IDENTIFICATION AND USE OF COMPOUNDS IN THE TREATMENT OR PREVENTION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2
20230346717 · 2023-11-02
Inventors
- Mihael H. Polymeropoulos (Potomac, MD)
- Sandra SMIESZEK (Washington, DC, US)
- Bartlomiej PRZYCHODZEN (Washington, DC, US)
- Vasilios M. POLYMEROPOULOS (Potomac, MD, US)
Cpc classification
G01N33/5008
PHYSICS
International classification
Abstract
Methods for identifying compounds useful in treating or preventing infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are described, as are methods of treating or preventing SARS-CoV-2 infection using such compounds.
Claims
1. A method of identifying compounds useful in treating or preventing infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising: screening at least one candidate compound for an ability to inhibit cleavage of a SARS-CoV-2 spike protein by a human protease at one or more target site.
2. The method of claim 1, further comprising: determining, for the at least one candidate compound, a level of inhibition of host cell peptide cleavage.
3. The method of claim 1, wherein the one or more target site is selected from a group consisting of: between threonine at position 768 and glycine at position 769 of SEQ ID 1; and between arginine at position 815 and serine at position 816 of SEQ ID 1.
4. The method of claim 1, wherein the at least one candidate compound includes a plurality of compounds.
5. The method of claim 4, wherein screening includes screening the plurality of compounds in an assay.
6. The method of claim 1, wherein the ability to inhibit cleavage includes one or more measure selected from a group consisting of: binding of the at least one candidate compound with the human protease, binding of the at least one candidate compound to block a target site on the spike protein, a change of expression of the human protease, and a change of function of a lysosome in which the human protease is contained in a host cell.
7. The method of claim 1, further comprising: determining whether the spike protein amino acid sequence includes one or more mutation from a wildtype sequence (SEQ ID 1), the one or more mutation being selected from a group consisting of: histidine at position 675 (SEQ ID 3), leucine at position 704 (SEQ ID 4), alanine at position 719 (SEQ ID 5), phenylalanine at position 752 (SEQ ID 6), leucine at position 765 (SEQ ID 7), leucine at position 772 (SEQ ID 8), glutamine at position 780 (SEQ ID 9), cysteine at position 797 (SEQ ID 10), and serine at position 812 (SEQ ID 11).
8-11. (canceled)
12. A method of treating or preventing infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an individual, the method comprising: administering to the individual amantadine in an amount sufficient to reduce lysosomal enzymatic activity in a cell of the individual.
13. The method of claim 12, wherein the amount of amantadine is sufficient to reduce expression of at least one gene in the individual, the at least one gene selected from a 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 of claim 13, wherein the at least one gene is CTSL.
15. The method of claim 12, wherein the cell is located in respiratory tract tissue.
16. The method of claim 12, wherein the amount of amantadine is sufficient to decrease a viral load in the individual.
17. The method of claim 12, further comprising: determining a genotype of the individual at the rs2378757 locus.
18. The method of claim 17, wherein the amount of amantadine administered to the individual is greater if the individual's rs2378757 genotype is determined to be AA than if the individual's rs2378757 genotype is determined to be AC or CC.
19. A method of decreasing a SARS-CoV-2 viral load in an individual infected therewith, the method comprising: administering to the individual amantadine in an amount sufficient to reduce lysosomal enzymatic activity in a cell of the individual.
20. The method of claim 19, wherein the amount of amantadine is sufficient to reduce expression of at least one gene in the individual, the at least one gene selected from a 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 of claim 20, wherein the at least one gene is CTSL.
22. The method of claim 19, wherein the cell is a lung tissue cell.
23. The method of claim 19, wherein the amount of amantadine is sufficient to decrease a viral load in the individual.
24. The method of claim 19, further comprising: determining a genotype of the individual at the rs2378757 locus.
25-122. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] These and other features of the invention will be more readily understood from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawings that depict various embodiments of the invention, in which:
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[0072] It is noted that the drawings are not to scale and are intended to depict only typical aspects of the invention, and therefore should not be considered as limiting the scope of the invention.
DETAILED DESCRIPTION
Screening Assay
[0073] Applicant has identified a number of novel cleavage sites for CatL in the SARS-CoV-2 S protein sequence that may be relevant in the treatment or prevention of SARS-CoV-2 infection. Any compound, including small molecules, having an ability to inhibit cleavage at one or more of these sites—whether by direct inhibition of enzymatic cleavage by binding to a host protease, binding to the S protein to block these cleavage sites, altering expression of the host protease, or altering a function of a lysosome in which the host protease is contained—may be useful in treating or preventing SARS-CoV-2 infection.
[0074] The novel cleavage sites identified by Applicant comprise locations of the wildtype amino acid sequence of the S protein (SEQ ID 1) at which protease cleavage is known or believed to occur, whether during viral entry, replication, packaging, or egress. One skilled in the art will recognize that other amino acid sequences for the SARS-CoV-2 S protein, representing minor deviations from the wildtype sequence, are known. The novel cleavage sites of the invention are found in these alternative sequences as well, although the particular amino acid positions may differ.
[0075] The novel cleavage sites of the invention include those located between the threonine and glycine at positions 768 and 769 (SEQ ID 1), respectively, and the arginine and serine at positions 815 and 816 (SEQ ID 1), respectively.
[0076] Embodiments of the invention include methods of screening one or more candidate compound for an ability to inhibit cleavage of the SARS-CoV-2 S protein at either of these novel cleavage sites. Such screening includes simultaneously or sequentially screening a plurality of candidate compounds using an assay.
[0077] Assays useful in practicing the invention include, for example, the HTSA described by Elshabrawy et al., which is hereby incorporated by reference for all that it teaches, as though fully set forth.
[0078] Other embodiments of the invention include inhibiting 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 as having such an inhibitory ability.
[0079] In fact, Applicant has discovered a sequence range within the S protein that appears susceptible both to enzymatic cleavage and potential binding to inhibit such cleaving. That sequence is 23 amino acids in length, extending from the glycine at position 757 (SEQ ID 1) to the glutamine at position 779 (SEQ ID 1). This sequence is shown in full below and identified specifically herein as SEQ ID 2.
TABLE-US-00002 (SEQ ID 2) GSFCTQLNRALTGIAVEQDKNTQ
[0080] Any amino acid sequence within SEQ ID 2 may be targeted for binding. As will be understood by one skilled in the art, such sequences should be of sufficient length to ensure proper binding specificity. Any octameric, nonameric, or decameric sequence within SEQ ID 2 would be expected to provide sufficient binding specificity.
[0081] Applicant has also discovered that one or more mutations in the SARS-CoV-2 S protein sequence may make inhibition of enzymatic cleavage more effective. It is believed that these mutations result in a conformational change in the S protein such that either of these novel cleavage sites is less susceptible to enzymatic cleavage. The presence of one or more of these mutations may also improve the inhibition of cleavage at the known CatL cleavage sites noted above, either by known compounds or those identified according to embodiments of the invention.
[0082] These mutations are shown below in Table 1. Mutations are described in terms of their mutation from an amino acid in the wildtype sequence (SEQ ID 1) at a particular position, as will be understood by one skilled in the art.
TABLE-US-00003 TABLE 1 SEQ ID name mutation 3 Q675H glutamine to histidine at position 675 4 S704L serine to leucine at position 704 5 T719A threonine to alanine at position 719 6 L752F leucine to phenylalanine at position 752 7 R765L arginine to leucine at position 765 8 V772L valine to leucine at position 772 9 E780Q glutamate to glutamine at position 780 10 F797C phenylalanine to cysteine at position 797 11 P812S proline to serine at position 812
[0083] Thus, embodiments of the invention may further include determining whether an individual is infected or at risk of being infected by a strain of the SARS-CoV-2 virus that includes one or more of the mutations in Table 1.
[0084] Of course, as should be apparent to one skilled in the art, determining whether the spike protein sequence includes one or more such mutations, and therefore is more susceptible to inhibition of enzymatic cleavage, is applicable to screening and treatment methods other than those described herein. Such a determining step may be used, for example, in treating an individual with a compound identified according to methods other than those described herein.
[0085] Understanding the mechanism of action of SARS-CoV-2 infection is a fundamental step in delineating the optimal therapeutic agents. For example, interfering with the S protein processing by the host cell, whether by affecting the environment or modulating gene expression levels, offers one potential therapeutic strategy.
[0086] Novel therapeutics identified by high throughput screening assay and shown to block the cleavage of SARS-CoV2-S protein by CTSL, CTSB, TMPRSS2, or any other host protease at predicted/selected binding sites will be a viable approach to functionally target and limit infection by SARS-CoV-2.
[0087] Other therapeutic mechanisms of action could involve lowering or modulating the expression of CTSL or affecting the conditions of the CTSL lysosomal environment by modulating pH.
[0088] Applicant has tested various agents that could help identify potential therapeutics with the capacity to decrease expression of the CTSL gene. Five such agents showed such potential, one of which is amantadine, and merit consideration as potentially useful in treating patients with COVID-19 infection.
Cell Culture and Drug Treatment
[0089] A drug screen was used to test agents having the potential to treat or prevent SARS-CoV-2 infection. The retinal pigment epithelia cell line, ARPE-19/HPV-16, was chosen to establish a database of drug profiles because of its non-cancerous, human origin, with a normal karyotype. ARPE-19/HPV-16 can be easily grown as monolayer in 96-well plates and expresses a variety of well known, neuronal, cell surface receptors that include the dopamine receptor D2, the serotonin receptors 1A, 2A, and 2C, the muscarinic receptor M3, and the histamine receptor H1.
[0090] Cell lines were propagated according to supplier's specifications (ATCC Manassas, VA). Compounds were obtained from Sigma (St. Louis, MO) or Vanda Pharmaceuticals (Washington, DC). Cells were aliquoted on 96-well plates (˜2×10.sup.5 cells/well) and incubated for 24 hours prior to providing fresh media with a drug, or the drug vehicle (water, dimethyl sulfoxide, ethanol, methanol, or phosphate-buffered saline solution). Drugs were diluted 1000-fold in buffered Advanced D-MEM/F-12 culture medium (Invitrogen, Carlsbad, CA) containing nonessential amino acids and 110 mg/L sodium pyruvate. In these conditions, no significant changes of pH were expected, which was confirmed by the monitoring of the pH indicator present in the medium.
[0091] A final 10 μM drug concentration was chosen because it is believed to fit in the range of physiological relevance. Microscopic inspection of each well was conducted at the end of the treatment to discard any samples where cells had morphological changes consistent with apoptosis. It was also verified that the drug had not precipitated in the culture medium.
Gene Expression Assay
[0092] Cells were harvested 24 hours after treatment and RNA was extracted using the RNeasy 96 protocol (Qiagen, Valencia, CA). Gene expression for 22,238 probe sets of 12,490 genes was generated with U133A2.0 microarrays following the manufacturer's instructions (Affymetrix, Santa Clara, CA). Drugs were profiled in duplicate or triplicate, with multiple vehicle controls on each plate. A total of 708 microarrays were analyzed including 74 for 18 antipsychotics, 499 for 448 other compounds, and 135 for vehicle controls.
[0093] The raw scan data were first converted to average difference values using MAS 5.0 (Affymetrix). The average difference values of both treatment and control data were set to a minimum threshold of 50 if below 50. For each treatment instance, all probe sets were then ranked based on their amplitude, or level of expression relative to the vehicle control (or average of controls when more than one was used). Amplitude was defined as the ratio of expression (t−v)/[(t+v)/2] where t corresponds to treatment instance and v to vehicle instance.
[0094] Each drug group profile was created using a novel Weighted Influence Model, Rank of Ranks (WIMRR) method which underscores the rank of each probe set across the entire gene expression profile rather than the specific change in expression level. WIMRR takes the average rank of each probe set across all of the members of the group and then re-ranks the probe sets from smallest average rank to largest average rank. A gene-set enrichment metric based on the Kolmogorov-Smirnov (KS) statistic. Specifically, for a given set of probes, the KS score gives a measure of how up (positive) or down (negative) the set of probes occurs within the profile of another treatment instance.
Results
[0095] Applicant analyzed expression profile of CTSL across all 466 drugs tested. In order to find positive hits and selected only those results that caused decrease of CTSL expression by at least 33% (1.5-fold down). Top drug targets (Table1) included drugs from various therapeutic areas—muscle relaxer, antihistamine, anti-epileptic, anticholirgenic, and antiviral. There was no drug that would decrease CTSL expression by more than 40%. Among the top results is amantadine, a known and safe antiviral agent that was previously used to treat patients with influenza A.
TABLE-US-00004 TABLE 2 List of top drugs affecting CTSL downregulation log2(treated) log2(control) log2 DrugID CTSL CTSL difference Baclofen 9.58 10.40 −0.82 Triprolidine Hydrochloride 9.54 10.33 −0.79 Brompheniramine Maleate 9.57 10.33 −0.75 Amantadine Hydrochloride 9.62 10.33 −0.70 Phenytoin 9.56 10.26 −0.70 Atropine Sulfate 9.63 10.33 −0.70
[0096] Amantadine hydrochloride is a lysosomotropic alkalinizing agent. The physical chemical properties of amantadine lead to lysosomal accumulation. Lysosomotropic drugs affect lysosomes by pH alteration, blocking Ca.sup.2+ signaling, lysosomal membrane permeabilizations, enzyme activity inhibition, and storage material accumulation. Amantadine behaves as a lysosomotropic substance that passes easily through the lysosome membrane and accumulates in the lysosome. It may lower the pH of the lysosome, thereby inhibiting protease activity.
[0097] Amantadine can also block the assembly of influenza virus during viral replication. Moreover, amantadine may directly affect viral entry by down-modulating CTSL and other lysosomal pathway genes.
[0098] Amantadine HCl IR is available as a 100-mg tablet (equivalent to 81 mg base amantadine) and 50 mg/S mL syrup (equivalent to 40 mg/S mL base amantadine) and is typically administered twice daily.
[0099] Since CTSL was not the top differentially expressed transcript, Applicant extended the analysis to all genes that were downregulated by amantadine. Among the top 500 differentially expressed probes (383 genes, all with at least 50% expression reduction) Applicant found 21 genes related to lysosomes (GO:005764, p=2.49×10.sup.−5). In addition, the top significant pathway by ENRICHR enrichment analysis is KEGG lysosome. Amantadine's significant effect on lysosome-associated membrane glycoprotein (LAMP) pathway genes is shown in
TABLE-US-00005 TABLE 3 % Fold P-value P-value Term Category Count Pathway Enrichment P-value Bonferroni FDR GO:0005764-lysosome GOTERM_CC_DIRECT 21 5.19 4.42 6.65E−08 2.49E−05 9.39E−09 Lysosome UP_KEYWORDS 19 4.69 3.75 3.77E−06 0.001389 0.005198 hsa04142:Lysosome KEGG_PATHWAY 14 3.46 4.37 1.55E−05 0.003425 0.019698
TABLE-US-00006 TABLE 4 Probe avg_tx avg_ctrl diff Gene ID (log2) (log2) (log2) CTSH 202295_s_at 9.62 12.13 −2.51 GALC 204417_at 6.58 8.64 −2.06 RNASET2 217983_s_at 8 9.82 −1.81 CTSK 202450_s_at 7.09 8.9 −1.81 GJA1 201667_at 11.23 12.99 −1.76 ceroid-lipofuscinosis 204085_s_at 5.93 7.67 −1.74 SCARB2 201647_s_at 6.97 8.6 −1.63 AGA 204333_s_at 6.57 8.17 −1.6 ceroid-lipofuscinosis 214252_s_at 6.13 7.71 −1.57 MAR3 213256_at 5.55 7.12 −1.57 PCYOX1 203803_at 7.15 8.71 −1.57 CPQ 203501_at 6.41 7.96 −1.55 CTSB 213274_s_at 7.75 9.23 −1.48 steroid sulfatase 203767_s_at 5.55 7.02 −1.47 (microsomal) LAMP1 201551_s_at 7.87 9.27 −1.4 AGA 204332_s_at 9.18 10.57 −1.39 AGA 216064_s_at 8.12 9.5 −1.38 PSAP 200866_s_at 10.59 11.95 −1.36 LGMN 201212_at 8.33 9.68 −1.35 CPQ 208454_s_at 7.3 8.65 −1.34 CD164 208654_s_at 8.7 10.04 −1.34 CTBS 218924_s_at 6.59 7.91 −1.32 RAB38 219412_at 6.51 7.81 −1.3 N-acetyl-6-sulfatase 212334_at 9.71 10.97 −1.27 BCL10 205263_at 7.44 8.69 −1.25
[0100] Applicant has also investigated the natural variation of CTSL expression across ethnicities, focusing on common and rare variants. The Genotype-Tissue Expression (GTEx) project provides genotype information and gene expression levels across 49 human tissues from 838 donors, allowing examination of the expression patterns of CTSL, both across tissues and across individuals.
[0101] Looking at eQTL variants in CTSL, Applicant found a very significant and lung specific (rs2378757) variant conferring highly variable expression. As shown in
[0102] Applicant notes a series of splice QTLs, with variants affecting splicing ratios of transcripts, such as rs114063116, which was significant and present in lung tissue. CTSL GTEx analysis points to potential protection or susceptibility of certain individuals.
[0103] Interestingly, the alternative splicing of the CTSL transcript in the lung further displays tissue-specific regulatory programs. Results for various tissue types are shown in
[0104] A recent functional study points to a common variant in CTSL in the proximal CTSL1 promoter (position C-171A) confirmed to alter transcription via alteration of the xenobiotic response element. This and similar other variants likely affect the natural diversity in baseline expression and therefore viral fitness at cell entry of SARS-CoV-2.
[0105] Additionally, Applicant notes, in the gnomAD database, a number of rare variants and variation tolerance statuses of CTSL. The results show that there are on average 167 missense variants in CTSL and the gene is predicted to be loss-of-function variant tolerant with a pLI of 0.01. Together with significant eQTLs, this indicates a large effect of genetic variation on CTSL expression and variation thereof.
[0106] TMPRSS2 is also widely expressed in multiple tissues, including those in the GI system, lung, and kidney. The high expression of CTSL and TRMPSS2 transcripts in a series of organs could explain the viral manifestation in these tissues. Recent studies, for example, have shown SARS-CoV-2 in stool samples from infected individuals and significant effects of the virus across tissues.
COVID-19 ARDS and Increased Human Leukocyte Elastase Activity
[0107] The pathophysiology of ARDS in COVID-19 has not been elucidated yet. Without being bound by any particular mechanism, Applicant hypothesizes that the viral infection leads to an inflammatory host response, especially in the lung, which leads to sequestration and activation of granulocytes in the lower respiratory tract and the alveoli. Significant evidence exists that HLE is responsible for the depletion of at least one of the surfactant proteins, surfactant protein D (SP-D) during inflammation of the lung. Surfactant proteins A, B, C and D are part of the surfactant which is produced by type II alveolar cells and functions to lower surface tension in the interphase between the liquid and air phases at the alveoli. It is hypothesized that an increase in the activity of HLE at the alveoli can lead to the rapid and catastrophic deterioration of respiratory function in patients with ARDS secondary to COVID-19 infection. If true, this theory presents a number of potential therapeutic opportunities including some that are immediate.
Alpha 1 Antitrypsin Deficiency (AAT) Allele Carriers and Risk for COVID-19 ARDS
[0108] Individuals who are carriers of genetic polymorphisms that lead to AAT would be predicted to be at increased risk of ARDS associated with COVID-19 infection. Given that the rapid pandemic has overwhelmed health care systems, it is important to identify individuals with the highest risk of mortality. It has been discussed that older individuals and individuals with underlying medical conditions are at higher risk for severe complications and death. However, differences in outcomes exist in this population and additionally, with the expansion of the infected population, it is now apparent that younger people without any apparent underlying conditions are becoming severely ill and some of them are dying of acute respiratory failure. It is imperative that the necessary epidemiological analyses be conducted rapidly, and the data broadly shared, in order to better assess and identify individuals at risk who may require additional and urgent interventions.
[0109] Europe has been the epicenter of the COVID-19 epidemic which is associated with a high prevalence of ARDS and associated mortality. The prevalence of AAT alleles reported in the literature as well as the emerging COVID-19 mortality data suggests a trend of higher mortality in populations that have higher allele frequency for either the S or the Z AAT alleles. In the review by Blanco et al., they reported the following: “In Europe, the mean SZ prevalence by regions (from the highest to the lowest) was 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.” Therefore, higher COVID-19 mortality would be expected in Southern Europe and lower mortality in Central and Eastern Europe.
[0110] The Pi*SZ genotype is more prevalent in Southern Europe and less prevalent in Central Europe. It should also be noted that the Italian peninsula shows higher prevalence in the North as compared to the South where the prevalence is very low.
[0111] The accumulating data for mortality during COVID-19 infections (March 31, 2020) shows a correlation between mortality rate (defined as deaths/cases confirmed) and the reported prevalence of the Pi*SZ A1AT gene allele by country. For robustness Applicant has included data only for the nine EU countries that had, at the time of filing, reported over 10,000 confirmed cases. Results of this correlation analysis are shown in Table 5. A significant correlation of R=0.66 (pvalue=0.05) was observed using all nine country mortality rates and Pi*SZ prevalence. From Blanco et al (2017), we observe that there is a significant difference in prevalence of the Pi*SZ genotype between North Italy (higher) and South Italy (lower). We have therefore reanalyzed the data this time excluding Italy. In this analysis also shown in Table 5, (excluding Italy), the correlation between mortality rate and Pi*SZ prevalence is even stronger R=0.88 (pvalue=0.003) Table 5.
TABLE-US-00007 TABLE 5 Correlation between COVID-19 mortality and Pi*SZ genotype Country Cases Deaths Deaths/Cases Pi*SZ (1 in:) Italy 105792 12428 0.117475802 967 Spain 94417 8269 0.087579567 278 France 44550 3024 0.067878788 413 Germany 68180 682 0.010002933 1337 Switzerland 16186 395 0.024403806 1152 Belgium 12775 705 0.05518591 551 Austria 10109 128 0.012661984 1680 Netherlands 12595 1039 0.082493053 617 UK 25150 1789 0.071133201 900 Pearson's Correlation All Countries R = 0.66 pvalue = 0.05 (n = 9) Excluding Italy R = 0.88 pvalue = 0.003 (n = 8)
[0112] These results suggest that a Pi*SZ genotype status may be a risk factor for COVID-19 ARDS and resulting mortality. With further confirmation, this observation may suggest that a different therapeutic approach is instituted for patients with COVID-19 infection and the Pi*SZ genotype that can include aggressive supportive therapy and the institution of elastase activity reducing treatments that may include small molecule inhibitors of the enzyme and/or supplementation of the A 1AT activity.
Human Leukocyte Elastase (HLE) Inhibitors in the Treatment of COVID-19 ARDS
[0113] There has been interest in the development of HLE inhibitors for the treatment of emphysema and the treatment of patients with inherited forms of alpha 1 antitrypsin (A1AT) deficiency.
Sivelestat
[0114] Sivelestat is currently available in Japan and Korea for the treatment of Acute Lung Injury (ALI) including ARDS. A number of clinical studies Aikawa and Kawasaki support the therapeutic utility of sivelestat in ARDS. Nonetheless the magnitude of the clinical benefit is still debated. In one Phase III study in 230 ventilated patients with ALI, sivelestat reduced duration of mechanical ventilation and shortened ICU stay, however, no significant effect was seen on the 30-day survival rate. In another study of 492 patients there was no effect on the ventilator free days or 28-day all-cause mortality. However, in a postmarketing study designed to reevaluate the efficacy of sivelestat in 404 ALI patients and 177 controls, sivelestat significantly improved the number of ventilator free days. While the differences in the results of these studies may have been due to differences in the study population and the design of the study, sivelestat is currently widely used in Japan and Korea in the ICU setting for patients with ARDS.
Zemaira®
[0115] Zemaira® is an alpha-proteinase inhibitor (A 1-Pi) approved by the U.S. Food and Drug Administration and is indicated for chronic augmentation and maintenance therapy in adults with A1AT deficiency and clinical evidence of emphysema. Zemaira® is not approved for lung disease patients for whom severe A1AT deficiency has not been established.
Alvelestat (MPH996)
[0116] Alvelestat is an experimental leukocyte elastase inhibitor under development in the US for the treatment of patients with alpha 1 antitrypsin deficiency of Pi*ZZ, Pi*SZ or Pi*Null/Null genotype. According to NCATS, “the drug's clinical profile suggests that it will be well tolerated with few, if any, side effects, and the existence of simple methods that can indirectly measure its activity in vivo.”
[0117] Unbalanced over-activity of the human leukocyte elastase is likely to play a role in the production and progression of the symptoms of acute respiratory distress in COVID-19 patients. Administration of small molecule, peptide or protein inhibitors that act to reduce the activity of HLE, including by direct interference with its enzymatic activity or down regulation of its coding neutrophil elastase gene (ELANE), is likely to be of therapeutic value to critically ill COVID-19 patients and as such it would be worth studying in controlled clinical trials. Moreover, Applicant's observation of higher mortality rate among COVID-19 patients with the Pi*SZ genotype, if confirmed, may suggest specific therapeutic options and treatment plan for these patients.
TMPRSS2
[0118] A TMPRSS2 single nucleotide polymorphism, rs8134378, has been shown to reduce binding and transactivation by the androgen receptor. It is therefore possible that TMPRSS2 protease levels on the cell surface may vary depending on androgen levels. Other genetic sequence variations may also lead to variability in TMPRSS2 expression on cell membrane surfaces.
[0119] For example, a number of expression quantitative trait loci (eQTL) are reported in the Genotype-Tissue Expression (GTEx) database that are specifically associated with variable expression of the TMPRSS2 gene. These include rs8134657, rs8134378, rs6517673, rs9979885, rs9984523, rs9978587, rs28360562, rs34205539, rs1041449, and rs3498323. In each of these nine polymorphisms, carriers of the minor allele were associated with lower TMPRSS2 expression, suggesting higher expression among carriers of the alternative major allele.
[0120] Given these findings and the role of TMPRSS2 as a cellular receptor for SARS-CoV-2, antiandrogen therapy may lower levels of TMPRSS2, thereby decreasing the ability of the virus to enter human cells, lowering viral loads, and leading to better clinical outcomes.
[0121] In practicing the various embodiments of the invention, an antiandrogenic agent, i.e., a medicine that blocks the action of androgens or male sex hormones, such as testosterone, may be employed. Suitable antiandrogenic agents include, for example, androgen receptor (AR) antagonists, i.e., medicines that directly block the effects of androgens. Examples of AR antagonists, as noted above, include steroidal antagonists—such as cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendolone, and osaterone acetate, dienogest, drospirenone, medrogestone, nomegestrol acetate, promegestone, and trimegestone, as well as non-steroidal antagonists—such as flutamide, bicalutamide, nilutamide, topilutamide, enzalutamide, and apalutamide.
[0122] Other suitable antiandrogenic agents include androgen synthesis inhibitors, i.e., medicines that act to lower androgen levels. Such agents include CYP17A1 inhibitors, such as ketoconazole, abiraterone acetate, and seviteronel, as well as aminoglutethimide, a CYP11A1 inhibitor. Other androgen synthesis inhibitors include 5α-reductase inhibitors, such as finasteride, dutasteride, epristeride, alfatradiol, and Serenoa repens (saw palmetto) extract.
[0123] Still other antiandrogenic agents include antigonadotropins, i.e., medicines that, like androgen synthesis inhibitors, act to lower androgen levels. These include the gonadotropin-releasing hormone (GnRH) modulators, such as cetrorelix; progestogens, such as allylestrenol, chlormadinone acetate, cyproterone acetate, gestonorone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate, and oxendolone; and estrogens, such as estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, and diethylstilbestrol.
[0124] Other antiandrogenic agents may be employed in addition to those described and exemplified above, as are known in the art.
[0125] In practicing aspects of the invention, one or more antiandrogenic agents, including one or more of the agents noted above, are administered to an individual in an amount sufficient to reduce TMPRSS2 activity in the individual. Such reduced TMPRSS2 activity may result from direct blocking of androgens (such as through use of an AR antagonist), reduced androgen production or synthesis (such as through the use of androgen synthesis inhibitors or antigonadotropins), or both. In some embodiments of the invention, both an AR antagonist and an androgen synthesis inhibitor or antigonadotropin are employed, including any number of or combination of the agents set out above.
[0126] In the practice of the method of the present invention, an individual is selected for treatment based upon manifestations of ARDS. ARDS results when fluid builds up in the alveoli of the lungs, resulting in oxygen repletion in the bloodstream, depriving organs of the oxygen needed to function. Severe shortness of breath is the main symptom of ARDS and can develops within hours to days following a precipitating infection, such as a diagnosed or suspected coronavirus 2 (SARS-CoV-2) infection. ARDS, once the syndrome develops, is often fatal. The risk of death increases with age and co-morbidities. ARDS can produce lasting damage to the lungs. The diagnosis of ARDS is accomplished using established diagnostic criteria known in the art, as is the determination of whether the individual is suffering from a coronavirus 2 (SARS-CoV-2) infection or is suspected to have such an infection based on symptoms or possible exposure to the virus. Diagnostic testing for the infection is known in the art.
[0127] In addition, in the practice of the present invention, the amount of the antiandrogenic agent administered to the individual is determined by the condition of the patient, the potency of the agent, the age and weight of the patient and other criteria known in the art for the producing the desired antiandrogenic effect. The treatment according to the present method is initiated at any point following a determination that the individual to be treated has a known or suspected coronavirus 2 (SARS-CoV-2) infection that has produced manifestations of ARDS or that, for an asymptomatic patient, that may result in ARDS. For example, patients at elevated risk for developing ARDS may be administered an antiandrogenic agent prophylactically prior to the diagnosis of ARDS. Such patients may include those of advanced age (e.g., over the age of 60 or 65) and those with co-morbidities, e.g., hypertension, diabetes, cardiovascular disease, asthma, or disorders of the immune system including immunosuppressed individuals.
[0128] Treatment of an individual in accordance with the present method can be sustained until the desired amelioration of one or more symptoms of ARDS is observed or sustained until the ARDS has fully resolved or so long as needed for the individual to regain lung function that may have been compromised as a result of the syndrome. Accordingly, treatment of the individual may be sustained for a period of days to weeks from the initiation of therapy or, if needed for months.
ACES2
[0129] Natural genetic variation in ACE2 may affect individual susceptibility to SARS-CoV-2 infection and the propagation capacity of the virus. Specifically, ACE2 genotype-tissue analysis points to rather rare population frequencies of potential variants that may constitute susceptibility or resilience to infection of SARS-CoV-2 in certain individuals. There are also age-related differences in the expression of ACE2 relative to ACE. The ACE2/ACE ratio is much higher among younger individuals. In addition, because the human ACE2 gene is located on the X chromosome, males who carry rare ACE2 coding variants will be hemizygous, expressing only those rare variants in all ACE2-expressing cells. Females carrying rare ACE2 coding variants, on the other hand, are significantly more likely to be heterozygous and will typically express those rare ACE2 variants in a mosaic distribution determined by early X-inactivation events.
[0130] Comparing the human ACE2 amino acid sequence (SEQ ID 12) to those of other animals (chicken, pig, dog, and cat) as well as to their reported susceptibilities to SARS-CoV-2 infection, and focusing on functional, contact amino acid residues, certain amino acids are likely important to viral entry. One amino acid in particular, the histidine at position 34 (His34; shown in blue in
[0131] It has been suggested that the His.sup.34 of the ACE2 sequence likely engages in hydrogen bonding with the tyrosine at position 453 (Tyr.sup.453), located within the RBD of the S protein sequence (SEQ ID 2). ASP.sup.30 of ACE2 is believed to similarly bond with Lys.sup.417 of the RBD, as are Tyr.sup.41, Gln.sup.42, LyS.sup.353, and Arg.sup.357 of ACE2 with Gln.sup.498, Thr.sup.500, and Asn.sup.501 of the RBD.
[0132] Conducting a in silico chemical library screen permits the identification of small molecules that may be capable of binding to or masking binding to any of the amino acids of the ACE2 or RBD sequences. Such binding or masking affords a mechanism for the inhibition of SARS-CoV-2 viral entry, either to treat or prevent infection.
[0133] The glide docking protocol is first applied. This includes the calculation of a GlideScore for candidate molecules to predict the binding of ACE2 and the RBD. GlideScores are calculated using the Glide software available from Schrodinger, LLC. The components and use of GlideScore calculations are known in the art, specifically the methodology described and marketed by Schrodinger, LLC in, for example, their Glide 6.7 User Manual, which is hereby incorporated herein as though fully set forth.
[0134] The docking score (GlideScore) is an empirical scoring function designed to maximize separation of compounds with strong binding affinity from those with little to no binding ability. As an empirical scoring function, it is comprised of terms that account for the physics of the binding process including a lipophilic-lipophilic term, hydrogen bond terms, a rotatable bond penalty, and contributions from protein-ligand coulomb-vdW energies. The lower the docking score the more optimal the docking. Candidate molecules are assessed for actual interactions at the docking site (also how the molecule anchors), hbond score (the hbond term will be lower if the hydrogen bonds are more optimal, for example at closer distance) and ligand efficiency (normalized version of the glide score (gscore) divided by a number of heavy atoms).
[0135] Next, a determination of pharmacokinetically relevant molecular descriptors of the candidate molecules is made. And, finally, molecular dynamics simulations are conducted to validate the stability of docked binding modes. A total of approximately 11,000 non-redundant candidate molecules are analyzed.
[0136] Of the candidate molecules screened, the 10 with the lowest (i.e., most favorable to binding) docking score are shown below in Table 6.
TABLE-US-00008 TABLE 6 glide glide rotatable docking ligand glide DrugID Drug CAS ID bonds score efficiency hbond ZINC000098052516 AY-NH.sub.2 352017-71-1 24 −8.541 −0.174 −0.916 ZINC000008214766 Nicotinamide 53-84-9 15 −7.979 −0.181 −0.852 adenine dinucleotide NAD+ ZINC000001542931 Reproterol 54063-54-6 10 −7.71 −0.275 −1.169 ZINC000245219534 Thymopentin 69558-55-0 28 −7.645 −0.159 −1.423 ZINC000009228252 CGS 21680 HCl 124431-80-7 13 −7.57 −0.21 −1.084 ZINC000008215403 Disodium NADH 606-68-8 15 −7.467 −0.17 −1.214 ZINC000098052511 Nociceptin (1-7) 178249-42-8 25 −7.249 −0.154 −0.83 ZINC000257482989 Mupirocin 12650-69-0 20 −7.078 −0.202 −1.124 ZINC000026468553 SLIGRL-NH2 171436-38-7 29 −6.973 −0.152 −0.949 ZINC000000001872 Oxiniacic Acid 2398-81-4 1 −6.948 −0.695 −0.16
[0137] AY-NH.sub.2 is a selective PAR4 receptor agonist peptide (H-Ala-Tyr-Pro-Gly-Lys-Phe-NH.sub.2) and yields the most favorable docking score.
[0138] NAD.sup.+ (oxidized nicotinamide adenine dinucleotide), a coenzyme involved in many metabolic reactions, yields the second most favorable docking score. NAD.sup.+ plasma levels have been reported to significantly decline with age. Recent studies indicate that SARS-CoV-2 infection of cell lines significantly dysregulates the NAD.sup.+ pathway with respect to NAD.sup.+ synthesis and utilization.
[0139] Reproterol (7-[3-[[2-(3,5-dihydroxyphenyl)-2-hydroxyethyl]amino]propyl]-1,3-dimethylpurine-2,6-dione) is a short-acting β.sub.2 adrenoreceptor agonist approved for use in the treatment of asthma. It yields the third most favorable docking score.
[0140] Thymopentin (H-Arg-Lys-Asp-Val-Tyr-OH) is a synthetic pentapeptide used to enhance the production of thymic T cells. It yields the fourth most favorable docking score.
[0141] The other compounds of Table 6 yielded lower docking scores but are capable of acting to inhibit binding at His34 and other of the ACE2 amino acids noted above, thereby inhibiting SARS-CoV-2 infection.
[0142] CGS 21680 HCl (2-p-(2-Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride) is a selective adenosine A2A-R agonist.
[0143] Disodium NADH (reduced disodium nicotinamide adenine dinucleotide) is a coenzyme of a large number of oxidoreductases.
[0144] Nociceptin (1-7) (nociception fragment 1-7; H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-OH) is an active metabolite of nociception.
[0145] 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-methylbut-2-enoyl]oxynonanoic acid; pseudomonic acid) is a naturally-occurring antibiotic currently used topically to treat impetigo and other staph infections of the skin. It has also been used in the treatment of methicillin-resistant S. aureus (MRSA) infections.
[0146] SLIGRL-NH.sub.2 (H-Ser-Leu-Ile-Gly-Arg-Leu-NH.sub.2) is a peptide derived from the N-terminus of protease-activated receptor-2 (PAR2) and acts as a PAR2 agonist.
[0147] Oxiniacic acid (3-pyridinecarboxylic acid 1-oxide; nicotinic acid 1-oxide) is a nicotinic acid derivative with hypolipidemic activity.
[0148] In the practice of the method of the present invention, an individual is selected for treatment based upon manifestations of symptoms associated with SARS-CoV-2 infection, including acute respiratory distress syndrome (ARDS), or the individual's risk of SARS-CoV-2 infection. Diagnostic testing for SARS-CoV-2 infection is known in the art.
[0149] Of particular concern in determining whether an individual is to be treated is the manifestation of ARDS, which results when fluid builds up in the alveoli of the lungs, resulting in oxygen repletion in the bloodstream, depriving organs of the oxygen needed to function. Severe shortness of breath is the main symptom of ARDS and can develop within hours to days following a precipitating infection, such as a diagnosed or suspected SARS-CoV-2 infection. ARDS, once the syndrome develops, is often fatal. The risk of death increases with age and co-morbidities. ARDS can produce lasting damage to the lungs. The diagnosis of ARDS is accomplished using established diagnostic criteria known in the art, as is the determination of whether the individual is suffering from a SARS-CoV-2 infection or is suspected to have such an infection based on symptoms or possible exposure to the virus.
[0150] In addition, in the practice of the present invention, the amount of the treating agent (e.g., AY-NH.sub.2, NAD.sup.+, Reproterol, Thymopentin, CGS 21680 HCl, disodium NADH, Nociceptin (1-7), Mupirocin, SLIGRL-NH.sub.2, or oxiniacic acid) administered to the individual is determined by the condition of the patient, the potency of the agent, the age and weight of the patient and other criteria known in the art.
[0151] Treatment according to the present method may be initiated at any point following a determination that the individual to be treated has a known or suspected SARS-CoV-2 infection that has produced manifestations of ARDS or, for an asymptomatic patient, that may result in ARDS. For example, patients at elevated risk for developing ARDS may be administered a treating agent prophylactically prior to the diagnosis of ARDS. Such patients may include those of advanced age (e.g., over the age of 60 or 65) and those with co-morbidities (e.g., hypertension, diabetes, cardiovascular disease, asthma, or disorders of the immune system including immunosuppressed individuals).
Coenzyme Q10 and COVID-19
[0152] A potential association may exist between reduced levels of CoQ10 and the population of people most severely affected by COVID-19. The causative mechanisms of creating a susceptibility to severe illness remain unclear, though they could be a result of a reduced ability to either prevent oxidative stress, attenuate coagulation, mitigate a hyper-immune response, or inhibit viral replication of entry directly. The deficiencies associated with disease may involve other antioxidants such as Vitamin C and E. Large studies should measure CoQ10 levels along with vitamins and lipids of infected people with COVID-19 at the time of presentation to examine whether correlations predict clinical outcomes and correlate with the levels of inflammatory cytokines and molecules such as IL-2, IL-6, TNF-alpha and D-dimer. Clinical outcomes of COVID-19 in individuals with genetically predisposed CoQ10 deficiency should also be examined. Given the complexity of SARS-CoV2 infection and heterogeneity in disease presentation, the reasons for severe illness are likely multifactorial. CoQ10 may serve as a correlated marker in severe illness, and potentially as a causative agent for susceptibility to worse clinical outcomes.
[0153] If an association is confirmed, a causative mechanism could further be explored and CoQ10 may potentially offer a protective therapy in the future. Dosing to replenish levels of CoQ10 in deficiency could begin between 100 mg to 200 mg daily to have physiological impact. As it is the case with many disease states, more impactful benefits can be made with prophylaxis. If lower levels of CoQ10 are correlated with severe COVID-19 illness, supplementation of deficient individuals could potentially offer a therapeutic solution to reduce the burden of disease and potentially improve the state of this pandemic.
Severe COVID-19 and FYCO1
[0154] The genomes of 80 COVID-19 patients (68% male, 32% female; ages 35-87) exhibiting severe symptoms were analyzed and compared to the genomes of 1,876 individuals from a 2000 genome-wide association study (GWAS). The results of this comparison not only confirm the previously reported association between severe COVID-19 infection and the six-gene Neanderthal haplotype, but also point to three mutations within the FYCO1 gene and severe COVID-19 infection, as well as their association with the rs73064425 SNP within the LZTFL1 gene. This more discrete haplotype showed the strongest association with severe COVID-19 infection and may be useful in predicting and diagnosing severe COVID-19 infection.
[0155] The LZTFL1 SNP, rs73064425, a C-to-T variant, has a reported minor allele frequency (MAF) of 0.05. This value was consistent with that found within the 2000 control genotypes. Among the COVID-19 patients, however, the MAF was 0.17. This SNP was in strong linkage disequilibrium with each of the three FYCO1 SNPS. These FYCO1 SNPs, two of which occur in the same codon represent three coding mutations, cause amino acid substitutions in the resulting mRNA.
[0156] Of the newly associated FYCO1 SNPs, the first, rs13079478, is a G/T variant resulting in an amino acid substitution of aspartic acid for asparagine; the second, rs13059238, is a T/C variant in the same codon as rs13079478, but results in an amino acid substitution of lysine for asparagine; and the third, rs33910087, is a G/A variant resulting in an amino acid substitution of cysteine for arginine.
[0157] Table 7 below shows the MAF for each FYCO1 SNP within the COVID-19 population and the 2000 control population.
TABLE-US-00009 TABLE 7 Control COVID-19 SNP WT MA MAF MAF P value rs73064425 C T 0.0533 0.1696 1.19 × 10.sup.−5 rs13079478 G T 0.07809 0.2143 7.84 × 10.sup.−6 rs13059238 T C 0.08316 0.2232 7.01 × 10.sup.−6 rs33910087 G A 0.08076 0.2143 1.34 × 10.sup.−5
[0158] As can be seen in Table 7, the frequency of the minor allele at each of the FYCO1 and LZTFL1 SNPs is significantly higher within the COVID-19 population. This offers a useful way of predicting whether an individual is predisposed to severe COVID-19 symptoms in the event of exposure, as well as a valuable treatment tool in the treatment of COVID-19 patients, enabling identification of those patients more likely to experience severe COVID-19 symptoms and earlier treatment of those patients for such symptoms.
The FYCO1 Gene
[0159] The FYCO1 gene encodes a protein involved in vesicle transport and autophagy. It has been suggested to be a key mediator linking endoplasmic reticulum-derived double membrane vesicle, the primary replication site for coronaviruses, with the microtubule network.
[0160] Through its LC3-interacting region (LIR) motif, FYCO1 has also been shown to be important for the fusion of autophagosomes with lysosomes. FYCO1 dimerizes via the CC region, interacts with PI3P via its FYVE domain, and forms a complex with Rab7 via a part of the CC region located in front of the FYVE domain. Specifically, FYCO1 was shown to act as a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. The depletion of Rab7 inhibits maturation of late endosomes/multivesicular bodies (MVBs) and leads to reduced lysosome numbers in cells. FYCO1 also mediates clearance of a-synuclein aggregates.
[0161] A genome-scale CRISPR loss-of-function screen in human alveolar basal epithelial carcinoma cells identified genes whose loss enabled resistance to SARS-CoV-2 infection. The loss of RAB7A reduces viral entry/egress by sequestering the ACE2 receptor inside cells. Depletion of FYCO1 antibodies against the N-terminus of LC3 blocks the subcellular redistribution of autophagosomes. Some rare FYCO1 variants containing missense mutations in the LIR domain are associated with inclusion body myositis, a disease characterized by impaired autophagic degradation.
[0162] Gain-of-function variants in FYCO1 confer a greater risk of severe COVID-19 infection and may confer a similarly greater risk with respect to other betacoronaviruses. As such, downregulation of FYCO1 would offer protection against such severe infection, offering a potential therapeutic against COVID-19.
[0163] A high-throughput gene expression analysis identified compounds capable of downregulating FYCO1. A total of 466 compounds from 14 therapeutic classes were subjected to such analysis using human retinal pigment epithelial cell line ARPE-19 and gene expression changes collected across 12,490 genes. The effect of all 466 compounds on FYCO1 expression is greatest for the four compounds shown in Table 8.
TABLE-US-00010 TABLE 8 Compound Treated Control Difference indomethacin 4.949 6.488 −1.539 primidone 6.4453 7.9412 −1.4959 triprolidine hydrochloride 6.4963 7.9898 −1.4935 baclofen 6.7222 7.9412 −1.2190
[0164] Indomethacin, a non-steroidal anti-inflammatory, exhibits 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 tendonitis. It may be administered orally, intravenously, or rectally. Oral dosages are typically 75-150 mg daily in up to four divided doses. Oral dosages are available in 20 mg, 25 mg, 40 mg, and 50 mg capsules, a 75 mg extended release capsule, and a 25 mg/5 mL oral suspension.
[0165] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless expressly states otherwise or the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. “Optional” or “optionally” means that the subsequently described element, event, or circumstance may or may not occur, and that the description includes instances where the element, event, or circumstance occurs or is present and instances where it does not occur or is not present.
[0166] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims are intended to include any structure, material, or act for performing a function in combination with other claimed elements as specifically claimed. The description of the present disclosure is presented for purposes of illustration and description but is not intended to be exhaustive or limited to the disclosure in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the disclosure. Any embodiments chosen and described herein appear in order to best explain the principles of the disclosure and their practical application, and to enable others of ordinary skill in the art to understand the disclosure for various embodiments with various modifications as are suited to the particular use contemplated.