ARAVAMUDAN MURALI (US)
PURANIK ARJUN (US)
ANAND PRAVEEN (IN)
VENKATAKRISHNAN AIVELIAGARAM (US)
WO2009023306A2 | 2009-02-19 |
AU2005334458B9 | 2012-06-07 |
What is claimed is: 1. A method of treating or reducing viral infection in a human subject, comprising administering to the subject a pharmaceutical composition comprising a protease inhibitor. 2. The method of claim 1, wherein the pharmaceutical composition comprises an inhibitor of Furin, PCSK2, PCSK 4, PCSK 5, PCSK 6, PCSK 7, PLG, or any combination thereof. 3. The method of claim 1 or 2, wherein the composition is administered to the subject directly to an organ selected from lung, small intestine, large intestine, pancreas, kidney, heart, or tongue. 4. The method of any one of claims 1 to 3, wherein the composition is administered to the subject directly to the nasopharynx and/or the oropharynx. 5. The method of any one of claims 1 to 4, wherein the composition is administered subcutaneously, intravenously, intramuscularly, intranasally, by inhalation, orally, sublingually, by buccal administration, topically, transdermally, or transmucosally. 6. The method of any one of claims 1 to 5, wherein the subject is suffering from SARS- CoV-2 infection. |
ENaC regulates Na+ and water homeostasis and its expression levels are controlled by aldosterone and the associated Renin-Angiotensin-Aldosterone System (RAAS) 6 . Similar to SARS-CoV2, ENaC-ɑ needs to be proteolytically activated for its function 7 . FURIN cleaves the equivalent peptide on mouse ENaC-ɑ between the Arginine and Serine residues in the 4 th and 5 th positions respectively (RSAR|SASS) 8,9 , akin to the recent report establishing FURIN cleavage at the S1/S2 site of SARS-CoV-2 (Figure 1B) 1 . Furthermore, a frameshift mutation leading to a premature stop codon in Serine-205 at the 5 th position of the ENaC-ɑ mimicked peptide (RRAR|SVAS) is known to cause the monogenic disease Pseudohypoaldosteronism type 1 (PHA1) 10 . This emphasizes the functional salience of the 8-mer peptide being mimicked by SARS-CoV-2. Mimicry of human ENaC-ɑ by the S1/S2 site suggests that SARS-CoV-2 is hijacking the protease network of ENaC-ɑ for viral activation. An overlap between putative SARS- CoV-2 infecting cells and ENaC-ɑ expressing cells was observed. Systematic single cell expression profiling of the ACE2 receptor and ENaC-ɑ was performed across human and mouse samples comprising ~1.3 million cells (Figure 1C) 11 . Notably, ENaC-ɑ is expressed in the nasal epithelial cells, type II alveolar cells of the lung, tongue keratinocytes, and colon enterocytes (Figure 1C and Figures 2-7), which are all implicated in COVID-19 pathophysiology 11,12 . Further, ACE2 and ENaC-ɑ are known to be expressed generally in the apical membranes of polarized epithelial cells 13,14 . Such overlap of cell-types expressing ACE2 and ENaC-ɑ, and similar spatial distributions at the apical surfaces, indicate that SARS-CoV-2 may be leveraging the protease network responsible for ENaC cleavage. A 160-dimensional vector space (20 amino acids x 8 positions on the peptide) was created for assessment of cleavage similarities between the 178 human proteases with biochemical validation from the MEROPS database 15 . FURIN (PCSK3) had overall proteolytic similarity to select PCSK family members, specifically PCSK5 (0.99), PCSK7 (0.99), PCSK6 (0.99), PCSK4 (0.98), and PCSK2 (0.94) (Table 2). It is also known that the protease PLG cleaves the ɣ-subunit of ENaC (ENaC-ɣ) 16 . Table 2: Protease cleavage propensities for FURIN and the other proteases identified as similar from the vector space analysis conducted. Similarity (FURIN) ranges from 0 to 1. Amino acids occuring in greater than 10% of the cleaved substrates at that position are in bold (compiled from MEROPS).
The tissue tropism of SARS-CoV-2, in view of the cleavage similarities found within the host proteolytic network, the co-expression of these proteases concomitant with the viral receptor ACE2, and ENaC-ɑ was assessed (Figure 8). FURIN was expressed with ACE2 and ENaC-ɑ in the colon (immature enterocytes, transit amplifying cells) and pancreas (ductal cells, acinar cells) of human tissues, as well as tongue (keratinocytes) of mouse tissues. PCSK5 and PCSK7 were broadly expressed across multiple cell types with ACE2 and ENaC- ɑ, suggesting they are broad-spectrum proteases that cleave the S1/S2 site and further infection by the SARS-CoV-2 viral particle. In humans, concomitant with ACE2 and ENaC- ɑ, PCSK6 appeared to be expressed in cells from the intestines, pancreas, and lungs, whereas PCSK2 was noted to be co-expressed in the respiratory tract and the pancreas (Figure 8). Notably, extracellular proteases need not necessarily be expressed in the same cells as ACE2 and ENaC-ɑ. Among the PCSK family members with the potential to cleave the mimicked 8- mer peptide, the same tissue can house multiple proteases. Also multiple tissues can share the same set of proteases. Redundancy may be wired into the mechanisms of host proteolytic activation of SARS-CoV-2, and may inform the development of selective human protease inhibitors as COVID-19 therapeutics. The mimicry of a cleavable host peptide central to cardiovascular, renal, and pulmonary function provides a further strategy for the treatment and/or prevention of viral infection. Example 2: Methods Alignment of coronavirus spike proteins The complete S-protein sequence for SARS-CoV (Uniprot ID: P59594) and SARS- CoV-2 was obtained from uniprot (ftp.uniprot.org/pub/databases/uniprot/pre_release) 16 . Sequence alignments using Clustal-W, and comparison of SARS-CoV-2 versus other coronavirus strains were performed using JalView 17 . 8-mer analysis of human proteome The number of 8-mers in Uniprot 20,350 reference sequences are 10,257,893 (10.26M). The previously identified SARS-CoV-28-mer ‘RRARSVAS’ was found in a Uniprot reference sequence (p-value ≈ 10.26M/20 8 = 4E-4; chance of finding that particular 8-mer anywhere in the reference sequences). Scoring cleavage sites Biochemical specificities are inferred from substrates of the proteases that have been determined using the evidence from (i) N-terminal sequencing, (ii) mass spectroscopy, (iii) mutational studies, (iv) consensus analysis or (v) liquid chromatography. The protein residues from the protease substrates spanning the scissile bond (±4 residues) is considered to define sequence specificity of the motif. The motif length thus spans across 8 residues (or positions), and frequency of all the 20 amino acids at each position is calculated from the substrates identified for each of the proteases to get a position frequency matrix (PFM, with a dimension of 8 x 20). This PFM was converted to a probability matrix by normalizing to the frequency distribution of all the 20 amino acids per position. The position frequency matrices were downloaded from the MEROPS database 15 . In total, probabilities matrices for 178 proteases were constructed. The number of distinct cleavages for each protease ranges from as few as 10 to over 3000, based on which probability matrices for the cleavage of the 8-mer polypeptide was estimated (Table 2). The probability matrix is then used to scan the peptide sequence (‘RRARSVAS’) using a window frame equal to the length of the motif. A log- likelihood ratio score is calculated for each sequence position in the scanned window with respect to background distribution of particular amino acid in the human proteome and summed up across all the positions to get a motif score. A random set of sequences are generated from the given background of amino acid distribution in the human proteome and used as a null model to evaluate the statistical significance of the scanned window. FIMO tool was used to obtain the scores and the corresponding statistical significance of the PWM match 18 . These were then used to estimate cleavage propensity by assuming background distribution of amino-acid frequencies from human proteome 19 using MEME 20 and FIMO 18 . A stringent cut-off of 1E-04 was used to identify the potential cleavage sites on S proteins of both SARS-CoV and SARS-CoV-2 at S1/S2 site. The hits obtained were further manually filtered to ensure that residue at the immediate vicinity of cleavage site is conserved. Calculating the cosine similarity metric for protease cleavage site The position frequency matrix (PFM) of the individual proteases obtained from the MEROPS database was converted to a probability weight matrix (PWM) (normalized and scaled). Out of 178 proteases, there were 146 proteases that had specificity information available on the 8 mer peptide spanning the cleavage site (±4). The 20 (amino acids) x 8 (position) matrix defined for each of the proteases were flattened into a single vector with 160 elements. A cosine similarity calculation was performed between all pairs (X,Y) of protease specificity vector. The similarity was derived as the normalized dot product of X and Y : K(X, Y) = <X, Y> / (||X||*||Y||)). Overlap of cell types expressing ENaC-ɑ, ACE2 and proteases from scRNA-seq datasets A systematic expression profiling of the ACE2 and ENaC-ɑ was performed across 65 published human and mouse single-cell studies comprising ~1.3 million cells using nferX Single Cell platform (Table 3, academia.nferx.com) 6 . The ACE2 expression could be detected in 67 studies (59 studies of human samples and 8 studies of mouse samples) spanning across ~50 tissues, over 450 cell-types and ~1.05 million cells. In order to call a given cell-type to be positive for both ACE2 and a protease we applied a cutoff of 1% of the cells in the total cell-type cluster population to have a non-zero count associated with both ACE2 and the respective protease. The mean expression of the proteases, ENaC-ɑ and ACE2 was derived for individual cell population within each of the studies. The cell-type information was obtained from the author annotations provided for each of the studies. The analysis was performed separately on the mouse and human datasets. For each protease, the mean expression of in a given cell-population (mean log[cp10k +1] counts) was Z-score normalized (to ensure the sd=1 and mean ~0 for all the genes) to obtain relative expression profiles across all the samples. The same normalization was applied to ACE2 and ENaC-ɑ and both human and mouse datasets were analyzed independently by generating heatmaps. The cell types having zero-expression values of ACE2 were also included as negative control to probe the expression of various proteases. Table 3: List of single-cell studies analyzed and incorporated into the nferX resource (https://academia.nferx.com/) An analysis to identify the cell types with significant overlap of ACE2 and ENaC-ɑ expression was performed. To this end, cell types in which ENaC-ɑ is expressed in a significantly higher proportion of ACE2-expressing cells than in the overall population of cells of that sub-type were shortlisted. 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The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Research 46, D624–D632 (2018). 16. Passero, C. J. et al. Plasmin Activates Epithelial Na+ Channels by Cleaving the γ Subunit. J. Biol. Chem.283, 36586–36591 (2008). Incorporation by Reference All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Equivalents While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.