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WO2021224803A1 - Inhibiteurs peptidiques contre l'infection par le sarsr-cov - Google Patents

Inhibiteurs peptidiques contre l'infection par le sarsr-cov Download PDF

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Publication number
WO2021224803A1
WO2021224803A1 PCT/IB2021/053763 IB2021053763W WO2021224803A1 WO 2021224803 A1 WO2021224803 A1 WO 2021224803A1 IB 2021053763 W IB2021053763 W IB 2021053763W WO 2021224803 A1 WO2021224803 A1 WO 2021224803A1
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peptide
protein
cov
seq
human
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Jonathan David Garman
Michael Tymianski
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NoNO Inc
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NoNO Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the novel coronavirus SARS-CoV-2 has become a dangerous, worldwide pathogen stressing health systems and risking global social upheaval at a previously never-seen scale 1 .
  • a major risk is that of overwhelming health systems due to a rapid surge of severely affected cases because of the limited capacity of intensive care unit departments 2, 3 .
  • the mortality associated with this pathogen is much higher than other endemic viruses, with realistic global estimates reaching as high as 5.7% 5 . Treatments are therefore urgently needed to mitigate the spread and virulence of SARS-CoV-2 for the current pandemic as well as for future years to come.
  • SARS-CoV-2 The clinical course of SARS-CoV-2 infection has a favorable trend in the majority of patients. In a percentage of cases still to be defined, after about a week there is a sudden worsening of clinical conditions due to fulminant inflammation with rapidly worsening respiratory failure and multi-organ dysfunction and failure. These patients overwhelm healthcare resources and have a high mortality 6 .
  • SARS-CoV viral pathology has been shown to affect multiple organs 7 and research is mounting that SARS-CoV-2 may be similar, affecting not only the lungs (pneumonia), but also heart (myocarditis) 8 and potentially the nervous system 9,
  • NRP1 neuropilin-1
  • the invention provides methods of treating a subject infected with a sudden acute respiratory syndrome related coronavirus (SARSr-CoV) virus, comprising administering to the subject an effective regime of an inhibitor peptide inhibiting the interaction of the viral S protein with a human angiotensin converting enzyme 2 receptor (ACE2).
  • ACE2 human angiotensin converting enzyme 2 receptor
  • the virus is a SARS-CoV-2 virus.
  • the inhibitor peptide comprises a segment of up to 75 residues, optionally 3-15 residues of the viral S protein that binds to ACE2.
  • the inhibitor peptide comprises a segment of up to 75 residues, optionally 3-15 residues of the human angiotensin-converting enzyme 2 receptor that binds to the S protein.
  • the inhibitor peptide is linked to an internalization peptide.
  • the internalization peptide is a tat peptide.
  • the inhibitor peptide and/or the internalization peptide includes one or more D-amino acids.
  • the inhibitor peptide and internalization peptide is all D-amino acids.
  • the subject has been diagnosed as infected with the SARS-CoV- 2 virus.
  • the administration reduces load of the virus in the subject, reduces inflammation in the subject, reduces an inflammatory marker selected from C-reactive protein, IL-6, lactate dehydrogenase, and D-dimer, or reduces damage to an organ of the subject or reduces one or more neurological signs and/or symptoms.
  • the inhibitor peptide has an amino acid sequence comprising or consisting of CNGVEGFNC (SEQ ID NO:10) or CNGVEGFNCYFPLQSYGFQPTNGVGYQ (SEQ ID NO:ll).
  • the invention further provides methods of treating a subject infected with a SARSr-CoV virus, comprising administering to the subject an effective regime of an inhibitor peptide inhibiting the interaction of a viral E protein with human syntenin, PALS1, or a PDZ identified as binding to the SARS E protein including the human form of any of TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2, NHERF1, MAST2, or RADIL (collectively "SARS-E-PDZs").
  • the virus is a SARS-CoV-2 virus.
  • the inhibitor peptide comprises a segment of 3-15 residues of the C-terminus of the viral E protein that binds to human syntenin, PALS1 or SARS-E-PDZs.
  • the inhibitor peptide comprises a segment of 3-15 residues of a SARS-E-PDZ protein that binds to the viral E protein.
  • the inhibitor peptide is linked to an internalization peptide.
  • the internalization peptide is a tat peptide.
  • the inhibitor peptide and/or the internalization peptide includes one or more D-amino acids.
  • the subject has been diagnosed as infected with the SARS-CoV-2 virus.
  • the administration reduces load of the virus in the subject, reduces inflammation in the subject, reduces an inflammatory marker selected from C-reactive protein, IL-6, lactate dehydrogenase, and D-dimer, reduces damage to an organ of the subject or reduces one or more neurological signs and/or symptoms.
  • the inhibitor peptide has a sequence comprising or consisting of any of DLLV (SEQ ID NO:18), PDLLV (SEQ ID NO:19), VPDLLV (SEQ ID NO:20), GVPDLLV (SEQ ID NO:21), EGVPDLLV (SEQ ID NO:22), SRVPDLLV (SEQ ID NO:75), vlldpvge (SEQ ID NO:27)or vlldp (SEQ ID NO:28) with lower case indicating D amino acids.
  • DLLV SEQ ID NO:18
  • PDLLV SEQ ID NO:19
  • VPDLLV SEQ ID NO:20
  • GVPDLLV SEQ ID NO:21
  • EGVPDLLV SEQ ID NO:22
  • SRVPDLLV SEQ ID NO:75
  • vlldpvge SEQ ID NO:27
  • vlldp SEQ ID NO:28
  • the inhibitor peptide linked to the internalization peptide has a sequence comprising or consisting of any of vlldpvgerrrqrrkkr (SEQ ID NO:29), vyvysrvknlnssegvPDLLV (SEQ ID NO:30), or vyvysrvknlnssegvpDLLV (SEQ ID NO:31), with lower case letters indicating D-amino acids.
  • the invention further provides methods of treating a subject infected with a SARSr-CoV virus, comprising administering to the subject an effective regime of an inhibitor peptide comprising a PBM motif of a SARSr-CoV nucleoprotein.
  • the inhibitor peptide comprises or consists of a sequence selected from any of SSADSTQA (SEQ ID NO:33), SADSTQA (SEQ ID NO:34), ADSTQA (SEQ ID NO:35), and DSTQA (SEQ ID NO:36).
  • the inhibitor peptide is linked to an internalization peptide.
  • the inhibitor peptide linked to the internalization peptide has a sequence selected from any of RKKRQRRRSSADSTQA (SEQ ID NO:37), RKKRQRRRSADSTQA (SEQ ID NO:38), RKKRQRRRADSTQA (SEQ ID NO:39), and RKKRQRRRDSTQA (SEQ ID NO:40).
  • the virus is a SARS-CoV-2 virus.
  • the inhibitor peptide and/or the internalization peptide includes one or more D-amino acids.
  • the inhibitor peptide and internalization peptide are all D-amino acids.
  • the subject has been diagnosed as infected with a SARSr-CoV-2 virus.
  • the administration reduces load of the virus in the subject, reduces inflammation in the subject, reduces an inflammatory marker selected from C-reactive protein, IL-6, lactate dehydrogenase, and D-dimer, or reduces damage to an organ of the subject or reduces one or more neurological signs and/or symptoms.
  • the invention further provides methods of detecting SARSr-CoV in a sample from a subject, comprising contacting the sample with a PDZ domain peptide and detecting specific binding, if any, between the PDZ domain peptide and an E protein or nucleoprotein of the SARSr-CoV to indicate presence, if any, of SARSr-CoV in the sample.
  • such methods further comprise contacting the sample with an antibody binding to the SARSr-CoV E protein or nucleoprotein, wherein the detecting comprising detecting formation of a sandwich complex, if any, formed by the E protein or nucleoprotein, the antibody and the PDZ domain peptide.
  • the invention further provides a method of treating a subject infected with a sudden acute respiratory syndrome related coronavirus (SARSr-CoV) virus, comprising administering to the subject an effective regime of an inhibitor peptide inhibiting the interaction of the viral SI protein with human neuropilin-1.
  • the virus is a SARS-CoV-2 virus.
  • the inhibitor peptide comprises a segment of up to 50 residues, optionally 3-15 residues of the viral SI protein that binds to neuropilin-1 .
  • the segment includes RRAR (SEQ ID NO:76) as its C-terminus.
  • the inhibitor peptide comprises a segment of up to 75 residues, optionally 3-15 residues of human neuropilin-1 that binds to the SI protein.
  • the inhibitor peptide is linked to an internalization peptide.
  • the internalization peptide is a tat peptide.
  • the inhibitor peptide and/or the internalization peptide includes one or more D-amino acids.
  • the inhibitor peptide and internalization peptide are all D-amino acids.
  • the subject has been diagnosed as infected with the SARS-CoV-2 virus.
  • the administration reduces load of the virus in the subject.
  • the administration reduces inflammation in the subject.
  • the administration reduces an inflammatory marker selected from C-reactive protein, IL-6, lactate dehydrogenase, and D- dimer.
  • the administration reduces damage to an organ of the subject or reduces one or more neurological signs and/or symptoms.
  • the inhibitor peptide has an amino acid sequence comprising or consisting of IGAGICASYQTQTNSPRRAR (SEQ ID NO:73) or a segment thereof that binds SI protein .
  • the inhibitor peptide when linked to the internalization peptide can cross the blood brain barrier to reduce viral infectivity or replication in the brain or central nervous system, and thereby reduce at least one neurological sign or symptom.
  • Fig. 1 shows SARS-CoV-2 E protein (SEQ ID NO:l, upper sequence) and SARS-CoV E protein (SEQ ID NO:2, lower sequence). Approximate demarcation into virion surface, helical and intravirion regions is indicated by single underlining, dotted underlining and double underlining respectively.
  • Fig. 2 shows interaction of SARS-CoV-2 S protein and human ACE2.
  • K353, D38, E35, K31, and M82 are ACE2 residues.
  • N501, S494, Q493, L455 and F486 are S protein residues.
  • Fig. 3 shows an alignment of SARS-CoV (SEQ ID NO:3) and -CoV-2 S protein (SEQ ID NO:72) RBD. Boxed residues are conserved between SARS-CoV and CoV-2, italicized text is the region involved with direct hACE2 binding, dark dots above are the SARS-CoV-2 hACE2 contacting residues and the lighter dots below are the SARS-CoV hACE2 contacting residues.
  • a "chimeric peptide” means a peptide having two component peptides not naturally associated with one another joined to one another as a fusion protein or by chemical linkage, as when an inhibitor peptide is joined to an internalization peptide.
  • Chimeric peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual chimeric peptides can be joined end-to-end or via a linker.
  • a "fusion" protein or polypeptide refers to a composite polypeptide, i.e., a single contiguous amino acid sequence, made up of sequences from two (or more) distinct, heterologous polypeptides which are not normally fused together in a single polypeptide sequence.
  • PDZ domain refers to a modular protein domain of about 90 amino acids, characterized by significant sequence identity (e.g., at least 60%) to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl).
  • PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF (SEQ ID NO:4) repeats.
  • DHRs Discs-Large homology repeats
  • GLGF SEQ ID NO:4 repeats.
  • PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76).
  • PBM means PDZ binding motif.
  • PBM protein or "PDZ Ligand protein” refers to a naturally occurring protein that forms a molecular complex with a PDZ-domain, or to a protein whose carboxy-terminus, when expressed separately from the full length protein (e.g., as a peptide fragment of 3-25 residues, e.g. 3, 4, 5, 8, 9, 10, 12, 14 or 16 residues), forms such a molecular complex.
  • the molecular complex can be observed in vitro using the "A assay” or "G assay” described, e.g., in U.S. application Ser. No. 10/714,537, or in vivo.
  • a "PBM motif” refers to the amino acid sequence of the C-terminus of a PL protein (e.g., the C- terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25 contiguous residues) ("C-terminal PL sequence") or to an internal sequence known to bind a PDZ domain ("internal PL sequence").
  • a “PBM peptide” is a peptide of comprising or consisting of, or otherwise based on, a PL motif that specifically binds to a PDZ domain.
  • Inhibitor peptides typically have lengths of up to 5, 10, 15, 20, 30, 40, 50, 60 or 75 amino acids. Some peptides have lengths of 3-30, 4-15 or 5-10 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the amino acids can be L or D amino acids or a combination thereof.
  • Peptides can be linear or cyclic. Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker.
  • amino acids of the inhibitor peptide represent sequence of natural protein or variant thereof
  • the amino acids of the inhibitor peptide can be a single contiguous segment of amino acids of the natural protein or two or more such segments, joined by one or more spacers.
  • Variants of natural sequences typically have at least 70, 80, or 90% identity with a natural sequence when maximally aligned not including gaps. Some variants have up to five substitutions, or internal deletions or insertions relative to a natural sequence.
  • isolated or purified means that the object species (e.g., a peptide) has been purified from contaminants that are present in a sample, such as a sample obtained from natural sources that contain the object species. If an object species is isolated or purified it is the predominant macromolecular (e.g., polypeptide) species present in a sample (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, an isolated, purified or substantially pure composition comprises more than 80 to 90 percent of all macromolecular species present in a composition.
  • macromolecular e.g., polypeptide
  • an isolated, purified or substantially pure composition comprises more than 80 to 90 percent of all macromolecular species present in a composition.
  • the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), in which the composition consists essentially of a single macromolecular species.
  • the term isolated or purified does not necessarily exclude the presence of other components intended to act in combination with an isolated species.
  • an internalization peptide can be described as isolated notwithstanding that it is linked to an active peptide or combined with a pharmaceutically acceptable excipient.
  • a "peptidomimetic” refers to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of a peptide consisting of natural amino acids.
  • the peptidomimetic can contain entirely synthetic, non-natural analogues of amino acids, or can be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the peptidomimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or inhibitory or binding activity.
  • Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, NjN-dicyclohexylcarbodiimide (DCC) or N.sub.5N-diisopropylcarbodiimide (DIC).
  • glutaraldehyde N-hydroxysuccinimide esters
  • bifunctional maleimides NjN-dicyclohexylcarbodiimide (DCC) or N.sub.5N-diisopropylcarbodiimide (DIC).
  • aminomethylene CH 2 --NH
  • ethylene olefin
  • ether CH. sub.2-0
  • Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-l, -2,3-, or A-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine;
  • D- or L-(4-isopropyl)-phenylglycine D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)- phenylalanine; D-p-fluorophenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p- methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids.
  • Aromatic rings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Mimetics of acidic amino acids can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
  • Carboxyl side groups e.g., aspartyl or glutamyl
  • Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
  • Nitrile derivative e.g., containing the CN-moiety in place of COOH
  • Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
  • Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
  • one or more conventional reagents including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
  • Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha- haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
  • alpha- haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines
  • Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l, 3-diazole.
  • cysteinyl residues e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid
  • chloroacetyl phosphate N-alkylmaleimides
  • 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
  • Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate.
  • imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate.
  • Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
  • Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
  • Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
  • mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl orthreonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
  • the peptidomimetics of the invention can also include a structural mimetic residue, particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like.
  • a structural mimetic residue particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like.
  • substitution of natural amino acid residues with D- amino acids; N-alpha-methyl amino acids; C-alpha-methyl amino acids; or dehydroamino acids within a peptide can induce or stabilize beta turns, gamma turns, beta sheets or alpha helix conformations.
  • Beta turn mimetic structures have been described, e.g., by Nagai (1985) Tet. Lett. 26:647-650; Feigl (1986) J. Amer. Chem. Soc.
  • Beta sheet mimetic structures have been described, e.g., by Smith (1992) J. Amer. Chem. Soc. 114:10672-10674.
  • a type VI beta turn induced by a cis amide surrogate, 1,5-disubstituted tetrazol is described by Beusen (1995) Biopolymers 36:181-200.
  • Peptidomimetics can contain any combination of nonnatural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a peptidomimetic of a chimeric peptide comprising an active peptide and an internalization peptide either the active moiety or the internalization moiety or both can be a peptidomimetic.
  • binding refers to binding between two molecules, for example, a ligand and a receptor, characterized by the ability of a molecule (ligand) to associate with another specific molecule (receptor) even in the presence of many other diverse molecules, i.e., to show preferential binding of one molecule for another in a heterogeneous mixture of molecules. Specific binding of a ligand to a receptor is also evidenced by reduced binding of a detectably labeled ligand to the receptor in the presence of excess unlabeled ligand (i.e., a binding competition assay).
  • subject includes humans and veterinary animals, such as mammals, as well as laboratory animal models, such as mice or rats used in preclinical studies.
  • Inhibitor peptides can be referred to as pharmacological agents.
  • the term "pharmacologic agent” means an agent having a pharmacological activity.
  • Pharmacological agents include compounds that are known drugs, compounds for which pharmacological activity has been identified but which are undergoing further therapeutic evaluation in animal models or clinical trials..
  • An agent can be described as having pharmacological activity if it exhibits an activity in a screening system that indicates that the active agent is or may be useful in the prophylaxis or treatment of a disease.
  • the screening system can be in vitro, cellular, animal or human.
  • Agents can be described as having pharmacological activity notwithstanding that further testing may be required to establish actual prophylactic or therapeutic utility in treatment of a disease.
  • a tat peptide means a peptide comprising or consisting of GRKKRRQRRR (SEQ ID NO:5), in which no more than 5 residues are deleted, substituted or inserted within the sequence, which retains the capacity to facilitate uptake of a linked peptide or other agent into cells.
  • any amino acid changes are conservative substitutions.
  • any substitutions, deletions or internal insertions in the aggregate leave the peptide with a net cationic charge, preferably similar to that of the above sequence. Such can be accomplished by not substituting or deleting the R and K residues.
  • the amino acids of a tat peptide can be derivatized with biotin or similar molecule to reduce an inflammatory response.
  • a tat peptide can include D-amino acids at one or more or all positions and the amino acids can be present in the reverse order.
  • Co-administration of a pharmacological agents means that the agents are administered sufficiently close in time for detectable amounts of the agents to present in the plasma simultaneously and/or the agents exert a treatment effect on the same episode of disease or the agents act co operatively or synergistically in treating the same episode of disease.
  • an anti-inflammatory agent acts cooperatively with an agent including a tat peptide when the two agents are administered sufficiently proximately in time that the anti-inflammatory agent can inhibit an inflammatory response inducible by the internationalization peptide.
  • Statistically significant refers to a p-value that is ⁇ 0.05, preferably ⁇ 0.01 and most preferably ⁇ 0.001.
  • amino acids may be grouped as follows: Group I (hydrophobic side chains); met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acid side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain conformation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same group. Non-conservative substitutions constitute exchanging a member of one of these groups for a member of another.
  • a peptide is maximally aligned with a reference sequence when the number of exact matches between the peptide and the reference sequence is maximized Aligned can be performed by eye.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • default program parameters can be used.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix See Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989)).
  • Sequence identity is compared between maximally aligned sequences over the length of the shorter sequence. The percentage sequence identity is the number of matched residues over the total number in the comparison window not including gaps.
  • a peptide occurring within a specified range of amino acids can include a peptide including either or both amino acids defining the limits of the range as well as a peptide including only amino acids in between the amino acids defining the range.
  • SARSr-CoV sudden acute respiratory syndrome related virus
  • SARS-CoV and SARS-CoV-2 are strains of this virus.
  • Description of inhibitor peptides and methods for SARS-CoV-2 should be understood as providing additional description of corresponding peptides and methods for SARS-CoV and vice versa, unless the context requires otherwise.
  • the invention provides peptides inhibitors for treating SARSr-CoV infection. Some inhibitors inhibit the interaction of the viral S protein receptor binding domain with a human angiotensin converting enzyme 2 receptor and others inhibit interaction of the viral E protein with human syntenin, PALS1 or other PDZ domain. Some inhibitor peptides inhibit interaction between viral nucleoprotein and cellular PDZ domains. Some inhibitor peptides inhibit interaction between SI, a proteolytic fragment of S, and human neuropilin-1. Inhibitor peptide can be linked to an internalization peptide to facilitate entry into tissues or cells.
  • Coronaviruses are a large family of single-stranded enveloped RNA viruses and can be divided into four major genera 11 .
  • Severe acute respiratory syndrome-related coronavirus SARSr-CoV is a species of coronavirus that infects humans, bats and certain other mammals. It is an enveloped positive-sense single-stranded RNA virus that enters its host cell by binding to the angiotensin converting enzyme 2 (ACE2) receptor. It is a member of the genus Betacoronavirus and subgenus Sarbecoronavirus.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • MERS Middle East Respiratory Virus
  • An envelope-anchored spike protein mediates coronavirus entry into host cells by first binding to a host receptor and then fusing viral and host membranes 13 .
  • a defined receptor-binding domain (RBD) of SARSr-CoV spike specifically recognizes its host receptor angiotensin- converting enzyme 2 (ACE2) 14, 15 .
  • ACE2 angiotensin- converting enzyme 2
  • Susceptibility to SARSr-CoV infection is primarily determined by the affinity between the viral RBD and host ACE2 in the initial viral attachment step 16 20 , making this protein- protein interaction a potential therapeutic target.
  • SARS-CoV-2 like other CoVs, mutates - including in-vivo after infection 21 . Therefore, therapies are preferably targeted against conserved sequences so that treatments are applicable for this and future SARS outbreaks.
  • GenBank accession number NC_045512 provides a reference sequence for SARS-CoV-2. About 1700 variants of this sequence have been assigned GenBank accession numbers as of April 2020. The vast majority of variants show greater than 99% identity to the reference sequences at amino acid and nucleic acid levels, and many show greater than 99.9% identity. Thus, typically SARS-CoV-2 viruses can be recognized by at least 99%, 99.5%, or 99.9% identity at the nucleic acid level to the indicated reference sequence. Some exemplary mutations involving amino acid substitutions in the receptor binding domain of the S protein are shown in Fig. 3. SARS-CoV-2 has 79.5% identity to SARS-CoV (Gen Bank accession number AY278741) at the nucleic acid level.
  • SARS-CoV viruses can typically be recognized by at least 99%, 99.5%, or 99.9% identity to a SARS-CoV reference sequence at the nucleic acid or amino acid level.
  • FIG. 2 shows binding of the SARS-CoV-2 S protein RBD to human ACE2. Perturbing the protein- protein interactions between SARS-Cov-2 S protein RBD and its target human ACE2 receptor (hACE2) can inhibit infection.
  • the RBD protein is large (residues 331-524 of SARS-CoV-2 S protein). It would be difficult to express such a biologic agent at large scale, properly-folded, in a human or other model expression system in a manner that meets regulatory requirements. A protein of this size is too large to be produced synthetically. Further, it is processed post translationally by other enzymes like furin, making it more difficult to produce the active form.
  • a protein of this size is expected to be immunogenic, which may be desirable in a vaccine but not in a therapeutic drug that might need to be administered more than once. Rather, a smaller agent would be desirable - ideally a peptide recapitulating the minimum sequence making up the hACE2 domain that binds the SARS-CoV-2 RBD. Such a sequence, which recapitulates the human protein, is unlikely to mutate, rendering it useful against any form of RBD that binds it. Alternatively, a peptide recapitulating protein sequence(s) of RBD binding to the hACE2 can be used.
  • Such peptides can be synthetically manufactured to enable a rapid production of high-purity agent at large scale, because it is much simpler to develop a controlled, scalable, manufacturing process for peptides than for large proteins. Additionally, immunogenicity is unlikely to be an issue with small peptides, making them useful for repeated administration. Further, peptides can be readily modified to increase affinity for a target, and peptides binding different regions that are spatially near each other can be linked to further increase the affinity and avidity of inhibitors
  • Some inhibitor peptides have sequences from the S protein RBD of a SARSr-CoV virus, e.g., SARS-CoV or SARS-CoV-2, and specifically bind to human ACE2.
  • Some inhibitor peptides have lengths of up to 5, 10, 15, 20, 30, 40, 50, 60 or 75 amino acids of an S protein RBD (wildtype or variant sequences shown in Fig. 3). The lengths can be a single contiguous segment or two or more segments joined by spacer(s).
  • Some such peptides have at least 70, 80, 90, 95, 98, 99 or 100% identity to a natural sequence when maximally aligned not including gaps.
  • Some such peptides have at least 70, 80, 90, 95, 98, 99 or 100% identity to a composite sequence including multiple mutations found separately in natural sequences. Some such peptides include up to five substitutions, internal insertions or deletions relative to a contiguous sequence of amino acids from the S-protein RBD or composite of such sequences from different strains. Thus, for example the sequence can include any combination of the four variants shown in Fig. 3 irrespective of whether a strain has been found including the particular combination. Some inhibitors have the same amino acid sequence as segment of an RBD but formed from one or more, or all D-amino acids. Inhibitor peptides formed from D-amino acids may or may not be in retro sequence order.
  • Inhibitor peptides can be linear or cyclic. Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker. Some peptides have lengths of 3-30, 4-15 or 5-10 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids of an S protein RBD. Some exemplary inhibitors comprise or consist of 3-15 contiguous amino acids from the S protein RBD.
  • inhibitor peptides have sequences from the human ACE2 protein and specifically bind to an S protein RBD. Some inhibitor peptides have lengths of up to 5, 10, 15, 20, 30, 40, 50, 60 or 75 amino acids of the human ACE2. The lengths can be a single contiguous segment or two or more segments joined by spacer(s). Some such peptides have at least 70, 80, 90 or 95, 98, 99 or 100% identity to a natural sequence when maximally aligned not including gaps. Some such peptides include up to five substitutions, internal insertions or deletions relative to a contiguous sequence of amino acids from the human ACE2 protein.
  • Some inhibitors have the same amino acid sequence as segment of the human ACE2 protein but formed from one or more, or all D-amino acids. Inhibitor peptides formed from D- amino acids may or may not be in retro sequence order. Inhibitor peptides can be linear or cyclic. Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker. Some exemplary inhibitors comprises or consist of 3-16 contiguous amino acids from human ACE2 and specifically bind to the RBD of S protein. Some such inhibitor peptides have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acids from human ACE2 proteins.
  • SEQ ID NO:6 shows a representative sequence of the SARS-CoV-2 RBD. Peptides corresponding to these sequences, including those present in Table 1, can bind ACE-2 and block viral recognition and cell entry.
  • SEQ ID NO:7 and SEQ ID NO:8 shows a core RBD sequence that contains much of the ACE-2 interacting sequences and a shortened sequence that removes N-terminal sequences. Either of these can be circularized with a linker to promote stability and optionally the addition or deletion of amino acid sequences on either end. Substitutions may be made in the sequences to improve affinity for ACE- 2, including peptidomimetic substitutions.
  • Protein transduction domains i.e., internalization peptides
  • lipidation can optionally be added to improve tissue penetration.
  • SEQ ID NO:9 contains the core RBD, and has section in the middle identified by bold typeface. These bold sequences have fewer contact points with ACE-2 and are therefore optionally substitutable or deletable to increase affinity of inhibitors for ACE-2. Constructs deleting this region or replacing it with a structure that brings the sequences before can inhibit interaction of ACE-2 with coronavirus S protein.
  • SEQ ID NOs:10 and 11 contain sequences predicted to bind the N-terminal sequences of ACE-2 that form an internal disulfide bridge. These sequences can be used to inhibit interactions between ACE-2 and coronaviruses, either alone or when linked to inhibitors of other RBD regions known to bind ACE-2.
  • sequences from the SARS-CoV Spike protein and RBD can be used to design inhibitors of ACE-2 (Table 2). conserveed regions between the SARS-CoV and SARS-CoV-2 strains can guide the design of inhibitors, while varying sequences can be targeted to increase affinity of inhibitors.
  • Table 3 provides an exemplary ACE-2 human sequence (SEQ ID NO:14). Sequences within the ACE-2 gene predicted to interact with the SARS-CoV-2 RBDs can be synthesized as short peptides and used as inhibitors. SEQ ID NO:15 provides a section of the first alpha helix after N-terminal processing that contains RBD-interacting sequences. This can be used as an inhibitor, with substitutions, additions or deletions of up to 5 amino acids. Similarly, these can be attached to other sequences predicted to bind the SARS RBDs with linkers to ensure the spacing between binding sequences is appropriate.
  • Table 1 Exemplary SARS-CoV-2 Spike protein, RBD and inhibitor constructs
  • Table 2 Exemplary SARS-CoV Spike, RBD and inhibitor peptides are shown in the tables below:
  • Table 3 An exemplary human ACE2 sequence and portion-terminal helical sequences thereof are shown below.
  • SARSr-CoV-2 E-protein plays a central role in virus morphogenesis and assembly. It elicits a strong influence on the interaction of SARSr-CoV with the host. In SARS-CoV, deletion mutants missing E protein have reduced replication by 20 and 200-fold, and have a dramatically attenuated virulence phenotype 2933 .
  • the E protein domains that contribute the most to SARS-CoV virulence are those covering the carboxy-terminus, comprising a functional PDZ domain-binding motif (PBM) 34 .
  • PBM PDZ domain-binding motif
  • PDZ motifs are abundant modules involved in protein-protein interaction, which consist of 80-90 amino acids that recognize a PBM motif found in the extreme C-termini of target proteins 35 .
  • cellular PDZs have emerged as a common targets of pathogenic viruses, including influenza virus, Dengue virus, tick- borne encephalitis virus, rabies virus, SARS CoV and human immunodeficiency virus 36 .
  • SARS-CoV and SARS-CoV-2 E proteins are shown in Fig. 1.
  • Some exemplary inhibitors comprise or consist of 3-15 contiguous amino acids from the SARSr- CoV E protein C terminus (i.e., PBM peptides) and specifically bind to a human syntenin, and/or PALS1, or other PDZ identified as binding to the SARS E protein including human forms of any of TJP1 (Q07157), PTPN13 (Q12923), HTRA1 (Q92743), PARD3 (Q8TEW0), MLLT4 (P55196), LNX2 (Q8N448) , NHERF1 (014745), MAST2 (Q6P0Q8), or RADIL (Q96JH8) (collectively "SARS-E-PDZs”).
  • Swiss-Prot accession numbers are included in parentheses .
  • Some such inhibitor peptides have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • Peptides can be linear or cyclic. Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker.
  • inhibitors comprises or consist of 3-16 contiguous amino acids from human syntenin (Swiss Prot 000560) or PALS1 (Swiss Prot Q8N3R9) or other human PDZ protein binding to the E protein.
  • Some such inhibitor peptides have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous amino acids from one of these sources.
  • Some such peptides include up to five substitutions, additions or deletions relative to a contiguous sequence of amino acids from these sources.
  • Some inhibitors have the same amino acid sequence as a natural C-terminal segment of one of these proteins but are formed from one or more, or all D-amino acids. Inhibitors formed of D-amino acids may or may not be in retro order.
  • Peptides can be linear or cyclic. Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker.
  • SARS-CoV-2 E protein 20 amino acid terminus, various PBM inhibitor peptides, internalization peptides, and inhibitor peptides linked to internalization peptides are shown in the Table 4.
  • the full-length sequences of SARS-CoV and SARS-CoV-2 E proteins are also provided in Fig. 1.
  • SARSr-CoV nucleoprotein also contains a PBM.
  • the SARS-CoV-2 nucleoprotein (SEQ ID NO:32), the full sequence of which is shown in Table 5 above, contains a C-terminal PBM.
  • Inhibitor peptides can be prepared comprising or consisting of e.g., the C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 amino acids. Examples of such inhibitor peptides along or linked to a tat internalization peptide as shown in the above table. Some such peptides include up to five substitutions, additions or deletions relative to a contiguous sequence of amino acids from the nucleoprotein C-terminus.
  • Some inhibitors have the same amino acid sequence as segment of the nucleoprotein, but formed from one or more, or all D-amino acids. Inhibitors formed of D-amino acids may or may not be in retro order.
  • Peptides can be linear or cyclic. Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker.
  • SI has a polybasic RRAR (SEQ ID NO:76) carboxyl sequence, which conforms with a CendR motif [R/K]XX[R/K] (SEQ ID NO:77) that binds to cell surface neuropilin-1 (NRP1) receptor.
  • Neuropilin-1 is assigned Swiss Prot 0147786 and is receptor of 923 amino acids of which residues 1-21 are a signal sequence, residues 22-856 are an extracellular domain, residues 857-879 are transmembrane and 880-923 are cytoplasmic.
  • a segment of neuropilin-1 between residues 297 and 427 and particularly a segment including residues Y297, W301, T316, D320, S346, T349 and Y353 are reported bind to bind a motif including RRAR (SEQ ID NO:76) at the C-terminus of SI.
  • the ligand binding portion of neuropilin-1 is sometimes referred to as the bl domain (Lee etal., Structure 11, 99-108 (2003)). Therefore, peptides including some or all of the respective motifs can be used to inhibit interaction of SI with neuropilin-1 receptor and thus entry of SARS-COV-2 into cells.
  • Table 6 below provides sequence from the relevant area of neuropilin-1, which corresponds to the neuropili-1 beta groove.
  • Some inhibitor peptides have sequences from the C-terminus of the SI protein RBD of a SARSr- CoV virus, e.g., SARS-CoV or SARS-CoV-2, and specifically bind to human neuropilin-1. Such inhibitor peptides can block viral recognition by neuropilin-1 and cell entry.
  • Some inhibitor peptides have lengths of up to 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or 75 amino acids of an SI protein C-terminus. The lengths can be a single contiguous segment or two or more segments joined by spacer(s). Some such peptides have at least 70, 80, 90, 95, 98, 99 or 100% identity to a natural sequence when maximally aligned not including gaps. Some such peptides include up to five substitutions, internal insertions or deletions relative to a contiguous sequence of amino acids from the Sl-protein C-terminus.
  • Substitutions can be made to improve affinity for neuropilin-1, including peptidomimetic substitutions.
  • Protein transduction domains i.e., internalization peptides, or lipidation can optionally be added to improve tissue penetration.
  • Some inhibitors have the same amino acid sequence as SI but formed from one or more, or all D-amino acids.
  • Inhibitor peptides formed from D-amino acids may or may not be in retro sequence order.
  • Inhibitor peptides can be linear or cyclic.
  • Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker.
  • Some peptides have lengths of up to e.g., 75 residues 3-30, 3-20, 3-15, 4-15 or 5-10 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20 amino acids of an SI protein C-terminus, preferably including RRAR (SEQ ID NO:76) as the four C-terminal amino acids.
  • Some exemplary inhibitors comprise or consist of 3-15 contiguous amino acids from the SI protein C-terminus. IGAGICASYQTQTNSPRRAR (SEQ ID NO:73), NSPRRAR (SEQ ID NO:74) and subsequences thereof provide exemplary sequences of inhibitor peptides.
  • inhibitor peptides have sequences from the human neuropilin-1 protein and specifically bind to SI protein at its C-terminus. Such inhibitor peptides can inhibit SI recognition of neuropilin-1 recognition and inhibit viral entry into cells.
  • Some inhibitor peptides have lengths of e.g., up to 5, 10, 15, 20, 30, 40, 50, 60 , or 75 amino acids of the human neuropilin-1. The lengths can be a single contiguous segment or two or more segments joined by spacer(s). Some such peptides have at least 70, 80, 90, 95, 98, 99 or 100% identity to a natural sequence when maximally aligned not including gaps.
  • Some such peptides include up to five substitutions, internal insertions or deletions relative to a contiguous sequence of amino acids from the human neuropilin-1 protein. Substitutions can increase affinity for SI. Substitutions can include peptidomimetic substitutions. Protein transduction domains, i.e., internalization peptides, or lipidation can be added to improve cell penetration. Some inhibitors have the same amino acid sequence as segment of the human neuropilin-1 protein but formed from one or more, or all D-amino acids. Inhibitor peptides formed from D-amino acids may or may not be in retro sequence order. Inhibitor peptides can be linear or cyclic.
  • Peptides can be in the form of monomers, dimers, trimers, tetramers or other multimers. If in the form of multimers, individual peptides can be joined end to end or through a linker.
  • Some exemplary inhibitor peptides comprises or consist of e.g., 75 residues, 3-30, 3-20, 3-15, 3-16, 4-15 or 5-10 contiguous amino acids from human neuropilin-1 and specifically bind to the SI protein at the C-terminus.
  • Some such inhibitor peptides have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids from a human neuropilin-1 protein.
  • a inhibitor peptide can be linked to an internalization peptide to facilitate uptake into cells and from the blood or other body fluid into tissues. Any of the above inhibitor peptides can be linked to any of the internalization peptides described below. Internalization peptides are a well-known class of relatively short peptides that allow many cellular or viral proteins to traverse membranes.
  • Internalization peptides also known as cell membrane transduction peptides or cell penetrating peptides can have e.g., 5-30 amino acids. Such peptides typically have a cationic charge from an above normal representation (relative to proteins in general) of arginine and/or lysine residues that is believed to facilitate their passage across membranes. Some such peptides have at least 5, 6, 7 or 8 arginine and/or lysine residues. Examples include the antennapedia protein (Bonfanti, Cancer Res.
  • tat protein of human immunodeficiency virus the protein VP22, the product of the UL49 gene of herpes simplex virus type 1, Penetratin, SynBl and 3, Transportan, Amphipathic, gp41NLS, polyArg, and several plant and bacterial protein toxins, such as ricin, abrin, modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labile toxins, and Pseudomonas aeruginosa exotoxin A (ETA).
  • ricin abrin
  • modeccin diphtheria toxin
  • cholera toxin cholera toxin
  • anthrax toxin heat labile toxins
  • Pseudomonas aeruginosa exotoxin A ETA
  • a preferred internalization peptide is tat from the HIV virus.
  • a tat peptide reported in previous work comprises or consists of the standard amino acid sequence YGRKKRRQRRR (SEQ ID NO:23) found in HIV Tat protein.
  • two preferred agents incorporating this tat peptide are the peptides comprising or consisting of the amino acid sequence .
  • residues flanking such a tat motif can be for example natural amino acids flanking this segment from a tat protein, spacer or linker amino acids of a kind typically used to join two peptide domains, e.g., gly (ser) 4 (SEQ ID NO:42), TGEKP (SEQ ID NO:43), GGRRGGGS (SEQ ID NO:44) or LRQRDGERP (SEQ ID NO:45) (see, e.g., Tang et al. (1996), J. Biol. Chem. 271, 15682-15686; Hennecke et al. (1998), Protein Eng.
  • gly (ser) 4 SEQ ID NO:42
  • TGEKP SEQ ID NO:43
  • GGRRGGGS SEQ ID NO:44
  • LRQRDGERP SEQ ID NO:45
  • flanking amino acids other than an active peptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:23).
  • One suitable tat peptide comprising additional amino acid residues flanking the C-terminus of YGRKKRRQRRR (SEQ ID NO:23) is YGRKKRRQRRRPQ (SEQ ID NO:46).
  • YGRKKRRQRRRPQ SEQ ID NO:46
  • no flanking amino acids are present.
  • Other tat peptides that can be used include GRKKRRQRRRPQ (SEQ ID NO:47) and GRKKRRQRRRP (SEQ ID NO:48).
  • variants of the above tat peptide having reduced capacity to bind to N-type calcium channels are described by W02008/109010.
  • Such variants can comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ ID NO:49), in which X is an amino acid other than Y or can comprise or consist of an amino acid sequence GRKKRRQRRR (SEQ ID NO:5).
  • a preferred tat peptide has the N-terminal Y residue substituted with F.
  • a tat peptide comprising or consisting of FGRKKRRQRRR (SEQ ID NO:50) is preferred.
  • Another preferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO:5).
  • Another preferred tat peptide comprises or consists of RRRQRRKKRG (SEQ ID NO:51) or RRRQRRKKRGY (SEQ ID NO:52).
  • Other tat derived peptides that facilitate uptake of an inhibitor peptide without inhibiting re type calcium channels include those below.
  • X can represent a free amino terminus, one or more amino acids, or a conjugated moiety.
  • Internalization peptides can be used in inverso or retro or inverso retro form with or without the linked peptide or peptidomimetic being in such form.
  • a chimeric peptide has an amino acid sequence comprising RRRQRRKKRGY (SEQ ID NO:52) linked to an inhibitor peptide.
  • Internalization peptides can be attached to inhibitor peptides by conventional methods. If the inhibitor peptide is a PBM peptide, then the internalization peptide is preferably attached to the N- terminus of the PBM peptide leaving a free C-terminus. Otherwise, attachment at either end or internally via conjugation is possible.
  • the agents can be joined to internalization peptides by chemical linkage, for instance via a coupling or conjugating agent. Numerous such agents are commercially available and are reviewed by S. S. Wong, Chemistry of Protein Conjugation and Cross- Linking, CRC Press (1991).
  • cross-linking reagents include J-succinimidyl 3 (2- pyridyldithio) propionate (SPDP) or N,N'-(l,3-phenylene) bismaleimide; N,N'-ethylene-bis- (iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges (which relatively specific for sulfhydryl groups); and l,5-difluoro-2, 4-dinitrobenzene (which forms irreversible linkages with amino and tyrosine groups).
  • SPDP J-succinimidyl 3 (2- pyridyldithio) propionate
  • N,N'-(l,3-phenylene) bismaleimide N,N'-ethylene-bis- (iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges (which relatively specific for sulfhydryl groups)
  • cross-linking reagents include p,p'-difluoro-m, m'- dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1, 4-disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p- diisocyanate (which reacts principally with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine).
  • inhibitor peptides that are peptides attachment to an internalization peptide can be achieved generating a fusion protein comprising the inhibitor peptide fused to the internalization peptide.
  • lipidation lipidation
  • lipidation lipidation
  • Suitable forms of lipidation include myristoylation, palmitoylation or attachment of other fatty acids preferably with a chain length of 10-20 carbons, such as lauric acid and stearic acid, as well as geranylation, geranylgeranylation, and isoprenylation. Lipidations of a type occurring in posttranslational modification of natural proteins are preferred.
  • Lipidation can be by peptide synthesis including a prelipidated amino acid, be performed enzymatically in vitro or by recombinant expression, by chemical crosslinking or chemical derivatization of the peptide. Amino acids modified by myristoylation and other lipid modifications are commercially available.
  • Lipidation preferably facilitates passage of a linked peptide without causing a transient reduction of blood pressure as has been found when a standard tat peptide is administered at high dosage (e.g., at or greater than 3 mg/kg), or at least with smaller reduction that than the same peptide linked to a standard tat peptide.
  • Pharmacologic peptides optionally fused to tat peptides, can be synthesized by solid phase synthesis or recombinant methods. Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al.
  • an inhibitor peptides is administered in an amount, frequency and route of administration effective to reduce, inhibit or delay one or more damaging effects of SARSr-CoV infection.
  • dosages for inhibitor peptides that are chimeric agents including an inhibitor peptide linked to an internalization peptide refer to the whole agent rather than just the inhibitor peptide component of the chimeric agent.
  • An effective amount means an amount of inhibitor peptide or pharmacological agent including an inhibitor peptide sufficient significantly to reduce, inhibit or delay onset or more damaging effects of SARSr-CoV infection in a population of subjects (or animal models) suffering from the disease treated with an inhibitor peptide of the invention relative to the damage in a control population of subjects (or animal models) suffering from SARSr-CoV infection who are not treated with the inhibitor peptide or pharmacological agent.
  • the control population can be contemporaneously treated with a placebo or can be a historical control.
  • the amount is also considered effective if an individual treated subject achieves an outcome more favorable than the mean outcome in a control population of comparable subjects not treated by methods of the invention.
  • An effective regime involves the administration of an effective dose at a frequency and route of administration needed to achieve the intended purpose.
  • administration can be parenteral, intravenous, nasal, oral, subcutaneous, intrapulmonary or inhaled, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.
  • Treated subjects for treatment preferably have a confirmed diagnosis of SARSr-CoV infection but treatment can also be administered to subjects suspected of infection or prophylactically to subjects at high risk of infection due to, for example, proximity to other infected subjects, or predisposing factors to incidence or severity of disease. Diagnosis can be performed by e.g., PCR with primers specific for a SARSr-CoV virus, or by immunoassay. [78] During and/or following treatment subjects can be monitored for changes in signs and symptoms relative to baseline before treatment.
  • a positive treatment outcome can be evidenced by reduced viral load, reduced inflammation, reduce levels of one or more inflammatory markers, such as any of C-reactive protein, IL-6, lactate dehydrogenase, and D-dimer, reduced blood clotting, and reduced organ damage, e.g., to lungs, heart, liver, kidneys and CNS and reduce one or more neurological signs and/or symptoms (e.g., impaired cognition, impaired memory, impaired sense of smell, headache, migraine, meningitis, myelitis, Guillain-Barre syndrome, or CNS vasculitis).
  • inflammatory markers such as any of C-reactive protein, IL-6, lactate dehydrogenase, and D-dimer
  • reduced blood clotting e.g., to lungs, heart, liver, kidneys and CNS
  • one or more neurological signs and/or symptoms e.g., impaired cognition, impaired memory, impaired sense of smell, headache, migraine, meningitis, myelitis, Guillain-Barre
  • Reduction of neurological signs and symptoms can occur as a result of reducing viral load generally in a patient or more specifically because an inhibitor peptide linked to an internalization peptide can cross the blood brain barrier and reduce infectivity or replication of SARSr-CoV in the brain or central nervous system.
  • chimeric agents including an internalization peptide particularly a HIV tat peptide
  • administration of the pharmacological agent may or may not be combined with an anti-inflammatory agent to reduce release or histamine and its downstream effects associated with high levels of the internalization peptide.
  • Preferred agents for co-administration are inhibitors of mast cell degranulation, such as cromolyn or lodoxamide or any others listed herein.
  • Anti-histamines or corticosteroids can also be used, particularly in combinations or higher dosages (see W02009/076105, and WO2010/14474261).
  • Inhibitor peptides can be administered as a single dose or multiple doses. For example, dosing can be initiated on diagnosis of a SARSr-CoV infection and continued until symptoms decline or resolve or until virus is no longer detectable. Dosing can be daily, , 2, 3, 4, 5 or 6 times per day, every other day, twice weekly or weekly, among other possibilities. Dosing can also be adjusted depending on a subject's response.
  • Inhibitor peptides or pharmacological agents including the same can be administered in the form of a pharmaceutical composition.
  • Pharmaceutical compositions are typically manufactured under GMP conditions.
  • Pharmaceutical compositions for parenteral administration are preferentially sterile (e.g., filter sterilization of peptide) and free of pyrogens.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
  • Pharmaceutical compositions can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of chimeric agents into preparations which can be used pharmaceutically. Proper formulation is dependent on the route of administration chosen.
  • An exemplary formulation of an inhibitor peptide or pharmacological agent incorporating the same includes the agent in normal saline (0.8-1.0% and preferably 0.9% saline) or phosphate buffered saline at a concentration of 1-100 mg/ml or 10-30 mg/ml, for example 16-20 or 18 mg/ml.
  • normal saline or phosphate buffered saline without such excipients is sufficient to obtain this stability.
  • Such a composition can be thawed and diluted into a larger volume of normal saline for infusion into a blood vessel.
  • the present inhibitor peptides can be used in combination therapy with each other (e.g., one agent targeting protein S-ACE2 interaction and another targeting protein E-syntenin or PALS1 interaction.
  • the present inhibitor peptides can also be used in combination therapy with other pharmacological agents effective against SARSr-CoV, for example, remdesivir, hydroxychloroquine, chloroquine, azithromycin, antibodies against SARSr-CoV, particularly against the S protein, convalescent plasma, actemra, kaletra, interferon-beta, and kevzara.
  • Such agents can be used in the present combination methods in accordance with their conventional formulations, doses, routes of administration, and frequency of administration (see Physician's Desk Reference and applicable package inserts).
  • mechanical methods of reperfusion can be employed in accordance with conventional practice.
  • PBM-PDZ interactions of SARSr-CoV E protein and nucleoprotein can also be used in diagnostic assays to detect such viruses in samples from subjects, such as saliva, nasal or throat swabs or blood, plasma or serum.
  • the assays detect specific binding between a peptide comprising or consisting of a PDZ domain and a viral protein.
  • a peptide comprising or consisting of a PDZ domain and a viral protein.
  • the human form of any of syntenin, PALS1, TJP1, PTPN13, FITRA1, PARD3, MLLT4, LNX2, NHERF1, MAST2,or RADIL PDZ domains can be used to detect the E protein.
  • Immunometric or sandwich assays are a suitable format (see U.S. Pat. Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375). Such assays use a PDZ domain immobilized to a solid phase as a capture agent, and an or population of antibodies in solution as detection agent. Typically, the detection agent is labeled. If an antibody population is used, the population typically contains antibodies binding to different epitope specificities within the target antigen.
  • Target antigen in a sample competes with exogenously supplied labeled target antigen for binding to PDZ detection reagent.
  • the amount of labeled target antigen bound to the detection reagent is inversely proportional to the amount of target antigen in the sample.
  • Lateral flow devices are a preferred format (see, e.g., U.S. Pat. Nos. 5,569,608; 6,297,020; and 6,403,383)
  • Lateral flow devices work by applying fluid to a test strip that has been treated with specific biologicals. Carried by the liquid sample, phosphors labeled with corresponding biologicals flow through the strip and can be captured as they pass into specific zones. The amount of phosphor signal found on the strip is proportional to the amount of the target analyte.
  • a sample suspected of containing SARSr-CoV is added to a lateral flow device, the sample is allowed to move by diffusion and a line or colored zone indicates the presence of the virus.
  • the lateral flow typically contains a solid support (for example nitrocellulose membrane) that contains three specific areas: a sample addition area, a capture area containing one or more PDZ peptides, and a read-out area that contains one or more zones, each zone containing one or more labels.
  • a solid support for example nitrocellulose membrane
  • Suitable detectable labels for use in the above methods include any moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means.
  • suitable labels include biotin for staining with labeled streptavidin conjugate, fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 l, 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex beads).
  • fluorescent dyes e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels e.g., 3 H, 125 l, 35 S
  • Patents that described the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. See also Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.). Radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.
  • Example 1 Therapeutics based on SARS-CoV-2 RBD/hACE2 interaction (viral attachment inhibitors)
  • the SARS-CoV-2 RBD has been recently identified as comprising residues 331 to 524 ( ⁇ 23kDa) of the S protein 12 .
  • Treatment of human ACE2-expressing 293T cells with recombinant SARS-CoV-2 RBD protein could inhibit SARS-CoV-2 pseudovirus entry into human ACE2-expressing 293T cells 12 .
  • a protein of this size is expected to be immunogenic - potentially causing an undesired hyperimmune response and inflammation. Proteins of this size are impractical parenterally (i.v.) administered therapies, as they do not easily distribute from the bloodstream into tissues 50 .
  • a small peptide coupled to a protein-transduction domain, i.e., internalization peptide, that makes it tissue-permeable and/or able to cross the blood brain barrier, would be preferred.
  • Peptide therapeutics can be screened for their capacity to inhibit the attachment of SARS-CoV-2 RBD to hACE2, by two approaches: A standard competition ELISA assay to enable screening of multiple peptide candidates in multiwell plates.
  • Recombinant ACE2 protein is available from multiple vendors, and Vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 Spike Glycoprotein Gene RBD with C- Terminal Hexa-Histidine Tag is also commercially available.
  • an expression vector for a protein of this size can also be assembled de-novo from the published genome of SARS-CoV-2 if necessary, using standard techniques.
  • a second viral attachment assay comprises expression of hACE2 in hACE2/293T cells.
  • Stably transfected cell lines are commercially available. These can be used to detect the binding of labelled SARS-CoV-2 RBD protein to cell associated hACE2 by flow cytometry analysis and immunofluorescence staining as described by Tai and colleagues 12 .
  • Peptides spanning different lengths of the 193 residues of the SARS-CoV-2 RBD are synthesized. Peptides can target either hACE2 or the RBD. The former is preferred because the RBM may mutate.
  • Initial peptides include around 50 amino acids. Biotinylated peptides are used, with approximately 50% overlapping sequences. These are tested initially in dot blots but also in the ELISA and receptor attachment assays as described above. The smallest peptide(s) showing binding to hACE2 re selected. It can be inferred from a model of protein-protein interaction 52 ( Figure 2) that the hACE2 receptor has several residues that can be targeted with relatively short peptides. Some of the core binding residues on hACE2 spanning from positions 31-38 suggest that a peptide as short as 7 residues could suffice to perturb binding.
  • Optimized interfering peptides are coupled with an internalization peptide 53 . Because in SARS- CoV, viral pathology has been shown to affect multiple organs 7 including the lungs but also heart (myocarditis) 8 and potentially the nervous system9, 10 , the internalization peptide is advantageous in ensuring these agents can reach their target tissues. The resulting inhibitors are re-screened in the ELISA assays to ensure that they have maintained their affinity and binding inhibition characteristics.
  • Example 2 Therapeutics based on SARS-CoV-2 E-protein PDZ binding domain (virulence inhibitors)
  • SARS-CoV deletion mutants missing the E protein have reduced replication by 20 and 200-fold, and have a dramatically attenuated virulence phenotype 29 33 .
  • the E protein domains that contribute the most to SARS-CoV virulence are those covering the carboxy-terminus, comprising a functional PDZ domain-binding motif (PBM) 34 .
  • PBM PDZ domain-binding motif
  • This E-protein PBM is highly conserved among coronaviruses, including SARS-CoV and SARS-CoV-2 (NCBI Blast Search), implying it is critical for its function.
  • the C-terminal 12 residues of the SARS-CoV and SARS-CoV-2 sequences in Fig. 1 can serve as the starting sequence.
  • This PBM sequence is already established to mediate virulence, and is agnostic to which PDZ domain(s) it may bind.
  • the C-terminal 6 residues are highly conserved, so are likely to make up the core binding to most PDZ domains.
  • the PBM has already been reported to bind two known PDZ-domain binding proteins: Syntenin, a relevant scaffolding protein that participates in the activation of p38 mitogen-activated protein kinase (MAPK) 34 and PALS1, a key component of the complex that controls polarity establishment and tight junction formation in epithelia 54 . More PDZ protein interactions with the SARS E protein have been identified in the scientific literature (e.g., Caillet-Saguy et al, 2021 FEBS J. 2021 Apr 17. doi: 10.1111/febs.l5881. Epub ahead of print. PMID: 33864728.). Therefore, the starting sequence can be modified to further optimize its binding to those targets irrespective of whether additional PDZ partners are identified.
  • additional PDZ targets of a given PBM can be determined in proteomic screens containing PDZ domains proteins individually cloned into expression vectors and produced as properly folded GST-fusion recombinant proteins 44 .
  • Such platforms are now available as commercially- produced PDZ domain arrays (e.g., Panomics/Affymetrix, USA). These are referred-to hereafter as PDZ- array, and will be used to identify further PDZ domain targets.
  • the SARS-CoV-2 E-protein PBM is first screened against recombinant human syntenin and PALS1 PDZ proteins, and also in the PDZ array to identify additional potential PDZ targets. Binding studies are then conducted to determine the EC50 of the PBM to its PDZ targets. We expect to obtain EC50 in the nanomolar range. Confirmatory experiments are conducted as necessary to determine the IC50 of the PBM in inhibiting the interactions of the full recombinant E-protein (obtained commercially, or purified in the lab) with the given PDZ target.
  • Modifications can be introduced into PBMs to further enhance their target binding affinity and specificity.
  • the fusion peptides are then screened in the PDZ arrays and their affinity and selectivity characterized in the binding assays.
  • Preferred peptides I have the characteristic of high affinity binding (EC50 ⁇ 50nm) to as many of the identified PDZ targets as possible.
  • the candidate peptide or SARS-CoV-2 RBD protein at serial dilutions are incubated with hACE2/293T target cells for 1 h at 37 °C. After removing medium containing the protein, the cells are infected with SARS-CoV-2 pseudovirus. Fresh medium is added 24 h later, and the cells are lysed in cell lysis buffer (Promega, Madison, Wl). The lysed cell supernatants are incubated with luciferase substrate (Promega) and detected for relative luciferase activity. With the capacity for only one-cycle infection, S protein-expressing pseudovirus cannot replicate in the target cells 55 . Therefore, the inhibition of pseudovirus infection represents inhibition of viral entry, as mediated by viral S protein.
  • the assay is safe as the pseudovirus is replication deficient. Inhibition of infection by the candidate peptide is compared to that of the full SARS-CoV-2 RBD, which is used as a positive control with an expected IC50 of ⁇ 1.35u/ml.
  • mice Either BALB/c and/or K18-hACE transgenic mice can be used.
  • a maximally-tolerated dose (MTD) study is performed to guide therapeutic dosing.
  • Studies can be initiated in 16 week-old specific-pathogen-free BALB/c Ola Hsd mice 57 , which are susceptible to SARS-CoV infection.
  • these studies are conducted in K18- hACE2 transgenic mice 18 which are available through Jackson Labs, and are more susceptible.
  • the MTD is the highest dose that does not produce unacceptable side-effects over the 7- day treatment period.
  • mice or K18-hACE2 transgenic mice are infected at an age of 16 weeks with 100,000 plaque forming units (pfu). Infection is achieved by light anesthesia of the mice, followed by the intranasal introduction of the indicated dosage of SARS-CoV-2 in 30 mI of Dulbecco's modified Eagle medium. Infected mice are examined and weighed daily. The infection begins in the airway epithelium, spreads to the alveoli and finally out of the lungs to the brain. The infection causes infiltration of macrophages and lymphocytes in the lungs and up-regulation of pro-inflammatory cytokines and chemokines in the lungs and brain. Three to five days following infection, K18-hACE2 mice are expected to begin to lose weight and become lethargic with labored breathing. Death follows within seven days 18 .
  • Candidate agents are used to treat the infected mice daily via the intravenous route beginning at day 0, at the maximum tolerated dose as determined by the MTD. Administration can be conducted in restrained animals via a tail vein injection. Effectiveness of a candidate therapeutic is determined by monitoring clinical observations, survival rates and daily weights. Animals are sacrificed at appropriate intervals (typically at 2 and 4 days post-infection and at the end of each study period) and organs are removed from mice at 2 and 4 days post-infection, fixed in zinc formalin, and processed. For routine histology, sections are stained with hematoxylin and eosin. Tissues and blood samples are also harvested for quantitative and qualitative determinations of inflammatory markers, especially along the NF-KB pathway which is the most frequently activated 23, 29
  • Statistical analyses include as appropriate Fisher's Exact Test or Chi-Square for binary outcomes (such as infection or mortality) or, for cardinal data, log-rank testing (survival curves) or one-sample t- tests (infectious units).
  • Li F Structure, function, and evolution of coronavirus spike proteins. Annu Rev Virol. 2016;3:237- 261.
  • Angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus. Nature. 2003;426:450-454.
  • Li F Receptor recognition mechanisms of coronaviruses: A decade of structural studies. Journal of virology. 2015;89:1954-1964.
  • DeDiego ML Alvarez E, Almazan F, Rejas MT, Lamirande E, Roberts A, et al.
  • a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo. Journal of virology. 2007;81:1701-1713.
  • DeDiego ML Nieto-Torres JL, Jimenez-Guardeno JM, Regla-Nava JA, Alvarez E, Oliveros JC, et al. Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis.
  • PLoS Pathog. 2011;7:el002315 Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis.
  • the sars coronavirus e protein interacts with palsl and alters tight junction formation and epithelial morphogenesis.
  • Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Science (New York, N.Y.), 370(6518), 856-860.
  • Neuropilin-1 is a host factor for SARS-CoV-2 infection. Science (New York, N.Y.), 370(6518), 861-865. https://doi.org/10.1126/science.abd3072. Jobe, A., & Vijayan, R. (2021). Neuropilins: C-end rule peptides and their association with nociception and COVID-19. Computational and structural biotechnology journal, 19, 1889-1895. World wide web doi.org/10.1016/j.csbj.2021.03.025. Davies, J., Randeva, H. S., Chatha, K., Hall, M., Spandidos, D. A., Karteris, E., & Kyrou, I. (2020).
  • Neuropilin-1 as a new potential SARS-CoV-2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID-19.

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Abstract

L'invention concerne des inhibiteurs peptidiques pour le traitement d'une infection par le SARSr-CoV. Certains inhibiteurs inhibent l'interaction du domaine de liaison du récepteur de la protéine S virale avec un récepteur 2 de l'enzyme de conversion de l'angiotensine humaine et d'autres inhibent l'interaction de la protéine E virale avec la synténine humaine ou PALS1. Le peptide inhibiteur peut être lié à un peptide d'internalisation pour faciliter l'entrée dans des tissus ou des cellules.
PCT/IB2021/053763 2020-05-05 2021-05-04 Inhibiteurs peptidiques contre l'infection par le sarsr-cov Ceased WO2021224803A1 (fr)

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WO2005060520A2 (fr) * 2003-11-25 2005-07-07 Dana-Farber Cancer Institute, Inc. Anticorps diriges contre sras-cov et methodes d'utilisation de ceux-ci

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WO2005060520A2 (fr) * 2003-11-25 2005-07-07 Dana-Farber Cancer Institute, Inc. Anticorps diriges contre sras-cov et methodes d'utilisation de ceux-ci

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