CN115867265A - E-protein channel blockers and ORF3 inhibitors as anti-COVID-19agents - Google Patents

E-protein channel blockers and ORF3 inhibitors as anti-COVID-19agents Download PDF

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CN115867265A
CN115867265A CN202180046512.XA CN202180046512A CN115867265A CN 115867265 A CN115867265 A CN 115867265A CN 202180046512 A CN202180046512 A CN 202180046512A CN 115867265 A CN115867265 A CN 115867265A
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I·阿尔金
P·P·辛格托马
M·克鲁格利亚克
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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Abstract

Pharmaceutical compositions comprising a SARS-CoV-2E protein channel blocker and an ORF3 inhibitor for treating or preventing SARS-CoV-2 virulence in a subject are provided. The invention also provides pharmaceutical compositions comprising a SARS-CoV-2E protein channel blocker or ORF3 inhibitor for use in preventing entry, uncoating and/or release of SARS-CoV-2 cells from cells.

Description

E-protein channel blockers and ORF3 inhibitors as anti-COVID-19agents
Cross Reference to Related Applications
Priority is claimed in U.S. provisional application nos. 63/018,598 entitled "E PROTEIN CHANNEL BLOCKERS AS ANTI-cove-19 AGENTS (E PROTEIN CHANNEL blocks AS ANTI-cove-19 AGENTS)" filed on day 5/1 of 2020 AND U.S. provisional application No. 63/117,619 entitled "E PROTEIN CHANNEL BLOCKERS AND ORF3 INHIBITORS (E PROTEIN CHANNEL blocks AND ORF3 INHIBITORS AS ANTI-cove-19 AGENTS" (filed on day 11/24 of 2020), the contents of both of which are incorporated herein by reference in their entirety.
Technical Field
The present invention is in the field of antiviral therapy.
Background
Coronaviruses are positive-sense, single-stranded RNA viruses that are commonly associated with minor respiratory infections in humans. However, three members of the same family are notorious for unusual virulence: SARS-CoV-1 is the causative agent of the SARS epidemic causing 774 deaths in 8098 cases in winter 2002/3; MERS-CoV is responsible for the prevalence of MERS-CoV that causes 862 deaths in 2506 infected persons from 2012; finally, SARS-CoV-2 is responsible for a persistent COVID-2019 pandemic, resulting in the death of 131 million people in 54, 068, 330 cases (11 months and 15 weeks as of 2020).
Genomic analysis has shown that SARS-CoV-1 and SARS-CoV-2 are very similar to each other (about 80%) but differ from most other members of the family Coronaviridae that infect humans. Two viruses have been classified in subgroup B of the genus β coronavirus of the orthocoronaviridae subfamily of the family coronaviridae.
Of the structural proteins of all coronaviruses, the E protein is the least understood in terms of mechanism of action and structure. Functionally, the E protein is involved in viral assembly, release and pathogenesis. However, and importantly, the coronavirus E protein is important for viral pathogenesis, and attenuated viruses lacking this protein have even been proposed as vaccine candidates.
The SARS-CoV-2 3a protein, also known as open reading frame 3a (ORF 3 a), is associated with a homotetrameric potassium-sensitive ion channel (viral porin) and can modulate viral release. In addition, it is associated with pathogenesis, including upregulation of expression of fibrinogen subunits FGA, FGB, and FGG in host lung epithelial cells, inducing apoptosis in cell culture.
Disclosure of Invention
According to a first aspect, there is provided a method of treating or preventing SARS-CoV-2 toxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of: a SARS-CoV-2E protein channel blocker and a SARS-CoV-2 3a protein inhibitor, thereby treating or preventing SARS-CoV-2 virulence in a subject.
According to another aspect, there is provided a pharmaceutical composition for treating or preventing SARS-CoV-2 virulence in a subject in need thereof, comprising a SARS-CoV-2E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor.
In some embodiments, preventing comprises preventing any one of: entry of SARS-CoV-2 into the subject cell, uncoating of SARS-CoV-2 in the subject cell, release of SARS-CoV-2 from the subject cell, and any combination thereof.
In some embodiments, the subject is infected with SARS-CoV-2 or suspected of being infected with SARS-CoV-2.
In some embodiments, the SARS-CoV-2E protein channel blocker is at least one molecule selected from the group consisting of: 5-Azacytidine (5-Azacytidine), memantine (Memantine), gliclazide (Gliclazide), mavorexafu (Mavorixafor), saroglitazar Magnesium (Saroglitazar Magnesium), mebrofenacin (mebrofenan), cyclen (Cyclen), kasugamycin (Kasugamycin), plexafof (Plerixafor) and any salts thereof.
In some embodiments, the SARS-CoV-2E protein channel blocker is used at a daily dose of 0.01 to 500 mg/kg.
In some embodiments, the SARS-CoV-2E protein channel blocker is ginsenoside.
In some embodiments, the SARS-CoV-2E protein channel blocker is memantine.
In some embodiments, the SARS-CoV-2 3a protein inhibitor is at least one molecule selected from the group consisting of: capreomycin (Capreomycin), pentamidine (Pentamidine), spectinomycin (Spectinomycin), kasugamycin, plerixafor (Plerixafor), flumatinib (Flumatinib), littrowel (litronib), daprolidine (Darapladib), floxuridine (Floxuridine) and Fludarabine (Fludarabine).
In some embodiments, the SARS-CoV-2 3a protein inhibitor is capreomycin.
In some embodiments, preventing comprises preventing any one of: entry of SARS-CoV-2 into a subject cell, uncoating of SARS-CoV-2, release of SARS-CoV-2 from a subject cell, and any combination thereof.
Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred forms of the invention, are given by way of illustration only, since various changes and modifications within the spirit of the invention will become apparent to those skilled in the art from this detailed description.
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FIGS. 1A-1B include graphs showing membrane permeabilization assays. Bacterial growth curves (n = 2) as a function of SARS-CoV-2E protein expression (1A, right) or as a function of SARS-CoV-2 3a protein expression (1B). Bacteria expressing maltose binding protein without binding (conjugated) viral ion channels are shown on the left as negative controls. Bacteria expressing influenza M2 viral porin are shown in the middle as a positive control. Induction at different IPTG concentrations (as indicated) occurred when the bacterial density reached 0.2 o.d.600nm. The growth O.D.600nm value was collected every 15 min. FIG. 1B shows bacterial growth curves as a function of SARS-CoV-3a protein expression. Negative control (no channel; NC); no Drug (ND).
FIGS. 2A-2B include displays K + Graph of conductivity measurements. Viral protein SARS-CoV-2E protein pair K + Uptake-deficient bacteria (K) + -effect of uptake specific bacteria) (left panel, 2A). Different protein expression levels were achieved by varying IPTG inducer concentrations, as indicated. Bacterial growth Rate as [ K ] + ]Is plotted on the right graph (2A). (2B) Describes the use of varying concentrations of IPTG inducer, SARS CoV-2 3a protein vs K + The effect of uptake of defective bacterial growth.
FIGS. 3A-3B include graphs showing fluorescence-based H + Conductivity measurementA graph of (a). The fluorescence of bacteria carrying pHluorin, a pH-sensitive GFP22, was detected as a function of SARS CoV-2E protein expression (3A) or SARS CoV-2 3a protein expression (3B). Protein levels were controlled by levels of induction agent (IPTG) as shown. The results are the mean of two independent experiments and the standard deviation is described as an error bar.
FIGS. 4A-4B include graphs showing the results of compound screening using positive and negative gene assays. As shown, the effect of different drugs and E protein expression on bacterial growth rate. (4A) Negative gene test (Negative genetic test) in which the SARS-CoV-2E protein is present at an elevated level (40mM 2. RTM. IPTG)]) Expressed and thus harmful to bacteria. In this case, the inhibitory drug promotes bacterial growth. (4B) Positive gene test (Positive genetic test), in which K is + Uptake of SARS-CoV-2E protein in deficient bacterium at a low level (10mM [2 ] IPTG)]). In this case, the inhibitory drug reduces bacterial growth. In both figures, the effect on growth compared to growth without any drug is listed.
Figures 5A-5C include graphs showing results of the marvorisavum screen using the following assays: negative assay (negative assay): viral channels harmful to bacteria, in which the blocker increases growth (5A), positive assay: viral channels essential for bacteria, wherein blockers reduce growth (5B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (5C).
Figures 6A-6C include graphs showing results of salogura magnesium screening using the following assay: and (3) negative determination: viral channels harmful to bacteria, in which blockers increase growth (6A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (6B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (6C).
Figures 7A-7C include graphs showing the results of the mebromelin screening using the following assays: and (4) negative determination: viral channels harmful to bacteria, in which blockers increase growth (7A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (7B), and fluorescence measurements: the viral channel changes fluorescence, with the blocker reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (7C).
Figures 8A-8C include graphs showing the results of the cyclenine screen using the following assays: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (8A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (8B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (8C).
FIGS. 9A-9C include graphs showing the results of a kasugamycin screen using the following assays: and (4) negative determination: viral channels harmful to bacteria, in which blockers increase growth (9A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (9B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (9C).
FIGS. 10A-10C include graphs showing the results of 5-azacytidine screening using the following assays: and (4) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (10A), positive assay: a bacterial-essential viral channel in which the blocker reduces growth (10B), and a fluorescence assay: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (10C).
FIGS. 11A-11C include graphs showing the results of a Plexaford (octahydrochloride) screen using the following assay: and (4) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (11A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (11B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (11C).
Figures 12A-12C include graphs showing the results of plerixafor screening using the following assays: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (12A), positive assay: viral channels essential for bacteria, wherein the blocker reduces growth (12B), and a fluorescence assay: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (12C).
Figures 13A-13C include graphs showing the results of a capreomycin (sulfate) screen using the following assay: and (4) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (13A), positive assay: viral channels essential for bacteria, in which blockers reduce growth (13B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing SARS-CoV-2E protein (13C).
FIGS. 14A-14C include graphs showing pentamidine (isethionate) screening results using the following assay: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (14A), positive assay: viral channels essential for bacteria, wherein the blocker reduces growth (14B), and a fluorescence assay: viral channels alter fluorescence, with blockers reducing the fluorescence change of bacteria expressing SARS-CoV-2 3a protein (14C).
FIGS. 15A-15C include graphs showing the results of spectinomycin (dihydrochloride) screening using the following assay: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (15A), positive assay: viral channels essential for bacteria, in which blockers reduce growth (15B), and fluorescence measurements: the viral channel changes fluorescence, with blockers reducing the change in fluorescence of bacteria expressing the SARS-CoV-2 3a protein (15C).
Figures 16A-16C include graphs showing the results of a kasugamycin (hydrochloride hydrate) screen using the following assay: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (16A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (16B), and fluorescence measurements: viral channels alter fluorescence, with blockers reducing the fluorescence change of bacteria expressing SARS-CoV-2 3a protein (16C).
Figures 17A-17C include graphs showing the results of plerixafor screening using the following assays: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (17A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (17B), and fluorescence measurements: the viral channel changes fluorescence, with the blocker reducing the change in fluorescence of bacteria expressing the SARS-CoV-2 3a protein (17C).
Figures 18A-18C include graphs showing the results of a flumatinib screen using the following assays: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (18A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (18B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing the SARS-CoV-2 3a protein (18C).
Fig. 19A-19C include graphs showing results of littrow-nico screening using the following assays: and (3) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (19A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (19B), and fluorescence measurements: the viral channel changes fluorescence, with the blocker reducing the change in fluorescence of bacteria expressing the SARS-CoV-2 3a protein (19C).
Figures 20A-20C include graphs showing the results of the daprolimus screen using the following assays: and (4) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (20A), positive assay: viral channels essential to bacteria, in which blockers reduce growth (20B), and fluorescence measurements: the viral channel changes fluorescence, with the blocker reducing the change in fluorescence of bacteria expressing the SARS-CoV-2 3a protein (20C).
FIGS. 21A-21C include graphs showing the results of floxuridine screening using the following assays: and (4) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (21A), positive assay: viral channels essential for bacteria, wherein the blocker reduces growth (21B), and a fluorescence assay: the viral channel changes fluorescence, with blockers reducing the change in fluorescence of bacteria expressing SARS-CoV-2 3a protein (21C).
FIGS. 22A-22C include graphs showing the results of a fludarabine screen using the following assays: and (4) negative determination: viral channels harmful to bacteria, in which the blocker increases growth (22A), positive assay: viral channels essential to bacteria, wherein the blocker reduces growth (22B), and fluorescence measurements: the viral channel changes fluorescence, with the blocking agent reducing the change in fluorescence of bacteria expressing the SARS-CoV-2 3a protein (22C).
FIG. 23 includes a vertical bar graph showing the effect of various test drugs on the viability of Vero-E6 cells infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01.
Detailed Description
In some embodiments, the invention provides compositions comprising a SARS-CoV-2E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor for use in treating or preventing SARS-CoV-2 virulence in a subject. In some embodiments, the invention provides compositions comprising a SARS-CoV-2E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor for use in preventing entry, uncoating, and/or release of a SARS-CoV-2 cell into, from, or from a cell.
SARS-CoV-2E protein channel blocker
The present invention is based, at least in part, on the discovery that SARS-CoV-2E protein is an ion channel using three bacteria-based assays. The present invention is also based, at least in part, on the following findings: gliclazide, memantine, mavorisavu, saroglitazar magnesium, mefenofibrate, cyclen, kasugamycin, azacytidine, and plexafofu inhibit SARS-CoV-2E protein, and thus can be used to treat and prevent SARS-CoV-2 virulence.
The SARS-CoV-2E protein is known to those skilled in the art as having GenBank accession number QIH45055.1. According to some embodiments, the SARS-CoV-2E protein comprises the amino acid sequence set forth in SEQ ID NO.1 MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNNVSLVKPFYVYSR VKNNSSRVPDLLV. According to some embodiments, the SARS-CoV-2E 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.
According to some embodiments, the present 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-2E protein channel blocker, thereby treating or preventing SARS-CoV-2 virulence in the subject.
In some embodiments, the present invention provides a method of treating or preventing 2019 coronavirus disease (covd-19) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SARS-CoV-2E protein channel blocker, thereby treating or preventing covd-19.
According to some embodiments, the present invention provides methods for preventing the release of SARS-CoV-2 from a cell. In some embodiments, the method comprises contacting the cell with a SARS-CoV-2E protein channel blocker, thereby preventing release of SARS-CoV-2 from the cell.
According to some embodiments, the present invention provides methods for preventing SARS-CoV-2 cell entry. In some embodiments, the method comprises contacting the cell with a SARS-CoV-2E protein channel blocker, thereby preventing entry of SARS-CoV-2 cell.
According to some embodiments, the present invention provides methods for preventing SARS-CoV-2 uncoating. In some embodiments, the method comprises contacting the cell with a SARS-CoV-2E protein channel blocker, thereby preventing SARS-CoV-2 uncoating.
According to some embodiments, the cell is a cell of a subject. According to some embodiments, the contacting comprises administering to the subject. According to some embodiments, the subject is a subject infected with SARS-CoV-2 or suspected of being infected with SARS-CoV-2.
According to some embodiments, there is provided 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 the subject.
According to some embodiments, there is provided 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.
According to some embodiments, the SARS-CoV-2E protein channel blocker is at least one molecule selected from the group consisting of: memantine, gliclazide, mavorisarff, sarogexel magnesium, mefenoxiline, cycleanine, kasugamycin, azacytidine, plexafop, or any salt thereof.
According to some embodiments, the present invention provides a SARS-CoV-2E protein channel blocker for use in treating or preventing SARS-CoV-2 virulence in a subject in need thereof.
According to some embodiments, the present invention provides SARS-CoV-2E protein channel blockers for preventing SARS-CoV-2 release from a cell.
According to some embodiments, the SARS-CoV-2E protein channel blocker is in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
According to some embodiments, the present invention provides a pharmaceutical composition comprising azacytidine, an analog or salt thereof, for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is azacytidine, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is 5-azacytidine.
As used herein, azacytidine includes azacytidine (CAS: 320-67-2, 4-amino-1- β -D-ribofuranosyl-s-triazin-2 (1H) -one, and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
According to some embodiments, the present invention provides a pharmaceutical composition comprising memantine, an analog, or a salt thereof, for use in the treatment of a viral infection. In some embodiments, the viral infection comprises a coronavirus infection. In some embodiments, the viral infection comprises a viral infection having an E protein as an ion channel. In some embodiments, the viral infection is a coronavirus infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is memantine, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is memantine hydrochloride.
As used herein, memantine includes memantine (CAS: 19982-08-2, 1-amino-3, 5-dimethyladamantane) and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof. Memantine is described, for example, in U.S. Pat. nos. 3,391,142, 5,891,885, 5,919,826, and 6,187,338.
According to some embodiments, the present invention provides a pharmaceutical composition comprising gliclazide, an analog or salt thereof, for use in the treatment of a viral infection. In some embodiments, the viral infection is a viral infection having an E protein as an ion channel.
According to some embodiments, the SARS-CoV-2E protein channel blocker is gliclazide or an analog or salt thereof.
As used herein, gliclazide includes gliclazide (CAS: 21187-98-4, 1- (3-azabicyclo (3.3.0) oct-3-yl) -3- (p-toluenesulfonyl) urea), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
According to some embodiments, the SARS-CoV-2E protein channel blocker is selected from ginsenoside.
According to some embodiments, the present invention provides a pharmaceutical composition comprising marvorexavir, an analog or salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is marvorexafu, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is marvorexafu.
As used herein, mavorisamide includes Mavorisamide (CAS: 558447-26-0, N- (1H-benzimidazol-2-ylmethyl) -N- [ (8S) -5,6,7, 8-tetrahydroquinolin-8-yl ] butane-1, 4-diamine), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Muvorisarft is described, for example, in US7332605, and in WO2003055876 as compound 89 from a series of 169 analogues.
According to some embodiments, the present invention provides a pharmaceutical composition comprising saroglitazar, an analog or salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is saroglitazar, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is saroglitamagnesium.
As used herein, saroglitaza includes Saroglitaza (CAS: 495399-09-2; (α S) - α -ethoxy-4- [2- [ 2-methyl-5- [4- (methylthio) phenyl ] -1H-pyrrol-1-yl ] ethoxy ] phenylpropionic acid), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Salogue is described in, for example, WO 2016181409.
According to some embodiments, the present invention provides a pharmaceutical composition comprising mefenoxaprop, an analog or salt thereof for treating a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is mefenolin, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is mefenolin.
As used herein, mebendazole includes mebendazole (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. Mefenoxaprop is described, for example, in US9,878,984.
According to some embodiments, the present invention provides a pharmaceutical composition comprising cyclen, an analog or salt thereof, for treating a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is cyclen, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is cyclen.
As used herein, cyclen includes cyclen (CAS: 294-90-6, 1,4,7, 10-tetraazacyclododecane), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Cyclen is described, for example, in US9421223B 2.
According to some embodiments, the present invention provides a pharmaceutical composition comprising kasugamycin, an analog or salt thereof, for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is kasugamycin, an analog, or a salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is kasugamycin hydrochloride hydrate (CAS: 19408-46-9).
Kasugamycin, as used herein, 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 ] oxypyran-3-yl ] iminoacetic acid) and pharmaceutically acceptable salts, solvates, hydrates or mixtures thereof. Kasugamycin is described, for example, in US 3358001A.
According to some embodiments, the present invention provides a pharmaceutical composition comprising plexafor, an analog or salt thereof, for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is plexafor, an analog or salt thereof. According to some embodiments, the SARS-CoV-2E protein channel blocker is plexafoe octa-hydrochloride.
As used herein, plerixafor includes plerixafor (CAS: 155148-31-5, (. Sup.4- (1, 4,8, 11-tetraazacyclotetradecan-1-ylmethyl) phenyl ] methyl ] -1,4,8, 11-tetraazacyclotetradecan), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Plexafor is described in e.g. WO2014125499 A1.
SARS-CoV-2 3a protein inhibitor
The present invention is based, at least in part, on the discovery that inhibitors of SARS-CoV-2 3a protein can be used as effective agents to treat and prevent SARS-CoV-2 virulence using three bacteria-based assays.
The SARS-CoV-2 3a protein, also known as open reading frame 3a (ORF 3 a), is known to those skilled in the art and has the UniProt accession number: p0DTC3.
The terms "3a protein" and "ORF3a" are used interchangeably herein.
<xnotran> , SARS-CoV-2 3a SEQ ID NO.2: MDLFMRIFTIGTVTLKQGEIKDATPSDFVRATATIPIQASLPFGWLIVGVALLAVFQSASKIITLKKRWQLALSKGVHFVCNLLLLFVTVYSHLLLVAAGLEAPFLYLYALVYFLQSINFVRIIMRLWLCWKCRSKNPLLYDANYFLCWHTNCYDYCIPYNSVTSSIVITSGDGTTSPISEHDYQIGGYTEKWESGVKDCVVLHSYFTSDYYQLYSTQLSTDTGVEHVTFFIYNKIVDEPEEHVQIHTIDGSSGVVNPVMEPIYDEPTTTTSVPL . </xnotran> According to some embodiments, the SARS-CoV-2 3a protein comprises an analog of SEQ ID NO:2, such as an analog having at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 2.
According to some embodiments, the present 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 3a protein inhibitor, thereby treating or preventing SARS-CoV-2 virulence in the subject.
In some embodiments, the present invention provides a method of treating or preventing 2019 coronavirus disease (covd-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 covd-19.
According to some embodiments, the invention provides a method of preventing the release of SARS-CoV-2 from a cell, the method comprising contacting the cell with an inhibitor of SARS-CoV-2 3a protein, thereby preventing the release of SARS-CoV-2 from the cell.
According to some embodiments, the method comprises contacting the cell with a SARS-CoV-2 3a protein inhibitor, thereby preventing entry of SARS-CoV-2 cells.
In some embodiments, the method comprises contacting the cell with an inhibitor of SARS-CoV-2 3a protein, thereby preventing SARS-CoV-2 uncoating.
According to some embodiments, the subject is a subject infected with SARS-CoV-2 or suspected of being infected with SARS-CoV-2.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is at least one molecule selected from the group consisting of: capreomycin, pentamidine, spectinomycin, kasugamycin, plerixafor, flumatinib, littrow, dapprandil, floxuridine, fludarabine, or salts thereof.
According to some embodiments, the present invention provides a SARS-CoV-2 3a protein inhibitor for use in treating or preventing SARS-CoV-2 virulence in a subject in need thereof.
According to some embodiments, the present invention provides SARS-CoV-2 3a protein inhibitors for preventing the release of SARS-CoV-2 from a cell.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is in a pharmaceutical composition.
In some embodiments, the viral infection is a viral infection having a 3a protein.
According to some embodiments, the present invention provides a pharmaceutical composition comprising capreomycin, an analogue or salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is capreomycin, an analog, or a salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is capreomycin sulfate.
As used herein, capreomycin includes capreomycin (CAS: 11003-38-6 IUPAC: (3S) -3, 6-diamino-N- [ [ (2S, 5S,8E,11S, 15S) -15-amino-11- [ (4R) -2-amino-3, 4,5, 6-tetrahydropyrimidin-4-yl ] -8- [ (carbamoylamino) methylene ] -2- (hydroxymethyl) -3,6,9,12, 16-pentaoxo-1, 4,7,10, 13-pentaazacyclohex-5-yl ] methyl ] hexanamide, (3S) -3, 6-diamino-N- [ (2S, 5S,8E, 1S, 15S) -15-amino-11- [ (4R) -2-amino-3, 4,5, 6-tetrahydropyrimidin-4-yl ] -8- [ (carbamoylamino) methylene ] -2-methyl-3, 6,9,12, 16-pentaoxo-1, 4,7, 10-pentaazacyclohex-5-yl ] methyl ] hexanamide, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates thereof, or mixtures thereof
According to some embodiments, the present invention provides a pharmaceutical composition comprising pentamidine, an analog or salt thereof, for use in the treatment of viral infections.
According to some embodiments, the SARS-CoV-2E protein channel blocker is pentamidine, an analog or salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is pentamidine isethionate.
Pentamidine, as used herein, includes pentamidine (CAS: 100-33-4.
According to some embodiments, the present invention provides a pharmaceutical composition comprising spectinomycin, an analogue or salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2E protein channel blocker is spectinomycin, an analog or a salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is spectinomycin dihydrochloride.
As used herein, spectinomycin includes spectinomycin (CAS: 1695-77-8.
According to some embodiments, the present invention provides a pharmaceutical composition comprising kasugamycin, an analog or salt thereof, for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is kasugamycin, an analog, or a salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is kasugamycin hydrochloride hydrate.
Kasugamycin, as used herein, includes kasugamycin (CAS: 69880-18-3.
According to some embodiments, the present invention provides a pharmaceutical composition comprising plexafor, an analog or salt thereof, for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is plerixafor, an analog or salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is plexafor.
As used herein, plerixafor includes plerixafor (CAS: 155148-31-5, IUPAC 1,1' - (1, 4-phenylenebismethylene) bis (1, 4,8, 11-tetraazacyclotetradecane)) and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
According to some embodiments, the present invention provides a pharmaceutical composition comprising flumatinib, an analogue or salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is flumatinib, an analog or salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is flumatinib.
As used herein, flumatinib includes flumatinib (CAS: 895519-90-1, iupac.
According to some embodiments, the present invention provides a pharmaceutical composition comprising littrow, an analog or a salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is ritonavir, an analog or a salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is ritonavir.
As used herein, littrow-hexicylate includes littrow-hexicylate (CAS: 910634-41-2 iupac.
According to some embodiments, the present invention provides a pharmaceutical composition comprising a dapagliflozin, an analog, or a salt thereof, for use in treating a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is dapiprodione, an analog, or a salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is dapipratropine.
As used herein, daprolidine includes daprolidine (CAS: 356057-34-6 iupac.
According to some embodiments, the present invention provides a pharmaceutical composition comprising floxuridine, an analog or salt thereof, for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is floxuridine, an analog or salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is floxuridine.
As used herein, floxuridine includes floxuridine (CAS: 50-91-9 iupac.
According to some embodiments, the present invention provides a pharmaceutical composition comprising fludarabine, an analog or salt thereof for use in the treatment of a viral infection.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is fludarabine, an analog or salt thereof. According to some embodiments, the SARS-CoV-2 3a protein inhibitor is fludarabine.
As used herein, 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), and pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
Pharmaceutical composition
As used herein, the terms "treating" or "treating" a disease, disorder or condition encompasses alleviating, reducing the severity of or inhibiting the progression of at least one symptom thereof. Treatment does not necessarily mean that the disease, disorder, or condition has been completely cured. To be an effective treatment, a composition useful herein need only reduce the severity of the disease, disorder or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.
As used herein, the term "preventing" a disease, disorder or condition encompasses delaying, preventing, suppressing or inhibiting the onset of the disease, disorder or condition. As used in accordance with the presently described subject matter, the term "preventing" relates to a prophylactic (prophyaline) process wherein a subject is exposed to a composition or formulation described herein prior to the induction or onset of a disease/condition process. The term "suppression" is used to describe a condition in which the disease/condition process has begun but significant symptoms of the condition have not yet been achieved. Thus, the cells of an individual may have a disease/condition, but no external signs of the disease/condition have been clinically identified. In either case, the term "control" can be used to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of an active agent to combat an already existing condition, the clinical manifestations of which have been achieved in a patient.
In some embodiments, preventing comprises reducing the severity of the disease, delaying the onset of the disease, reducing the cumulative incidence of the disease, or any combination thereof.
As used herein, the terms "administering", "administration" and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice.
As used herein, the term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, such as a human, for which treatment is desired.
In some embodiments, a therapeutically effective dose of a 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 "therapeutically effective amount" means an amount effective to achieve the desired therapeutic or prophylactic effect at the dosages and for the periods of time necessary. The exact dosage form and regimen will be determined by a physician in view of the condition of the patient.
The dosage administered will depend upon the age, health and weight of the subject, the type of concurrent treatment (if any), the frequency of treatment and the nature of the effect desired. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injection, such as intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal and any other injection means known in the art. Although the bioavailability of peptides administered by other routes may be lower than when administered by parenteral injection, by using suitable compositions it is envisaged that it will be possible to administer the compositions of the invention by transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular therapeutic means. Furthermore, it may be desirable to introduce the pharmaceutical compositions of the present invention by any suitable route, including intraventricular and intrathecal injections; intraventricular injection may be facilitated by, for example, an intraventricular catheter attached to a reservoir.
In some embodiments, the compositions of the present invention are delivered orally. In some embodiments, the compositions of the present invention are oral compositions. In some embodiments, the compositions of the present invention further comprise an orally acceptable carrier, excipient, or diluent.
According to some embodiments, the active agent of the invention (e.g., a SARS-CoV-2E protein channel blocker or a protein 3a inhibitor) is used at a daily dose of 0.01 to 500 mg/kg.
According to some embodiments, the SARS-CoV-2E protein channel blocker is memantine or a salt thereof and is used in a daily dose of about 1 mg/day to about 50 mg/day, about 1 mg/day to 45 mg/day, and 5 mg/day to 3 5mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker is gliclazide or a salt thereof, and is used at a daily dose of about 1 mg/day to 350 mg/day, 10 mg/day to 350 mg/day, 50 mg/day to 350 mg/day, 1 mg/day to 300 mg/day, 10 mg/day to 300 mg/day, 50 mg/day to 250 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker is mavorisavu or a salt thereof, and is used at a daily dose of about 50 mg/day to about 100 mg/day, about 50 mg/day to 200 mg/day, and 50 mg/day to 400 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker is saroglitazar or a salt thereof and is used at a daily dose of about 0.1 mg/day to about 5 mg/day, about 1 mg/day to 4 mg/day, and 1.5 mg/day to 4.5 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker is mefenolin or a salt thereof and is used at a daily dose of about 1 mg/day to about 50 mg/day, about 1 mg/day to 45 mg/day, and 5 mg/day to 35 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker is cyclen or a salt thereof, and is used at a daily dose of about 0.01 mg/day to about 0.5 mg/day, about 0.1 mg/day to 0.5 mg/day, and 0.05 mg/day to 0.3 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker, SARS-CoV-2 3a protein inhibitor, or both are kasugamycin or a salt thereof, and are used in a daily dose of about 1 mg/day to about 500 mg/day, about 5 mg/day to 250 mg/day, and 10 mg/day to 350 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker is azacytidine, or a salt thereof, and is used in a daily dose of about 1 mg/day to about 100 mg/day, about 1 mg/day to 200 mg/day, and 1 mg/day to 300 mg/day.
According to some embodiments, the SARS-CoV-2E protein channel blocker, SARS-CoV-2 3a protein inhibitor, or both are plerixafor or a salt thereof, and are used in daily doses of about 1 mg/day to about 50 mg/day, about 1 mg/day to 45 mg/day, and 5 mg/day to 35 mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is capreomycin or a salt thereof, and is used in a daily dose of about 50 mg/day to about 1,000mg/day, about 10 mg/day to 700 mg/day, and 20 mg/day to 800 mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is pentamidine or a salt thereof, and is used in a daily dose of about 50 mg/day to about 500 mg/day, about 30 mg/day to 400 mg/day, and 100 mg/day to 300 mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is spectinomycin or a salt thereof, and is used at a daily dose of about 500 mg/day to about 5,000mg/day, about 250 mg/day to 2,500mg/day, and 100 mg/day to 4,500mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is flumatinib, or a salt thereof, and is used in a daily dose of about 50 mg/day to about 1,000mg/day, about 100 mg/day to 1,500mg/day, and 50 mg/day to 5,000mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is littrow or a salt thereof, and is used in a daily dose of about 10 mg/day to about 3,000mg/day, about 50 mg/day to 2,500mg/day, and 20 mg/day to 2,000mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is dapiprodione or a salt thereof, and is used in a daily dose of about 10 mg/day to about 1,000mg/day, about 50 mg/day to 500 mg/day, and 100 mg/day to 800 mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is floxuridine or a salt thereof, and is used in a daily dose of about 1 mg/day to about 100 mg/day, about 5 mg/day to 80 mg/day, and 10 mg/day to 100 mg/day.
According to some embodiments, the SARS-CoV-2 3a protein inhibitor is fludarabine or a salt thereof, and is used in a daily dose of about 1 mg/day to about 100 mg/day, about 2 mg/day to 80 mg/day, and 5 mg/day to 60 mg/day.
In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, or excipient.
As used herein, the term "carrier," "adjuvant," or "excipient" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic inert solid, a semi-solid liquid filler, a diluent, an encapsulating material, any type of formulation aid, or a simple sterile aqueous medium, such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are 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, corn oil and soybean oil; glycols, such as propylene glycol, polyols, such as glycerol, 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; ethanol and phosphate buffered solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that may be used as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solutions, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible materials used in 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. Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein. In this regard, suitable pharmaceutically acceptable carriers, excipients and diluents are well known to those skilled in The art, such as those described in The Merck Index, thirteeth Edition, budavari et al, eds., merck & co, inc., rahway, n.j. (2001); CTFA (Cosmetic, toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, tenth Edition (2004); and "active Ingredient Guide," U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the entire contents of which are incorporated herein by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents that may be used in the composition of the present invention include distilled water, physiological saline, ringer's solution, dextrose solution, hank's solution and DMSO. These additional inactive ingredients, as well as effective formulations and application procedures, are well known in The art and are described in standard texts, such as Goodman and Gillman's: the Pharmacological Bases of Therapeutics,8th Ed., gilman et al Eds. Pergamon Press (1990); remington's Pharmaceutical Sciences,18th Ed., mack Publishing co., easton, pa. (1990); and Remington The Science and Practice of Pharmacy,21st Ed., lippincott Williams & Wilkins, philadelphia, pa. (2005), each of which is incorporated herein by reference in its entirety. The presently described compositions may also be contained in artificially created structures such as liposomes, ISCOMS, slow release particles, and other vehicles (vehicles) that increase the half-life of the peptide or polypeptide 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 typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is generally determined by factors such as liposome size and stability in blood. There are various methods available for preparing liposomes, see for example, coligan, J.E.et al, current Protocols in Protein Science,1999, john Wiley and sons, inc., new York, and also U.S. Pat. No. 4,235,871,4,501,728,4,837,028, and 5,019,369.
The carrier may comprise from about 0.1% to about 99.99999% of the total weight of the pharmaceutical composition described herein.
Screening assays
According to some embodiments, there is provided a method of screening an agent for effectiveness in treating or preventing a coronavirus infection, the method comprising providing a cell comprising a membrane permeabilized (membrane permeabilized) coronavirus E or 3a protein, contacting the cell with the agent, and determining the effect of the agent on cell growth, wherein a significant effect of the agent on cell growth indicates that the agent is effective in treating or preventing a coronavirus infection, thereby screening an agent for effectiveness in treating or preventing a coronavirus infection.
In some embodiments, the method is a negative assay. In some embodiments, the cell is characterized by growth retardation due to membrane permeabilized E protein or 3a protein. In some embodiments, the agent that alleviates growth retardation is indicated to be effective in treating or preventing a coronavirus infection.
In some embodiments, the method is a positive assay. In some embodiments, the cell is at low [ K ] due to a channel formed by the E protein or the 3a protein + ]K for the growth of the culture medium + Uptake of defective cells. In some embodiments, the agent that induces growth retardation is indicated to be effective for treating or preventing a coronavirus infection.
In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the SARS-CoV is any one of SARS-CoV-1 and SARS-CoV-2. In some embodiments, the coronavirus E protein or 3a protein is a SARS-CoV-1E protein or 3a protein, respectively. In some embodiments, the coronavirus E protein or 3a protein is a SARS-CoV-2E protein or 3a protein, respectively.
In some embodiments, the method comprises performing a negative assay and a positive assay.
In some embodiments, the cell is a bacterial cell. In some embodiments, the cell lacks endogenous potassium uptake in addition to the exogenously supplied (e.g., expressed) membrane permeabilized SARS-CoV E protein or 3a protein.
Non-limiting examples of bacterial cell growth suitable for use in the screening methods provided herein include: astrahan, p.et al, acta 1808,394-8 (2011); santner, p.et al. Biochemistry 57,5949-5956 (2018), and Taube, r., alhadeff, r., assa, d., krugliak, M. & Arkin, i.t. plos One 9, e105387 (2014).
In some embodiments, the assay is used to determine the susceptibility of a virus to develop resistance to the agent.
As used herein, the term "about" when combined with a value refers to plus or minus 10% of the referenced value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1000nm ± 100 nm.
It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the or said polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that drafting of the claims may exclude any optional elements. Accordingly, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements or use of a "negative" limitation.
In general, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.)). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one, either, or both of these terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments related to the invention are specifically embraced by the present invention and are disclosed herein just as if each combination were individually and explicitly disclosed. Moreover, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each such sub-combination were individually and explicitly disclosed herein.
Other objects, advantages and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the present invention described above and claimed in the appended claims section is supported experimentally in the following examples.
Various embodiments and aspects of the present invention as described above and claimed in the appended claims section are supported experimentally in the following examples.
Examples
Generally, nomenclature used herein and laboratory procedures utilized in the invention include molecular, biochemical, microbial, and recombinant DNA techniques. These techniques are described in detail in the literature. For example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); ausubel et al, "Current Protocols in Molecular Biology", john Wiley and Sons, baltimore, maryland (1989); perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, new York (1988); watson et al, "Recombinant DNA", scientific American Books, new York; birren et al (eds) "Genome Analysis: A Laboratory Manual Series", vols.1-4, cold Spring Harbor Laboratory Press, new York (1998); methodologies as set forth for in U.S. Pat. No. 4,666,828;4,683,202;4,801,531;5,192,659and 5,272,057; "Cell Biology: A Laboratory Handbook", volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freesney, wiley-Liss, N.Y. (1994), third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); stits et al, (eds), "Basic and Clinical Immunology" (8 th Edition), apple & Lange, norwalk, CT (1994); mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of these documents are incorporated by reference. Other general references are also provided in this document.
Materials and methods
Bacterial strains
Three strains of K12 E.coli (Escherichia coli) were used in this study: DH10B, LB650 and LR1.DH10B cells were purchased from Invitrogen (Carlsbad, CA). LB650 bacteria (Δ trkG, Δ trkH, and Δ kdppabc 5 systems) contain a gene deletion (Stumpe, S) associated with potassium uptake.&Bakker, E.P. Arch Microbiol 167,126-36 (1997)). LR1 bacteria contain a chromosomal copy of pH sensitive Green Fluorescent Protein (GFP), called PHluorin: (
Figure BDA0004025355100000161
G and De Angelis,D A and Rothman,J E.Nature 394,192–5(1998))。
Plasmids
The SARS-CoV-2E protein, 3a protein and influenza M2 channel were expressed as fusion proteins of maltose binding protein using pMAL-p2X plasmid (New England Biolabs, ipswich, mass.). The gene for the viral protein has added at the 3' end a nucleotide sequence encoding a linker of 7 amino acids, 6 histidines and a stop codon. EcoRI and XbaI restriction sites were located at the 5 'and 3' ends, respectively. The sequence was synthesized by GenScript (Piscataway, NJ). Protein expression was achieved by addition of isopropyl β -D-1-thiogalactoside (IPTG) to the growth medium as shown.
Chemical product
IPTG was purchased from Biochemika-Fluka (Buchs, switzerland). All other chemicals were purchased from Sigma-Aldrich laboratory (Rehovot, israel).
Growth medium
All bacterial growth was performed using Lysogenic Broth (LB) (Bertani, G.J. Bacteriol 62, 293-300 (1951)) unless otherwise indicated. LBK is similar to LB except KCl is replaced with 10gr/L for NaCl. All media contained 50. Mu.g/ml ampicillin.
Bacterial growth
Coli DH10B bacteria carrying or lacking (as reference) the virus chimera were cultured overnight in LB medium at 37 ℃. Thereafter, the growth culture was diluted and the bacteria were allowed to grow until their o.d. 600 Up to 0.2. Fifty (50) μ l of bacterial culture were then dispensed into 96-well flat-bottom plates (Nunc, roskilde, denmark) containing 50 μ l of different treatments. Unless otherwise stated, IPTG was added to the cells to a final concentration in the range of 0 to 100 μ M. D-glucose was added to bring the concentration to 1%. Ninety-six (96) well plates at 37 ℃ Infinite 200 from Tecan Group (C.) (
Figure BDA0004025355100000171
Switzerland) was incubated at a constant high shaking rate for 16 hours. O.d was recorded every 15 minutes. 600 And (6) reading. For each measurement, either a duplicate or three replicates were performed.
For E.coli LB650 bacteria, the same protocol was used except for overnight growth in LBK. Thereafter, LB was used in place of the growth medium, and the cells were dilutedThe bacteria were grown until their o.d. 600 Up to 0.2 and diluted two-fold with each treatment in each well. Unless otherwise stated, IPTG was added to LB650 bacteria to reach a final concentration of 10 μ M.
pHlux assay
Transformed LR1 cells were cultured overnight in LB medium containing 1% glucose and 50 μ M ampicillin. The primary culture was diluted 1: 500 in LB medium and grown to an O.D. of 0.6-0.8. 600 Subcultures were prepared. Protein synthesis was induced for 2 hours by the addition of 50. Mu.M IPTG. Cultures not subjected to IPTG induction were used as controls. After 2 hours of induction, the o.d of all cells was measured. 600 After 10 minutes of granulation at 3,500g, the bacteria were resuspended in McIlvaine buffer (200 mM Na) 2 HPO 4 0.9% NaCl, 0.9% NaCl adjusted to pH 7.6 with 0.1M citric acid) so that the optical density at 600nm was 0.25. Subsequently 200. Mu.l of the cell suspension were transferred to a 96-well plate together with 30. Mu.l McIlvaine buffer. The plate included a row of assay buffer and culture only without induction. Fluorescence measurements were performed in a microplate reader (Infinite F200 pro, tecan), with the emission set at 520nm and excitation shifted between 390nm and 466 nm.
To the bacteria was added 70 μ l of 300mM citric acid containing 0.9% NaCl using a liquid handling system (Tecan). The fluorescence emission of each well after addition of acid was measured by alternately reading out two filter pairs for 50 seconds. Calculating the ratio of the two different excitation emissions F = F 390nm /F 466nm And converted to proton concentration using the following formula:
Figure BDA0004025355100000172
Vero-E6 cells were pretreated with test compound (drug, including its concentration depicted in FIG. 23) for 20 hours and infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.001 and 0.01 in the presence of the indicated compound. Medium of the same DMSO concentration was used as a non-drug control. Drug efficacy in controlling toxicity was assessed by MTT assay 24 hours post infection. All infection experiments were performed in the BSL-3 facility.
Results
Three recently developed bacteria-based assays (Astrahan, p.et al. Biochem biophysis Acta 1808,394-8 (2011); santner, p.et al. Biochemistry 57,5949-5956 (2018); taube, r., alandeff, r., assa, d.d., krugliak, M. & Arkin, i.t. plos One 9, E105387 (2014); tomar, p.p.s., oren, r., krugliak, M. & Arkin, i.t. visises 11 (2019)) were adjusted to examine whether the SARS-CoV-2E protein is an ion channel.
These assays are quantitative, easy to perform quickly, and suitable for high throughput screening to identify inhibitors. Furthermore, all three assays were used for known viral porins and showed differentiation of non-conducting transmembrane regions (Tomar, p.p.s., oren, r., krugliak, M. & Arkin, i.t., viruses 11 (2019)). Finally, one of these assays can also be used to predict the option that the virus must be resistant to any particular channel inhibitor prior to clinical use (Assa, d., alhadeff, r., krugliak, M. & Arkin, i.t., j.mol Biol 428,4209-4217 (2016)).
To ensure correct membrane incorporation (membrane incorporation), the inventors utilized the pMAL protein fusion and purification system (New England Biolabs). In this construct, which has been successfully used for various viral porins, the SARS-CoV-2E protein or the 3a protein is fused to the carboxy terminus of the periplasmic maltose-binding protein. As a positive control, the present inventors compared the activity of these proteins with the M2 channel of influenza a virus (a typical viral porin that can be inhibited by aminoadamantane) (Pinto, l.h., holsinger, l.j. & Lamb, r.a. cell 69,517-28 (1992)).
The first test performed was to examine whether the channel activity of SARS-CoV-2E and 3a proteins could lead to membrane permeabilization, thereby negatively affecting bacterial growth (negative gene test). As seen in many other viral porins, when expressed at increased levels, channel activity hinders growth due to its deleterious effects on proton motility. Subsequently, channel-blocking drugs can be easily identified due to their ability to alleviate growth retardation. The data in FIG. 1 show that expression of SARS-CoV-2E or 3a protein results in significant bacterial growth retardation proportional to the level of protein expression. This behavior is similar to the known proton channel, the M2 influenza a virus protein.
The present inventors recognized that growth retardation is not a rare consequence of heterologous protein expression in bacteria. In other words, in addition to channel activity, pseudofactors (spurious factors) can cause bacterial death. Thus, the inventors demonstrated that bacterial death is due to protein channel activity by three means, (I) the inventors identified E and 3a protein channel blockers and showed that they can reactivate bacterial growth; (ii) The present inventors developed a complementary bacterial growth assay in which channel activity is essential for growth (positive gene testing); and (iii) the inventors show that protein expression is increased H in assays involving pH sensitive GFP + Conductivity (antner, P.et al. Biochemistry 57,5949-5956 (2018)).
The second experimental test conducted by the present inventors examined K + Electrical conductivity. Specifically, K + Uptake of defective bacteria (Stumpe, S.&Bakker, E.P. Arch Microbiol 167,126-36 (1997)) could not grow unless the medium was supplemented with K + . However, when K can be transported + When the channels of (3) are expressed heterologously, the bacteria may even be at low K + Thriving in culture (Taube, R.et al PLoS One 9, e105387 (2014); tomar, P.P.S., oren, R., krugliak, M.&Arkin, i.t. viruses 11 (2019)). Thus, in this case, the viral channel is essential for bacterial growth (positive gene testing. Finally, the results shown in FIG. 2 indicate that the expression of SARS-CoV-2E or 3a protein can be under other restrictive conditions (i.e.low [ K ]) + ]) Lower increase of K + Uptake of growth rate of the deficient bacteria.
The final assay to examine channel activity was based on the detection of protein-mediated H in bacteria expressing pH sensitive Green fluorescent protein + Flux (Santner, P.et al. Biochemistry 57,5949-5956 (2018)). Capable of transporting H if expressed by bacteria + And adding an acidic solution to the medium will result in acidification of the cytoplasm. Thus, as seen in FIG. 3, expression of the E or 3a protein from SARS-CoV-2 results in significant cellular hyaluronic acidIndicating its ability to transport protons. Similar acidification was detected in other viral porins such as influenza a M2 channels.
Considering that all three bacterial assays indicate that SARS-CoV-2E and 3a protein is a potential viral porin, the inventors set out to screen a small data set of known channel blockers. First, in the field of "membrane transporters/ion channels", a 372 compound library was obtained from medchemfast (NJ, USA). Each of these chemicals was tested in the positive and negative gene tests for E protein described above.
In the negative assay, the bacteria experienced significant growth retardation due to the expression of SARS-CoV-2E protein at elevated levels (FIG. 1). Therefore, channel blockers can be easily identified because they can alleviate this growth retardation. Note that this screen inherently reduces potential toxicity because it selects chemicals that are not toxic to bacteria. Specifically, each chemical in the pilot library (pilot library) was added to the medium, followed by growth recording and comparison to bacteria that did not receive any treatment. In the 372 compound drug library, several chemicals mitigate the growth inhibition experienced by bacteria due to SARS-CoV-2E protein activity. Of particular note are gliclazide and memantine, which promote bacterial growth, as seen in fig. 4A.
In the positive assay screen, a reverse map (reverse picture) was obtained. At low [ K ] + ]K growth in culture Medium + Uptake-deficient bacteria underwent growth promotion due to (low level) expression of SARS-CoV-2E protein (FIG. 2). Therefore, channel blockers can be identified as they cause growth retardation. In a manner similar to the negative assay, each chemical in the test point library was added to the medium and then growth recordings were made. Gliclazide and memantine again gave positive scores in the test (sorted positiveiy) as they both inhibited growth (fig. 4B).
The above results are encouraging because the same chemical gives a positive score in a reverse assay (recyclical assays). Both assays gave positive scores, excluding any spurious factors. When the E protein is harmful to bacteria, the chemical promotes growth. However, when the E protein is essential for the bacteria, the same compound is detrimental to growth.
Our results indicate that the SARS-CoV-2E protein is an ion channel. Since the coronavirus E protein is essential for virulence, it represents an attractive drug target. Our screening work identified two inhibitors that block the activity of the E protein channel. Since both drugs are approved for human use for other indications, they represent drug candidates that rapidly alleviate the COVID-19 crisis.
In addition, the screening range is expanded, 3000 molecules are screened against SARS-CoV-2E protein, and 3,000 molecules are screened against SARS-CoV-2 3a protein.
The results show that: any of memantine, gliclazide, mavorisavu, saroglitazar magnesium, mefenofibrate, cyclen, kasugamycin, azacytidine, and plexafu can be used as a SARS-CoV-2E protein channel blocker, and thus can be used as an attractive covi-19 drug.
The results further indicate that any of capreomycin, pentamidine, spectinomycin, kasugamycin, plexaford, flumatinib, littrowel, dapiprodione, floxuridine, and fludarabine can be used as a SARS-CoV-2 3a protein inhibitor and therefore as an attractive covi-19 drug.
Furthermore, the inventors show the effect of the compounds tested herein on the survival of cells infected with SARS-CoV-2 virus. Specifically, the results show that Vero-E6 cells show a cell viability decrease of-60% when infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01, while cells pretreated with drug show a cell viability decrease of-10-50% after infection with virus (FIG. 23).
While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variations as fall within the spirit and broad scope of the appended claims.
Sequence listing
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<120> E protein channel blocker and ORF3 inhibitor as anti-COVID-19agent
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<151> 2020-05-01
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<151> 2020-11-24
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<213> Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
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Claims (14)

1. 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: a SARS-CoV-2E protein channel blocker and a SARS-CoV-2 3a protein inhibitor, thereby treating or preventing SARS-CoV-2 virulence in said subject.
2. The method of claim 1, wherein the preventing comprises preventing any one of: entry of SARS-CoV-2 into cells of the subject, uncoating of the SARS-CoV-2 in the cells of the subject, release of the SARS-CoV-2 from the cells of the subject, and any combination thereof.
3. The method of claim 1 or 2, wherein the subject is infected or suspected of being infected with SARS-CoV-2.
4. The method of any one of claims 1 to 3, wherein the SARS-CoV-2E protein channel blocker is at least one molecule selected from the group consisting of: 5-azacytidine, memantine, gliclazide, mavorexavir, sarogexel magnesium, mefenofibrate, cycleanine, kasugamycin, plexafu and any salts thereof.
5. The method of any one of claims 1 to 4, wherein the SARS-CoV-2E protein channel blocker is used at a daily dose of 0.01 to 500 mg/kg.
6. The method of any one of claims 1 to 5, wherein the SARS-CoV-2E protein channel blocker is ginsenoside.
7. The method of any one of claims 1 to 5, wherein the SARS-CoV-2E protein channel blocker is memantine.
8. The method of any one of claims 1 to 7, wherein the SARS-CoV-2 3a protein inhibitor is at least one molecule selected from: capreomycin, pentamidine, spectinomycin, kasugamycin, plerixafor, flumatinib, littrowel, dapprandil, floxuridine, and fludarabine.
9. The method of claim 8, wherein the SARS-CoV-2 3a protein inhibitor is capreomycin.
10. A pharmaceutical composition comprising a SARS-CoV-2E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor for treating or preventing SARS-CoV-2 virulence in a subject in need thereof.
11. The pharmaceutical composition of claim 10, wherein said preventing comprises preventing any one of: entry of SARS-CoV-2 into cells of said subject, uncoating of said SARS-CoV-2, release of said SARS-CoV-2 from cells of said subject, and any combination thereof.
12. The pharmaceutical composition of claim 10 or 11, wherein the SARS-CoV-2E protein channel blocker is at least one molecule selected from the group consisting of: 5-azacytidine, memantine, gliclazide, mavorexavir, sarogexel magnesium, mefenofibrate, cycleanine, kasugamycin, plexafu and any salts thereof.
13. The pharmaceutical composition of claim 10 or 11, wherein the SARS-CoV-2E protein channel blocker is ginsenoside.
14. The pharmaceutical composition of any one of claims 10 to 13, wherein the SARS-CoV-2 3a protein inhibitor is at least one molecule selected from the group consisting of: capreomycin, pentamidine, spectinomycin, kasugamycin, plerixafor, flumatinib, littrowel, dapprandil, floxuridine, and fludarabine.
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