Resistant clones were expanded, reinfected with the corresponding virus at MOI 0.1, and re-expanded. Genomic DNA was extracted from surviving cells, sgRNAs amplified, and sequenced.

[150] MAGeCK analysis of multiple replicates compared to uninfected control library replicates yielded log2fold changes (log2FC) that were plotted on the x-axis. Negative logio transformed false discovery rates (FDR) were plotted on the y-axis. Data are presented for SARS-CoV-2 and OC43 Vero E6 infections in FIG. 2A and 2B and Tables 1 A and IB. The heat maps display the log2FC for the 20 top-scoring genes comparing results for SARS- CoV-2 and OC43 infections (Tables 2A and 2B). Genes targeted by significantly enriched sgRNAs (FDR<0.25) were segregated into functional categories using PANTHER for SARS-CoV-2.

[151] We identified multiple candidate host factors previously demonstrated to play a functional role in SARS-CoV-2 and OC43 infections. For example, ACE2 was identified in the SARS-CoV-2 screen. Furthermore, TMEM41B was a top-scoring gene in the OC43 screen, supporting recent work by Schneider et al. demonstrating that TMEM41B is a pan- HCoV host factor. We also identified interferon (IFN)-induced transmembrane (IFITM) proteins that have been reported to regulate HCoV infection. In addition, there were six genes identified in common with a study by Wei et al.: ACE2, DPF2, DYRK1A, RAD54L2, SMARCA4, and TP53.

[152] Genes targeted by significantly enriched sgRNAs were next segregated into functional categories listed in Tables 1 and 2. Of note, CDK4, a master regulator of cell cycle, was identified as a key host factor for both viruses. Disruption of additional genes encoding regulators of cell cycle progression, including CDK1NA, DYRK1A, EIRK and P53, similarly increased cellular resistance to SARS-CoV-2 infection.

[153] Host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. A subset of genes identified in these screens identified as playing a role in HCoV replication was validated in multiple cell types, including a respiratory cell line. Notably, several of the host factors identified in our study provide insight into SARS-CoV-2 replication processes that could be targeted with antiviral drugs.

Table 1 A. Genes targeted by significantly enriched sgRNAs (FDR<0.25) following SARS-

CoV-2 infection.

Table IB. Genes targeted by significantly enriched sgRNAs (FDRO.25) following OC43 infection.

Table 2 A. Heat Map results

FIG. 2B Heat Map results.

Example 3. Genome-wide CRISPR screens in HEK293T-hACE2 cells identify host factors required for UCo V infection.

[154] We similarly performed CRISPR screens in human HEK293T cells ectopically expressing the human ACE2 receptor (HEK293T-hACE2) transduced with the Brunello sgRNA library. Transduced cells were infected with SARS-CoV-2 or OC43 at MOI 0.01. SARS-CoV-2-infected cultures developed -40% CPE and OC43-infected cultures developed >85% CPE. Resistant cell populations propagated to confluence were reinfected with the corresponding virus at either MOI 0.01 or MOI 0.1 and re-expanded. Genomic DNA was extracted and sgRNAs from both the initial and secondary infections were sequenced.

[155] MAGeCK analysis of multiple replicates compared to uninfected control library replicates yielded log2fold changes (log2FC) that were plotted on the x-axis. Negative log 10 transformed FDR were plotted on the y-axis. The genes targeted by the most highly enriched sgRNAs in each of the SARS-CoV-2 infections are indicated in FIGs. 3A-3C. EDC4, a gene encoding a scaffold protein that functions in programmed mRNA decay, was the overall top-scoring gene. Interestingly, XRN1 encodes another key player in this pathway and was also top-scoring. We further categorized the genes encoding candidate host factors (FDR<0.1) into functional categories depicted in heat maps in Table 3A. Consistent with other published screens, we identified multiple components of the endocytic pathway including CCZ1, DNM2, and WASL. Other functional categories in which multiple genes were identified include cell adhesion, cell cycle, integrator complex, lysosome, mTOR regulation, and ubiquitination/proteolysis. We carried out an independent SARS-CoV-2 screen using the higher MOI of 0.3 for initial infection which resulted in -80% CPE, and MOI 0.03 for secondary infection. Genes targeted by the most significantly enriched sgRNAs in this study are presented in FIGs. 3D-E and are segregated into functional categories depicted in heat maps in Table 3B. ACE2 was a top-scoring gene in this screen. Functional categories with multiple targeted genes include amphisome, autophagy, endosome, exocytosis, lysosome, peroxisome, transcription/transcriptional regulation, and ion transporters. C18orf8, CCZ1, CD! 12, and TMEM251 wcvc identified in both the low- and high-MOI SARS-CoV-2 screens.

Table 3A. The heat map displaying the log2FC for top-scoring genes (FDR<0.1) across the three infections.

Table 3B. The heat map displaying the log2FC for top-scoring genes (FDR<0.25) across the two infections of FIGs. 3E-F.

[156] MAGeCK analysis was performed as described above. The genes targeted by the most highly enriched sgRNAs in the OC43 HEK293T-hACE2 screens are indicated in FIGs. 4A-4C and segregated into functional categories in Table 4. Genes encoding IFITM proteins were identified as proviral factors for OC43. TMEM41B was a top-scoring gene along with the functionally related VMP1, as were CCZ1, CCZ1B, SLC35B2, and WDR81 which have all been reported in other recent OC43 genome-wide screens. When comparing the SARS-CoV-2 and OC43 HEK293T-hACE2 datasets, there were 6 genes in common targeted by significantly enriched sgRNAs (C18orf8, CCZ1, CCZ1B, RAB7A, WDR81, and WDR91 . Notably, all of the corresponding gene products function in vesicle-mediated transport.

Table 4. Heat map showing the log2FC for top-scoring genes (FDR<0.25) across the three infections of FIGs. 4A-C

[157] Similar SARS-CoV-2 screens have been performed in human Huh-7.5 and A549 cells. We compared our data sets to those of screens and analyzed them using a common analysis framework (MAGeCK) and stringency (FDR<0.25). Using this stringency, no genes were identified in all five studies, 1 gene was identified in four studies ( CE2), 6 genes were identified in three studies (XPS35, CTSL, DNM2, CCZIB, TMEM106B, and VAC14), and 25 genes were identified in two studies (ALG5, ARVCF, ATP6V1A, ATP6V1G1, B3GAT3, CNOT4, EPT1, EXOC2, EXT1, EXTL3, GDI2, LUC7L2, MBTPS2, PIK3C3, RAB7A, RNH1, SCAF4, SCAP, SLC30A1, SLC33A1, SNX27, TMEM41B, TMEM251, WDR81, and WDR91) (Table 5A). It should be noted that these genes were top- scoring across studies performed in different human cell lines, suggesting they are broadly important in SARS-CoV-2 replication. Shared pathways include vesicle-mediated transport (CCZIB, DNM2, EXOC2, GDI2, PIK3C3, RAB7A, SNX27, VAC14, VPS35, WDR81, WDR91 , vacuolar ATPases important in organelle acidification (ATP6V1A, ATP6V1E, ATP6V1GF), and heparan sulfate biosynthesis genes (EXT1, EXTL3, B3GAT3). We identified 53 genes targeted by enriched sgRNAs in our study that were not identified in published studies (Table 5B), including EDC4 an XRNl.

Table 5 A. The heat map displaying the log2FC for the 32 genes found in common across two or more published studies with FDRO.25.

A. Daniloski et. al., MOI 0.01

B. Daniloski et. al., MOI 0.3

C. Schneider et. al., 33 °C

D. Schneider et. al., 37°C E. Wang et. al., combined

F. Baggen et. al., high stringency: TMEM106B score = 2.5646

Table 5B. Heat map displaying the log2FC for the 53 genes uniquely identified as significant in our studies compared to their observed log2FC across the other published studies

Example 4. Validation of a subset of genes that promote human coronavirus replication [158] To confirm that genes identified in our screens promote HCoV replication, HEK293T-hACE2 cells were engineered to stably express gene-specific shRNAs targeting CCZ1 or EDC4. Lentivirus-packaged shRNA clones directed to CTSL, CCZ1, and EDC4NQ Q transduced into HEK293T-hACE2 cells and selected with puromycin. CTSL knockdown was tested as a positive control for SARS-CoV-2. Efficiency of gene knockdown assessed by western blotting was robust for all three genes (FIG. 5A). Knockdown cells were then infected with SARS-CoV-2 or OC43 and viral genome copy number determined at 2 days post-infection (dpi). Viral genome copy numbers were determined by RT -qPCR and normalized to GAPDH levels as a housekeeping control (done in triplicate). All three genes were required for optimal SARS-CoV-2 infection while CCZ1 and EDC4, but not CTSL, promoted OC43 infection (FIG. 5B). Because EDC4 was unique to our screens, we next tested whether it plays a role in promoting HCoV replication in respiratory cells. The small airway epithelial cell (SAEC) line was engineered to express human ACE2 (hACE2) and then EDC4 was targeted for knockout using a CRISPR/Cas9- based approach. As mentioned above, Xrnl functions in the same mRNA decay pathway as Edc4 and was also identified in our screens. We therefore engineered Xrnl^' SAEC- hACE2 expressing cells in parallel. After verifying gene knockout (FIG. 5C), cells were infected with either SARS-CoV-2 or OC43 and virus titers measured at Ohpi, 1dpi, 2dpi and 3 dpi by TCID50 assay. EDC4 and XRN1 were observed to be necessary for efficient replication of both viruses FIG. 5D.

Example 5. CRISPR screening reveals novel antiviral drugs displaying in vitro efficacy [159] We next determined whether gene products and pathways identified in our screens could be targeted with commercially available inhibitors to block HCoV infection. Numerous genes involved in cell cycle regulation were identified in our screens. The following inhibitors targeting this class of host factors were tested: abemaciclib (ABE; Cdk4, Cdk6 inhibitor), UC2288 (UC2; CDKNlA/p21 inhibitor), harmine (HAR) and INDY (DyrklA/B inhibitors), AZ1 (Usp25/28 inhibitor), olaparib (OPB; ARID1A inhibitors were not available so inhibition of PARP-mediated DNA repair was investigated to see if DNA damage repair was involved in SARS-CoV-2 replication), and nintedanib (NIN; FGFR1/2/3, VEGFR1/2/3, and PEGFRa/p inhibitor). Host factors involved in endocytosis have been widely reported to regulate HCoV replication and were identified in our and others’ CRISPR screens. We therefore tested several drugs targeting this process, including CID 1067700 (CID; Rab7a inhibitor), and chlorpromazine (CPZ) and promethazine (PMZ), which both suppress clathrin function in cells through signaling receptor inhibition. Finally, we tested amlexanox (AMX) which inhibits TANK binding kinase 1 (TBK1) and its adaptor protein TBK-b inding protein 1 (TBKBP1) which has been reported to variously regulate Rab7a activity or induction of IFN response genes. The heat map in Table 6 shows the fold-enrichment of sgRNAs targeting the genes of interest across the screens performed in this study.

49648-573151 able 6. Heat map illustrating log2FC for the indicated gene targets

[160] In an initial experiment of the entire panel of small molecules, inhibitors were added to culture supernatants at the initiation of SARS-CoV-2 infection and evaluated for their capacity to inhibit virus-induced cytopathic effect at 3 dpi in Vero E6 cells. The concentrations of inhibitors used, based on available toxicity data, were generally nontoxic in Vero E6 cells (FIG. 6A, white bars). ABE, AMX, HAR, NIN, OPB, PMZ, and UC2 significantly inhibited virus- induced cytotoxicity while AZ1, CPZ, INDY, and CID did not (FIG. 6A, gray bars). Our CID results are consistent with prior work which showed reduced CoV egress, but no effect on cell viability or viral replication, in response to CID treatment. Although INDY and HAR both target Dyrkl A, only HAR displayed activity in this assay. The enzymatic ECso of HAR for DyrklA is 0.08 pM. The enzymatic ECso of INDY for DyrklA is 0.24 pM. As a complementary approach to measure antiviral activity of these compounds, we quantified viral genome copies by RT-qPCR in cells treated with each compound at 2 dpi. ABE, AMX, HAR, PMZ and UC2 significantly decreased viral genome copy number (FIG. 6B), consistent with their ability to protect from virus-induced cytotoxicity. On the other hand, NIN and OPB had no effect on viral genome copy number despite their moderate inhibition of SARS-CoV-2 - induced cytotoxicity. Conversely, AZ1 completely inhibited viral genome replication in spite of having no significant effect on cytotoxicity.

[161] For compounds displaying activity in one or both of these assays, we next determined their ECso and CC50 against SARS-CoV-2 infection by cytotoxicity measurements in the presence or absence of virus across a series of inhibitor dilutions (FIGs. 6C-I). Several of the inhibitors had EC50 values below 20 pM (10.86 pM for ABE, 14.1 pM for NIN, and 2.16 pM for UC2), with the p21 inhibitor UC2 being the most potent. AMX is typically used as topical treatment and had a high EC50 at 342.96 pM. AZ1 (37.49 pM), HAR (61.44 pM) and PMZ (88.41 pM) showed intermediate EC50 levels. The selectivity indices (SI; ratios of CC50 to EC50) of the investigated compounds from highest to lowest are: AMX >53.23, ABE >5.68, UC2 >7.40 with the SI of AZ1, HAR, NIN, and PMZ falling below 2. For comparison, the SI of clinically relevant antiviral drugs are as follows: remdesivir is >129.87, nafamostat >4.44, and ribavirin >3.65. Generally, determining ECso with cytotoxicity measurements results in overestimation of EC50, leading to a conservative estimate of SI.

[162] A subset of inhibitors displaying efficacy in Vero E6 cells was further assessed for their capacity to inhibit SARS-CoV-2 and OC43 replication in SAEC-hACE2 or SAEC, respectively, using TCID50 assay as a readout of infectious virus titers. ABE, AMX, AZ1, HAR, NIN, and PMZ inhibited OC43 replication (FIG. 7B) and all compounds except for AZ1 inhibited SARS-CoV-2 replication (FIG. 7A). Phospholipidosis of cell membranes by drug treatment has been implicated as a confounding issue during in vitro viral inhibition screens (22). While others have disputed this claim (23) we decided to test our compounds for phospholipidosis induction. In FIGs. 7C-H, phospholipidosis was measured in SAEC-hACE2 cells treated with each inhibitor. Compared to the positive phospholipidosis control amiodarone (AMD), induction of phospholipidosis by PMZ was strongest followed by HAR. The phospholipidosis curve for HAR was biphasic, indicating a potential therapeutic window between 2.5 and 20 pg/ml. NIN induced minimal levels of phospholipidosis while ABE and AMX did not induce phospholipidosis. Overall, these findings reveal novel candidates for anti- HCoV treatment.

Example 6. Viral inhibitory activity of amlexanox and. archazolid in human lung epithelial cell line H23-hACE.

[163] Inhibitory activities of amlexanox and archazolid were also tested in human lung epithelial cell line H23-hACE. Archazolid may inhibit pathways that involve EDC4 and was effective at very low doses. Archazolid A was effective in inhibiting HCoV infection in both H23 and SAEC cells (FIGs. 8A-D).

Example 7. Use of amlexanox in decreasing viral load.

[164] K18-hACE2 expressing mice were infected with 25 mg/kg amlexanox 4 h prior to infection with 2.66^x10⁴ PFU of SARS-CoV-2, then treated again 24 and 48 h post-infection. 72 h post-infection mice were euthanized and lungs were homogenized to determine virus concentration in the isolated lungs by TCID50 measurement. One mouse had a 72% reduction in viral load while two others had 99.9 %and 99.94% reductions (FIG. 9).

Example 8. Analysis of results

[165] We performed screens for both SARS-CoV-2 and the common cold-causing HCoV OC43 to increase the probability of identifying pan-HCoV proviral factors representing strong targets for developing broad-spectrum antivirals.

[166] The factors in Table 7 were found to promote SARS-CoV-2 and HCoV OC43 infection. These factors represent host targets suitable for targeting in developing SARS and coronavirus antiviral therapeutics. Inhibition of these factors may be used to treat or prevent SARS-CoV-2 and coronavirus infection and one or more symptoms associate with SARS-CoV-2 and coronavirus infection. Table 7. Candidate pan-HCoV host factors.

[167] In Vero E6 cells, cell cycle regulation was important to SARS-CoV-2 replication. CDK4 was a top-scoring gene for both SARS-CoV-2 and OC43, suggesting it is broadly required for HCoV replication. CoVs utilize diverse strategies to manipulate the host cell cycle to promote their replication. Identification of specific cell cycle-related host factors required for HCoV replication could provide clues to dissecting viral regulatory mechanisms. We also identified IFITM proteins in both SARS-CoV-2 and OC43 screens in Vero E6 cells. Interestingly, recent work suggests that IFITM proteins promote HCoV entry when it occurs at the plasma membrane but inhibit HCoV entry when it occurs in the endocytic pathway, suggesting that HCoVs enter Vero E6 cells primarily at the plasma membrane instead of using the endosomal pathway. This finding is consistent with the paucity of factors involved in endocytosis identified in these screens, in stark contrast to our and others’ results in screens performed in human cell lines.

[168] In addition to CDK4 and IFITM proteins, targeting of SLC35B2 in both SARS-CoV-2 and OC43 Vero E6 screens increased resistance to infection, suggesting that it is a pan-HCoV host factor in this cell line. SLC35B2 encodes 3 '-phosphoadenosine 5 '-phosphosulfate transporter 1 (PAPST1) which plays an important role in heparan sulfate biosynthesis. PAPST1 is required for optimal replication of a variety of viruses including HIV, dengue virus, and bunyaviruses, enabling heparan sulfate-mediated viral entry or sulfating a viral receptor that enables virion binding. It is hence logical to expect that it functions in HCoV entry in Vero E6 cells as well. Additional candidate pan-HCoV factors identified in the Vero E6 studies include PLN encoding phospholamban and C16orf74, which are both implicated in maintaining calcium homeostasis, and C3orf80, encoding a protein of unknown function. None of these gene products have been previously identified as viral host factors to our knowledge.

[169] The functional categories with the most top-scoring genes were vesicle transport, cell cycle regulation, autophagy, and ubiquitination/proteolysis. For the OC43 screens, the most abundant functional categories were vesicle transport, transcriptional regulation including the SWI/SNF complex, innate immunity, and transporters. The host factors identified in the HEK293T-hACE2 screens for both SARS-CoV-2 and OC43 (C18orf8, CCZ1, CCZ1B, RAB7A, WDR81, and WDR91) are all involved in vesicle-mediated transport and particularly in endosomal maturation, underscoring the importance of this process for HCoV infection.

[170] When comparing our SARS-CoV-2 data sets in HEK293T-hACE2 cells to published data sets in other human cell lines, there were 21 genes in common with other studies (FDR<0.25; ACE2, ALG5, ARVCF, CCZ1B, CTSL, DNM2, EPT1, GDI2, LUC7L2, RAB7A, RNH1, SCAF4, SLC30A1, SLC33A1, SNX27, TMEM41B, TMEM251, VAC14, VPS35, WDR81, and WDR91 which highlight key functional pathways required for viral infection, including endocytosis, glycosylation, and exocytosis. Remarkably though, we identified 53 unique genes. Certain unique genes function in previously identified pathways such as vesicle transport (e.g. , CCZ1, C18orfF) and ER/Golgi-localized proteins (e.g., SEC63, ERGIC3). Other unique genes function in processes that have not been previously described as proviral in HCoV infections. For example, EDC4 was a top-scoring gene in our SARS-CoV-2 screens in HEK293T-hACE2 cells. EDC4 functions as a scaffold protein for the assembly of the programmed mRNA decay complex. Although it has not been reported to play a role in HCoV infection before, it does promote rotavirus replication complex assembly. Another component of the programmed mRNA decay pathway, XRN1, was also modestly enriched, suggesting that this pathway promotes SARS-CoV-2 replication. EDC4 and XRN1 are both P-body components. Many RNA viruses interact with and hijack P-bodies in order to promote viral replication and SARS- CoV-2 has recently been reported to disrupt P-bodies so it is possible that the virus interacts with these host factors to disassemble P-bodies and facilitate viral replication. We also identified three unique genes encoding factors involved in targeting proteins to lysosomes - GNPTAB, GNPTG, and NAGPA. HCoVs may interact with these proteins to facilitate virion release from the infected cell. [171] Two approaches were taken to validate the proviral role of a subset of unique host factors identified in our screens. First, shRNA-mediated knockdown of CCZ1, EDC4, and XRN1 resulted in reduced SARS-CoV-2 and OC43 replication. Second, drugs targeting selected host factors displayed antiviral efficacy in vitro against SARS-CoV-2. These include cell cycle inhibitors ABE targeting Cdk4, AZ1 targeting Usp25/28, HAR targeting DyrklA, NIN targeting Fgfrl/2/3, and UC2 targeting p21; the endocytosis inhibitor PMZ targeting Wdr81; and the Tbkl inhibitor AMX. Chen et al. recently reported similar activity of ABE against SARS-CoV-2, validating our findings. To our knowledge, the discovery that AMX, AZ1, HAR, NIN, PMZ, and UC2 possess antiviral activity against SARS-CoV-2 has not been reported. AMX, PMZ, and NIN are currently available drugs which could potentially be repurposed, while HAR is a natural product being investigated for the treatment of a variety of diseases. While no clinical therapeutics are currently available targeting p21 or Usp25/28, our data suggest that these could be worthwhile targets for drug development.

Claims

47 Claims:

1. A method of treating a subject suffering from infection by or susceptible to infection by a coronavirus comprising administering to a subject a therapeutically effective amount of one or more host-targeted anti-viral therapeutics selected from the group consisting of: amlexanox, USP25/28 inhibitor AZ 1, harmine, nintedanib, UC2288, promethazine, archazolid, olaparib, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

2. The method of claim 1, wherein the coronavirus is selected from the group consisting of: a betacoronavirus, a SARS-CoV-related betacoronavirus, a SARS, SARS-CoV-2, and a MERS, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, and KHU4.

3. A method of treating a subject suffering from a coronavirus-related illness comprising administering to the subject a therapeutically effective amount of one or more host- targeted anti-viral therapeutics selected from the group consisting of: amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

4. The method of claim 3, wherein the coronavirus-related illness is selected from the group consisting of: a betacoronavirus, a SARS-CoV -related betacoronavirus, a SARS, SARS-CoV-2, and a MERS, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, and KHU4.

5. A method of preventing infection by a coronavirus comprising administering to a subject a therapeutically effective amount of one or more host- targeted anti-viral therapeutics selected from the group consisting of: amlexanox, USP25/28 inhibitor AZ1, harmine, nintedanib, UC2288, promethazine, archazolid, abemaciclib, an RNA function inhibitor targeting the CCZ1 gene, an RNA function inhibitor targeting the EDC4 gene, and an RNA function inhibitor targeting the XRN1 gene.

6. The method of claim 5, wherein the coronavirus is selected from the group consisting of: a betacoronavirus, a SARS-CoV-related betacoronavirus, a SARS, SARS-CoV-2, and a 48

MERS, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, and KHU4.

7. The method of any one of claims 1-6, wherein the RNA function inhibitor comprises an antisense oligonucleotide, a peptide-conjugated phosphorodiamidate, an siRNA, or a shRNA.

8. The method of any one of claims 1-6, wherein the method further comprises administering one or more additional active ingredients.

9. The method of claim 4, wherein the additional active ingredients are selected from the group consisting of: azelastine, diphenhydramine, and hydroxyzine.

10. The method of any one of claims 1-4 and 4-8, wherein the subject has tested positive for a coronavirus, has been exposed to a coronavirus, is suspected of having been exposed to a coronavirus, is at risk of being exposed to a coronavirus, is suffering from or diagnosed with a coronavirus-related illness, or is suffering from acute lung injury due to a coronavirus-related illness.

11. The method of any one of claims 2 and 4-7, wherein the coronavirus-related illness is a common cold, SARS, MERS, or COVID- 19.

12. The method of claim 1, wherein treating a subject suffering from coronavirus infection comprises decrease coronavirus disease burden, decreasing viral load, decreasing viral transmission, decreasing the severity of infection, and/or decreasing the duration of infection, decreasing coronavirus entry into host cells, or decreasing viral replication.

13. The method of claim 3, wherein treating a subject suffering from a coronavirus-related illness comprises: decreasing the severity of a symptom or decreasing the duration of a symptom.

14. The method of claim 5, wherein preventing infection by a coronavirus comprises inhibiting viral entry into host cells.

15. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: amlexanox. 49

16. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: USP25/28 inhibitor AZ 1.

17. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: harmine.

18. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: nintedanib.

19. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: UC2288.

20. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: promethazine.

21. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: archazolid.

22. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: abemaciclib.

23. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: an RNA function inhibitor targeting the CCZ1 gene.

24. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: an RNA function inhibitor targeting the EDC4 gene.

25. The method of any one of claims 1-14, wherein the one or more host-targeted anti-viral therapeutics comprises: and an RNA function inhibitor targeting the XRN1 gene.