title: Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay.
abstract: Recent studies have clearly underscored the association between Zika virus (ZIKV) and severe neurological diseases such as microcephaly and Guillain-Barre syndrome. Given the historical complacency surrounding this virus, however, no significant antiviral screenings have been performed to specifically target ZIKV. As a result, there is an urgent need for a validated screening method and strategy that is focused on highlighting potential anti-ZIKV inhibitors that can be further advanced via rigorous validation and optimization. To address this critical gap, we sought to test whether a cell-based assay that measures protection from the ZIKV-induced cytopathic effect could serve as a high-throughput screen assay for discovering novel anti-ZIKV inhibitors. Employing this approach, we tested the anti-ZIKV activity of previously known broad-spectrum antiviral compounds and discovered several compounds (e.g., NITD008, SaliPhe, and CID 91632869) with anti-ZIKV activity. Interestingly, while GTP synthesis inhibitors (e.g., ribavirin or mycophenolic acid) were too toxic or showed no anti-ZIKV activity (EC50> 50 μM), ZIKV was highly susceptible to pyrimidine synthesis inhibitors (e.g., brequinar) in the assay. We amended the assay into a high-throughput screen (HTS)-compatible 384-well format and then screened the NIH Clinical Compound Collection library, which includes a total of 727 compounds organized, using an 8-point dose response format with two Zika virus strains (MR766 and PRVABC59, a recent human isolate). The screen discovered 6-azauridine and finasteride as potential anti-ZIKV inhibitors with EC50levels of 3.18 and 9.85 μM for MR766, respectively. We further characterized the anti-ZIKV activity of 6-azauridine and several pyrimidine synthesis inhibitors such as brequinar in various secondary assays including an antiviral spectrum test within flaviviruses and alphaviruses, Western blot (protein), real-time PCR (RNA), and plaque reduction assays (progeny virus). From these assays, we discovered that brequinar has potent anti-ZIKV activity. Our results show that a broad anti-ZIKV screen of compound libraries with our CPE-based HTS assay will reveal multiple chemotypes that could be pursued as lead compounds for therapies to treat ZIKV-associated diseases or as molecular probes to study the biology of the ZIKV replication mechanism.
authors.affiliation: Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, KY, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Adcock
authors.affiliation: Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, KY, USA.
authors.forename: Yong-Kyu
authors.initials: YK
authors.lastname: Chu
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Golden
authors.affiliation: Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, KY, USA; Department of Microbiology and Immunology, School of Medicine, University of Louisville, KY, USA. Electronic address: dhchun01@louisville.edu.
authors.forename: Dong-Hoon
authors.initials: DH
authors.lastname: Chung
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001380:Azauridine / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D055808:Drug Discovery; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D012254:Ribavirin / Q000494:pharmacology; D054852:Small Molecule Libraries; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects; D000071244:Zika Virus / Q000187:drug effects*; D000071243:Zika Virus Infection / Q000821:virology; D011798:Quinaldines
chemical_list: D000998:Antiviral Agents; D001380:Azauridine; D001713:Biphenyl Compounds; D012254:Ribavirin; D054852:Small Molecule Libraries; C046943:brequinar; D011798:Quinaldines