Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

ARTICLES npg © 2016 Nature America, Inc. All rights reserved. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen Miao Xu1,2,16, Emily M Lee3,16, Zhexing Wen4–7,16, Yichen Cheng3, Wei-Kai Huang7,8, Xuyu Qian7,9, Julia TCW10, Jennifer Kouznetsova1, Sarah C Ogden3, Christy Hammack3, Fadi Jacob7,11, Ha Nam Nguyen7,12, Misha Itkin1, Catherine Hanna3, Paul Shinn1, Chase Allen3, Samuel G Michael1, Anton Simeonov1, Wenwei Huang1, Kimberly M Christian7,12, Alison Goate10, Kristen J Brennand13, Ruili Huang1, Menghang Xia1, Guo-li Ming7,9,11,12,14,15,17, Wei Zheng1,17, Hongjun Song7,9,11,12,15,17 & Hengli Tang3,17 In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ~6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development. ZIKV, a mosquito-borne flavivirus, has spread across the Western Hemisphere in the past year. First isolated in 1947 from a sentinel rhesus macaque in the Ziika Forest region of Uganda1, ZIKV remained in relative obscurity for many years until outbreaks occurred in the Pacific islands and then in the Americas in the past decade. A large outbreak started in Brazil in late 2014, and it is a growing public health concern2. Active transmission has been reported in approximately 60 countries and territories globally. Most human infections are transmitted by mosquitos; however, ZIKV can also spread directly through sexual contact3–6 and vertically from mother to fetus7–9. About 20% of ZIKV-infected individuals develop symptoms, which mostly resemble those caused by other arboviruses, such as dengue viruses or chikungunya virus. Unlike these viruses, however, ZIKV causes congenital defects, including microcephaly10,11, and is associated with Guillain–Barré syndrome, meningoencephalitis and myelitis in infected adults8,11–14. Since the recent declaration by the World Health Organization (WHO) that ZIKV is a global health concern, rapid progress has been made to understand its pathogenesis and to develop human in vitro models and animal in vivo models15–23. Following clinical observations of ZIKV in fetal brains obtained from infected women9,10, we reported that ZIKV efficiently targets human neural progenitor cells (hNPCs) and attenuates their growth15. This finding provides a potential mechanism for ZIKV-induced microcephaly as hNPCs drive the development of the cortex in the human brain. Furthermore, we and others have shown that ZIKV infection of brain organoids, which are used as three-dimen- 1National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA. 2Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China. 3Department of Biological Science, Florida State University, Tallahassee, Florida, USA. 4Department of Psychiatry and Behavioral Science, Emory University School of Medicine, Atlanta, Georgia, USA. 5Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA. 6Department of Neurology, Emory University School of Medicine, Atlanta, Georgia, USA. 7Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 8Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 9Biomedical Engineering Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 10Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 11The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 12Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 13Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 14Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 15The Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 16These authors contributed equally to this work. 17These authors jointly directed this work. Correspondence should be addressed to H.S. (), G.M. (), W.Z. (wzheng@mail. nih.gov) or H.T. (). Received 21 June; accepted 22 August; published online 29 August 2016; doi:10.1038/nm.4184 NATURE MEDICINE VOLUME 22 | NUMBER 10 | OCTOBER 2016 1101 ARTICLES F O OH N H F O F O O Caspase activity (RLU) IC50 (μM) FSS13025 0.17 MR766 0.13 PRVABC59 0.19 100 50 0 −8 −7 −6 −5 FSS13025 0.84 MR766 1.06 PRVABC59 0.45 60 40 20 Mock 0 −8 −7 −6 −5 ZIKV FSS13025 ZIKV + emricasan Mock 6 ZIKV ** 4 2 0 0 15 30 Emricasan (μM) ZIKVE CAS3 DAPI Normalized cell viability (%) © 2016 Nature America, Inc. All rights reserved. c IC50 (μM) 80 Mock −4 log(emricasan), M npg b CAS3+ cells (%) H N N H F ZIKV FSS13025 ZIKV + emricasan Mock CAS3+ cells (%) O O ZIKVE CAS3 DAPI a Emricasan 0.3 ZIKV *** 0.2 0.1 0.0 0 10 Emricasan (μM) −4 log(emricasan), M Figure 1 Emricasan suppresses cell death of ZIKV-infected human astrocytes and hNPCs in 2D monolayer cultures and in 3D brain organoids. (a) Top, chemical structure of emricasan. Bottom, dose-response curves showing the effect of emricasan treatment on caspase activity (middle) and cell viability (bottom) in SNB-19 glioblastoma cells exposed to three different ZIKV strains. Values represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating the IC50 values, and the insets in each graph report the calculated IC50 value against each strain. RLU, relative luminescence units. (b) Left, representative images of 2D monolayer cultures of forebrain-specific hNPCs immunostained for ZIKV protein (ZIKVE; green), cleaved caspase-3 (CAS3; red) and nuclei (with DAPI; gray) 72 h after ZIKV FSS-13025 exposure (with or without emricasan treatment) or m (...truncated)