10.1074/jbc.M609919200. no currently available vaccines or approved therapeutics for this computer virus, there is a crucial unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed TG 100572 a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral access or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses recognized single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C computer virus (HCV). Taken together, SBI-0090799 represents a encouraging lead candidate for the development of an antiviral treatment against ZIKV contamination for the mitigation of severe complications and potential resurgent outbreaks of the computer virus. IMPORTANCE This study explains the elucidation TG 100572 of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika computer virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C computer virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been a part of a curative strategy for HCV. genus in the family and is related to dengue computer virus (DENV), yellow fever computer virus (YFV), Japanese encephalitis computer virus (JEV), Kunjin computer virus (KUNV), and West Nile computer virus (WNV) (6, 7). The TG 100572 11-kb genome codes for any polyprotein that is proteolytically cleaved by cellular and viral proteases into three structural (capsid protein, premembrane/membrane protein, and envelope glycoprotein) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (8). The structural TG 100572 proteins constitute the components of the computer virus particle, TG 100572 protecting the viral genome and representing a major target for antibody-mediated immune responses, while most of the nonstructural proteins are involved in other functions, including restriction of immune response and viral RNA replication (8, 9). RNA replication of flaviviruses occurs in virus-induced vesicle-like membrane rearrangements corresponding to endoplasmic reticulum (ER) invaginations and designated vesicle packets (VPs) (10, 11). These structures contain viral nonstructural proteins and intermediates of viral genome replication (double-stranded RNA) and are the sites where viral RNA replication most likely takes place (10, 11). Nonstructural protein 4A (NS4A) has been explained to induce membrane curvatures and, thus, is considered a crucial initiator of membrane rearrangements required for Rabbit polyclonal to FTH1 assembly of the viral replicase complexes (12,C16). In this report, using a large-scale high-content screening approach, we recognized (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a specific inhibitor of ZIKV replication. It was found to exert antiviral activity through blocking the formation of the vesicle packets. Single point mutations in the N-terminal region of NS4A conferred resistance to the drug and restored formation of vesicle packets, suggesting a mechanism of action mediated by the role of NS4A as an initiator of the formation of the membranous ZIKV replication compartment. Thus, blocking the biogenesis of these membranous replication factories represents a encouraging antiviral approach against ZIKV. RESULTS To identify antivirals targeting ZIKV contamination, we performed a high-content phenotypic screen of 51,520 chemical compounds covering diverse chemical structures and chemotypes in Huh-7.5 cells infected with the.