Classifications

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  • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
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  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
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  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
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  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
  • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
  • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
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  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses

Definitions

• the present invention is in the field of anti- viral therapy.
• Coronavimses are positive-sense, single-stranded RNA viruses that are often associated with mild respiratory tract infections in humans.
• SARS-CoV-1 was the etiological agent of the SARS epidemic in the winter of 2002/3 that caused 774 deaths amongst 8,098 cases
• MERS-CoV was responsible for the MERS epidemic that started from 2012 with 862 deaths from 2506 infections
• SARS-CoV-2 is responsible for the ongoing COVID-2019 pandemic resulting in 1.31 million deaths out of 54,068,330 cases (as of Sun Nov 15, 2020.
• SARS-CoV-1 and SARS-CoV-2 are very similar to one another (ca. 80%) but are distinct from most other Coronaviridae members that infect humans. Both viruses have been placed in subgroup B in the Betacoronavims genus within the Orthocoronavirinae subfamily of the Coronaviridae.
• SARS-CoV-2 3a protein also known as open reading frame 3a (ORF3a)
• ORF3a open reading frame 3a
• Vroporin homotetrameric potassium sensitive ion channels
• pathogenesis including up- regulation of expression of fibrinogen subunits FGA, FGB and FGG in host lung epithelial cells, inducement of apoptosis in cell culture.
• a method for treating or preventing SARS-CoV-2 virulence in a subject in need thereof comprising administering to the subject a therapeutically effective amount of any one of: SARS-CoV-2 E protein channel blocker and a SARS-CoV-2 3a protein inhibitor, thereby treating or preventing SARS-CoV-2 virulence in the subject.
• a pharmaceutical composition comprising a SARS-CoV-2 E protein channel blocker and/or SARS-CoV-2 3a protein inhibitor for use in the treatment or prevention of SARS-CoV-2 virulence in a subject in need thereof.
• preventing comprises preventing any one of: SARS-CoV-2 entry to a cell of the subject, uncoating of the SARS-CoV-2 in a cell of the subject, release of the SARS-CoV-2 from a cell of the subject, and any combination thereof.
• the subject is infected or suspected of being infected by SARS-CoV-2.
• the SARS-CoV-2 E protein channel blocker is at least one molecule selected from the group consisting of: 5-Azacytidine, Memantine, Gliclazide, Mavorixafor, Saroglitazar Magnesium, Mebrofenin, Cyclen, Kasugamycin, Plerixafor, and any salt thereof.
• the SARS-CoV-2 E protein channel blocker is for use at a daily dose of 0.01 to 500 mg/kg.
• the SARS-CoV-2 E protein channel blocker is Ginsenoside. [014] In some embodiments, the SARS-CoV-2 E protein channel blocker is Memantine.
• the SARS-CoV-23a protein inhibitor is at least one molecule selected from the group consisting of: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine, and Fludarabine.
• the SARS-CoV-2 3a protein inhibitor is Capreomycin.
• the prevention comprises prevention of any one of: SARS-CoV-2 3a protein inhibitor
• CoV-2 from a cell of the subject, and any combination thereof.
• Figures 2A-2B include graphs showing K + conductivity assay. Impact of viral protein SARS-CoV-2 E protein on the growth of K + -uptake deficient bacteria (left panel, 2A). Different protein expression levels are achieved by varying the concentration of the IPTG inducer, as noted. Bacterial growth rate as a function of [K + ] is plotted in the right panel (2A). (2B) depicts the impact of SARS CoV-2 3a protein on the growth of K + -uptake deficient bacteria, using varying concentration of the IPTG inducer.
• Figures 3A-3B include graphs showing fluorescence-based H + conductivity assay.
• Figures 4A-4B include graphs showing compound screening results using the positive and negative genetic tests. Impact of different drugs, as noted, and E protein expression on the growth rates of bacteria.
• Figures 5A-5C include graphs showing Mavorixafor screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (5A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (5B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (5C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 6A-6C include graphs showing Saroglitazar magnesium screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (6A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (6B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (6C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 7A-7C include graphs showing Mebrofenin screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (7A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (7B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (7C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 8A-8C include graphs showing Cyclen screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (8A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (8B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (8C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 9A-9C include graphs showing Kasugamycin screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (9A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (9B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (9C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 10A-10C include graphs showing 5-Azacytidine screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (10A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (10B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (IOC) of SARS-CoV-2 E protein expressing bacteria.
• Figures 11A-11C include graphs showing Plerixafor (octahydrochloride) screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (11A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (11B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (11C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 12A-12C include graphs showing Plerixafor screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (12A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (12B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (12C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 13A-13C include graphs showing Capreomycin (sulfate) screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (13A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (13B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (13C) of SARS-CoV-2 E protein expressing bacteria.
• Figures 14A-14C include graphs showing Pentamidine (isethionate) screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (14A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (14B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (14C) of SARS-CoV-23a protein expressing bacteria.
• Figures 15A-15C include graphs showing Spectinomycin (dihydrochloride) screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (15A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (15B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (15C) of SARS-CoV-2 3a protein expressing bacteria.
• Figures 16A-16C include graphs showing Kasugamycin (hydrochloride hydrate) screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (16A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (16B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (16C) of SARS-CoV-2 3a protein expressing bacteria.
• Figures 17A-17C include graphs showing Plerixafor screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (17A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (17B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (17C) of SARS-CoV-23a protein expressing bacteria.
• Figures 18A-18C include graphs showing Flumatinib screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (18A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (18B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (18C) of SARS-CoV-23a protein expressing bacteria.
• Figures 19A-19C include graphs showing Litronesib screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (19A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (19B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (19C) of SARS-CoV-23a protein expressing bacteria.
• Figures 20A-20C include graphs showing Darapladib screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (20A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (20B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (20C) of SARS-CoV-23a protein expressing bacteria.
• Figures 21A-21C include graphs showing Floxuridine screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (21A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (21B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (21C) of SARS-CoV-23a protein expressing bacteria.
• Figures 22A-22C include graphs showing Fludarabine screening results using the negative assay: Viral channel harmful to bacteria, wherein blocker increases growth (22A), positive assay: Viral channel essential to bacteria, wherein blocker decreases growth (22B), and fluorescence assay: Viral channel alters Fluorescence, wherein blocker decreases fluorescence change (22C) of SARS-CoV-23a protein expressing bacteria.
• Figure 23 includes a vertical bar graph showing the effect of various tested drugs on the viability of Vero-E6 cells which were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. DETAILED DESCRIPTION
• the present invention in some embodiments, provides compositions comprising a SARS-CoV-2 E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor for treating or preventing SARS-CoV-2 virulence in a subject.
• the present invention in some embodiments, provides compositions comprising a SARS-CoV-2 E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor, for preventing SARS-CoV-2 2 cell entry, uncoating and/or release from a cell.
• the invention is based, at least in part, on the finding using three bacteria-based assays, that SARS-CoV-2 E protein is an ion channel.
• the invention is further based, at least in part, on a finding that Gliclazide, Memantine, Mavorixafor, Saroglitazar Magnesium, Mebrofenin, Cyclen, Kasugamycin, Azacytidine, and Plerixafor, inhibit SARS-CoV-2 E protein and therefore can be used to treat and prevent SARS-CoV-2 virulence.
• SARS-CoV-2 E protein is known to one skilled in the art and has a GenBank Accession no: QIH45055.1.
• the SARS-CoV-2 E protein comprises the amino acid sequence as set forth in SEQ ID NO 1: MY S F V S EETGTLIVN S VLLFL AF V VFLL VTLAILT ALRLC A Y CCNIVN V S L VKPS FY VYSRVKNLNSSRVPDLLV.
• the SARS-CoV-2 E protein comprises an analog of SEQ ID NO: 1, such as an analog having at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 1.
• the invention provides a method of treating or preventing SARS-CoV-2 virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SARS-CoV-2 E protein channel blocker, thereby treating or preventing SARS-CoV-2 virulence in the subject.
• the invention provides a method of treating or preventing Coronavims disease 2019 (COVID-19) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SARS-CoV-2 E protein channel blocker, thereby treating or preventing COVID-19.
• the invention provides a method of preventing SARS-CoV-2 release from a cell.
• the method comprises contacting a cell with a SARS-CoV-2 E protein channel blocker, thereby preventing SARS-CoV-2 release from the cell.
• the invention provides a method of preventing
• the method comprises contacting a cell with a SARS-CoV-2 E protein channel blocker, thereby preventing SARS-CoV-2 cell entry.
• the invention provides a method of preventing SARS-CoV-2 uncoating.
• the method comprises contacting a cell with a SARS-CoV-2 E protein channel blocker, thereby preventing SARS-CoV-2 uncoating.
• a cell is a cell of a subject.
• contacting comprises administering to the subject.
• the subject is a subject infected or suspected as being infected by SARS-CoV-
• a method for treating or preventing SARS-CoV-2 virulence in a subject in need thereof comprising administering to the subject a therapeutically effective amount of 5-Azacytidine, thereby treating or preventing SARS-CoV-2 virulence in said subject.
• a pharmaceutical composition comprising 5-Azacytidine, for use in the treatment and/or prevention of SARS-CoV-2 virulence in a subject in need thereof.
• the SARS-CoV-2 E protein channel blocker is at least one molecule selected from: Memantine, Gliclazide, Mavorixafor, Saroglitazar Magnesium, Mebrofenin, Cyclen, Kasugamycin, Azacytidine, Plerixafor, or any salt thereof.
• the invention provides a SARS-CoV-2 E protein channel blocker for use in treating or preventing SARS-CoV-2 virulence in a subject in need thereof.
• the invention provides a SARS-CoV-2 E protein channel blocker for use in preventing SARS-CoV-2 release from a cell.
• the SARS-CoV-2 E protein channel blocker is within a pharmaceutical composition.
• the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
• the invention provides a pharmaceutical composition comprising Azacytidine, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Azacytidine, an analog or a salt thereof.
• the SARS-CoV- 2 E protein channel blocker is 5-Azacytidine.
• Azacytidine as used herein, includes Azacytidine (CAS: 320-67-2; 4-Aih ⁇ ho-1-b-0- ribofuranosyl-s-triazin-2(lH)-one), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Azacytidine is described, for example in WO2012135405A1.
• the invention provides a pharmaceutical composition comprising Memantine, an analog or a salt thereof, for use in the treatment of a viral infection.
• the viral infection comprises a coronaviruses infection.
• the viral infection comprises an infection by virus having an E protein being an ion channel.
• the viral infection is a coronaviruses infection.
• the SARS-CoV-2 E protein channel blocker is Memantine, an analog or a salt thereof. According to some embodiments, the SARS-CoV-2 E protein channel blocker is memantine hydrochloride.
• Memantine as used herein, includes memantine (CAS: 19982-08-2; l-amino-3,5- dimethyladamantane), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Memantine is described, for example, in U.S. Patents 3,391,142, 5,891,885, 5,919,826, and 6,187,338.
• the invention provides a pharmaceutical composition comprising Gliclazide, an analog or a salt thereof, for treating a viral infection.
• the viral infection is an infection by virus having an E protein being an ion channel.
• the SARS-CoV-2 E protein channel blocker is Gliclazide an analog or a salt thereof.
• Gliclazide as used herein, includes gliclazide (CAS: 21187-98-4; l-(3- azabicyclo(3.3.0)oct-3-yl)-3-(p-tolylsulfonyl)urea) as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the SARS-CoV-2 E protein channel blocker is selected from a group including Ginsenoside.
• the invention provides a pharmaceutical composition comprising Mavorixafor, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is
• Mavorixafor includes Mavorixafor (CAS: 558447-26-0; N-(1H- benzimidazol-2-ylmethyl)-N-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]butane- 1,4-diamine), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• Mavorixafor is described, for example, in U.S. Patent US7332605, and as compound 89 from a series of 169 analogues in W02003055876.
• the invention provides a pharmaceutical composition comprising Saroglitazar, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Saroglitazar, an analog or a salt thereof. According to some embodiments, the SARS-CoV- 2 E protein channel blocker is Saroglitazar Magnesium.
• Saroglitazar includes Saroglitazar (CAS: 495399-09-2; (aS)-a- Ethoxy-4-[2-[2-methyl-5-[4-(methylthio)phenyl]-lH-pyrrol-l-yl]ethoxy]benzenepropanoic Acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Saroglitazar is described, for example, in W02016181409.
• the invention provides a pharmaceutical composition comprising Mebrofenin, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Mebrofenin, an analog or a salt thereof. According to some embodiments, the SARS-CoV- 2 E protein channel blocker is Mebrofenin.
• Mebrofenin includes Mebrofenin (CAS: 78266-06-5; 2-[[2-(3- bromo-2,4,6-trimethylanilino)-2-oxoethyl]-(carboxymethyl)amino]acetic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Mebrofenin is described, for example, in US9,878,984.
• the invention provides a pharmaceutical composition comprising Cyclen, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Cyclen, an analog or a salt thereof. According to some embodiments, the SARS-CoV-2 E protein channel blocker is Cyclen. [078] Cyclen, as used herein, includes Cyclen (CAS: 294-90-6; 1,4,7,10-
• Tetraazacyclododecane as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Cyclen is described, for example in US9421223B2.
• the invention provides a pharmaceutical composition comprising Kasugamycin, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Kasugamycin, an analog or a salt thereof. According to some embodiments, the SARS-CoV- 2 E protein channel blocker is Kasugamycin hydrochloride hydrate (CAS: 19408-46-9).
• Kasugamycin includes Kasugamycin (CAS: 6980-18-3; 2-amino-2- [(2R,3S,5S,6R)-5-amino-2-methyl-6-[(2R,3S,5S,6S)-2,3,4,5,6- pentahydroxycyclohexyl]oxyoxan-3-yl]iminoacetic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Kasugamycin is described, for example in US3358001A.
• the invention provides a pharmaceutical composition comprising Plerixafor, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Plerixafor, an analog or a salt thereof. According to some embodiments, the SARS-CoV-2 E protein channel blocker is Plerixafor octahydrochloride.
• Plerixafor includes Plerixafor (CAS: 155148-31-5; l-[[4-(l,4,8,ll- tetrazacyclotetradec-l-ylmethyl)phenyl]methyl]-l,4,8,l 1-tetrazacyclotetradecane), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• Plerixafor is described, for example in WO2014125499A1.
• the invention is based, at least in part, on the finding using three bacteria-based assays, that SARS-CoV-23a protein inhibitors can serve as effective agents for treating and preventing SARS-CoV-2 virulence.
• SARS-CoV-2 3a protein also known as open reading frame 3a (ORF3a)
• ORF3a open reading frame 3a
• P0DTC3 UniProt Accession no: P0DTC3.
• the SARS-CoV-23a protein comprises the amino acid sequence as set forth in SEQ ID NO 2: MDLFMRIFTIGTVTLKQGEIKDATPSDFVRATATIPIQASLPFGWLIVGVALLAVFQ S AS KIITLKKRW QL ALS KG VHFVCNLLLLFVT V Y S HLLL V A AGLE APFLYL Y ALV Y
• the invention provides a method of treating or preventing SARS-CoV-2 virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SARS-CoV-23a protein inhibitor, thereby treating or preventing SARS-CoV-2 virulence in the subject.
• the invention provides a method of treating or preventing Coronavims disease 2019 (COVID-19) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SARS-CoV-2 3a protein inhibitor, thereby treating or preventing COVID-19.
• the invention provides a method of preventing SARS-CoV-2 release from a cell, the method comprising contacting the cell with a SARS- CoV-2 3a protein inhibitor, thereby preventing SARS-CoV-2 release from the cell.
• the method comprising contacting the cell with a SARS-CoV-2 3a protein inhibitor, thereby preventing SARS-CoV-2 cell entry.
• the method comprising contacting the cell with a SARS-CoV- 2 3a protein inhibitor, thereby preventing SARS-CoV-2 uncoating.
• the subject is a subject infected or suspected as being infected by SARS-CoV-2.
• the SARS-CoV-2 3a protein inhibitor is at least one molecule selected from: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine, Fludarabine, or salts thereof.
• the invention provides a SARS-CoV-2 3a protein inhibitor for use in the treatment or prevention of SARS-CoV-2 virulence, in a subject in need thereof.
• the invention provides a SARS-CoV-2 3a protein inhibitor for use in the prevention of SARS-CoV-2 release from a cell.
• the SARS-CoV-2 3a protein inhibitor is within a pharmaceutical composition.
• the viral infection is an infection by virus having a 3a protein.
• the invention provides a pharmaceutical composition comprising Capreomycin, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is Capreomycin, an analog or a salt thereof.
• the SARS-CoV- 23a protein inhibitor is Capreomycin sulfate.
• Capreomycin includes Capreomycin (CAS: 11003-38-6; IUPAC: (3S)-3,6-diamino-N-[[(2S,5S,8E,l IS, 15S)-15-amino-l l-[(4R)-2-amino-3, 4,5,6- tetrahydropyrimidin-4-yl]-8-[(carbamoylamino)methylidene]-2-(hydroxymethyl)- 3,6,9,12,16-pentaoxo-l,4,7,10,13-pentazacyclohexadec-5-yl]methyl]hexanamide; (3S)-3,6- diamino-N-[[(2S,5S,8E,llS,15S)-15-amino-ll-[(4R)-2-amino-3,4,5,6- tetrahydropyrimidin-4-yl]-8-[(carbamo)
• the invention provides a pharmaceutical composition comprising Pentamidine, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Pentamidine, an analog or a salt thereof.
• the SARS-CoV- 23a protein inhibitor is Pentamidine isethionate.
• Pentamidine as used herein, includes Pentamidine (CAS: 100-33-4; IUPAC: 4,4'- [pentane-l,5-diylbis(oxy)]dibenzenecarboximidamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Spectinomycin, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 E protein channel blocker is Spectinomycin, an analog or a salt thereof.
• the SARS- CoV-23a protein inhibitor is Spectinomycin dihydrochloride.
• Spectinomycin includes Spectinomycin (CAS: 1695-77-8; IUPAC: lR,3S,5R,8R,10S,llS,12S,13R,14S)-8,12,14-trihydroxy-5-methyl-ll,13- bis(methylamino)-2,4,9-trioxatricyclo[8.4.0.03,8]tetradecan-7-one), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Kasugamycin, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• the SARS-CoV- 23a protein inhibitor is Kasugamycin hydrochloride hydrate.
• Kasugamycin includes Kasugamycin (CAS :6980- 18-3; IUPAC: 2- amino-2-[(2R,3S,5S,6R)-5-amino-2-methyl-6-[(2R,3S,5S,6S)-2,3,4,5,6- pentahydroxycyclohexyl]oxyoxan-3-yl]iminoacetic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Plerixafor, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-23a protein inhibitor is Plerixafor, an analog or a salt thereof.
• the SARS-CoV-2 3a protein inhibitor is Plerixafor.
• Plerixafor includes Plerixafor (CAS: 155148-31-5; IUPAC: 1,1’- (l,4-phenylenebismethylene)bis(l,4,8,ll- tetraazacyclotetradecane)), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Flumatinib, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• the SARS-CoV-2 SARS-CoV-2
• 3a protein inhibitor is Flumatinib.
• Flumatinib includes Flumatinib (CAS: 895519-90-1; IUPAC: 4-[(4- methylpiperazin-l-yl)methyl]-N-[6-methyl-5-[(4-pyridin-3-ylpyrimidin-2- yl)amino]pyridin-3-yl]-3-(trifluoromethyl)benzamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Litronesib, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is
• the SARS-CoV-2 Litronesib, an analog or a salt thereof. According to some embodiments, the SARS-CoV-2
• 3a protein inhibitor is Litronesib.
• Litronesib includes Litronesib (CAS: 910634-41-2; IUPAC: N- [(5R)-4-(2,2-dimethylpropanoyl)-5-[[2-(ethylamino)ethylsulfonylamino]methyl]-5-phenyl- l,3,4-thiadiazol-2-yl]-2,2-dimethylpropanamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Darapladib, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• the SARS-CoV-2 3a protein inhibitor is Darapladib.
• Darapladib includes Darapladib (CAS: 356057-34-6; IUPAC: N-(2- Diethylaminoethyl)-2-[2-[(4-fluorophenyl)methylsulfanyl]-4-oxo-6,7-dihydro-5H- cyclopenta[d]pyrimidin- 1 -yl] -N- [ [4- [4-(trifluoromethyl)phenyl]phenyl] methyl] acetamide) , as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Floxuridine, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• the SARS-CoV- 2 3a protein inhibitor is Floxuridine.
• Floxuridine includes Floxuridine (CAS: 50-91-9; IUPAC: 5-Fluoro- l-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-lH-pyrimidine-2,4-dione), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• the invention provides a pharmaceutical composition comprising Fludarabine, an analog or a salt thereof, for treating a viral infection.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• the SARS-CoV- 2 3a protein inhibitor is Fludarabine.
• Fludarabine includes Fludarabine (CAS: 21679-14-1; IUPAC: [(2R,3S,4S,5R)-5-(6-amino-2-fluoro-purin-9-yl)- 3,4-dihydroxy-oxolan-2- yl]methoxyphosphonic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• Pharmaceutical compositions include Fludarabine (CAS: 21679-14-1; IUPAC: [(2R,3S,4S,5R)-5-(6-amino-2-fluoro-purin-9-yl)- 3,4-dihydroxy-oxolan-2- yl]methoxyphosphonic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
• treatment encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
• a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
• prevention of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition.
• prevention relates to a process of prophylaxis in which a subject is exposed to the presently described compositions or formulations prior to the induction or onset of the disease/disorder process.
• suppression is used to describe a condition wherein the disease/disorder process has already begun but obvious symptoms of the condition have yet to be realized.
• the cells of an individual may have the disease/disorder, but no outside signs of the disease/disorder have yet been clinically recognized.
• the term prophylaxis can be applied to encompass both prevention and suppression.
• treatment refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.
• preventing comprises reducing the disease severity, delaying the disease onset, reducing the disease cumulative incidence, or any combination thereof.
• administering refers to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
• the terms “subject” or “individual” or “animal” or “patient” or “mammal,” refers to any subject, particularly a mammalian subject, for whom therapy is desired, for example, a human.
• a therapeutically effective dose of the composition of the invention is administered.
• the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
• the term “a therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen would be determined by the physician according to the patient's condition.
• the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• the route of administration of the pharmaceutical compositions will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art.
• compositions of the invention can be lower than when administered via parenteral injection, by using appropriate compositions it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment.
• the composition of the invention is delivered orally.
• the composition of the invention is an oral composition.
• the composition of the invention further comprises orally acceptable carrier, excipient, or a diluent.
• the active agents of the invention e.g., SARS- CoV-2 E protein channel blocker or protein 3a inhibiter
• the active agents of the invention is for use at a daily dose of 0.01 to 500 mg/kg.
• the SARS-CoV-2 E protein channel blocker is Memantine or a salt thereof and is for use at a daily dose of between about 1 mg/day and about 50 mg/day, about 1 mg/day and 45 mg/day, and 5 mg/day and 3 5mg/day.
• the SARS-CoV-2 E protein channel blocker is Gliclazide or a salt thereof and is for use at a daily dose of between about 1 mg/day and 350 mg/day, 10 mg/day and 350 mg/day, 50 mg/day and 350 mg/day, 1 mg/day and 300 mg/day, 10 mg/day and 300 mg/day, and 50 mg/day and 250 mg/day.
• the SARS-CoV-2 E protein channel blocker is Mavorixafor or a salt thereof and is for use at a daily dose of between about 50 mg/day and about 100 mg/day, about 50 mg/day and 200 mg/day, and 50 mg/day and 400 mg/day.
• the SARS-CoV-2 E protein channel blocker is Saroglitazar or a salt thereof and is for use at a daily dose of between about 0.1 mg/day and about 5 mg/day, about 1 mg/day and 4 mg/day, and 1.5 mg/day and 4.5 mg/day. [0143] According to some embodiments, the SARS-CoV-2 E protein channel blocker is
• Mebrofenin or a salt thereof is for use at a daily dose of between about 1 mg/day and about 50 mg/day, about 1 mg/day and 45 mg/day, and 5 mg/day and 35 mg/day.
• the SARS-CoV-2 E protein channel blocker is Cyclen or a salt thereof and is for use at a daily dose of between about 0.01 mg/day and about 0.5 mg/day, about 0.1 mg/day and 0.5mg/day, and 0.05 mg/day and 0.3 mg/day.
• the SARS-CoV-2 E protein channel blocker, the SARS-CoV-23a protein inhibitor, or both is Kasugamycin or a salt thereof and is for use at a daily dose of between about 1 mg/day and about 500 mg/day, about 5 mg/day and 250mg/day, and 10 mg/day and 350 mg/day.
• the SARS-CoV-2 E protein channel blocker is Azacytidine or a salt thereof and is for use at a daily dose of between about 1 mg/day and about 100 mg/day, about 1 mg/day and 200 mg/day, and 1 mg/day and 300 mg/day.
• the SARS-CoV-2 E protein channel blocker, the SARS-CoV-2 3a protein inhibitor, or both is Plerixafor or a salt thereof and is for use at a daily dose of between about 1 mg/day and about 50 mg/day, about 1 mg/day and 45 mg/day, and 5 mg/day and 35 mg/day.
• the SARS-CoV-2 3a protein inhibitor is Capreomycin or a salt thereof and is for use at a daily dose of between about 50 mg/day and about 1,000 mg/day, about 10 mg/day and 700 mg/day, and 20 mg/day and 800 mg/day.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• Pentamidine or a salt thereof is for use at a daily dose of between about 50 mg/day and about 500 mg/day, about 30 mg/day and 400 mg/day, and 100 mg/day and 300 mg/day.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• Spectinomycin or a salt thereof is for use at a daily dose of between about 500 mg/day and about 5,000 mg/day, about 250 mg/day and 2,500 mg/day, and 100 mg/day and 4,500 mg/day.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• Flumatinib or a salt thereof is for use at a daily dose of between about 50 mg/day and about 1,000 mg/day, about 100 mg/day and 1,500 mg/day, and 50 mg/day and 5,000 mg/day.
• the SARS-CoV-23a protein inhibitor is Litronesib or a salt thereof and is for use at a daily dose of between about 10 mg/day and about 3,000 mg/day, about 50 mg/day and 2,500 mg/day, and 20 mg/day and 2,000 mg/day.
• the SARS-CoV-2 3a protein inhibitor is Litronesib or a salt thereof and is for use at a daily dose of between about 10 mg/day and about 3,000 mg/day, about 50 mg/day and 2,500 mg/day, and 20 mg/day and 2,000 mg/day.
• the SARS-CoV-2 3a protein inhibitor is Litronesib or a salt thereof.
• Darapladib or a salt thereof is for use at a daily dose of between about 10 mg/day and about 1,000 mg/day, about 50 mg/day and 500 mg/day, and 100 mg/day and 800 mg/day.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• Floxuridine or a salt thereof is for use at a daily dose of between about 1 mg/day and about 100 mg/day, about 5 mg/day and 80 mg/day, and 10 mg/day and 100 mg/day.
• the SARS-CoV-2 3a protein inhibitor is N-(2-aminoethyl)-2-a protein inhibitor
• Fludarabine or a salt thereof is for use at a daily dose of between about 1 mg/day and about 100 mg/day, about 2 mg/day and 80 mg/day, and 5 mg/day and 60 mg/day.
• the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant or excipient.
• carrier refers to any component of a pharmaceutical composition that is not the active agent.
• pharmaceutically acceptable carrier refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
• sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethy
• substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations.
• Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present.
• compositions contemplated herein Any non toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein.
• Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index,
• FDA Food and Drug Administration
• CDER Center for Drug Evaluation and Research
• compositions examples include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO.
• additional inactive components as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s:
• compositions may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum.
• Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc.,
• the carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.
• a method of screening effectiveness of an agent in treating or preventing a coronavirus infection comprising providing a cell comprising a membrane permeabilized coronavirus E or 3a protein, contacting the cell with the agent, and determining effect of the agent on growth of the cell, wherein a substantial effect of the agent on cellular growth is indicative of the agent as being effective for treating or preventing a coronavims infection, thereby screening effectiveness of an agent in treating or preventing a coronavims infection.
• the method is a negative assay.
• the cell is charecterized by growth retardation due to the membrane permeabilized E protein or 3 a protein.
• an agent that alleviates growth retardation is indicative as being effective for treating or preventing a coronavims infection.
• the method is a positive assay.
• the cell is a K + -uptake deficient cell grown in low [K + ] media experience growth, due to the channel formed by the E protein or 3a protein.
• an agent that induces growth retardation is indicative as being effective for treating or preventing a coronavims infection.
• the coronavims is SARS-CoV.
• SARS- CoV is any one of SARS-CoV- 1 and SARS-CoV-2.
• the coronavims E protein or 3a protein is a SARS-CoV- 1 E protein or 3a protein, respectively.
• the coronavims E protein or 3a protein is a SARS-CoV-2 E protein, or 3a protein, respectively.
• the method comprises performing both the negative assay and the positive assay.
• the cell is a bacterial cell.
• the cell is devoid of endogenous potassium uptake, besides an exogenously provided (e.g., expressed) membrane permeabilized SARS-CoV E protein or by the 3a protein.
• Non-limiting examples for growing a bacterial cell applicable for the screening methods provided herein include: Astrahan, P. et al., Acta 1808, 394-8 (2011); Santner, P. et al. Biochemistry 57, 5949-5956 (2016), and Taube, R., Alhadeff, R., Assa, D., Kmgliak, M. & Arkin, I. T. PLoS One 9, el05387 (2014).
• the assay is for determining susceptibility of the vims to develop resistance against the agent.
• the term "about” when combined with a value refers to plus and minus 10% of the reference value.
• a length of about 1,000 nanometers (nm) refers to a length of 1000 nm ⁇ 100 nm.
• DH10B Three strains of K12 Escherichia coli were used in the current study: DH10B, LB650, and LR1.
• DH10B cells were purchased from Invitrogen (Carlsbad, CA).
• LB650 bacteria trkG , EtrkH, and AkdpABCS system
• LR1 bacteria contained a chromosomal copy of a pH sensitive green fluorescence protein (GFP) called pHluorin (Miesenbock, G and De Angelis, D A and Rothman, J E. Nature 394, 192— 5 (1998)).
• GFP pH sensitive green fluorescence protein
• the SARS-CoV-2 E protein, 3a protein, and the influenza M2 channel were expressed as fusion proteins to the maltose binding protein using the pMAL-p2X plasmid (New England Biolabs, Ipswich, MA). Genes for the viral proteins have been added with a nucleotide sequence coding for linker of seven amino-acids, six histidines, and a stop codon at the 3 ’ end. EcoRI and Xbal restriction sites were located at the 5 ’ and 3 ’ ends, respectively. The sequences were synthesized by GenScript (Piscataway, NJ). Protein expression was achieved by adding isopropyl b-D- 1 -thiogalactopyranosidc (IPTG) to the growth media, as indicated.
• IPTG isopropyl b-D- 1 -thiogalactopyranosidc
• IPTG was purchased from Biochemika-Fluka (Buchs, Switzerland). All other chemicals were purchased from Sigma- Aldrich laboratories (Rehovot, Israel).
• Lysogeny Broth was used for all bacterial growth (Bertani, G. J Bacteriol 62, 293-300 (1951)) unless noted otherwise. LBK was similar to LB expect that KC1 replaces NaCl at 10 gr/L. All media contained ampicillin at 50 pg /ml.
• Escherichia coli DH10B bacteria bearing or lacking (as a reference) the viral chimera were grown overnight in LB at 37 °C. Thereafter, the growth culture was diluted and the bacteria were grown until their O.D.eoo reached 0.2. Fifty (50) pi of bacterial culture were subsequently dispensed into 96-well flat-bottomed plates (Nunc, Roskilde, Denmark) containing 50 m ⁇ of the different treatments. Unless stated otherwise, IPTG was added to the cells to final concentrations ranging from 0 to 100 mM. D-glucose was added to a concentration of 1%.
• Transformed LR1 cells were cultured overnight in LB media containing 1% glucose and 50 mM ampicillin. Secondary cultures were prepared by diluting the primary culture by 1:500 in LB media and allowing it to grow to an O.D.eoo of 0.6-0.8. Protein synthesis was induced by the addition of 50 mM IPTG for two hours. Cultures without IPTG induction were used as control. Following two hours of induction, the O.D.eoo of all cells were measured, and after pelleting at 3,500 g for 10 min, the bacteria were resuspended in Mcllvaine buffer (200 mM Na 2HPO4, 0.9% NaCl adjusted to pH 7.6 with 0.1 M citric acid,
• a liquid handling system (Tecan) was used to add 70 m ⁇ of 300 mM citric acid with
• Vero-E6 cells were pretreated for 20 h with tested compounds (drugs including their concentration are described in Fig.23) and were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.001 and 0.01 in the presence of the compounds as indicated.
• MOI multiplicity of infection
• the medium with the same DMSO concentration was used as the no-drug control.
• Drug efficacies in control of toxicity were assessed at 24 hours post infection by MTT assay. All infection experiments were performed in a BSL-3 facility.
• the inventors made use of of the pMAL protein fusion and purification system (New England Biolabs). In this construct which has been used successfully with multiple viroporins, SARS-CoV-2 E protein or 3a protein are fused to the carboxyl terminus of the periplasmic maltose binding protein. As a positive control, the inventors compared the activity of the proteins to the M2 channel from the influenza A virus, the archetypical viroporin that can be inhibited by aminoadamantanes
• the first test that was undertaken was to examine if the SARS-CoV-2 E and 3a proteins’ channel activity can lead to membrane permeabilization and thereby negatively impact bacterial growth (negative genetic test). As seen in numerous other viroporins, when expressed at increasing levels, channel activity hampers growth due to its deleterious impact on the proton motive force. Subsequently, channel-blocking drugs may be identified readily due to their ability to alleviate growth retardation.
• the data in Fig. 1 show that expression of the SARS-CoV-2 E or 3a protein causes significant bacterial growth retardation proportional to the protein’s expression levels. This behaviour is similar to that of a known proton channel, the M2 influenza A protein.
• the inventors recognized that growth retardation is not an uncommon consequence of heterologous protein expression in bacteria. In other words, spurious factors could lead to bacterial death in addition to channel activity.
• the inventors thus demonstrate that bacterial death is due to protein channel activity in the following three ways: (i) The inventors identify E and 3a protein channel blockers and show that they can revive bacterial growth; (ii) The inventors developed a complementary bacterial growth assay, where channel activity is essential for growth (positive genetic test); and (iii) The inventors show that protein expression increases H + conductivity in an assay involving a pH sensitive GFP (Santner, P. et al. Biochemistry 57, 5949-5956 (2016)).
• K + conductivity The second experimental test that the inventors have performed examines K + conductivity. Specifically, K + -uptake deficient bacteria (Stumpe, S. & Bakker, E. P. Arch Microbiol 167, 126-36 (1997)) are incapable of growth, unless the media is supplemented by K + . However, when a channel capable of K + transport is heterologously expressed, the bacteria can thrive even under low K + media (Taube, R. et al. PLoS One 9, el05387 (2014); Tomar, P. P. S., Oren, R., Krugliak, M. & Arkin, I. T. Viruses 11 (2019)).
• results shown in Fig. 2 indicate that expression of SARS-CoV-2 E or 3a proteins are able to increase the growth rate of K + -uptake deficient bacteria in otherwise limiting conditions (i.e., low [K + ]).
• the final test to examine channel activity was based on detecting protein-mediated
• the screen was broadend and 3000 moleclues were screened against the SARS-CoV-2 E protein, and 3,000 moleclues were screened against SARS-CoV-2 3a protein.
• the resluts indicate that any one of: Memantine, Gliclazide, Mavorixafor, Saroglitazar Magnesium, Mebrofenin, Cyclen, Kasugamycin, Azacytidine, and Plerixafor can be used as SARS-CoV-2 E protein channel blocker, and accordingly be used as attractive COVID-19 drugs.
• the inventors showed the effect of the compounds tested herein on the viability of cells infected with SARS-CoV-2 vims. Specifically, the results show there was -60% reduction in the cell viability of the Vero-E6 cells when infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01, whereas the cells pretreated with the drugs showed -10-50% reduction of the cell viability after infected with the virus (Fig. 23).
• MOI multiplicity of infection

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