Abstract
ABSTRACTA high-throughput screen of a Roche internal chemical library based on inhibition of the respiratory syncytial virus (RSV)-induced cytopathic effect (CPE) on HEp-2 cells was performed to identify RSV inhibitors. Over 2,000 hits were identified and confirmed to be efficacious against RSV infection in vitro. Here, we report the discovery of a triazole-oxadiazole derivative, designated triazole-1, as an RSV replication inhibitor, and we characterize its mechanism of action. Triazole-1 inhibited the replication of both RSV A and B subtypes with 50% inhibitory concentration (IC50) values of approximately 1 μM, but it was not effective against other viruses, including influenza virus A, human enterovirus 71 (EV71), and vaccinia virus. Triazole-1 was shown to inhibit RSV replication when added at up to 8 h after viral entry, suggesting that it inhibits RSV after viral entry. In a minigenome reporter assay in which RSV transcription regulatory sequences flanking a luciferase gene were cotransfected with RSV N/P/L/M2-1 genes into HEp-2 cells, triazole-1 demonstrated specific and dose-dependent RSV transcription inhibitory effects. Consistent with these findings, deep sequencing of the genomes of triazole-1-resistant mutants revealed a single point mutation (A to G) at nucleotide 13546 of the RSV genome, leading to a T-to-A change at amino acid position 1684 of the L protein, which is the RSV RNA polymerase for both viral transcription and replication. The effect of triazole-1 on minigenome transcription, which was mediated by the L protein containing the T1684A mutation, was significantly reduced, suggesting that the T1684A mutation alone conferred viral resistance to triazole-1.
Highlights
A high-throughput screen of a Roche internal chemical library based on inhibition of the respiratory syncytial virus (RSV)-induced cytopathic effect (CPE) on HEp-2 cells was performed to identify RSV inhibitors
The N protein binds to genomic and antigenomic RNA to form helical nucleocapsids, which serve as the templates for RNA synthesis; the L protein is an RNAdependent RNA polymerase that is responsible for synthesis of viral RNAs and mRNA capping; the P protein interacts with the N, L, and matrix protein 2-1 (M2-1) proteins and facilitates the replication complex formation; and the M2-1 protein is a transcription antitermination factor [20,21,22,23,24,25]
To identify novel small-molecular inhibitor scaffolds that target RSV replication, we designed an high-throughput screening (HTS) based on the RSV CPE assay
Summary
A high-throughput screen of a Roche internal chemical library based on inhibition of the respiratory syncytial virus (RSV)-induced cytopathic effect (CPE) on HEp-2 cells was performed to identify RSV inhibitors. RSV604 is a small-molecule inhibitor targeting the N protein, and it inhibits both viral RNA synthesis and infectivity of the released virus [26, 27]. The nucleoside analog ALS-8176, which targets the RSV polymerase but does not inhibit human RNA polymerase, showed excellent efficacy in humans in a phase IIa RSV challenge study [32] These findings demonstrated the efficacy of an RSV replication inhibitor in infected patients for the first time. YM-53403, another RSV L inhibitor, was identified through a HeLa cell-based cytopathic effect (CPE) assay screen This compound targets L polymerase, as shown by the identification of a single point mutation in the Antimicrobial Agents and Chemotherapy aac.asm.org 2
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
