REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[001] The contents of the electronic sequence listing (VRB -P-003 -PCT ST26.xml; size: 2,936 bytes; and date of creation: July 3, 2023) is herein incorporated by reference in its entirety.

CROSS-REFERENCE TO RELATED APPLICATIONS

[002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/358,659, titled “VIROPORINS BLOCKERS/INHIBITORS AS ANTIFLA VIVIRUSES AGENTS”, filed 6 July 2022, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[003] The present invention is in the field of anti-viral therapy.

BACKGROUND

[004] The genus Flaviviruses belongs to the Flaviviridae family of enveloped positivestrand RNA viruses. It includes several important arboviral pathogens that represent a specific health burden, such as dengue virus, West Nile virus, Zika virus, yellow fever virus, and tick-borne encephalitis, to name a few. Dengue fever, as an example, is transmitted by Aedes mosquitos and exacts an appreciable toll with ca. 40,000 annual deaths stemming from 390 million infections and half a million hospitalizations. Despite the fact that risk of infection exists in 129 countries, 70% of the actual burden is in Asia. According to World Health Organization (WHO) dengue cases has been increased 8 fold over past two decades.

[005] West Nile fever, transmitted by Culex mosquitos, is another cause of concern. Identified in Uganda in 1937, it is now spread globally. Since its discovery in 1999, there have been 52,532 cases in the US, of which 25,849 have been neuroinvasive, leading to 2,456 deaths. Consequently, West Nile fever is the leading cause of mosquito -borne disease in the continental US.

[006] No antiviral drugs are currently approved against flaviviruses, reducing treatment options to supportive care. There is an urgent need for methods and compounds for ameliorating or treating flaviviruses, such as Dengue fever and West Nile fever. SUMMARY

[007] According to a first aspect, there is provided a method of treating or preventing a flavivirus virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a flavivirus M protein channel blocker, thereby treating or preventing flavivirus virulence in the subject.

[008] According to another aspect, there is provided a method of preventing flavivirus entry, uncoating and/or release from a cell, the method comprising contacting the cell with a flavivirus M protein channel blocker, thereby preventing a flavivirus cell entry, uncoating and/or release from the cell.

[009] According to another aspect, there is provided a pharmaceutical composition comprising a flavivirus M protein channel blocker for use in treating or preventing flavivirus virulence in a subject in need thereof.

[010] According to another aspect, there is provided a pharmaceutical composition comprising a flavivirus M protein channel blocker for use in preventing a flavivirus cell entry, uncoating and/or release from a cell.

[Oi l] In some embodiments, the cell is a cell of a subject, and the contacting is administering to the subject.

[012] In some embodiments, the subject is a subject infected or suspected as being infected by a flavivirus virulence.

[013] In some embodiments, the flavivirus is a Dengue virus, and the flavivirus M protein channel blocker is a Dengue virus DPI channel blocker.

[014] In some embodiments, the flavivirus is a West Nile virus, and the flavivirus M protein channel blocker is a West Nile virus MgM channel blocker.

[015] In some embodiments, the Dengue virus DPI channel blocker is at least one molecule selected from the group consisting of: plerixafor, glecaprevir streptomycin, mesna, kasugamycin, tranexamic acid, CI- 1040, or a salt thereof.

[016] In some embodiments, the West Nile virus MgM channel blocker is at least one molecule selected from the group consisting of: idasanutlin, benzbromarone, 5 -azacytidine, and plerixafor, or a salt thereof.

[017] In some embodiments, the flavivirus M protein channel blocker is for use at a daily dose of 0.01 to 500 mg/kg. [018] In some embodiments, the pharmaceutical composition comprises a Dengue virus DPI channel blocker for use in treating or preventing Dengue virulence in a subject in need thereof.

[019] In some embodiments, the pharmaceutical composition comprises a West Nile virus MgM channel blocker for use in treating or preventing West Nile virulence in a subject in need thereof.

[020] In some embodiments, the pharmaceutical composition comprises a Dengue virus DPI channel blocker for use in preventing a Dengue virus cell entry, uncoating and/or release from a cell.

[021] In some embodiments, the pharmaceutical composition comprises a West Nile virus MgM channel blocker for use in preventing a West Nile virus cell entry, uncoating and/or release from a cell.

[022] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[023] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[024] Figure 1 includes bar graphs demonstrating a negative assay of Dengue Virus DPI and West Nile Virus MgM viroporins. Maximal growth rates of DH10B bacteria expressing each viroporin are depicted as a function of the inducer concentration (IPTG).

[025] Figures 2A-2D include bar graphs demonstrating a positive assay of Dengue Virus DPI and West Nile Virus MgM viroporins. (2A-2B) Maximal growth rates of K ⁺ -uptake deficient bacteria expressing each viroporin, monitored as a function of different K ⁺ concentrations. Growth enhancement can be observed upon comparing induced and uninduced bacteria in orange and blue, respectively. (2C-2D) Growth rates at different inducer concentrations (IPTG), as noted.

[026] Figure 3 includes graphs showing an acidity assay of Dengue Virus DPI and West Nile Virus MgM viroporins. Cytoplasmic H ⁺ concentration is monitored as a function of time whereby at time 0, an acidic solution is injected into the media. Results are shown from bacteria in which viroporin induction is changed a as function of IPTG concentration as noted.

[027] Figures 4A-4F include graphs showing blocker screening results against Dengue Virus DPI and West Nile Virus MgM viroporins. The negative assay results are depicted in (4A) and (4B), whereas the results from the positive assay are presented in (4C) and (4D). The acidity assay results are presented in (4E) and (4F). Bacteria in which the channel expression was not induced, or the drug was not added were used as controls. Blocker concentration was 50 pM.

[028] Figures 5A-5D include graphs showing blocker screening results against Dengue Virus DPI and West Nile Virus MgM viroporins. Maximal growth rates are depicted as a function of different compound concentrations (0.78-100 pM), as noted in the color legend. Bacteria in which the channel expression was not induced, or the drug was not added were used as controls. (5A-5B) Negative assay results for Dengue Virus DPI and West Nile Virus MgM viroporins, respectively. (5C-5D) Positive assay results for Dengue Virus DPI and West Nile Virus MgM viroporins, respectively.

[029] Figure 6 includes curves showing inhibitory constants of blocker screening results against Dengue Virus DPI (left column) and West Nile Virus MgM (right column) viroporins using the negative assays (as provided in Figures 5A-5B). Normalized growth enhancements are depicted as a function of different compound concentrations (0.78-100 pM). Bacteria in which the channel expression was not induced or the drug was not added are used as controls. The inhibitory constants are listed in each panel as well as the fitting coefficient.

[030] Figures 7A-7B include a three-dimensional bar graph demonstrating blocker combinations against (7A) West Nile Virus MgM viroporins and (7B) Dengue Virus DPI. Results from the negative assay in which active blockers should improve growth rate relative to untreated samples. Different combinations are presented according to the color scale on the left and numerically on the right. Each drug was at 100 pM. Diagonal values in white indicate individual treatments. [031] Figure 8 includes a vertical bar graphs showing cell viability of mammalian cells (Vero E6 cells) after 5 days post infection with Dengue virus. Drug concentration = 10 pM, and multiplicity of infection = 3.

DETAILED DESCRIPTION

[032] The present invention, in some embodiments, provides compositions comprising a M protein channel blocker for treating or preventing a flavivirus virulence in a subject. The present invention, in some embodiments, provides compositions comprising a flavivirus M protein channel blocker for preventing flavivirus cell entry, uncoating and/or release from a cell.

[033] In some embodiments, a flavivirus comprises a Dengue virus. In some embodiments, the M (membrane) protein of a Dengue virus is termed herein “DPI” (Dengue Pl). In some embodiments, the present invention provides compositions comprising a DPI protein channel blocker for treating or preventing Dengue virus virulence in a subject. The present invention, in some embodiments, provides compositions comprising a DPI protein channel blocker for preventing Dengue virus cell entry, uncoating and/or release from a cell.

[034] In some embodiments, a flavivirus comprises a West Nile virus. In some embodiments, the M protein of West Nile virus is termed herein “MgM”. In some embodiments, the present invention provides compositions comprising a MgM protein channel blocker for treating or preventing West Nile virus virulence in a subject. The present invention, in some embodiments, provides compositions comprising a MgM protein channel blocker for preventing West Nile virus cell entry, uncoating and/or release from a cell.

[035] The present invention, according to three bacteria-based assays, provides that Dengue virus DPI is an ion channel. The invention, in some embodiments, is further based, at least in part, on a finding that at least one molecule selected from: mesna, kasugamycin, glecaprevir, plerixafor, CI- 1040, streptomycin, tranexamic acid, or any combination thereof, inhibits Dengue virus DPI, and therefore, can be used to treat and/or prevent Dengue virus virulence.

[036] The invention, in some embodiments, is further based, at least in part, on a finding that at least one molecule selected from: plerixafor, glecaprevir, streptomycin, mesna, and kasugamycin, or any combination thereof, increased cell viability (e.g., of a mammalian cell) upon Dengue virus infection, and therefore, can be used to treat and/or prevent Dengue virus virulence. Further, tranexamic acid was found to increase, at least partially, the viability of a mammalian cell upon infection of Dengue virus. [037] The present invention, according to three bacteria-based assays, provides that West Nile virus MgM is an ion channel. The invention, in some embodiments, is further based, at least in part, on a finding that at least one molecule selected from: benzbromarone, idasanutlin, 5-azacytidine, and plerixafor, or any combination thereof, inhibited West Nile virus MgM, and therefore, can be used to treat and prevent West Nile virus virulence.

[038] In some embodiments, Dengue virus DPI is disclosed under GenBank Accession no: NP_722459.2.

[039] The terms “DPI” and “membrane glycoprotein (Mg) of Dengue virus” are used herein interchangeably.

[040] According to some embodiments, Dengue virus DPI comprises the amino acid sequence:

SVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGT SITQKGIIFILLMLVTPSMA (SEQ ID NO: 1).

[041] According to some embodiments, the Dengue virus DPI comprises an analog of SEQ ID NO: 1, having at least 85%, at least 90%, at least 95% sequence identity or homology thereto, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. According to some embodiments, the DPI comprises an analog of SEQ ID NO: 1 having homology within the range of: 85-100%, 91-100%, and 96- 100%. Each possibility represents a separate embodiment of the invention.

[042] In some embodiments, West Nile virus MgM is disclosed under GenBank Accession no: YP_001527879.1. According to some embodiments, the West Nile virus MgM comprises the amino acid sequence:

SLTVQTHGESTLANKKGAWMDSTKATRYLVKTESWILRNPGYALVAAVIGWML GSNTMQRVVFVV LLLLVAPAYS (SEQ ID NO: 2).

[043] According to some embodiments, the West Nile virus MgM comprises an analog of SEQ ID NO: 2, having at least 85%, at least 90%, at least 95% sequence identity or homology thereto, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. According to some embodiments, the West Nile virus MgM comprises an analog of SEQ ID NO: 2 having homology within the range of: 85-100%, 91- 100%, and 96-00%. Each possibility represents a separate embodiment of the invention.

[044] The term “analog” as used herein, refers to a polypeptide that is similar, but not identical, to the polypeptide of the invention that still is capable of binding succinate or still comprises the succinate binding pocket. [045] An analog may have deletions or mutations that result in an amino acids sequence that is different than the amino acid sequence of the polypeptide of the invention. It should be understood that all analogs of the polypeptide of the invention would still be capable of generating an ion channel. Further, an analog may be analogous to a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 30 consecutive amino acids of the polypeptide of the invention.

[046] According to some embodiments, the invention provides a method of treating or preventing a flavivirus virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a flavivirus M protein channel blocker, thereby treating or preventing a flavivirus virulence in the subject.

[047] According to some embodiments, the invention provides a method of treating or preventing a Dengue virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a Dengue virus DPI channel blocker, thereby treating or preventing a Dengue virulence in the subject.

[048] According to some embodiments, the invention provides a method of treating or preventing a West Nile virulence in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a West Nile MgM channel blocker, thereby treating or preventing a West Nile virulence in the subject.

[049] According to some embodiments, the invention provides a method of preventing a flavivirus release from a cell, the method comprising contacting the cell with a flavivirus M protein channel blocker, thereby preventing a flavivirus release from the cell.

[050] According to some embodiments, the invention provides a method of preventing a Dengue virus release from a cell, the method comprising contacting the cell with Dengue virus DPI channel blocker, thereby preventing a dengue virus release from the cell.

[051] According to some embodiments, the invention provides a method of preventing a West Nile virus release from a cell, the method comprising contacting the cell with West Nile virus MgM channel blocker, thereby preventing a West Nile virus release from the cell.

[052] According to some embodiments, the invention provides a method of preventing a flavivirus cell entry, the method comprising contacting the cell with a flavivirus M protein channel blocker, thereby preventing a flavivirus cell entry.

[053] According to some embodiments, the invention provides a method of preventing a Dengue virus cell entry, the method comprising contacting the cell with a Dengue virus DPI channel blocker, thereby preventing a Dengue virus cell entry. [054] According to some embodiments, the invention provides a method of preventing a West Nile virus cell entry, the method comprising contacting the cell with a West Nile virus MgM channel blocker, thereby preventing a West Nile virus cell entry.

[055] According to some embodiments, the invention provides a method of preventing a flavivirus uncoating, the method comprising contacting the cell with a flavivirus M channel blocker, thereby preventing a flavivirus uncoating.

[056] According to some embodiments, the invention provides a method of preventing a Dengue virus uncoating, the method comprising contacting the cell with a Dengue virus DPI channel blocker, thereby preventing a Dengue virus uncoating.

[057] According to some embodiments, the invention provides a method of preventing a West Nile virus uncoating, the method comprising contacting the cell with a West Nile virus MgM channel blocker, thereby preventing a West Nile virus uncoating.

[058] According to some embodiments, the cell is a cell of a subject. According to some embodiments, contacting is administering to the subject. According to some embodiments, the subject is a subject infected or suspected as being infected by a Dengue virus. According to other embodiments, the subject is a subject infected or suspected as being infected by a West Nile virus.

[059] According to some embodiments, the flavivirus M channel blocker is at least one molecule selected from: plerixafor or a salt thereof, streptomycin or a salt thereof, tranexamic acid or a salt thereof, CI- 1040 or a salt thereof, glecaprevir or a salt thereof, kasugamycin, or a salt thereof, mesna or a salt thereof, idasanutlin or a salt thereof, benzbromarone or a salt thereof, 5-azacytidine or a salt thereof, or any combination thereof.

[060] According to some embodiments, the Dengue virus DPI channel blocker is at least one molecule selected from: plerixafor or a salt thereof, streptomycin or a salt thereof, tranexamic acid or a salt thereof, CI- 1040 or a salt thereof, glecaprevir or a salt thereof, kasugamycin, or a salt thereof, mesna or a salt thereof, or any combination thereof.

[061] According to some embodiments, the West Nile virus MgM channel blocker is at least one molecule selected from: idasanutlin or a salt thereof, benzbromarone or a salt thereof, 5-azacytidine or a salt thereof, plerixafor or a salt thereof, or any combination thereof.

[062] According to some embodiments, the invention provides a Dengue virus DPI channel blocker for use in treating or preventing a Dengue virulence in a subject in need thereof. According to some embodiments, the invention provides a West Nile virus MgM channel blocker for use in treating or preventing a West Nile virulence in a subject in need thereof.

[063] In some embodiments, the Dengue Virus DPI blocker comprises a combination comprising tranexamic acid and mesna. In some embodiments, the Dengue Virus DPI blocker comprises a combination comprising kasugamycin and CI- 1040. In some embodiments, the Dengue Virus DPI blocker comprises a combination comprising kasugamycin and tranexamic acid. In some embodiments, the combination of Dengue Virus DPI blocker comprises, exhibits, or characterized by additivity of the individual Dengue Virus DPI blockers. In some embodiments, the Dengue Virus DPI blocker comprises a combination comprising plerixafor and at least one additional Dengue Virus DPI blocker. In some embodiments, the Dengue Virus DPI blocker comprises a combination comprising plerixafor and at least one molecule selected from: streptomycin, tranexamic acid, CI- 1040, glecaprevir, kasugamycin, mesna, or any combination thereof.

[064] In some embodiments, a Dengue virus DPI channel blocker comprises any salt thereof.

[065] According to some embodiments, the invention provides a Dengue virus DPI channel blocker for use in preventing Dengue virus release from a cell. According to some embodiments, the invention provides a West Nile virus MgM channel blocker for use in preventing West Nile virus release from a cell.

[066] According to some embodiments, the Dengue virus DPI channel blocker is within a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier. According to some embodiments, the West Nile virus MgM channel blocker is within a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[067] According to some embodiments, the invention provides a pharmaceutical composition comprising plerixafor, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises West Nile virus infection. In some embodiments, the viral infection comprises an infection by a virus comprising a flavivirus M protein. In some embodiments, the viral infection comprises an infection by a flavivirus comprising a Dengue virus DPI protein. In some embodiments, the viral infection comprises an infection by a flavivirus comprising a West Nile virus MgM protein. [068] According to some embodiments, the Dengue virus DPI channel blocker comprises plerixafor, an analog or a salt thereof. According to other embodiments, the West Nile virus channel blocker comprises plerixafor, an analog or a salt thereof.

[069] Plerixafor, as used herein, includes plerixafor (CAS: 110078-46-1, IUPAC: l-[[4- (1,4,8,11 -tetrazacyclo tetradec - 1 -y Imethy l)pheny 1] methyl] -1,4,8,11- tetrazacyclotetradecane), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof. Plerixafor, is described, for example, in U.S. Patent US20200268850A1.

[070] According to some embodiments, the invention provides a pharmaceutical composition comprising streptomycin, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises an infection by virus having a dengue virus DPI being an ion channel.

[071] According to some embodiments, the Dengue virus DPI channel blocker comprises streptomycin, an analog or a salt thereof.

[072] Streptomycin, as used herein, includes streptomycin (CAS: 57-92-1, IUPAC: 2- [(17?,27?,3S,47?,57?,6S)-3-(diaminomethylideneamino)-4-[(27? ,37?,47?,55')-3- [(2S,3S,4S,57?,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methyl amino)oxan-2-yl]oxy-4- formyl-4-hydroxy-5-methyloxolan-2-yl]oxy-2,5,6-trihydroxycyc lohexyl]guanidine), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[073] According to some embodiments, the invention provides a pharmaceutical composition comprising tranexamic acid, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises an infection by virus having a Dengue virus DPI being an ion channel.

[074] According to some embodiments, the Dengue virus DPI channel blocker comprises tranexamic acid an analog or a salt thereof.

[075] Tranexamic acid, as used herein, includes tranexamic acid (CAS: 701-54-2, IUPAC: 4-(aminomethyl)cyclohexane- 1 -carboxylic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[076] According to some embodiments, the invention provides a pharmaceutical composition comprising CI- 1040, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises an infection by virus having a Dengue virus DPI being an ion channel. [077] According to some embodiments, the Dengue virus DPI channel blocker comprises CI- 1040, an analog or a salt thereof.

[078] CI-1040, as used herein, includes CI-1040 (CAS: 212631-79-3, IUPAC: 2-(2-chloro- 4-iodoanilino)-A-(cyclopropylmethoxy)-3,4-difluorobenzamide) , as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[079] According to some embodiments, the invention provides a pharmaceutical composition comprising glecaprevir, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises an infection by virus having a Dengue virus DPI being an ion channel.

[080] According to some embodiments, the Dengue virus DPI channel blocker comprises glecaprevir, an analog or a salt thereof.

[081] Glecaprevir, as used herein, includes glecaprevir (CAS: 1365970-03-1; IUPAC: (l/?,14E,18/?,22/?,265,295)-26-fert-butyl-A-[(l/?,2/?)-2-(di fluoromethyl)-l-[(l- methylcyclopropyl) sulfonylcarbamoyl] cyclopropyl] -13,13 -difluoro-24,27 -dioxo-2, 17,23- trioxa-4,l l,25,28-tetrazapentacyclo[26.2.1.0 ³ ’ ¹² .0 ⁵ ’ ¹⁰ .0 ¹⁸ ’ ²² ] hentriaconta-3,5,7,9,11,14- hexaene-29-carboxamide), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[082] According to some embodiments, the invention provides a pharmaceutical composition comprising kasugamycin, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises an infection by virus having a Dengue virus DPI being an ion channel.

[083] According to some embodiments, the Dengue virus DPI channel blocker is kasugamycin, an analog or a salt thereof.

[084] Kasugamycin, as used herein, includes kasugamycin (CAS: 6980-18-3; IUPAC: 2- amino-2-[(27?,3S,5S,67?)-5-amino-2-methyl-6-[(27?,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.

[085] According to some embodiments, the invention provides a pharmaceutical composition comprising mesna, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a Dengue virus infection. In some embodiments, the viral infection comprises an infection by virus having a Dengue virus DPI being an ion channel. [086] According to some embodiments, the Dengue virus DPI channel blocker comprises mesna, an analog or a salt thereof.

[087] Mesna, as used herein, includes mesna (CAS: 19767-45-4; IUPAC: sodiumssulf any lethanesulfonate), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[088] According to some embodiments, the invention provides a pharmaceutical composition comprising idasanutlin, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a West Nile virus infection. In some embodiments, the viral infection comprises an infection by virus having a West Nile MgM being an ion channel.

[089] According to some embodiments, the West Nile MgM channel blocker comprises idasanutlin, an analog or a salt thereof.

[090] Idasanutlin, as used herein, includes idasanutlin (CAS: 1229705-06-9; IUPAC: 4- [[(27?,3S,47?,5S)-3-(3-chloro-2-fhiorophenyl)-4-(4-chloro-2- fluorophenyl)-4-cyano-5-(2,2- dimethylpropyl)pyrrolidine-2-carbonyl] amino] -3 -methoxybenzoic acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[091] According to some embodiments, the invention provides a pharmaceutical composition comprising benzbromarone, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a West Nile virus infection. In some embodiments, the viral infection comprises an infection by virus having a West Nile MgM being an ion channel.

[092] According to some embodiments, the West Nile MgM channel blocker comprises benzbromarone, an analog or a salt thereof.

[093] Benzbromarone, as used herein, includes benzbromarone (CAS: 3562-84-3; IUPAC: (3,5-dibromo-4-hydroxyphenyl)-(2-ethyl-l-benzofuran-3-yl)met hanone), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

[094] According to some embodiments, the invention provides a pharmaceutical composition comprising 5-azacytidine, an analog or a salt thereof, for treating a viral infection. In some embodiments, the viral infection comprises a West Nile virus infection. In some embodiments, the viral infection comprises an infection by virus having a West Nile MgM being an ion channel.

[095] According to some embodiments, the West Nile MgM channel blocker is 5- azacytidine, an analog or a salt thereof. [096] 5 -Azacytidine, as used herein, includes 5-azacytidine (CAS: 320-67-2; IUPAC: 4- amino-l-[(27?,37?,4S,5S)-3,4-dihydroxy-5-(hydroxyamino)oxola n-2-yl]pyrimidin-2-one), as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.

Pharmaceutical compositions

[097] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition 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. To be an effective treatment, 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.

[098] As used herein, the terms “administering”, “administration” and like terms refer 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.

[099] As used herein, 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.

[0100] In some embodiments, 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.

[0101] 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. Although the bioavailability of peptides administered by other routes 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. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.

[0102] In some embodiments, the composition of the invention comprising oral delivery. In some embodiments, the composition of the invention comprises an oral composition. In some embodiments, the composition of the invention further comprises orally acceptable carrier, excipient, or a diluent.

[0103] According to some embodiments, the Dengue virus DPI channel blocker is for use at a daily dose of 0.01 to 500 mg/kg.

[0104] According to some embodiments, the West Nile virus MgM channel blocker is for use at a daily dose of 0.01 to 500 mg/kg.

[0105] According to some embodiments, the Dengue virus DPI channel blocker comprises plerixafor or a salt thereof and is for use at a daily dose between about 0.1 mg/kg/day and about 10 mg/kg/day, 0.1 mg/kg/day and 5 mg/kg/day, and 0.1 mg/kg/day and 1 mg/kg/day.

[0106] According to some embodiments, the Dengue virus DPI channel blocker comprises streptomycin or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/day, about 5 mg/kg/day and 60 mg/kg/day, and 10 mg/kg/day and 40 mg/kg/day.

[0107] According to some embodiments, the Dengue virus DPI channel blocker comprises tranexamic acid or a salt thereof and is for use at a daily dose of between about 1 mg//kg/day and about 100 mg/kg/day, about 1 mg/kg/day and 50 mg/kg/day, and 5 mg/kg/day and 40 mg/kg/day.

[0108] According to some embodiments, the Dengue virus DPI channel blocker comprises CI- 1040 or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 500 mg/kg/day, about 5 mg/kg/day and 400 mg/kg/day, and 40 mg/kg/day and 200 mg/kg/day.

[0109] According to some embodiments, the Dengue virus DPI channel blocker comprises glecaprevir or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 150 mg/kg/day, about 10 mg/kg/day and 100 mg/kg/day, and 1 mg/day and 10 mg/kg/day.

[0110] According to some embodiments, the Dengue virus DPI channel blocker comprises kasugamycin or a salt thereof and is for use at a daily dose of between about 1 mg/day and about 100 mg/kg/day, about 5 mg/kg/day and 50 mg/kg/day, and 10 mg/kg/day and 40 mg/kg/day. [0111] According to some embodiments, the Dengue virus DPI channel blocker comprises mesna or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 100 mg/kg/day, about 5 mg/kg/day and 80 mg/kg/day, and 5 mg/kg/day and 50 mg/kg/day.

[0112] According to some embodiments, the West Nile virus MgM channel blocker comprises plerixafor or a salt thereof and is for use at a daily dose between about 0.1 mg/kg/day and about 10 mg/kg/day, 0.1 mg/kg/day and 5 mg/kg/day, and 0.1 mg/kg/day and 1 mg/kg/day.

[0113] According to some embodiments, the West Nile virus MgM channel blocker comprises idasanutlin or a salt thereof and is for use at a daily dose of between about 10 mg/kg/day and about 100 mg/kg/day, about 10 mg/kg/day and 50 mg/kg/day, and 5 mg/kg/day and 40 mg/kg/day.

[0114] According to some embodiments, the West Nile virus MgM channel blocker comprises benzbromarone or a salt thereof and is for use at a daily dose of between about 1 mg/kg/day and about 200 mg/kg/day, about 2 mg/kg/day and 150 mg/kg/day, and 3 mg/kg/day and 100 mg/kg/day.

[0115] According to some embodiments, the West Nile virus MgM channel blocker comprises 5-azacytidine or a salt thereof and is for use at a daily dose of between about 0.5 mg/kg/day and about 500 mg/day, about 1 mg/kg/day and 200 mg/day, and 1 mg/day and 100 mg/day.

[0116] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0117] As used herein, the term “carrier”, “adjuvant” or “excipient” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “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. Some examples of the materials that can serve 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, 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; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of 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. 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, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These 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: 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 by reference herein in its entirety. The presently described composition 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., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0118] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

Screening assays

[0119] By another aspect, there is provided a method of screening effectiveness of an agent in treating or preventing a Dengue virus infection. According to some embodiments, the method comprising providing a cell comprising a membrane permeabilized with Dengue DPI, 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 Dengue virus infection, thereby screening effectiveness of an agent in treating or preventing a Dengue virus infection.

[0120] In some embodiments, the method comprises a negative assay. In some embodiments, the cell is characterized by growth retardation due to the membrane permeabilized with Dengue DPI. In some embodiments, an agent that alleviates growth retardation is indicative as being effective for treating or preventing a Dengue virus infection.

[0121] In some embodiments, the method comprises a positive assay. In some embodiments, the cell is a K ⁺ -uptake deficient cell grown in low [K ⁺ ] media experience growth, due to the channel formed by a Dengue DPI. In some embodiments, an agent that induces growth retardation is indicative as being effective for treating or preventing a Dengue virus infection.

[0122] In some embodiments, the method comprises performing both the negative assay and the positive assay.

[0123] In some embodiments, the method further comprises an acidity assay. In some embodiments, the cell is a pH-sensitive cell comprising a reporter gene or a protein product thereof, such as, but not limited to, green fluorescent protein (GFP). In some embodiments, H ⁺ influx to a pH-sensitive cell, grown in media supplemented with an acidic solution, is induced. In some embodiments, an agent that elevates pH (reduces acidity) within the pH- sensitive cell is suitable for treating or preventing a Dengue virus infection.

[0124] In some embodiments, there is provided a method of screening effectiveness of an agent in treating or preventing a West Nile virus infection. According to some embodiments, the method comprising providing a cell comprising a membrane permeabilized with West Nile MgM, 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 West Nile virus infection, thereby screening effectiveness of an agent in treating or preventing a West Nile virus infection.

[0125] In some embodiments, the method comprises a negative assay. In some embodiments, the cell is characterized by growth retardation due to the membrane permeabilized with West Nile MgM. In some embodiments, an agent that alleviates growth retardation is indicative as being effective for treating or preventing a West Nile virus infection.

[0126] In some embodiments, the method comprises a positive assay. In some embodiments, the cell is a K ⁺ -uptake deficient cell grown in low [K ⁺ ] media experience growth, due to the channel formed by a West Nile MgM. In some embodiments, an agent that induces growth retardation is indicative as being effective for treating or preventing a West Nile virus infection.

[0127] In some embodiments, the method comprises performing both the negative assay and the positive assay.

[0128] In some embodiments, the method further comprises an acidity assay. In some embodiments, the cell is a pH-sensitive cell comprising a reporter gene or a protein product thereof, such as, but not limited to, green fluorescent protein (GFP). In some embodiments, H ⁺ influx to a pH-sensitive cell, grown in media supplemented with an acidic solution, is induced. In some embodiments, an agent that elevates pH (reduces acidity) within the pH- sensitive cell is suitable for treating or preventing a West Nile virus infection.

[0129] 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 (2018), and Taube, R., Alhadeff, R., Assa, D., Krugliak, M. & Arkin, I. T. PLoS One 9, el05387 (2014).

[0130] In some embodiments, the assay comprises determining susceptibility of the virus to develop resistance against the agent.

[0131] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0132] It is 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 polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, 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.

[0133] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general 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 of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0134] 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 sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0135] Additional 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. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0136] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0137] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, 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 in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 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 Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and Methods

Bacteria-based channel assays

[0138] Three bacteria-based channel assays were employed for investigating the viroporin channel activity and blockers thereof. In all assays The MBP (Maltose binding protein) fusion purification system (New England BioLabs; Ipswich, MA, USA) was used, where Dengue Virus DPI and West Nile Virus MgM were expressed as chimera by fusion to the carboxy terminal of the maltose binding protein. Previous results have shown that utilizing this construct results in functional expression in bacteria .

Negative assay

[0139] Bacteria cultures (DH10B) were grown overnight and finally diluted 500 fold and again set to grow until their O.D.600 reached 0.2. From the culture 50 pl were added in 96- well flat-bottomed plates which was pre-treated with 50 pl of required staff. Induction was achieved by employing different concentration of isopropyl-P-d-1 -thiogalactopyranoside. A multi-plate incubator (infinite M200 pro from Tecan Group; Mannedorf, Switzerland or LogPhase 600 from BioTek; Santa Clara, CA, USA) was used to incubate the plates for 16 hours at 37 °C at a constant, shaking rate (700 rpm). Bacterial growth was monitored by measuring O.D.600 every 15 min. Duplicates, or triplicates were conducted for every measurement.

Positive assay

[0140] The positive assay employed the same protocol as the negative assay was but in this instance a K ⁺ -uptake deficient bacteria strain was used . Additionally, overnight growth was conducted in LB media in which 100 mM KC1 replaced NaCl.

[0141] The acidity assay is based on bacteria expressing a chromosomal copy of a pH sensitive GFP. Overnight bacterial cultures were diluted to 1:500 in LB media and subsequently grown to an O.D.600 of 0.6-0.8. Protein expression was induced by isopropyl- P-D-l- thiogalactopyranoside at different concentrations, as noted. After one hour of induction, the cells were diluted to an O.D.600 of 0.2 and pelleted at 3500 g for 10 min. Subsequently, the cells were resuspended in McILvaine Buffer, which contains 200 mM Na2HPO4 and 0.9% NaCl adjusted to pH 7.6 with 0.1 M citric acid. 200 pl of cell suspension were added to a 96-well plate (Nunclon f96 Microwell Black Polystyrene, Thermo Fisher Scientific; Waltham, MA, USA), each well containing 30 pl of McILvaine Buffer. The plate included three wells with McILvaine buffer and three with culture without induction as control. The fluorescent measurements were carried out at an ambient temperature in a microplate reader (Infinite F200 Pro, Tecan Group; Mannedorf, Switzerland) with two pairs of bandpass filters: 520 nm emission filter and 390 and 466 nm excitation filters. At the starting point, 70 pl of 300 mM citric acid was added to the bacterial culture, and a fluorescent read-out was taken for 30 s for each wavelength. Finally, proton concentration was calculated from the ratio of two wavelengths.

Chemical Screening

[0142] A chemical library of 2,839 compounds was purchased from MedChem Express (HY- L035, Monmouth Junction, NJ, USA). All chemicals were screened at 100 pM using the negative screening at first, whereby 100 pM isopropyl-P-D-l-thiogalactopyranoside was used to induce DPI and MgM expression. Bacteria without isopropyl-P-D-1- thiogalactopyranoside (z.e., no protein induction) were used as a positive control, while bacteria that received only DMSO served as the negative control. Drugs that increased bacterial growth up to a certain threshold were selected for further duplicate analyses. Subsequently, drugs that passed duplicate analysis in the negative assays were examined by positive assay, where 5 pM and 10 pM isopropyl-P-D-l-thiogalactopyranoside were used to induce DPI and MgM expression, respectively. Compounds that passed the negative and positive assays were subjected to dose-response analyses and fluorescence-based assays. Finally, the negative assay tested the additive effects of drugs by employing equal concentrations of all possible combinations.

Chemicals

[0143] Isopropyl-P-d-1 -thiogalactopyranoside was purchased from Biochemika-Fluka (Buchs, Switzerland). All other chemicals were purchased from Sigma- Aldrich laboratories (Rehovot, Israel).

Bacterial growth media

[0144] Lysogeny broth (LB) was used for most of the cases only LBK was used for positive assay, where NaCl is replaced with KCL at lOgm/lt. All media contained ampicilin at 100 g/ml.

Cell viability assay

[0145] Growth of Vero E6 cells was tested in the presence of Dengue virus (type 1, BC89/94). Cells were infected with a muliplicity of infection (MOI) of 3, and their viability was monitored by MTS post five days of infection. At time 0, different drugs were added (each diluted in 0.1% DMSO) at a concentration of 10 pM. Results were normalized relative to uninfected cells (100%) and cells that received only 0.1% DMSO (0%).

EXAMPLE 1

Bacteria-based assays for assessment of ion channels activity

[0146] In order to evaluate the activity of both viroporins, the inventors used bacteria-based assays in which the channel’s functionality changes the bacteria’s phenotype. The advantages of the assays are that they are amenable to high-throughput screening, and the ease of genetic manipulations in bacteria enables a rapid transition from one sequence/variant to another. Finally, these assays have been tested on numerous viroporins from various viruses (Assa, D. et al. J Mol Biol 2016, 428, 4209-4217, Astrahan, P. et al. 2011, 1808, 394-398, Taube, R, PLoS One 2014, 9, el05387, Tomar, P.P.S. et al. Viruses 2019, 11, Tomar, P.P.S. et al. Viruses 2021, 13, Tomar, P.P.S. et al. Pharmaceuticals (Basel) 2021, 14, Tomar, P.P.S. et al. Krugliak, M.; Singh, A.; Arkin, I.T. Biomedicines 2022).

Negative assay

[0147] The first assay used to characterize both viroporins is one in which the protein is expressed at increasing levels in bacteria. As seen in other channels, elevated expression levels are detrimental to bacterial growth due to excessive permeabilization of the bacterial inner membrane. Therefore, in this assay, the viral protein eponymously impacts bacteria negatively.

[0148] As seen in Figure 1, both proteins are able to retard bacterial growth appreciably. At inducer concentrations of 50-100 pM the growth rate is roughly half of what is observed without induction. Finally, the inventors recognize that spurious factors can contribute to toxicity upon heterologous protein expression in bacteria. Therefore the inventors employed two additional bacteria-based assays to evaluate the channel activity of the viral proteins.

Positive assay

[0149] The second bacteria-based assay that was employed was reciprocal in nature to the negative assay listed above. K ⁺ -uptake deficient bacteria are incapable of growing in regular bacteriological media unless it is enriched with potassium. However, the bacteria are able to thrive in low K ⁺ media if they express a channel capable K ⁺ transport. Hence, in this instance the activity of the channel impacts growth positively.

[0150] Results shown in Figures 2A-2D indicate clearly that both viroporins are able to enhance the growth of K ⁺ -uptake deficient bacteria. The experiments were repeated at several different K ⁺ concentrations in order to demonstrate the reproducible nature of the effect. Note that in this assay the channel was expressed at a low level, since higher levels were once more detrimental to the bacteria, as seen in Figures 2C-2D.

Acidity assay

[0151] The final assay that was employed measured H ⁺ conductivity by using bacteria that express a pH-sensitive GFP. In this assay, an acidic solution is injected into the media while monitoring bacterial fluorescence that is indicative of its cytoplasmic pH. A change in fluorescence would indicate H ⁺ - flux due to a channel conductivity. Results shown in Figure 3 demonstrate that both viroporins were able to facilitate H ⁺ transport with Dengue Virus DPI exhibiting higher activity than West Nile Virus MgM.

Blocker screening

[0152] Following confirmation of channel activity of both viral proteins, the inventors sought to identify drugs that could block their function. The negative assay described above was empolyed and a 2,839 repurposed drug library was screened. The inventors subsequently followed by cross-checking every hit using the positive and pH-based assays. In the negative assay, a successful hit is expected to increase bacterial growth by abrogating the detrimental impact the viroporin has. In contrast, in the positive assay, successful blockers would impair bacterial growth since they would counteract the beneficial effect of the viral channel. [0153] The reciprocal nature of both assays decreases the possibility of erroneous results. Specifically, compounds were classified as hits only if they increased bacterial growth in the negative assay while concomitantly reducing growth in the positive assay.

[0154] The results of the negative assay screen are shown Figures 4A-4B in which seven blockers were identified against Dengue Virus DPI and four against West Nile Virus MgM. Subsequently, the positive assay was employed to examine the potency of the hits identified in the negative assay: out of the seven blockers active against Dengue Virus DPI, four were shown to be active in the positive assay, conducted herein (Figure 4C). In contrast, all hits in the negative assay against West Nile Virus MgM were confirmed in the positive assay (Figure 4D).

[0155] Finally, the hits identified in the negative assay were examined in the acidity assay: Out of the seven blockers active against Dengue Virus DPI, six were confirmed in the acidity assay (Figure 4E). Once more, all hits from the negative and positive assay against West Nile Virus MgM were active in the acidity assay (Figure 4F).

[0156] The final step in the screening process involved dose-response analyses of the compounds that passed the negative and positive assays for each viroporin: plerixafor, streptomycin, tranexamic acid, and CI-1040 against Dengue Virus DPI, and idasanutlin, benzbro-marone, 5-azacytidine, and plerixafor against West Nile Virus MgM. The results are depicted in Figures 5A-5D, and the fitted inhibitory contestants are provided in Figure 6.

Combination testing

[0157] The inventors next sought to determine potential additivity or synergism between the different hits. To that end, they examined all possible combinations of drugs that were active against Dengue Virus DPI and West Nile Virus MgM channels in equal concentrations. From Figure 7B it can be seen that several Dengue Virus DPI blocker combinations, like tranexamic acid + mesna, kasugamycin + CI- 1040, and kasugamycin + tranexamic acid exhibited additivity. Tranexamic acid + mesna in particular, demonstrated 98% enhancement of growth, whereas the impact of individual components was lower (32% and 82% for tranexamic acid and mesna, respectively). All the compounds that were combined with plerixafor exhibited better activity due to its masking effect. In the case of West Nile Virus MgM (Figure 7A), combinations such as plerixafor + idasanutlin (128%) and plerixafor + benzbromarone (181%) exhibited additivity, while the effect of combining 5-azacytidine with idasanutlin (105%) reduced activity.

Cell viability [0158] Further, the invenotrs have examined cell viability Vero E6 cells infected with the Dengue virus. The results show that Plerixafor, Mesna, Glecaprevir, Kasugamycin, and Streptomycin presented a clear anti-dengue virus activity, as provided by increased cell viability in vitro (Figure 8).

[0159] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.