Identification and use of compounds in the treatment or prevention of severe acute respiratory syndrome coronavirus 2

Abstract

To provide a method for identifying compounds useful for treating or preventing infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a method for treating or preventing SARS-CoV-2 infection using such compounds. [Solution] A method for identifying compounds useful for treating or preventing infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) includes screening at least one candidate compound for its ability to inhibit the cleavage of the SARS-CoV-2 spike protein by human proteases at one or more target sites.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of the concurrently pending U.S. Provisional Patent Applications No. 63 / 001,114 filed on 27 March 2020, No. 63 / 004,916 filed on 3 April 2020, No. 63 / 003,187 filed on 31 March 2020, No. 63 / 007,261 filed on 8 April 2020, No. 63 / 025,631 filed on 15 May 2020, No. 63 / 025,837 filed on 15 May 2020, and No. 63 / 142,392 filed on 27 January 2021, each of which is incorporated herein by reference as if described in its entirety.

[0002] Sequence List The sequence listing contained in the electronic file titled "VAND-0208-PCT_sequence_listing_ST25.txt", which was created on March 27, 2021, and consists of 131kb, is incorporated herein by reference. [Background technology]

[0003] SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, sometimes called 2019-nCoV), a spherical, positive-sense single-stranded RNA virus, is causing the current COVID-19 pandemic. To date, SARS-CoV-2 has infected more than 126 million people worldwide and killed more than 2.7 million. SARS-CoV-2 is a strain of the SARS-CoV species, belonging to the genus Betacoronavirus.

[0004] Like other coronaviruses, SARS-CoV-2 has four structural proteins: S (spike), E (envelope), M (membrane), and N (nucleocapsid). The S, E, and M proteins form the viral envelope, while the N protein is located within the core of the viral particle and is involved in binding to viral RNA and encapsulating the three-dimensional structure of the viral RNA within the viral particle. Some beta-coronaviruses also contain hemagglutinin-esterase (HE) proteins on the particle surface, and these HE proteins may enhance entry into host cells.

[0005] The spike protein is a highly glycosylated trimer class I fusion protein that protrudes from the virion surface, facilitating attachment and entry into host cells. In some coronaviruses, the S protein consists of two subunits, S1 and S2, on the surface of the viral particle. In other coronaviruses, including SARS-CoV-2, the S protein contains both S1 and S2 domains, but remains intact on the viral particle surface until it is cleaved within an endosite vesicle upon viral entry.

[0006] Significant structural rearrangement of the S protein is involved in the fusion of the S protein with the host cell membrane. Specifically, the receptor-binding domain (RBD) of the S1 subunit of the S protein undergoes a conformational shift from a "downward" conformation, where the receptor bound to the S1 subunit is inaccessible, to an "upward" conformation, where the receptor is accessible. The "upward" conformation is thought to be less stable than the "downward" conformation.

[0007] Recently, the precise three-dimensional structure of the S protein was determined using cryo-electron microscopy (cryo-EM). Figure 7 shows a three-dimensional representation of the S protein structure.

[0008] The S2 domain controls the entry of the virus into host cells. Angiotensin-converting enzyme 2 (ACE2) is a type I membrane protein that is widely expressed throughout human tissues, including the lungs, heart, kidneys, intestines, and adipose tissue. ACE2 was identified as a host cell receptor for early SARS-CoV strains and is a target of RBD for the SARS-CoV-2 S protein.

[0009] RBD binding is thought to occur on the outer surface of the ACE2 protein, while angiotensin substrate binding occurs in the deep depressions of the ACE2 protein. Figure 8 shows a three-dimensional representation of the ACE2 structure.

[0010] Recently, the S protein structure at a resolution of 3.5 angstroms was reported. As mentioned above, the S protein is cleaved into two subunits: the S1 subunit and the S2 subunit. This cleavage of the S protein by host proteases is important for viral infection and for the virus to exit the cell via lysosomes.

[0011] During infection, the S protein is cleaved by the host cell's protease, exposing the fusion peptide in the S2 domain. The cleavage of the S protein occurs between the S1 and S2 domains, and then within the S2 domain (S2') near the fusion peptide. This leads to fusion of the viral membrane with the cell membrane, releasing the viral genome into the host cell's cytoplasm. Both cleavage sites are thought to be necessary for viral entry into the host cell.

[0012] The S1 / S2 cleavage site of SARS-CoV-2 is located between threonine at position 696 and methionine at position 697 of the S protein amino acid sequence provided herein as Sequence ID No. 1. This S1 / S2 cleavage site is identical to that of SARS-CoV and has been shown to be cleaved by cathepsin L (CatL or CTSL), a lysosomal cysteine ​​protease encoded by the CTSL1 gene. CatL is a C1 peptidase dimer containing disulfide-bonded heavy and light chains derived from a protein precursor.

[0013] The cleavage site in the S2 domain of SARS-CoV-2, the S2' site, is the same as that of SARS-CoV and is located between arginine at position 815 and serine at position 816 (SEQ ID NO: 1). This site is also thought to be cleaved by CatL or transmembrane protease, serine 2 (TMPRSS2) upon virus entry. Inhibition of CatL or TMPRSS2 has been shown to suppress SARS-CoV infection.

[0014] SARS-CoV-2 also has a furin-like protease cleavage site between arginine at position 685 and serine at position 686 (SEQ ID NO: 1), which is not present in SARS-CoV. This site may be cleaved by furin upon virus release. The S protein of SARS-CoV-2 may also be stimulated by TMPRSS2. Inhibition of TMPRSS2 has been shown to suppress SARS-CoV infection. Furthermore, TMPRSS2 expression is correlated with SARS-CoV infection in the upper lobe of the lung.

[0015] The function of TMPRSS2 itself is unclear. TMPRSS2 is known to be highly expressed in the prostate. It has also been found that TMPRSS2 is upregulated by male hormones in prostate cancer cells. In addition, 50% of prostate cancers involve genomic rearrangements in which the androgen-inducible promoter of the TMPRSS2 gene is juxtaposed near the E26 transformation-specific (ETS) oncogene and the ETS oncogene is placed under androgen control. From these facts, the potential role of TMPRSS2 in the growth and clinical course of prostate cancer has been studied.

[0016] Similar to SARS-CoV, SARS-CoV-2 enters cells by binding to cell receptors including ACE2. Decreased expression of ACE2 is associated with cardiovascular diseases.

[0017] Recently, the structural basis of this recognition has been mapped, and the cryo-EM structure of the full-length viral spike protein targeting the human ACE2 complex has been reported. The SARS-CoV-2 S protein binds to ACE2 at least 10 times more tightly than the SARS-CoV ACE2, mediating receptor recognition.

[0018] Although the S1 / S2 and S2' cleavage sites are similar between SARS-CoV and SARS-CoV-2, it is unclear whether treatments applicable to SARS-CoV are similarly applicable to SARS-CoV-2. One study showed that several SARS-CoV RBD-specific monoclonal antibodies did not bind to the SARS-CoV-2 S protein. Another study showed that CR3022, a SARS-CoV-specific monoclonal antibody, bound to the SARS-CoV-2 RBD with high affinity.

[0019] The genomes of SARS-CoV-2 samples isolated from patients in the current pandemic have shown only slight differences, possibly less than 0.2%, suggesting a recent emergence in humans and rapid detection of the virus since its appearance. In other words, by timely identifying effective strategies for treating SARS-CoV-2 before further mutations occur as the virus spreads among human populations, overall effectiveness will be improved in response to the current pandemic.

[0020] As a result of infections caused by this virus, on March 11, 2020, the World Health Organization declared a pandemic of coronavirus disease 2019 (COVID-19). The most likely fatal outcome of SARS-CoV-2 infection in humans is acute respiratory distress syndrome or ARDS.

[0021] Severe COVID-19 infection / ARDS Common COVID-19 symptoms include cough, fever, fatigue, muscle aches, and diarrhea. Severe COVID-19 symptoms typically begin a week after initial symptoms and include dyspnea and hypoxemia. Patients presenting with these severe symptoms often experience progressive respiratory failure in the form of acute respiratory distress syndrome (ARDS). Severe COVID-19 infection can lead to acute damage to cardiac, renal, and hepatic tissue, including failure of these organ systems. Once ARDS develops, it is often fatal.

[0022] In the first few months of the pandemic, it began to become clear that older age and comorbidities were associated with a higher risk of severe COVID-19 infection. However, neither of these fully explained the heterogeneous course of infection observed.

[0023] More recently, gene sequencing studies of COVID-19 patients have suggested that the gene locus 3p21.31 is associated with the severity of COVID-19 infection. All of the gene variants on chromosome 3 most strongly associated with severe COVID-19 are in a state of high linkage disequilibrium (LD). Furthermore, recent phylogenetic analyses have shown that the haplotypes of these six genes (SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, and XCR1) associated with the risk of severe COVID-19 infection entered the modern human genome from Neanderthals.

[0024] Host protease The fact that SARS-CoV-2 entry depends on host proteases is of significant importance to the efficiency of SARS-CoV-2 infection. SARS-CoV utilizes the endosomal cysteine ​​proteases cathepsin B (CTSB) and cathepsin L (CTSL). CTSL is a peptidase that preferentially cleaves peptide bonds, with an aromatic residue at the P2 position and a hydrophobic residue at the P3 position. CTSL is active in the presence of thiols at pH 3-6.5, and its enzymatic stability depends on ionic strength. CTSL proteolysis has been shown to be an important mechanism in the processing of viral glycoproteins before cell membrane fusion during previous Ebola and SARS-CoV outbreaks.

[0025] Genetic variations in the CTSL gene may affect the replication capacity of SARS-CoV-2. For example, CTSL polymorphisms may influence an individual's susceptibility to SARS-CoV-2, such as individuals with certain gene variants exhibiting reduced CTSL expression. Such individuals may be relatively better protected or have lower viral titers compared to individuals with other CTSL variants. Furthermore, components of the host's MHC I-mediated and CTSL-mediated immune responses may also influence viral replication. Susceptibility factors range from ethnic background to age-related groups and comorbidities.

[0026] High-Processing Screening Assay (HTSA) Elshabrawy et al., “Identification of a Broad-Spectrum Antiviral Small Molecule against Sever Acute Respiratory Syndrome Coronavirus and Ebola, Hendra, and Nipah Viruses by Using a Novel High-Throughput Screening Assay,” Journal of Virology, Vol. 88 (No. 8): pp. 4353-4365 (2014), describes a high-throughput screening assay (HTSA) for identifying small molecules that may be useful in treating SARS-CoV, Ebola virus (EBOV), Hendra virus (HeV), and Nipah virus (NiV), all highly infectious zoonotic viruses. All of these viruses possess an envelope and require host proteases for the processing, cleavage, and entry into host cells of their glycoproteins.

[0027] The HTSA described by Elshabrawy et al. helped identify several compounds capable of inhibiting pseudotyped viral entry into host cells by selectively inhibiting CatL cleavage of viral fusion peptides. These preferred compounds strongly inhibited such entry, while inhibition of host peptide cleavage was significantly weaker (e.g., proNYP-derived peptides).

[0028] Human leukocyte elastase, D-dimer, and α1 antitrypsin D-dimers are clot degradation products, also known as fibrin degradation products (FDPs), that result from the breakdown of blood clots through fibrinolysis. D-dimers are found in inflammatory states and are thought to reflect the activity of two important proteases, plasmin and elastase. Human leukocyte elastase (HLE) is involved in fibrinolysis, and its activity leads to D-dimer production and the cleavage of several other proteins, including elastin. Leukocyte elastase originates from granulocytes, which often increase during inflammation, and is part of the host immune response. Increased elastase activity in the lungs is associated with emphysema. A common hypothesis for the pathogenesis of chronic obstructive pulmonary disease (COPD) is an imbalance between proteases and their inhibitors, specifically HLE and its natural inhibitor, alpha-1 antitrypsin (A1AT). Furthermore, extensive genetic evidence has shown that individuals with hereditary alpha-1 antitrypsin deficiency develop emphysema in adulthood.

[0029] Coenzyme Q10 In viral infections, oxidative stress plays a damaging role through multiple pathways, attenuating antioxidant responses. The international scientific community is attempting to rapidly clarify the pathophysiology of disease associated with SARS-CoV-2 infection, biomarkers associated with severe disease, and promising treatments. Promising associations may serve as markers of disease severity and may play a causal role, therefore, it is necessary to investigate the relationship between levels of key antioxidants, such as coenzyme Q10 (CoQ10), and the severity of SARS-CoV-2 infection.

[0030] CoQ10 is a lipid-soluble molecule that is a member of the ubiquinone family (Figure 13). CoQ10 is ubiquitous in humans, present in most cells, and can be both endogenously synthesized and exogenously acquired. CoQ10 levels are highest in organs with the highest metabolic demands, such as the heart, lungs, kidneys, and liver. CoQ10 has several important physiological roles, including acting as an essential cofactor in the electron transport chain to produce ATP, and acting as a lipid antioxidant to neutralize free radicals and the resulting damage to the body.

[0031] CoQ10 levels can decrease for several reasons, including aging, foreign compounds that interfere with synthesis, and genetic conditions that predispose individuals to low levels. Statins inhibit HMG-CoA reductase, thereby inhibiting a common synthesis pathway and reducing cholesterol synthesis and CoQ10 levels. Atorvastatin has been shown to reduce CoQ10 levels by 49% during 14 days of treatment. CoQ10 levels peak around age 20 and then gradually decline in an age-dependent manner. At age 80, the largest tissue-specific declines occur in the lungs (51.7% from peak) and the heart (42.9% from peak). Mutations in several genes involved in CoQ10 biosynthesis can result in deficiency.

[0032] CoQ10 plays an essential anti-inflammatory role in the body as a free radical scavenger and has been extensively studied in the treatment of various inflammatory diseases. CoQ10 supplementation improved survival and pulmonary edema in rats with sepsis-induced acute lung injury. In patients with septic shock, low CoQ10 levels were found to correlate with high levels of inflammatory markers. CoQ10 inhibition of platelet aggregation can occur through multiple pathways, including the upregulation of cAMP and PKA, or through the inhibition of vitronectin (CD51 / CD61). CoQ10 has been shown to be beneficial in attenuating fibrosis in the lungs and livers of rats through the upregulation of the autophagy process. CoQ10 supplementation improves hepatic and systemic inflammatory markers in patients with non-alcoholic fatty liver disease. CoQ10 supplementation improves mortality and cardiac markers in patients with heart failure. In diabetic patients, total cholesterol levels and low-density lipoprotein levels improve with CoQ10 supplementation. CoQ10 supplementation has been shown to improve endothelial dysfunction in patients with dyslipidemia.

[0033] Regarding its role in viral infection, CoQ10 levels are known to be lower in patients with acute influenza. A study of 65 children with influenza revealed that children with H1N1 had significantly lower CoQ10 levels compared to those with seasonal influenza.

[0034] CoQ10 levels decrease over time and through the use of exogenous drugs such as statins, but several genetic disorders are also known to cause CoQ10 deficiency. Patients with Down syndrome have been found to have low CoQ10 levels and high levels of TNFα and IL-6. Furthermore, patients with Down syndrome are more susceptible to viral and bacterial infections, have a higher incidence of autoimmune diseases (diabetes, hypothyroidism), and have a higher incidence of acute lung injury. Acute respiratory distress syndrome (ARDS) in patients with Down syndrome is thought to be due to an imbalance in free radical scavenging. [Overview of the project]

[0035] One aspect of the present invention is a method for identifying compounds useful for the treatment or prevention of infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising screening at least one candidate compound for its ability to inhibit the cleavage of the SARS-CoV-2 spike protein by human proteases at one or more target sites.

[0036] Another aspect of the present invention provides a method for predicting the effectiveness of a compound determined to have inhibitory activity in inhibiting the enzymatic cleavage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, comprising: determining whether the amino acid sequence of the spike protein contains one or more mutations from the wild-type sequence (SEQ ID NO: 1), wherein the one or more mutations are selected from the group consisting of histidine at position 675 (SEQ ID NO: 3), leucine at position 704 (SEQ ID NO: 4), alanine at position 718 (SEQ ID NO: 5), phenylalanine at position 752 (SEQ ID NO: 6), leucine at position 765 (SEQ ID NO: 7), leucine at position 772 (SEQ ID NO: 8), glutamine at position 780 (SEQ ID NO: 9), cysteine ​​at position 797 (SEQ ID NO: 10), and serine at position 812 (SEQ ID NO: 11); and predicting that the compound is effective in inhibiting the enzymatic cleavage of the SARS-CoV-2 spike protein if the amino acid sequence of the spike protein contains one or more such mutations.

[0037] A further aspect of the present invention provides a method for inhibiting the enzymatic cleavage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, comprising administering a compound capable of binding to the octamer, nonamer, or decamer sequence of SEQ ID NO: 2 to an individual infected with SARS-CoV-2 or at risk of infection by SARS-CoV-2: GSFCTQLNRALTGIAVEQDKNTQ (Sequence ID 2).

[0038] Another aspect of the present invention provides a method for treating or preventing infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an individual, comprising administering to the individual an amount of amantadine sufficient to reduce lysosomal enzyme activity in the individual's cells.

[0039] Another aspect of the present invention provides a method for reducing the SARS-CoV-2 viral load in an individual infected with SARS-CoV-2, comprising administering to the individual an amount of amantadine sufficient to reduce the lysosomal enzyme activity in the individual's cells.

[0040] Another aspect of the present invention provides a method for predicting an individual's susceptibility to SARS-CoV-2 infection, comprising: determining the individual's genotype at the rs2378757 locus; predicting that the individual is relatively resistant to SARS-CoV-2 infection if the rs2378757 genotype of the individual is AC or CC; or predicting that the individual is relatively susceptible to SARS-CoV-2 infection if the rs2378757 genotype of the individual is AA.

[0041] A further aspect of the present invention provides a method for predicting the effectiveness of amantadine in treating an individual infected with SARS-CoV-2, comprising: determining the genotype of the individual at the rs2378757 locus; predicting that treatment of the individual with amantadine will be relatively effective if the rs2378757 genotype of the individual is AC or CC; or predicting that treatment of the individual with amantadine will be relatively ineffective if the rs2378757 genotype of the individual is AA.

[0042] Yet another aspect of the present invention provides a method for reducing the risk of infection by SARS-CoV-2 in an individual, comprising administering to the individual an amount of amantadine sufficient to reduce the lysosomal enzyme activity in the individual's cells.

[0043] Another aspect of the present invention provides a method for treating an individual who is infected with SARS-CoV-2 and has or is at risk of having acute respiratory distress syndrome (ARDS), comprising administering to the individual an amount sufficient to reduce HLE activity in the individual with at least one human leukocyte elastase (HLE) inhibitor.

[0044] Another aspect of the present invention provides a method for treating an individual who is infected with SARS-CoV-2 and has or is at risk of having acute respiratory distress syndrome (ARDS), comprising administering to the individual an amount sufficient to increase alpha-1 antitrypsin (A1AT) activity in the individual with at least one A1AT inducer or A1AT substitute.

[0045] Another aspect of the present invention provides a method for predicting the susceptibility of an individual infected with SARS-CoV-2 to acute respiratory distress syndrome (ARDS), comprising: determining or having determined the alpha-1 antitrypsin (A1AT) genotype of the individual; predicting that the individual has an increased susceptibility to ARDS if the A1AT genotype of the individual contains the S allele, the Z allele, or both; or predicting that the individual does not have an increased susceptibility to ARDS if the A1AT genotype of the individual does not contain either the S allele or the Z allele.

[0046] Another aspect of the present invention provides a method for treating a patient diagnosed with or suspected of having SARS-CoV-2 infection that has progressed to acute respiratory distress syndrome (ARDS), based on symptoms and potential exposure. The method comprises administering an antiandrogen to the individual in an amount effective in reducing the type II transmembrane serine protease (TMPRSS2) activity in the individual to a level sufficient to improve the manifestation of ARDS symptoms (i.e., one or more symptoms or other physiological effects). A method for reducing type II transmembrane serine protease (TMPRSS2) activity in an individual is provided, comprising administering at least one antiandrogen to the individual.

[0047] More specifically, the above method is a method for treating an individual diagnosed with or susceptible to developing severe acute respiratory distress syndrome as a result of diagnosis or suspected coronavirus 2 (SARS-CoV-2) infection, comprising administering an antiandrogen to the individual in an amount effective in reducing the type II transmembrane serine protease (TMPRSS2) activity to a level sufficient to improve the manifestation of ARDS symptoms.

[0048] Another embodiment of the present invention provides a method for reducing type II transmembrane serine protease (TMPRSS2) activity in an organism, the method comprising administering at least one antiandrogenic agent to the organism.

[0049] For the purposes of the above method, the antiandrogenic agent is one or more androgen receptor (AR) blockers, androgen synthesis inhibitors, or antigonadotropins. In this regard, in one embodiment, the antiandrogenic agent is one or more AR blockers selected from the group consisting of cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, osaterone acetate, dienogest, drospirenone, medrogestone, nomegestrol acetate, promegestone, trimegestone, flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, and apalutamide. In another embodiment, the antiandrogenic agent is one or more androgen synthesis inhibitors selected from the group consisting of ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, alpha-estradiol, and saw palmetto (Serenoa repens) extract. In yet another embodiment, the antiandrogenic agent is one or more antigonadotropins selected from the group consisting of cetrorelix, allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, oxendrone, estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, and diethylstilbestrol.

[0050] In another embodiment, the present invention relates to a method for identifying a compound that is a candidate that may be useful in treating infection of an individual or preventing infection of an individual or cell by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein subsequent binding by the SARS-CoV-2 spike protein is selected from the group consisting of histidine at position 34 of SEQ ID NO: 1, aspartic acid at position 30 of SEQ ID NO: 1, tyrosine at position 41 of SEQ ID NO: 1, glutamine at position 42 of SEQ ID NO: 1, lysine at position 353 of SEQ ID NO: 1, and arginine at position 453 of SEQ ID NO: 1. The present invention provides a method comprising: confirming that the compound binds to angiotensin-converting enzyme 2 (ACE2) so as to be blocked at one or more amino acids; or confirming that the compound binds to the SARS-CoV-2 spike protein so as to be blocked at one or more amino acids selected from the group consisting of tyrosine at position 453 of SEQ ID NO: 2, glutamine at position 498 of SEQ ID NO: 2, threonine at position 500 of SEQ ID NO: 2, asparagine at position 501 of SEQ ID NO: 2, and lysine at position 417 of SEQ ID NO: 2. Such confirmation may include calculating one or more terms selected from the group consisting of van der Waals energy terms, Coulomb energy terms, lipophilicity terms, hydrogen bonding terms, metallic bonding terms, reward terms, and penalty terms.

[0051] In another embodiment, the present invention provides a method for inhibiting sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of cells exhibiting angiotensin-converting enzyme 2 (ACE2) on their cell membrane, the method comprising exposing the cells to a compound at a concentration capable of preventing the SARS-CoV-2 spike protein from binding to ACE2 on the cells.

[0052] In another embodiment, the present invention provides a method for treating an individual infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising administering to the individual a certain amount of a compound effective in preventing the SARS-CoV-2 spike protein from binding to angiotensin-converting enzyme 2 (ACE2) on the cells of the individual that exhibit ACE2 on the cell membrane.

[0053] In yet another aspect, the present invention provides a method for preventing an individual at risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from becoming infected with SARS-CoV-2, comprising administering to the individual a certain amount of a compound effective in preventing the SARS-CoV-2 spike protein from binding to angiotensin-converting enzyme 2 (ACE2) on the cells of the individual that exhibit ACE2 on the cell membrane.

[0054] Another aspect of the present invention provides a method for treating or preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an individual, comprising determining that the level of coenzyme Q10 in the individual is lower than expected; and administering a certain amount of coenzyme Q10 to the individual.

[0055] A further aspect of the present invention provides a method for treating a patient with betacoronavirus infection, comprising: determining, or having determined, whether the patient carries at least one genetic marker associated with severe betacoronavirus infection; and, if the patient carries at least one genetic marker associated with severe betacoronavirus infection, administering to the patient an effective amount of at least one compound capable of inhibiting the expression of the FYCO1 gene, wherein the at least one compound is selected from the group consisting of indomethacin, primidone, triprolidine hydrochloride, and baclofen.

[0056] Another aspect of the present invention provides a method for determining a predisposition to severe betacornonavirus infection in an individual, comprising determining, or having determined, whether the individual carries at least one marker associated with severe betacornonavirus infection; and determining, if the individual carries at least one marker associated with severe betacornonavirus infection, that the individual is susceptible to severe betacornonavirus infection. [Brief explanation of the drawing]

[0057] These and other features of the present invention will be better understood from the following detailed description of various embodiments of the invention, in conjunction with the accompanying drawings illustrating various embodiments of the present invention. [Figure 1] Figure 1 shows the effect of amantadine on genes in the lysosomal pathway. [Figure 2A] Figure 2 shows the expression of CTSL across various organs and related cell types. [Figure 2B] Figure 2 shows the expression of CTSL across various organs and related cell types. [Figure 2C] Figure 2 shows the expression of CTSL across various organs and related cell types. [Figure 2D] Figure 2 shows the expression of CTSL across various organs and related cell types. [Figure 3] Figure 3 shows the relative expression of each rs2378757 variant in lung tissue. [Figure 4A] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4B] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4C] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4D] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4E] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4F] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4G] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4H] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 4I] Figure 4 shows alternative splicing of CTSL transcripts in various tissue types. [Figure 5] Figure 5 shows the three-dimensional structure of human neutrophil elastase protein in panel (a) and the three-dimensional structure of α1 antitrypsin in panel (b). [Figure 6] Figure 6 shows a heatmap of Pi*SZ in Europe, as reported in one study. [Figure 7] Figure 7 shows the three-dimensional structure of the SARS-CoV-2S protein. [Figure 8] Figure 8 shows the three-dimensional structure of ACE2. [Figure 9A] Figures 9A and 9B illustrate the binding affinity analysis of the AY-NH2 compound to ACE2 and the three-dimensional structure of the AY-NH2 compound bound to ACES, respectively. [Figure 9B] Figures 9A and 9B illustrate the binding affinity analysis of the AY-NH2 compound to ACE2 and the three-dimensional structure of the AY-NH2 compound bound to ACES, respectively. [Figure 10A] Figures 10A and 10B illustrate the binding affinity analysis of NAD+ compounds to ACE2 and the three-dimensional structure of NAD+ compounds bound to ACES, respectively. [Figure 10B] Figures 10A and 10B illustrate the binding affinity analysis of NAD+ compounds to ACE2 and the three-dimensional structure of NAD+ compounds bound to ACES, respectively. [Figure 11A] Figures 11A and 11B illustrate the binding affinity analysis of the leproterol compound to ACE2 and the three-dimensional structure of the leproterol compound bound to ACES, respectively. [Figure 11B] Figures 11A and 11B illustrate the binding affinity analysis of the leproterol compound to ACE2 and the three-dimensional structure of the leproterol compound bound to ACES, respectively. [Figure 12A] Figures 12A and 12B illustrate the binding affinity analysis of the thymopentin compound to ACE2 and the three-dimensional structure of the thymopentin compound bound to ACES, respectively. [Figure 12B] Figures 12A and 12B illustrate the binding affinity analysis of the thymopentin compound to ACE2 and the three-dimensional structure of the thymopentin compound bound to ACES, respectively. [Figure 13] Figure 13 shows the molecular structure of coenzyme Q10. These figures are not to exact scale and are intended to illustrate only typical embodiments of the present invention; therefore, they should not be considered to limit the scope of the invention. [Modes for carrying out the invention]

[0058] Screening tests The applicant has identified several novel cleavage sites for CatL within the SARS-CoV-2 S protein sequence that may be relevant to the treatment or prevention of SARS-CoV-2 infection. Any compound, including small molecules, that has the ability to inhibit cleavage at one or more of these sites may be useful in the treatment or prevention of SARS-CoV-2 infection, whether by direct inhibition of enzymatic cleavage by binding to host proteases, by binding to the S protein and shielding these cleavage sites, by altering the expression of host proteases, or by altering the function of lysosomes in which host proteases are stored.

[0059] The novel cleavage sites identified by the applicant include locations in the wild-type amino acid sequence of the S protein (SEQ ID NO: 1) where protease cleavage is known or thought to occur, whether during viral entry, replication, packaging, or release. Those skilled in the art will recognize that other amino acid sequences of the SARS-CoV-2 S protein that deviate somewhat from the wild-type sequence are known. The novel cleavage sites of the present invention are also present in these other sequences, although they may differ in specific amino acid positions.

[0060] The novel cleavage sites of the present invention include a cleavage site located between threonine at position 768 and glycine at position 769 (SEQ ID NO: 1), and a cleavage site located between arginine at position 815 and serine at position 816 (SEQ ID NO: 1).

[0061] Embodiments of the present invention include methods for screening one or more candidate compounds with respect to their ability to inhibit the cleavage of the SARS-CoV-2 S protein at either of these novel cleavage sites. Such screening includes using assays to screen multiple candidate compounds simultaneously or sequentially.

[0062] An assay useful for carrying out the present invention is, for example, the HTSA reported by Elshabrawy et al., which is incorporated herein by reference as if everything it teaches were fully described.

[0063] Other embodiments of the present invention include inhibiting the enzymatic cleavage of the SARS-CoV-2 spike protein, treating SARS-CoV-2 infection, or preventing SARS-CoV-2 infection by administering to an individual at least one compound identified to have the following inhibitory activity.

[0064] In fact, the applicant has discovered a sequence range within the S protein that appears to be sensitive to both enzymatic cleavage and binding that is promising for inhibiting such cleavage. This sequence is 23 amino acids long and extends from glycine at position 757 (SEQ ID NO: 1) to glutamine at position 779 (SEQ ID NO: 1). This sequence is shown below in full length and is specifically identified herein as SEQ ID NO: 2. GSFCTQLNRALTGIAVEQDKNTQ(Sequence ID 2)

[0065] Any amino acid sequence within SEQ ID NO: 2 can be a binding target. As will be understood by those skilled in the art, such a sequence should be long enough to ensure adequate binding specificity. Any octamer, nonamer, or decamer sequence within SEQ ID NO: 2 is expected to provide sufficient binding specificity.

[0066] The applicant has also found that one or more mutations in the SARS-CoV-2 S protein sequence may make the inhibition of enzymatic cleavage more effective. These mutations are thought to result in conformational changes within the S protein that reduce the sensitivity of any of these novel cleavage sites to enzymatic cleavage. In the presence of one or more of these mutations, inhibition of cleavage at the aforementioned known CatL cleavage sites by any known compound or the compounds identified in embodiments of the present invention may also be improved.

[0067] These mutations are shown in Table 1 below. As those skilled in the art will understand, the mutations are described in terms of mutations from the amino acids of the wild-type sequence (SEQ ID NO: 1) at specific positions.

[0068] [Table 1]

[0069] Therefore, embodiments of the present invention may further include determining whether an individual is infected with or at risk of infection with a SARS-CoV-2 virus strain containing one or more mutations of Table 1.

[0070] Needless to say, as should be obvious to those skilled in the art, determining whether a spike protein sequence contains one or more such mutations and is therefore more sensitive to inhibition of enzymatic cleavage can be applied to screening and therapeutic methods other than those described herein. Such a determination step may be used, for example, when treating an individual with a compound identified by a method other than those described herein.

[0071] Understanding the mechanism of action of SARS-CoV-2 infection is a fundamental step in determining the optimal therapeutic agent. For example, interfering with the host cell's processing of the S protein, whether through environmental influences or altering gene expression levels, offers a promising therapeutic approach.

[0072] Novel therapeutic agents, identified by high-throughput screening assays and shown to block cleavage of the SARS-CoV2-S protein by CTSL, CTSB, TMPRSS2, or any other host protease at predicted / selected binding sites, would be an effective approach to functionally target and limit SARS-CoV-2 infection.

[0073] Other therapeutic mechanisms of action may include reducing or regulating CTSL expression, or influencing the conditions of the CTSL lysosomal environment by adjusting pH.

[0074] The applicant examined various drugs that could potentially help identify promising therapeutic agents capable of reducing CTSL gene expression. Five such drugs showed such potential, one of which, amantadine, deserves to be considered potentially useful in the treatment of patients with COVID-19.

[0075] Cell culture and drug treatment Drug screening was used to investigate drugs that may treat or prevent SARS-CoV-2 infection. To establish a drug profile database, the retinal pigment epithelial cell line ARPE-19 / HPV-16 was selected because it is non-cancerous, human-derived, and has a normal karyotype. ARPE-19 / HPV-16 can be easily grown as a monolayer in 96-well plates and expresses various well-known neuronal surface receptors, including dopamine receptor D2, serotonin receptors 1A, 2A, and 2C, muscarinic receptor M3, and histamine receptor H1.

[0076] Cell lines were grown according to the supplier's specifications (ATCC, Manassas, Virginia). Compounds were obtained from Sigma (St. Louis, Missouri) or Vanda Pharmaceuticals (Washington, Washington, D.C.). Cells were dispensed into 96-well plates (approximately 2 × 10⁶). 5 Cells were incubated for 24 hours, and then given fresh medium containing the drug or drug solvent (water, dimethyl sulfoxide, ethanol, methanol, or phosphate-buffered saline). The drug was diluted 1000-fold in buffered Advanced D-MEM / F-12 medium (Invitrogen, Carlsbad, California) containing essential amino acids and 110 mg / L sodium pyruvate. Under these conditions, a significant change in pH was not expected, but this was confirmed by monitoring the pH indicator present in the medium.

[0077] A final drug concentration of 10 μM was selected because it was considered to fall within a physiologically relevant range. Microscopic examination of each well was performed at the end of the treatment, and any samples were discarded if the cells had undergone morphological changes consistent with apoptosis. Confirmation was also made that the drug had not precipitated in the culture medium.

[0078] Gene expression assay Cells were harvested 24 hours after treatment, and RNA was extracted using the RNeasy 96 protocol (Qiagen, Valencia, California). Gene expression of 12,490 genes and 22,238 probe sets was prepared using a U133A2.0 microarray according to the manufacturer's instructions (Affymetrix, Santa Clara, California). Drugs were profiled in duplicate or triplicate form, and multiple solvent controls were placed on each plate. A total of 708 microarrays were analyzed, including 74 for 18 antipsychotics, 499 for 448 other compounds, and 135 for solvent controls.

[0079] First, raw scan data was converted to mean difference values ​​using MAS5.0 (Affymetrix). The mean difference values ​​for both the treatment and control groups were set to a minimum threshold of 50 if they were less than or equal to 50. Next, for each treatment example, all probe sets were ranked based on the amplitude or level of expression compared to the solvent control (or the average of the controls if more than one was used). Amplitude was defined as the expression ratio (tv) / [(t+v) / 2] (where t corresponds to the treatment example and v corresponds to the solvent example).

[0080] Each drug group profile was created using a novel WIMRR (Weighted Influence Model, Rank of Ranks) method, which emphasizes the rank of each probe set across the entire gene expression profile, rather than changes at specific expression levels. WIMRR obtains the average rank of each probe set across all members of the group, and then re-ranks the probe sets from the lowest mean rank to the highest mean rank. Gene set enrichment metrics were based on the Kolmogorov-Smirnov (KS) statistic. Specifically, for a given probe set, the KS score indicates how high (positive) or low (negative) that probe set is within the profile of another treatment example.

[0081] result The applicant analyzed the expression profiles of CTSL across all 466 test drugs. To find positive hits, only results that caused at least a 33% reduction in CTSL expression (a two-thirds reduction) were selected. The top drug targets (Table 1) included drugs from various therapeutic areas (muscle relaxants, antihistamines, antiepileptics, anticholinergics, and antivirals). No drugs reduced CTSL expression by more than 40%. Among the top results, amantadine is a known and safe antiviral agent that has been previously used to treat patients with influenza A.

[0082] [Table 2]

[0083] Amantadine hydrochloride is a lysosomal alkalizing agent. Due to the physicochemical properties of amantadine, accumulation in lysosomes occurs. Lysosomal agents affect pH, Ca 2+Amantadine affects lysosomes by blocking signal transduction, permeabilizing the lysosomal membrane, inhibiting enzyme activity, and accumulating storage substances. Amantadine acts as a lysosomal agent, readily passing through the lysosomal membrane and accumulating within lysosomes. It may also inhibit protease activity by lowering the pH of lysosomes.

[0084] Amantadine may also block the assembly of the influenza virus during viral replication. Furthermore, amantadine may directly affect viral entry by downregulating CTSL and other lysosomal pathway genes.

[0085] Amantadine hydrochloride IR is available as a 100 mg tablet (equivalent to 81 mg of base amantadine) and a 50 mg / 5 mL syrup (equivalent to 40 mg / 5 mL of base amantadine), and is typically administered twice daily.

[0086] Since CTSL was not the transcript with the most differing expression levels, the applicant extended the analysis to all genes downregulated by amantadine. Among the top 500 probes with differing expression levels (383 genes, all with at least a 50% reduction in expression), the applicant found 21 lysosome-related genes (GO:005764, p=2.49×10⁻⁶). -5 In addition, the most important pathway identified by ENRICHR enrichment analysis is the KEGG lysosome. The important effects of amantadine on lysosomal membrane protein (LAMP) pathway genes are shown in Figure 1, and in Tables 3 and 4.

[0087] [Table 3]

[0088] [Table 4]

[0089] The applicant also investigated ethnic, innate, and variability in CTSL expression, focusing on common and rare variants. The GTEx (Genotype-Tissue Expression) project provides genotypic information and gene expression levels for 49 human tissues obtained from 838 donors, enabling the examination of CTSL expression patterns both across tissues and individuals. Figure 2 shows CTSL expression across various organs and related cell types. CTSL is widely expressed in many vital organs (highly expressed in the lungs, nerve tibial, fat, arteries, and whole blood, among others).

[0090] By examining eQTL variants of CTSL, the applicant found a highly significant, lung-specific variant (rs2378757) exhibiting highly variable expression. As shown in Figure 3, the CC genotype is associated with low baseline expression and a potentially favorable treatment response, while the AA genotype is associated with high expression and is likely susceptible to high viral loads.

[0091] The applicant has focused on a series of splicing QTLs, including variants such as rs114063116 that affect the transcript splicing ratio and were significant and present in lung tissue. CTSL GTEx analysis suggests potential protection or susceptibility in certain individuals.

[0092] Interestingly, alternative splicing of CTSL transcripts in the lung further reveals a tissue-specific regulatory program. Results for various tissue types are shown in Figure 4.

[0093] Recent functional studies have identified a common variant in CTSL at the proximal CTSL1 promoter (position C-171A), which has been shown to alter transcription through changes in xenobiotic response elements. This variant, along with other similar variants, may influence viral adaptability in SARS-CoV-2 cell entry by affecting innate diversity in baseline expression.

[0094] Furthermore, the applicant focused on several rare variants and mutation resistance statuses of CTSL in the gnomAD database. The results showed that CTSL has an average of 167 missense variants, and the gene is predicted to be resistant to loss-of-function variants with a pLI of 0.01. Combined with significant eQTLs, this indicates a strong influence of genetic variation on CTSL expression and its variability.

[0095] TMPRSS2 is also widely expressed in multiple tissues, including those of the digestive system, lungs, and kidneys. High expression of CTSL and TRMPSS2 transcripts in a range of organs could explain the emergence of the virus in these tissues. Recent studies, for example, have shown SARS-CoV-2 in fecal samples obtained from infected individuals, demonstrating the significant inter-tissue influence of the virus.

[0096] ARDS and increased human leukocyte elastase activity in COVID-19 The pathophysiology of ARDS in COVID-19 is still not fully understood. While not bound by a specific mechanism, the applicant hypothesizes that the aforementioned viral infection triggers an inflammatory host response, particularly in the lungs, leading to the sequestration and activation of granulocytes in the lower respiratory tract and alveoli. There is significant evidence that HLE is involved in the depletion of at least one pulmonary surfactant protein, pulmonary surfactant protein D (SP-D), during lung inflammation. Pulmonary surfactant proteins A, B, C, and D are part of surfactant, produced by type II alveolar cells, and function to reduce surface tension at the interface between the liquid and gas phases of the alveoli. It is hypothesized that increased HLE activity in the alveoli could lead to a rapid and catastrophic deterioration of respiratory function in patients with ARDS secondary to COVID-19 infection. If true, this theory offers many promising therapeutic opportunities, some of which are fast-acting.

[0097] α1 antitrypsin deficiency (AAT) allele carriers and those at risk of COVID-19 ARDS Individuals carrying the genetic polymorphism that causes AAT are predicted to have an increased risk of ARDS associated with COVID-19 infection. Given the overwhelming healthcare systems due to the rapid pandemic, identifying individuals at highest risk of death is crucial. The high risk of severe complications and death for the elderly and those with underlying medical conditions has been discussed. However, in addition to differences in prognosis within this population, as the infection population expands, it is now becoming clear that young people with no apparent underlying medical conditions are also becoming more severely ill, some of whom are dying from acute respiratory failure. To better assess and identify individuals at risk who may require additional emergency intervention, it is necessary to rapidly conduct the necessary epidemiological analyses and widely share the data.

[0098] Europe has been the epicenter of the COVID-19 epidemic, with high incidence of ARDS and associated mortality. In addition to emerging COVID-19 mortality data, the prevalence of AAT alleles reported in the literature also suggests a tendency for higher mortality in populations with high allele frequencies of either the S AAT allele or the Z AAT allele. Blanco et al. reported the following in their review: "In Europe, the mean SZ carrier rates by region (from highest to lowest) were as follows: Southern Europe, 1 SZ per 483 subjects (1:483); Western Europe, 1:581; Northern Europe, 1:1,492; Central Europe, 1:1,712; and Eastern Europe, 1:11,81.8." From this, it is expected that mortality rates from COVID-19 will be higher in Southern Europe and lower in Central and Eastern Europe.

[0099] Carrier rates for the Pi*SZ genotype are high in Southern Europe and low in Central Europe. It should also be noted that in the Italian peninsula, carrier rates are high in the north compared to the south, where they are very low.

[0100] Cumulative data on mortality rates during COVID-19 infection (as of March 31, 2020) shows a correlation between mortality rates (defined as the number of deaths / number of confirmed cases) and the carrier rate of the Pi*SZ A1AT gene allele reported by country. For robustness, the applicant included data only from nine EU countries that had reported more than 10,000 confirmed cases at the time of application. The results of this correlation analysis are shown in Table 5. A significant correlation of R=0.66 (p=0.05) was found using mortality rates and Pi*SZ carrier rates for all nine countries. Blanco et al. (2017) confirmed a significant difference in Pi*SZ genotype carrier rates between northern Italy (high) and southern Italy (low). Therefore, the above data was re-analyzed, this time excluding Italy. In this additional analysis (excluding Italy), shown in Table 5, the correlation between mortality and Pi*SZ carrier rate is even stronger, at R=0.88 (p-value=0.003).

[0101] [Table 5]

[0102] These results suggest that Pi*SZ genotype status may be a risk factor for COVID-19 ARDS and subsequent death. Further confirmation may lead to the suggestion of different therapeutic approaches for COVID-19 patients with the Pi*SZ genotype, including aggressive supportive care and the introduction of elastase-reducing therapies, which may include small molecule elastase inhibitors and / or A1AT activity replacement.

[0103] Human leukocyte elastase (HLE) inhibitors in the treatment of COVID-19 ARDS There has been interest in developing HLE inhibitors for the treatment of emphysema and for the treatment of patients with genotype alpha-1 antitrypsin (A1AT) deficiency.

[0104] Sibelestad Sivelestat is currently available in Japan and South Korea for the treatment of acute lung injury (ALI), including ARDS. Several clinical studies, including those by Aikawa and Kawasaki, have supported the therapeutic usefulness of sivelestat in ARDS. Nevertheless, the extent of its clinical benefit remains debated. In one phase III trial of 230 mechanically ventilated ALI patients, sivelestat reduced the duration of mechanical ventilation and shortened ICU stay, but did not show a significant effect on 30-day survival. Another trial of 492 patients showed no effect on ventilator-free days or 28-day overall mortality. However, in a post-marketing trial designed to re-evaluate the efficacy of sivelestat in 404 ALI patients and 177 controls, sivelestat significantly improved ventilator-free days. While the differences in these trial results may be due to differences in the trial populations and trial design, sivelestat is now widely used in ICU settings for ARDS patients in Japan and South Korea.

[0105] Zemaira (registered trademark) Zemyra® is an alpha-protease inhibitor (A1-Pi) approved by the U.S. Food and Drug Administration for long-term augmentation and maintenance therapy in adults with clinical evidence of A1AT deficiency and emphysema. Zemyra® is not approved for patients with lung disease in whom severe A1AT deficiency has not been established.

[0106] Alberta (MPH996) Alberestat is an experimental leukocyte elastase inhibitor currently under development in the United States for the treatment of patients with α1 antitrypsin deficiency of the Pi*ZZ, Pi*SZ, or Pi*Null / Null genotypes. According to NCATS, "The clinical profile of this drug suggests that it is well-tolerated with minimal side effects, and that a simple method exists for indirectly measuring its activity in vivo."

[0107] Imbalanced hyperactivity of human leukocyte elastase may be involved in the development and progression of acute respiratory distress symptoms in COVID-19 patients. Administration of small molecule inhibitors, peptide inhibitors, or protein inhibitors that work to reduce HLE activity, including by direct interference with HLE enzymatic activity or by downregulation of the neutrophil elastase gene (ELANE) encoding HLE, may be therapeutically effective in critically ill COVID-19 patients and is therefore worth investigating in controlled clinical trials. Furthermore, the applicant's finding of higher mortality among COVID-19 patients with the Pi*SZ genotype, if confirmed, may suggest specific treatment options and treatment plans for these patients.

[0108] TMPRSS2 The single nucleotide polymorphism rs8134378 in TMPRSS2 has been shown to reduce androgen receptor binding and transactivation. Therefore, TMPRSS2 protease levels on the cell surface may vary depending on androgen levels. Other gene sequence variability may also contribute to variability in TMPRSS2 expression on the cell membrane surface.

[0109] For example, several eQTLs (external quantifiable trait loci) specifically associated with variable expression of the TMPRSS2 gene have been reported in the GTEx (Genotype-Tissue Expression) database. These include rs8134657, rs8134378, rs6517673, rs9979885, rs9984523, rs9978587, rs28360562, rs34205539, rs1041449, and rs3498323. In each of these nine polymorphisms, carriers of the minor allele are associated with low TMPRSS2 expression, while carriers of the other major allele are associated with high expression.

[0110] Given these findings and the role of TMPRSS2 as a cellular receptor for SARS-CoV-2, antiandrogen therapy may reduce the virus's ability to invade human cells by lowering TMPRSS2 levels, thereby reducing viral load and improving clinical outcomes.

[0111] In carrying out various embodiments of the present invention, antiandrogenic agents, that is, pharmaceuticals that block the action of androgens, i.e., male hormones such as testosterone, may be used. Suitable antiandrogenic agents include, for example, androgen receptor (AR) blockers, i.e., pharmaceuticals that directly block the action of androgens. Examples of AR blockers include steroidal antagonists, such as cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, and osaterone acetate, dienogest, drospirenone, medroguestone, nomegestrol acetate, promegestone, and trimegestone, as well as nonsteroidal antagonists, such as flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, and apalutamide.

[0112] Other suitable antiandrogenic agents include androgen synthesis inhibitors, i.e., drugs that act to lower androgen levels. Such drugs include CYP17A1 inhibitors such as ketoconazole, abiraterone acetate, and ceviteronel, and CYP11A1 inhibitors such as aminoglutethimide. Other androgen synthesis inhibitors include 5α-reductase inhibitors such as finasteride, dutasteride, epristeride, alfatradiol, and saw palmetto (Serenoa repens) extract.

[0113] Other antiandrogenic agents include antigonadotropins, or drugs that work to lower androgen levels, similar to androgen synthesis inhibitors. These drugs include gonadotropin-releasing hormone (GnRH) modifiers such as cetrorelix; progestogens such as allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, and oxendrone; and estrogens such as estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, and diethylstilbestrol.

[0114] In addition to those described and illustrated above, other antiandrogen agents known in the art may be used.

[0115] In carrying out aspects of the present invention, one or more antiandrogenic agents, including one or more of the above-described agents, are administered to an individual in an amount sufficient to reduce the TMPRSS2 activity in the individual. Such reduction of TMPRSS2 activity may result from direct androgen blockade (e.g., through the use of AR blockers), reduction of androgen production or synthesis (e.g., through the use of androgen synthesis inhibitors or antigonadotropins), or both. In some embodiments of the present invention, both AR blockers and androgen synthesis inhibitors or antigonadotropins are used and include any number or combination of the above-described agents.

[0116] In carrying out the method of the present invention, individuals are selected for treatment based on signs of ARDS. ARDS occurs when fluid accumulates in the alveoli, leading to oxygen replenishing in the bloodstream and depriving organs of the oxygen necessary for function. Severe shortness of breath is the main symptom of ARDS and can develop within hours to days following an induced infection, such as a diagnosis or suspicion of coronavirus 2 (SARS-CoV-2) infection. Once ARDS develops, it is often fatal. The risk of death increases with age and with complications. ARDS can cause persistent damage to the lungs. The diagnosis of ARDS is achieved using established diagnostic criteria known in the art, as is the determination of whether an individual has a coronavirus 2 (SARS-CoV-2) infection or is suspected of having such an infection based on symptoms or potential exposure to the virus. Diagnostic tests for infections are known in the art.

[0117] Furthermore, in the implementation of the present invention, the amount of antiandrogenic agent administered to an individual is determined by the patient's condition, the potency of the drug, the patient's age and weight, and other criteria known in the art for producing the desired antiandrogenic effect. Treatment by this method is initiated at any point after it has been determined that the individual being treated is known or suspected to have coronavirus 2 (SARS-CoV-2) infection, exhibiting signs of ARDS, or, in asymptomatic patients, potentially developing ARDS. For example, an antiandrogenic agent may be administered prophylactically to a patient at increased risk of developing ARDS, before a diagnosis of ARDS is made. Such patients may include elderly patients (e.g., 60 years or older or 65 years or older), and patients with comorbidities, such as patients with hypertension, diabetes, cardiovascular disease, asthma, or immunocompromised individuals with immune system disorders.

[0118] Treatment of an individual using this method may be continued until a desirable improvement in one or more symptoms of ARDS is observed, or until ARDS is completely resolved, i.e., for as long as necessary to restore the lung function that may have been impaired as a result of the syndrome. Therefore, treatment of an individual may be continued for several days to several weeks from the start of treatment, or for several months if necessary.

[0119] ACES2 Spontaneous genetic variations in ACE2 may influence an individual's susceptibility to SARS-CoV-2 infection and viral replication capacity. Specifically, histological analysis of ACE2 genotypes suggests that relatively rare candidate variants may constitute susceptibility or resilience to SARS-CoV-2 infection in certain individuals. There are also age-related differences in ACE2 expression relative to ACE. The ACE2 / ACE ratio is higher in younger individuals. In addition, because the human ACE2 gene is located on the X chromosome, males carrying rare ACE2 coding variants are hemizygous, meaning that all ACE2-expressing cells will express only that rare variant. Females carrying rare ACE2 coding variants, on the other hand, are considerably more likely to be heterozygous, and typically express that rare ACE2 variant in a mosaic distribution determined by an early X chromosome inactivation event.

[0120] By comparing the human ACE2 amino acid sequence (SEQ ID NO: 12) with the ACE2 amino acid sequences of other animals (chickens, pigs, dogs, and cats), and further comparing them with the reported SARS-CoV-2 infection susceptibility of the above-mentioned animals, and focusing on functional contact amino acid residues, certain amino acids may be important for viral entry. In particular, one amino acid is histidine at position 34 (His 34 (Shown in blue in Figure 8) and appears to be important for the entry of all types of viruses. Other ACE2 amino acids that may be important for SARS-CoV-2 virus entry include ASP. 30 (Shown in red in Figure 8), Tyr41 , Gln 42 , Lys 353 , and Arg 357 are exemplified.

[0121] His of the ACE2 sequence 34 is suggested to be potentially involved in the hydrogen bond with tyrosine (Tyr 453 ) at position 453 located within the RBD of the S protein sequence (SEQ ID NO: 2). Similarly, ASP of ACE2 30 is considered to bind to Lys of the RBD 417 , and Tyr 41 , Gln 42 , Lys 353 , and Arg 357 of ACE2 and Gln 498 , Thr 500 , and Asn 501 of the RBD are the same.

[0122] By performing in-silico chemical library screening, it becomes possible to identify small molecules that may be capable of binding to any of the amino acids of the ACE2 sequence or the RBD sequence, or may be capable of blocking such binding. Such binding or blocking provides a mechanism for inhibiting SARS-CoV-2 virus entry for treating or preventing infectious diseases.

[0123] First, apply the Glide docking protocol. This includes calculating the GlideScore of candidate molecules for predicting the binding of ACE2 and RBD. The GlideScore is calculated using Glide software available from Schrodinger, LLC. The components and usage of GlideScore calculation are known in the art, and specifically, the methodology is described and sold by Schrodinger, LLC in the user manual of Glide6.7, etc., which is incorporated herein as if fully described.

[0124] The docking score (GlideScore) is an empirical scoring function designed to maximize the distance between compounds with strong binding affinity and compounds with virtually no binding ability. As an empirical scoring function, it consists of terms that describe the physical properties of the binding process, including lipophilicity-lipophilicity terms, hydrogen bonding terms, rotatable binding penalties, and contributions from Coulomb-van der Waals energy between protein and ligand. A lower docking score indicates higher docking optimality. Candidate molecules are evaluated for their actual interactions at the docking site (including how the molecule is anchored), hydrogen bonding score (higher hydrogen bonding optimality, for example, as the distance decreases, the hydrogen bonding term decreases), and ligand efficiency (Glide score (G score) normalized by dividing by the number of heavy atoms).

[0125] Next, we determine the molecular descriptors related to the pharmacokinetics of the candidate molecules. Finally, we perform molecular dynamics simulations to verify the stability of the binding mode after docking. In total, we analyze approximately 11,000 non-overlapping candidate molecules.

[0126] Table 6 below shows the 10 candidate molecules with the lowest docking scores (i.e., those most favorable for binding) among the screened molecules.

[0127] [Table 6]

[0128] AY-NH2 is a selective PAR4 receptor agonist peptide (H-Ala-Tyr-Pro-Gly-Lys-Phe-NH2) and exhibits the most favorable docking score. Figure 9A illustrates the AY-NH2 compound annotated according to the above determination regarding the predicted interaction between the AY-NH2 compound and the relevant amino acids of ACE2. These include the ACE2 amino acid Asp 30 , Ala 387 , Gln 388, and Glu 564 This includes the predicted hydrogen bond. Figure 9B shows AY-NH2 bound to ACE2 in three dimensions as predicted above. When bound in this way, the SARS-CoV-2 S protein RBD causes His of ACE2 34 (Blue) or ASP 30 The binding to (red) is effectively blocked by NY-HN2 (gray).

[0129] NAD + Oxidized nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in many metabolic reactions, but it exhibits the second-best docking score. + Plasma levels have been reported to decrease significantly with age. Recent studies have shown that SARS-CoV-2 infection of cell lines leads to NAD + Regarding the synthesis and use of NAD + Significant dysregulation of the pathway has been shown. Figure 10A shows NAD + Regarding the predicted interaction between the compound and the related amino acids of ACE2, NAD was similarly annotated according to the above determination. + The compound is illustrated. Figure 10B shows the NAD bound to ACE2, as predicted. + This is displayed in three dimensions. When bound in this way, the SARS-CoV-2 S protein RBD causes His of ACE2 34 (Blue) or ASP 30 The binding to (red) is NAD + It is effectively blocked by (gray).

[0130] Reproterol (7-[3-[[2-(3,5-dihydroxyphenyl)-2-hydroxyethyl]amino]propyl]-1,3-dimethylpurine-2,6-dione) is a short-acting β2 adrenergic receptor agonist approved for the treatment of asthma. Reproterol exhibits the third-favorable docking score. Figure 11A illustrates the reproterol compound, annotated according to the above determination, with respect to the predicted interaction between the reproterol compound and the relevant amino acids of ACE2. Figure 11B shows a three-dimensional representation of reproterol bound to ACE2 as predicted. When bound in this manner, the His of ACE2 by the SARS-CoV-2 S protein RBD 34 (Blue) or ASP 30 Binding to (red) is effectively blocked by reproterol (gray).

[0131] Timopentin (H-Arg-Lys-Asp-Val-Tyr-OH) is a synthetic pentapeptide used to enhance thymic T cell production. Timopentin exhibits the fourth-favorable docking score. Figure 12A illustrates the timopentin compound, annotated according to the above determination, with respect to the predicted interaction of the timopentin compound with the relevant amino acids of ACE2. Figure 12B shows a three-dimensional representation of timopentin bound to ACE2 as predicted. When bound in this manner, the His of ACE2 by the SARS-CoV-2 S protein RBD... 34 (Blue) or ASP 30 Binding to (red) is effectively blocked by thymopentin (gray).

[0132] Other compounds in Table 6 showed lower docking scores, but they can inhibit SARS-CoV-2 infection by inhibiting the binding of the ACE2 amino acid to His34, among others.

[0133] CGS21680HCl (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamide adenosine hydrochloride) is a selective adenosine A2A-R agonist. NADH disodium (reduced nicotinamide adenine dinucleotide disodium) is a coenzyme for many oxidoreductases.

[0134] Nociceptin (1-7) (nociceptin fragment 1-7; H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-OH) is an active metabolite of nociceptin.

[0135] Mupirocin (9-[(E)-4-[(2S,3R,4R,5S)-3,4-dihydroxy-5-[[(2S,3S)-3-[(2S,3S)-3-hydroxybutan-2-yl]oxiran-2-yl]methyl]oxan-2-yl]-3-methylbuta-2-enoyl]oxynonanoic acid; pseudomonate) is a natural antibiotic currently used topically to treat staphylococcal infections such as impetigo of the skin. Mupirocin is also used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections.

[0136] SLIGRL-NH2 (H-Ser-Leu-Ile-Gly-Arg-Leu-NH2) is a peptide derived from the N-terminus of protease-activated receptor 2 (PAR2) and acts as a PAR2 agonist. Oxyneic acid (3-pyridinecarboxylic acid 1-oxide; nicotinic acid 1-oxide) is a nicotinic acid derivative with lipid-lowering activity.

[0137] In carrying out the method of the present invention, individuals are selected for treatment based on signs of symptoms associated with SARS-CoV-2 infection, including acute respiratory distress syndrome (ARDS), or on the individual's risk of SARS-CoV-2 infection. Diagnostic tests for SARS-CoV-2 infection are known in the art.

[0138] A particular concern when determining whether an individual should receive treatment is the signs of ARDS. ARDS occurs when fluid accumulates in the alveoli, leading to oxygen replenishing in the bloodstream and depriving organs of the oxygen necessary for their function. Severe shortness of breath is the main symptom of ARDS, and it can develop within hours to days after an induced infection, such as a diagnosis or suspicion of SARS-CoV-2 infection. Once ARDS develops, it is often fatal. The risk of death increases with age and with complications. ARDS can cause persistent damage to the lungs. The diagnosis of ARDS is achieved using established diagnostic criteria known in the art, as is the determination of whether an individual has a SARS-CoV-2 infection or is suspected of having such an infection based on symptoms or potential exposure to the virus.

[0139] In addition, when implementing the present invention, the therapeutic agent administered to the individual (e.g., AY-NH2, NAD) + The amount of leproterol, timopentin, CGS21680HCl, NADH disodium, nociceptin (1-7), mupirocin, SLIGRL-NH2, or oxyneopropyl acid) is determined by the patient's condition, the potency of the drug, the patient's age and weight, and other criteria known in the art.

[0140] Treatment using this method may be initiated at any point after the individual being treated is known to have or is suspected to have SARS-CoV-2 infection, either showing signs of ARDS or, in the case of asymptomatic patients, potentially developing ARDS. For example, the drug may be administered prophylactically to patients at increased risk of developing ARDS, before they are diagnosed with ARDS. Such patients may include elderly patients (e.g., 60 years or older or 65 years or older) and patients with comorbidities, such as patients with hypertension, diabetes, cardiovascular disease, asthma, or immunocompromised individuals with immune system disorders.

[0141] Coenzyme Q10 and COVID-19 A potential link may exist between reduced CoQ10 levels and the population most severely affected by COVID-19. While the causal mechanisms contributing to increased susceptibility to severe illness remain unclear, it may result from a reduction in one of the following: the ability to prevent oxidative stress, reduce coagulation, mitigate hyperimmune responses, or inhibit direct viral replication. Other antioxidants, such as vitamins C and E, may also be involved in disease-associated deficiencies. Large-scale studies should measure CoQ10 levels along with vitamin and lipid levels at presentation in COVID-19 patients to examine whether the correlation predicts clinical outcomes and whether it correlates with levels of inflammatory cytokines and molecules such as IL-2, IL-6, TNFα, and D-dimer. Clinical outcomes of COVID-19 in individuals genetically predisposed to CoQ10 deficiency should also be investigated. Given the complexity of SARS-CoV-2 infection and the heterogeneity of disease presentation, the reasons for severe illness may be multifactorial. CoQ10 may act as a correlated marker for severe disease, and possibly as a causative agent of susceptibility to worsening clinical outcomes.

[0142] If a correlation is confirmed, further investigation into the causal mechanism will be possible, and CoQ10 may potentially offer a protective treatment for infection in the future. Medication to supplement deficient CoQ10 levels, starting at 100-200 mg per day, can yield physiological effects. Given the multiple symptoms present in the case, prevention could yield more significant benefits. If low CoQ10 levels correlate with severe COVID-19 disease, supplementing deficient individuals may offer a therapeutic solution to reduce the disease burden and potentially improve the situation during this pandemic.

[0143] Severe COVID-19 and FYCO1 The genomes of 80 COVID-19 patients (68% male, 32% female; aged 35–87 years) exhibiting severe symptoms were analyzed and compared with the genomes of 1,876 individuals from 2000 genome-wide association studies (GWAS). The results of this comparison not only confirm previously reported associations between severe COVID-19 infection and six Neanderthal haplotypes, but also highlight the associations between three mutations in the FYCO1 gene and severe COVID-19 infection, as well as the rs73064425 SNP in the LZTFL1 gene. This more isolated haplotype showed the strongest association with severe COVID-19 infection, suggesting it may be useful in predicting and diagnosing severe COVID-19 infection.

[0144] The C / T variant, LZTFL1 SNP, rs73064425, has been reported to have a minor allele frequency (MAF) of 0.05. This value was consistent with those found within 2000 control genotypes. However, among COVID-19 patients, the above MAF was 0.17. This SNP is in strong linkage disequilibrium with each of three FYCO1 SNPs. These FYCO1 SNPs, two of which arise at the same codon, exhibit three coding mutations, causing amino acid substitutions in the resulting mRNA.

[0145] Of the newly associated FYCO1 SNPs, the first, rs13079478, is a G / T variant in which asparagine is substituted with aspartic acid; the second, rs13059238, is at the same codon as rs13079478 but is a T / C variant in which asparagine is substituted with lysine; and the third, rs33910087, is a G / A variant in which arginine is substituted with cysteine.

[0146] Table 7 below shows the MAF (Multiple Amount Factor) for each FYCO1 SNP within the COVID-19 population and the 2000 control population.

[0147] [Table 7]

[0148] As can be seen from Table 7, the minor allele frequencies of FYCO1 SNP and LZTFL1 SNP, respectively, are significantly higher within the COVID-19 population. This not only provides a useful method for predicting whether an individual is predisposed to developing severe COVID-19 symptoms upon exposure, but also offers a valuable therapeutic tool in the treatment of COVID-19 patients, enabling the identification of patients at higher risk of developing severe COVID-19 symptoms and the early treatment of such symptoms in those patients.

[0149] FYCO1 gene The FYCO1 gene encodes a protein involved in vesicular transport and autophagy. This protein is suggested to be an important intermediary that connects bimembrane vesicles derived from the endoplasmic reticulum, the primary replication site of coronavirus, to the microtubule network.

[0150] Furthermore, FYCO1 has been shown to be important in autophagosome-lysosome fusion via its LC3 interaction region (LIR) motif. FYCO1 dimerizes via its CC region, interacts with PI3P via its FYVE domain, and forms a complex with Rab7 via a portion of the CC region located anterior to the FYVE domain. Specifically, FYCO1 has been shown to bind to LC3 and PI3P and act as a Rab7 effector mediating microtubule-plus-end vesicle transport. Rab7 deficiency inhibits late endosome / multiple endoplasmic reticulum (MVB) maturation and reduces the number of lysosomes in the cell. FYCO1 also mediates the clearance of α-synuclein aggregates.

[0151] Genome-scale CRISPR function deletion screening in human alveolar basal cell carcinoma cells identified genes whose deletion enables resistance to SARS-CoV-2 infection. RAB7A deletion reduces viral entry / release by sequestering the ACE2 receptor within the cell. Depletion of FYCO1 antibodies against the N-terminus of LC3 blocks the intracellular redistribution of autophagosomes. Rare FYCO1 variants containing missense mutations in the LIR domain are associated with inclusion body myositis, a disease characterized by impaired autophagy.

[0152] The FYCO1 gain-of-function variant may increase the risk of severe COVID-19 infection and similarly increase the risk of other beta-coronavirus infections. In other words, downregulating FYCO1 could provide protection against these severe infections and offer promising treatments for COVID-19.

[0153] High-throughput gene expression analysis identified compounds capable of downregulating FYCO1. A total of 466 compounds from 14 therapeutic classes were subjected to this analysis using the human retinal pigment epithelial cell line ARPE-19, and changes in gene expression were collected for 12,490 genes. The effect of all 466 compounds on FYCO1 expression was greatest in the case of the four compounds shown in Table 8.

[0154] [Table 8]

[0155] Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) that has the greatest ability to inhibit or downregulate FYCO1 expression. Indomethacin is approved for the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, gouty arthritis, bursitis, and tendinitis. Indomethacin may be administered orally, intravenously, or rectally. The oral dose is usually 75-150 mg per day, divided into up to four doses. Oral doses are available in 20 mg, 25 mg, 40 mg, and 50 mg capsules, a 75 mg sustained-release capsule, and a 25 mg / 5 mL oral suspension.

[0156] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the disclosure. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless expressly stated otherwise or the context makes otherwise obvious. Furthermore, where used herein, the terms “comprises” and / or “comprising” indicate the presence of the described features, integers, processes, operations, elements, and / or components, but are understood not to exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof. The terms “optional” or “optional” mean that the element, phenomenon, or situation described thereafter may or may not occur, and that the description encompasses both the cases in which the element, phenomenon, or situation occurs or exists, and the cases in which it does not occur or does not exist.

[0157] The corresponding structures, materials, actions, and equivalents of all means or process elements and functional elements in the claims are intended to include any structures, materials, or actions for performing a function in combination with other specifically claimed elements. The descriptions in this disclosure are presented for illustrative and explanatory purposes, but are not intended to be exhaustive or to limit the disclosure to the disclosed forms. Many modifications and variations that do not depart from the spirit of this disclosure will be apparent to those skilled in the art. Any embodiment selected and described herein is presented to best illustrate the principles and practical applications of this disclosure, and to enable other those skilled in the art to understand that the disclosure of various modified embodiments is suitable for specific applications in which such applications are intended.

Claims

1. A method for identifying compounds useful for the treatment or prevention of infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), The method comprising screening at least one candidate compound for its ability to inhibit the cleavage of the SARS-CoV-2 spike protein by a human protease at one or more target sites.

2. The method according to claim 1, further comprising determining the level of inhibition of host cell peptide cleavage for at least one candidate compound.

3. The method according to claim 1, wherein the one or more target sites are selected from the group consisting of the space between threonine at position 768 and glycine at position 769 of SEQ ID NO: 1, and the space between arginine at position 815 and serine at position 816 of SEQ ID NO:

1.

4. The method according to claim 1, wherein the at least one candidate compound comprises a plurality of compounds.

5. The method according to claim 4, wherein the screening includes screening the plurality of compounds in an assay.

6. The method according to claim 1, wherein the ability to inhibit cleavage includes one or more means selected from the group consisting of: the at least one candidate compound binding to the human protease; the at least one candidate compound binding and blocking a target site on the spike protein; altering the expression of the human protease; and altering the function of the lysosome that stores the human protease in a host cell.

7. The method further includes determining whether the amino acid sequence of the spike protein contains one or more mutations from the wild-type sequence (SEQ ID NO: 1), The method according to claim 1, wherein the one or more mutations are selected from the group consisting of histidine at position 675 (SEQ ID NO: 3), leucine at position 704 (SEQ ID NO: 4), alanine at position 719 (SEQ ID NO: 5), phenylalanine at position 752 (SEQ ID NO: 6), leucine at position 765 (SEQ ID NO: 7), leucine at position 772 (SEQ ID NO: 8), glutamine at position 780 (SEQ ID NO: 9), cysteine ​​at position 797 (SEQ ID NO: 10), and serine at position 812 (SEQ ID NO: 11).

8. A method for inhibiting the cleavage of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), The method comprising administering to an individual at least one compound identified as having the ability to inhibit the cleavage by the method according to any one of claims 1 to 7.

9. A method for treating or preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an individual, The method comprising administering to the individual at least one compound identified as having the ability to inhibit the cleavage of the SARS-CoV-2 spike protein by the method according to any one of claims 1 to 7.

10. A method for predicting the effectiveness of a compound determined to have inhibitory activity in inhibiting the enzymatic cleavage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, To determine whether the amino acid sequence of the spike protein contains one or more mutations from the wild-type sequence (SEQ ID NO: 1), Here, the one or more mutations are selected from the group consisting of histidine at position 675 (SEQ ID NO: 3), leucine at position 704 (SEQ ID NO: 4), alanine at position 719 (SEQ ID NO: 5), phenylalanine at position 752 (SEQ ID NO: 6), leucine at position 765 (SEQ ID NO: 7), leucine at position 772 (SEQ ID NO: 8), glutamine at position 780 (SEQ ID NO: 9), cysteine ​​at position 797 (SEQ ID NO: 10), and serine at position 812 (SEQ ID NO: 11); It is predicted that the compound will be effective in inhibiting the enzymatic cleavage of the SARS-CoV-2 spike protein when the amino acid sequence of the spike protein contains one or more such mutations. The method, including the method described above.

11. A method for inhibiting the enzymatic cleavage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, Administering a compound capable of binding to the octamer, nonamer, or decamer sequence of Sequence ID No. 2 to individuals infected with SARS-CoV-2 or at risk of SARS-CoV-2 infection. GSFCTQLNRALTGIAVEQDKNTQ (Sequence ID 2) The method, including the method described above.

12. A method for treating or preventing infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an individual, The method comprising administering to the individual an amount of amantadine sufficient to reduce the lysosomal enzyme activity in the cells of the individual.

13. The method according to claim 12, wherein the amount of amantadine is sufficient to reduce the expression of at least one gene in the individual, and the at least one gene is selected from the group consisting of CTSL, AGA, BCL10, DC164, CLN5, CPQ, CTBS, CTSB, CTSH, CTSK, GALC, GJA1, GNS, LAMP1, LGMN, PCYOX1, PSAP, RAB38, RNASET2, SCARB2, STS, and MARCH3.

14. The method according to claim 13, wherein the at least one gene is CTSL.

15. The method according to claim 12, wherein the cells are located in airway tissue.

16. The method according to claim 12, wherein the amount of amantadine is sufficient to reduce the viral load in the individual.

17. The method according to claim 12, further comprising determining the genotype of the individual at the rs2378757 locus.

18. The method according to claim 17, wherein if the rs2378757 genotype of the individual is determined to be AA, the amount of amantadine administered to the individual is greater than if the rs2378757 genotype of the individual is determined to be AC ​​or CC.

19. A method for reducing the SARS-CoV-2 viral load in an individual infected with SARS-CoV-2, The method comprising administering to the individual an amount of amantadine sufficient to reduce the lysosomal enzyme activity in the cells of the individual.

20. The method according to claim 19, wherein the amount of amantadine is sufficient to reduce the expression of at least one gene in the individual, and the at least one gene is selected from the group consisting of CTSL, AGA, BCL10, DC164, CLN5, CPQ, CTBS, CTSB, CTSH, CTSK, GALC, GJA1, GNS, LAMP1, LGMN, PCYOX1, PSAP, RAB38, RNASET2, SCARB2, STS, and MARCH3.

21. The method according to claim 20, wherein the at least one gene is CTSL.

22. The method according to claim 19, wherein the cells are lung tissue cells.

23. The method according to claim 19, wherein the amount of amantadine is sufficient to reduce the viral load in the individual.

24. The method according to claim 19, further comprising determining the genotype of the individual at the rs2378757 locus.

25. The method according to claim 24, wherein, when the rs2378757 genotype of the individual is determined to be AA, the amount of amantadine administered to the individual is greater than when the rs2378757 genotype of the individual is determined to be AC ​​or CC.

26. A method for predicting an individual's susceptibility to SARS-CoV-2 infection, Determining the genotype of the individual at the rs2378757 locus; and, If the rs2378757 genotype of the said individual is AC or CC, it is predicted that the said individual is relatively less susceptible to infection with SARS-CoV-2; or, If the rs2378757 genotype of the aforementioned individual is AA, it is predicted that the individual is relatively susceptible to infection with SARS-CoV-2. The method, including the method described above.

27. A method for predicting the effectiveness of amantadine in treating individuals infected with SARS-CoV-2, Determining the genotype of the individual at the rs2378757 locus; and, If the rs2378757 genotype of the said individual is AC or CC, it is predicted that treatment of the said individual with amantadine will be relatively effective; or, If the rs2378757 genotype of the aforementioned individual is AA, it is predicted that treatment of the aforementioned individual with amantadine will be relatively ineffective. The method, including the method described above.

28. A method for reducing the risk of infection by SARS-CoV-2 in an individual, The method comprising administering to the individual an amount of amantadine sufficient to reduce the lysosomal enzyme activity in the cells of the individual.

29. The method according to claim 28, wherein reducing lysosomal enzyme activity reduces CTSL expression or CTSL activity in the individual.

30. A method for treating individuals who are infected with SARS-CoV-2 and who have or are at risk of developing acute respiratory distress syndrome (ARDS), The method comprising administering to the individual an amount sufficient to reduce the human leukocyte elastase (HLE) activity in the individual by at least one HLE inhibitor.

31. The method according to claim 30, further comprising determining whether the individual has an A1AT genotype associated with alpha-1 antitrypsin (A1AT) deficiency.

32. The method according to claim 31, wherein the A1AT genotype associated with A1AT deficiency includes the S allele, the Z allele, or both the S allele and the Z allele.

33. The method according to claim 30, wherein the at least one HLE inhibitor is selected from a group of drugs that have been shown to have HLE activity inhibitory activity, including sivelestat and alberestat.

34. The method according to claim 30, further comprising administering to the individual an amount sufficient to increase the α1 antitrypsin (A1AT) activity in the individual by at least one A1AT inducer or A1AT substitute.

35. The method according to claim 34, wherein the A1AT inducer is tamoxifen.

36. The method according to claim 34, wherein the A1AT substitute is a purified A1AT preparation.

37. A method for treating individuals who are infected with SARS-CoV-2 and who have or are at risk of developing acute respiratory distress syndrome (ARDS), The method comprising administering to the individual an amount sufficient to increase the α1 antitrypsin (A1AT) activity in the individual by at least one A1AT inducer or A1AT substitute.

38. The method according to claim 37, wherein the A1AT inducer is tamoxifen.

39. The method according to claim 37, wherein the A1AT substitute is a purified A1AT preparation.

40. The method according to claim 37, further comprising determining whether the individual has an A1AT genotype associated with alpha-1 antitrypsin (A1AT) deficiency.

41. The method according to claim 40, wherein the A1AT genotype associated with A1AT deficiency includes the S allele, the Z allele, or both the S allele and the Z allele.

42. A method for predicting the susceptibility of individuals infected with SARS-CoV-2 to developing acute respiratory distress syndrome (ARDS), Determining or having determined the α1 antitrypsin (A1AT) genotype of the said individual; and, If the A1AT genotype of the individual contains the S allele, the Z allele, or both, it is predicted that the individual will have an increased susceptibility to ARDS; or, If the A1AT genotype of the individual does not contain either the S allele or the Z allele, it is predicted that the individual will not have an increased susceptibility to ARDS. The method, including the method described above.

43. The method according to claim 42, further comprising administering to the individual, if the individual's A1AT genotype includes the S allele, the Z allele, or both, an amount sufficient to reduce the human leukocyte elastase (HLE) activity in the individual to a degree that reduces the human leukocyte elastase (HLE) activity in the individual.

44. The method according to claim 43, wherein the at least one HLE inhibitor is selected from a group of drugs that have been shown to have HLE activity inhibitory activity, including sivelestat and alberestat.

45. The method according to claim 42, further comprising administering to the individual, if the individual's A1AT genotype includes the S allele, the Z allele, or both, at least one A1AT inducer or A1AT substitute in an amount sufficient to increase the α1 antitrypsin (A1AT) activity in the individual.

46. The method according to claim 45, wherein the A1AT inducer is tamoxifen.

47. The method according to claim 45, wherein the A1AT substitute is a purified A1AT preparation.

48. A method for treating individuals diagnosed with or susceptible to developing severe acute respiratory distress syndrome (ARDS) as a result of diagnosed or suspected coronavirus 2 (SARS-CoV-2) infection, The method comprising administering an antiandrogen to the individual in an amount effective in reducing the activity of type II transmembrane serine protease (TMPRSS2) in the individual to a level sufficient to improve the manifestation of ARDS symptoms.

49. The method according to claim 48, wherein the antiandrogen agent is selected from the group consisting of androgen receptor (AR) blockers, androgen synthesis inhibitors, and antigonadotropins.

50. The method according to claim 49, wherein the antiandrogen agent is an AR blocker selected from the group consisting of cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, osaterone acetate, dienogest, drospirenone, medrogestone, nomegestrol acetate, promegestone, trimegestone, flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, and apalutamide.

51. The method according to claim 49, wherein the antiandrogen agent is an androgen synthesis inhibitor selected from the group consisting of ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, alfatradiol, and saw palmetto (Serenoa repens) extract.

52. The method according to claim 49, wherein the antiandrogen is an antigonadotropin selected from the group consisting of cetrorelix, allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, oxendrone, estradiol, estradiol ester, ethinylestradiol, conjugated estrogen, and diethylstilbestrol.

53. A method for reducing type II transmembrane serine protease (TMPRSS2) activity in an individual, comprising administering at least one antiandrogenic agent to the individual.

54. The method according to claim 53, wherein the at least one antiandrogen agent includes an androgen receptor (AR) blocker.

55. The method according to claim 54, wherein the AR blocker is selected from the group consisting of cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, osaterone acetate, dienogest, drospirenone, medrogestone, nomegestrol acetate, promegestone, trimegestone, flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, and apalutamide.

56. The method according to claim 53, wherein the at least one antiandrogen agent comprises an androgen synthesis inhibitor.

57. The method according to claim 56, wherein the androgen synthesis inhibitor is selected from the group consisting of ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, alpha-estradiol, and saw palmetto (Serenoa repens) extract.

58. The method according to claim 53, wherein the at least one antiandrogen agent comprises an antigonadotropin.

59. The method according to claim 58, wherein the antigonadotropin is an antigonadotropin selected from the group consisting of cetrorelix, allylestrenol, chlormadinone acetate, cyproterone acetate, gestolone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, oxendrone, estradiol, estradiol ester, ethinylestradiol, conjugated estrogen, and diethylstilbestrol.

60. A method for identifying a compound as a candidate that may be useful in treating infection of an individual with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or in preventing infection of an individual or cell, Confirm that the compound binds to angiotensin-converting enzyme 2 (ACE2) such that subsequent binding by the SARS-CoV-2 spike protein is blocked at one or more amino acids selected from the group consisting of histidine at position 34 of SEQ ID NO: 1, aspartic acid at position 30 of SEQ ID NO: 1, tyrosine at position 41 of SEQ ID NO: 1, glutamine at position 42 of SEQ ID NO: 1, lysine at position 353 of SEQ ID NO: 1, and arginine at position 453 of SEQ ID NO: 1; or, Confirm that the compound binds to the SARS-CoV-2 spike protein such that subsequent binding is blocked at one or more amino acids selected from the group consisting of tyrosine at position 453 of SEQ ID NO: 2, glutamine at position 498 of SEQ ID NO: 2, threonine at position 500 of SEQ ID NO: 2, asparagine at position 501 of SEQ ID NO: 2, and lysine at position 417 of SEQ ID NO:

2. Includes, The aforementioned verification involves calculating one or more terms selected from the group consisting of van der Waals energy terms, Coulomb energy terms, lipophilicity terms, hydrogen bonding terms, metallic bonding terms, reward terms, and penalty terms. The aforementioned method.

61. A method for inhibiting sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of cells showing angiotensin-converting enzyme 2 (ACE2) on the cell membrane, The method comprising exposing the cells to a compound at a concentration capable of preventing the SARS-CoV-2 spike protein from binding to ACE2 on the cells.

62. The method according to claim 61, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the histidine at position 34 of SEQ ID NO:

1.

63. The method according to claim 61, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the aspartic acid at position 30 of SEQ ID NO:

1.

64. The method according to claim 61, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the tyrosine at position 41 of SEQ ID NO:

1.

65. The method according to claim 61, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to glutamine at position 42 of SEQ ID NO:

1.

66. The method according to claim 61, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to lysine at position 353 of SEQ ID NO:

1.

67. The method according to claim 61, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the arginine at position 453 of SEQ ID NO:

1.

68. The method according to claim 61, wherein the compound is capable of preventing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from binding to ACES2.

69. The method according to claim 68, wherein the compound can prevent the glutamine at position 498 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

70. The method according to claim 68, wherein the compound is capable of preventing the threonine at position 500 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

71. The method according to claim 68, wherein the compound can prevent the asparagine at position 501 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

72. The method according to claim 68, wherein the compound is capable of preventing the lysine at position 417 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

73. The method according to claim 68, wherein the compound can prevent the tyrosine at position 453 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

74. The compound is AY-NH ２ , NAD ＋ Reproterol, Timopentine, CGS21680HCl, NADH disodium, Nociceptin (1-7), Mupirocin, SLIGRL-NH ２ The method according to claim 61, selected from the group consisting of , and oxyneous acid.

75. A method for treating individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), The method comprising administering to an individual an effective amount of a compound to prevent the SARS-CoV-2 spike protein from binding to angiotensin-converting enzyme 2 (ACE2) on the cells of an individual that exhibits ACE2 on the cell membrane.

76. The method according to claim 75, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the histidine at position 34 of SEQ ID NO:

1.

77. The method according to claim 75, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the aspartic acid at position 30 of SEQ ID NO:

1.

78. The method according to claim 75, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the tyrosine at position 41 of SEQ ID NO:

1.

79. The method according to claim 75, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to glutamine at position 42 of SEQ ID NO:

1.

80. The method according to claim 75, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to lysine at position 353 of SEQ ID NO:

1.

81. The method according to claim 75, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the arginine at position 453 of SEQ ID NO:

1.

82. The method according to claim 75, wherein the compound is capable of preventing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from binding to ACES2.

83. The method according to claim 82, wherein the compound can prevent the glutamine at position 498 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

84. The method according to claim 82, wherein the compound is capable of preventing the threonine at position 500 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

85. The method according to claim 82, wherein the compound can prevent the asparagine at position 501 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

86. The method according to claim 82, wherein the compound is capable of preventing the lysine at position 417 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

87. The method according to claim 82, wherein the compound is capable of preventing the tyrosine at position 453 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

88. The compound is AY-NH ２ , NAD ＋ Reproterol, Timopentine, CGS21680HCl, NADH disodium, Nociceptin (1-7), Mupirocin, SLIGRL-NH ２ The method according to claim 75, selected from the group consisting of , and oxyneous acid.

89. A method to prevent individuals at risk of infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from becoming infected with SARS-CoV-2, The method comprising administering to an individual an effective amount of a compound to prevent the SARS-CoV-2 spike protein from binding to angiotensin-converting enzyme 2 (ACE2) on the cells of an individual that exhibits ACE2 on the cell membrane.

90. The method according to claim 89, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the histidine at position 34 of SEQ ID NO:

1.

91. The method according to claim 89, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the aspartic acid at position 30 of SEQ ID NO:

1.

92. The method according to claim 89, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the tyrosine at position 41 of SEQ ID NO:

1.

93. The method according to claim 89, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to glutamine at position 42 of SEQ ID NO:

1.

94. The method according to claim 89, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to lysine at position 353 of SEQ ID NO:

1.

95. The method according to claim 89, wherein the compound is capable of preventing the SARS-CoV-2 spike protein from binding to the arginine at position 453 of SEQ ID NO:

1.

96. The method according to claim 89, wherein the compound is capable of preventing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from binding to ACES2.

97. The method according to claim 96, wherein the compound can prevent the glutamine at position 498 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

98. The method according to claim 96, wherein the compound is capable of preventing the threonine at position 500 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

99. The method according to claim 96, wherein the compound can prevent the asparagine at position 501 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

100. The method according to claim 96, wherein the compound is capable of preventing the lysine at position 417 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

101. The method according to claim 96, wherein the compound is capable of preventing the tyrosine at position 453 of SEQ ID NO: 2 from binding to ACE2 on a host cell.

102. where the compound is AY-NH ２ , NAD ＋ , reproterol, timopentin, CGS21680HCl, NADH disodium, nociceptin (1-7), mupirocin, SLIGRL-NH ２ , and oxynicotic acid, the method according to claim 89.

103. A method for treating or preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an individual, Determining that the level of coenzyme Q10 in the said individual is lower than predicted; and Administer a certain amount of coenzyme Q10 to the aforementioned individual. The method, including the method described above.

104. The method according to claim 103, wherein the amount of coenzyme Q10 is 100 mg / day to 200 mg / day.

105. A method for treating patients with beta coronavirus infection, Determining, or having determined, whether the patient carries at least one genetic marker associated with severe betacoronavirus infection; and, If the patient carries at least one genetic marker associated with the severe beta-coronavirus infection, administer an effective amount of at least one compound capable of inhibiting the expression of the FYCO1 gene to the patient. Includes, The at least one compound is selected from the group consisting of indomethacin, primidone, triprolidine hydrochloride, and baclofen. The aforementioned method.

106. The method according to claim 105, wherein the beta-coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

107. The method according to claim 105, wherein at least one genetic marker associated with the severe beta-coronavirus infection is selected from the group consisting of a non-CC genotype at the rs73064425 single nucleotide polymorphism (SNP) locus; a non-GG genotype at the rs13079478 SNP locus; a non-TT genotype at the rs13059238 SNP locus; and a non-GG genotype at the rs33910087 SNP locus.

108. The method according to claim 107, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-CC genotype at the rs73064425 SNP locus.

109. The method according to claim 107, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-GG genotype at the rs13079478 SNP locus.

110. The method according to claim 107, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-TT genotype at the rs13059238 SNP locus.

111. The method according to claim 107, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-GG genotype at the rs33910087 SNP locus.

112. The method according to claim 105, wherein the at least one compound is indomethacin.

113. The method according to claim 112, wherein the effective amount of indomethacin is 3 mg / day to 300 mg / day.

114. The method according to claim 112, wherein the effective amount of indomethacin is administered in divided doses up to four times a day.

115. The method according to claim 112, wherein the administration comprises orally administering the effective amount of indomethacin.

116. The method according to claim 112, wherein the administration comprises administering an effective amount of indomethacin rectally.

117. A method for determining the predisposition to severe betacoronavirus infection in an individual, Determining, or having determined, whether the said individual carries at least one marker associated with severe betacoronavirus infection; and, If the individual carries at least one marker associated with severe beta-coronavirus infection, it is determined that the individual is susceptible to severe beta-coronavirus infection. The method, including the method described above.

118. The method according to claim 117, wherein at least one genetic marker associated with the severe beta-coronavirus infection is selected from the group consisting of a non-CC genotype at the rs73064425 single nucleotide polymorphism (SNP) locus; a non-GG genotype at the rs13079478 SNP locus; a non-TT genotype at the rs13059238 SNP locus; and a non-GG genotype at the rs33910087 SNP locus.

119. The method according to claim 117, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-CC genotype at the rs73064425 SNP locus.

120. The method according to claim 117, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-GG genotype at the rs13079478 SNP locus.

121. The method according to claim 117, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-TT genotype at the rs13059238 SNP locus.

122. The method according to claim 117, wherein at least one genetic marker associated with the severe beta-coronavirus infection includes a non-GG genotype at the rs33910087 SNP locus.

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