Nucleoside Inhibitors of Zika Virus

The Journal of Infectious Diseases BRIEF REPORT Nucleoside Inhibitors of Zika Virus Luděk Eyer,1 Radim Nencka,2 Ivana Huvarová,1 Martin Palus,1,3,4 Maria Joao Alves,5 Ernest A. Gould,6 Erik De Clercq,7 and Daniel Růžek1,3,4 1 Department of Virology, Veterinary Research Institute, Brno, 2Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, 3Institute of Parasitology, Biology Center of the Czech Academy of Sciences, and 4Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic; 5National Institute of Health Dr Ricardo Jorge–CEVDI/INSA, Águas de Moura, Portugal; 6Aix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 Emergence des Pathologies Virales, France; and 7Rega Institute for Medical Research, KU Leuven, Belgium On 1 February 2016, the World Health Organization declared a public health emergency of international concern regarding neurological disorders associated with the rapid emergence of Zika virus (ZIKV) in Oceania and the Americas [1]. Previously, ZIKV was a relatively neglected mosquito-borne arbovirus in the genus Flavivirus, family Flaviviridae. ZIKV infections have been known in Africa and Asia since the 1940s. During the last years, the virus caused several outbreaks of infection across Oceania [2]. In May 2015, a ZIKV outbreak was first reported in Brazil, and within months most countries in Latin America and the Caribbean had reported local transmission of the virus [1, 3]. Until recently, ZIKV was associated with benign infection in humans, with common symptoms that include fever, rash, joint pain, and conjunctivitis. The illness was usually mild, with symptoms lasting for several days. However, there is growing evidence in Oceania and the Americas that ZIKV can cause devastating brain birth defects, most prominently Received 7 April 2016; accepted 24 May 2016; published online 27 May 2016. Correspondence: D. Růžek, Veterinary Research Institute, Hudcova 70, Brno CZ-62100, Czech Republic (). The Journal of Infectious Diseases® 2016;214:707–11 © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail . DOI: 10.1093/infdis/jiw226 METHODS Vero cells (ATCC CCL-81, African Green Monkey, adult kidney, epithelial) were used for determining ZIKV multiplication, for antiviral assays, and for conducting plaque assays. The cells were cultured at 37°C in 5% CO2 in Dulbecco′s modified Eagle′s medium supplemented with 10% fetal bovine serum and a 1 % mixture of antibiotics (Sigma-Aldrich, Prague, Czech Republic). ZIKV strain MR766 ( prototype strain, isolated from blood from experimental forest sentinel rhesus monkey, Uganda, 1947; GenBank accession no. AY632535) from the collection of the National Institute of Health Dr Ricardo Jorge– CEVDI/ INSA (Águas de Moura, Portugal) and from the European Virus Archive was used for evaluation of the antiviral activity of the test compounds. The virus was passaged >100 times in suckling mice and/or in Vero cells prior to this study. The following nucleoside analogues were purchased: 2′-Cmethyl–, 2′-O-methyl–, and 3′-O-methyl–substituted nucleosides, 3′-deoxynucleosides, sofosbuvir, and 6-azauridine from Carbosynth (Compton, United Kingdom); 4′-azidocytidine, balapiravir, and RO-9187 from Medchemexpress (Stockholm, Sweden); neplanocin A from Cayman Chemical (Ann Arbor, Michigan); 3-deazaneplanocin A from Selleckchem (Munich, Germany); mericitabine from ChemScene (Monmouth Junction, New Jersey); PSI-6206 from ApexBio (Boston, Massachusetts); and tubercidin, toyocamycin, sangivamycin, ribavirin, and 2′deoxynucleosides from Sigma-Aldrich (Prague, Czech Republic); rigid amphipathic. The test compounds were solubilized BRIEF REPORT • JID 2016:214 (1 September) • 707 There is growing evidence that Zika virus (ZIKV) can cause devastating infant brain defects and other neurological disorders in humans. However, no specific antiviral therapy is available at present. We tested a series of 2′-C- or 2′-O-methyl–substituted nucleosides, 2′-C-fluoro-2′-C-methyl–substituted nucleosides, 3′-O-methyl–substituted nucleosides, 3′-deoxynucleosides, derivatives with 4′-C-azido substitution, heterobase-modified nucleosides, and neplanocins for their ability to inhibit ZIKV replication in cell culture. Antiviral activity was identified when 2′-C–methylated nucleosides were tested, suggesting that these compounds might represent promising lead candidates for further development of specific antivirals against ZIKV. Keywords. Zika virus; flavivirus; nucleoside analogue; antiviral; therapy. microcephaly [4], and neurological disorders in adults, including Guillain-Barré syndrome, meningoencephalitis [5], and myelitis [6]. At present, neither vaccination nor specific antiviral therapies are available to prevent or treat ZIKV infections, making a search for effective viral inhibitors an international research priority. Nucleoside analogues are an important class of antiviral agents now commonly used as therapeutics for human viral infections, including AIDS and hepatitis B virus, cytomegalovirus, and herpes simplex virus infections [7]. These agents are generally safe and well tolerated since they target viral but not cellular polymerases and cause premature termination of viral nucleic acid synthesis [7]. In the present study, we evaluated 2′-C- and 2′-O-methyl–substituted nucleosides, 2′-C-fluoro-2′-C-methyl– substituted nucleosides, 3′-O-methyl–substituted nucleosides, 3′-deoxynucleosides, derivatives with a 4′-C-azido substitution, heterobase-modified nucleosides, and neplanocins for their ability to inhibit ZIKV replication in cell culture, with the objective of identifying promising lead candidates for further development of specific antivirals against ZIKV. RESULTS A series of 29 nucleoside analogues (Supplementary Figure 1) was tested at a concentration of 50 µM for their ability to inhibit CPE mediated by ZIKV infection on Vero cells. Inhibition of ZIKV-induced CPE was monitored by light microscopy from days 1 to 5 after infection and quantified at the end of the experiment, using the colorimetric cell death in an in vitro assay. Five of the nucleoside analogues, 7-deaza-2′-C-methyladenosine (7-deaza-2′-CMA), 2′-C-methyladenosine (2′-CMA), 2′708 • JID 2016:214 (1 September) • BRIEF REPORT C-methylcytidine (2′-CMC), 2′-C-methylguanosine (2′-CMG), and 2′-C-methyluridine (2′-CMU), were found to inhibit ZIKVmediated CPE in cell culture at a concentration of 50 µM and to reduce significantly the cell death ratio in the test wells when compared with mock-treated ZIKV-infected cells (P < .05, by a 2-tailed Student t test). All other compounds had no or little effect on ZIKV-induced CPE and cell death. Tubercidin, toyocamycin, and sangivamycin were found to be cytotoxic, causing cell death in all cells at micromolar concentrat (...truncated)