WO 2018/136559 Al 26 July 2018 (26.07.2018) W !P O PCT
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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/136559 Al 26 July 2018 (26.07.2018) W !P O PCT (51) International Patent Classification: A61K 31/70 (2006.01) A61K 38/00 (2006.01) A61K 31/47 (2006.01) (21) International Application Number: PCT/US2018/014108 (22) International Filing Date: 17 January 2018 (17.01.2018) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/447,290 17 January 2017 (17.01.2017) US (71) Applicant: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA [US/US]; 1111Franklin Street, 12th Floor, Oakland, CA 94607-5200 (US). (72) Inventor: RANA, Tariq, M.; 10610 Briarlake Woods Dri ve, San Diego, CA 92130 (US). (74) Agent: JENKINS, Kenneth, E. et al; Mintz Levin Cohn Ferris Glovsky And Popeo, P.C., 3580 Carmel Mountain Road, Suite 300, San Diego, CA 92130-6768 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: < — with international search report (Art. 21(3)) * * 54 Title: METHODS FOR TREATING FLAVIVIRUSES AND ZIKA INFECTIONS 00 (57) Abstract: Provided are methods of treatment of Zika virus infection. Specifically, the disclosure provides methods of treating a Zika viral infection in a subject in need thereof, said methods comprising administering to said subject an effective amount of a therapeutic composition including an NS5 (non-structural (NS) protein 5) polymerase inhibitor. Further disclosed are NS5 inhibitors that can be used for the methods. METHODS FOR TREATING FLAVIVIRUSESAND ZIKA INFECTIONS CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/447,290 filed January 17, 2017, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0002] Human infection with ZIKA virus (ZIKV), a mosquito-borne flavivirus, has spread rapidly since the 2015 outbreak in Brazil, and the World Health Organization declared ZIKV infection an International Public Health Emergency in 2016. ZIKV was discovered in 1947 and, although it had previously caused only sporadic disease in Africa and Asia, more recent outbreaks occurred in Micronesia in 2007 and in French Polynesia in 2013. ZIKV infection has been identified as the etiological agent of severe neurological defects, including microcephaly during fetal development and neuronal injury associated with Guillain-Barre syndrome in adults. New modes of viral transmission, including maternal-fetal and sexual transmission have been reported. ZIKV can infect human skin explants, peripheral blood mononuclear cells, human neuroprogenitor cells, and human cerebral organoids. In mouse models, ZIKV may be neurotropic. [0003] ZIKV and other members of the Flaviviridae family, such as dengue (DENV), West Nile (WNV), yellow fever (YFV), and Japanese encephalopathy (JEV), are positive (+) single-stranded RNA viruses. The ZIKV genome encodes a single polyprotein precursor that is cleaved by viral and host proteases to produce three structural and seven nonstructural proteins. Although our understanding of the molecular mechanisms involved in ZIKV infection of human cells has increased dramatically in the past few years, key determinants of ZIKV pathogenicity, such as cell- type specificity, mode of entry, and host factors essential for replication, are still largely unknown. In particular, there is a large gap in our understanding of the genetic and epigenetic regulatory mechanisms governing the viral life cycle and viral interactions with host cells. [0004] Solutions to this and other problems in the art are provided. Specifically, we discovered a number of FDA approved drugs that can be used to treat ZIKV infections and possibly other flaviviruses including Dengue, West Nile, JEV, and HCV. Specifically, we performed cell-based screens using libraries of compounds containing antiviral drugs and other available potential antiviral like compounds. We have accordingly identified FDA approved drugs that inhibit ZIKV in vitro and in vivo. These drugs are well tolerated in many cell lines including stem cells and mice and potently inhibit ZIKV infection. BRIEF SUMMARY OF THE INVENTION [0005] In a first aspect, there is provided a method of treating a Zika viral infection. The method includes administering to a subject in need thereof an effective amount of a compound as set forth in any of FIGS. 1A, IB, 2, 3, 6, 8, or 9 . [0006] In another aspect, there is provided a method of treating a Zika viral infection. The method includes administering to a subject in need thereof an effective amount of an NS5 polymerase inhibitor. [0007] In another aspect, there is provided a method of treating a Zika viral infection. The method includes administering to a subject in need thereof an effective amount of an HIV protease inhibitor. [0008] In another aspect, there is provided a method of treating a Zika viral infection. The method includes administering to a subject in need thereof an effective amount of a calcium channel blocker. [0009] In another aspect, there is provided a method of treating a Zika viral infection. The method includes administering to a subject in need thereof a combined effective amount of a therapeutic composition including an NS5 polymerase inhibitor and a HIV protease inhibitor. [0010] In another aspect, there is provided a method of treating a Zika viral infection. The method includes administering to a subject in need thereof an effective amount of a protein or a gene encoding the protein. In embodiments, the protein is a ZIKV non-structural (NS) protein. In embodiments, the ZIKV non-structural protein is NS5. In embodiments, the protein is NS5 RNA polymerase. In embodiment, the ZIKV non-structural protein is NS2B-NS3. In embodiments, the protein is NS2B-NS3 protease. BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIGS. 1A-1B. FIG. 1A: Antiviral activity of approved drugs/clinical molecules library was tested against Zika virus in human 293 T cells at the MOI of 5. Cells were pretreated with drugs (10 µΜ) for 1 hour and infected with ZIKV (MOI=5). After 24 hours of infection, mRNA was isolated and the levels of cellular ZIKV was determined by qRT-PCR. FIG IB: Methyl transferase inhibitors potently reduced ZIKV replication. Methods were as described in FIG. 1A . [0012] FIGS. 2A-2D. Dose dependent inhibition of Zika virus in 293 T cells by selected antiviral molecules (at the MOI of 5 using different concentration of drugs, IC50 was calculated). FIG. 2A - Lopinavir. FIG. 2B - Daclatasvir. FIG. 2C -Oxyclozanide. FIG. 2D - Rafoxanide. [0013] FIGS. 3A-3B. Synergistic activity of lopinavir and ritonavir. 293 T cells were pretreated with indicated drugs and after 1 hour, the cells were infected with ZIKV at the MOI of 5. After 24 hours, proteins were analyzed by western blotting (FIG. 3A) and mRNA was isolated and the levels of cellular ZIKV was determined by qRT-PCR (FIG. 3B). [0014] FIG. 4 . Experimental outline to determine the drug efficacy for ZIKV inhibition in vivo. [0015] FIGS. 5A-5D. Protease inhibitors reduced ZIKV in animals (brain - FIG. 5A; blood - FIG. 5B; testes - FIG. 5C; spleen - FIG. 5D) both as prophylaxis and therapeutic regimens. [0016] FIG. 6 . Screening of additional compounds for ZIKA levels using Vero cells (in 10 µΜ concentration). Methods of screening were as described herein. Viral RNA released was analyzed by qRT-PCR for ZIKA. Compounds (left to right): control, berberine, fluoxetine, formoterol, genistine, U0126, dibucaine, pirlindole, suramin, mycophenolic acid, polyhydroxyalkanoates, (PHA) and niclosamide. [0017] FIG. 7 . Screening of additional compounds for ZIKA levels using Vero cells (in 10 µΜ concentration) as described for FIG. 6 . Compounds (left to right): clomiphene, amphotericin B, and toremifene. [0018] FIG. 8. Screening of additional compounds for ZIKA levels using Vero cells (in 10 µΜ concentration) as described for FIG. 6 . Compounds providing hits: ganciclovir, procaine hydrochloride, zidovudine, aciclovir, drostanolone propionate, dapivirine (TMC120), tilorone hydrochloride (2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one, HC1), and Docosanol. [0019] FIGS. 9A-9K. ZIKA inhibition by various drugs in 293 cells at 20 µΜ , visualized by immune staining. Drugs: mock (FIG. 9A), DMSO (FIG. 9B), PHA-690509 (FIG. 9C), formoterol (FIG. 9D), fluoxetine (FIG. 9E), genistein (FIG. 9F), U0126 (FIG. 9G), berberine (FIG. 9H), dibucaine (FIG. 91), pirlindole (FIG. 9J), and suramin (FIG. 9K). Left panels: DAPI; right panels: ZIKV. [0020] FIGS. 10A-10E. Variable ZIKV Infection and Replication Levels in Different Cell Types. Immunohistochemistry of ZIKV gene expression in microglial (FIG. 10A), fibroblast (BJ) (FIG. 10B), kidney (293FT) (FIG.