Discovery of a Novel Respiratory Syncytial Virus Replication Inhibitor

Abstract

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. 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.

Keywords: inhibitor; replication; respiratory syncytial virus.

FIG 1

Discovery of triazole-1 as an RSV inhibitor from high-throughput screening. (A) The screen cascade for identifying RSV replication inhibitors using CPE assay-based high-throughput screening and the structure of Triazole-1. (B) Triazole-1 inhibition of RSV subtypes A and B in CPE assays and the effect of triazole-1 on cell viability. (C) Triazole-1 inhibition of the F_K394R variant virus in the CPE assay. Anti-RSV activity of the compounds is expressed as a percentage of viable cells compared with dimethyl sulfoxide (DMSO)-treated cells Each data point represents mean ± standard deviation from three replicates. The experiments were repeated in three independent experiments.

FIG 2

Time of addition experiment of triazole-1. HEp-2 cells were infected with RSV (multiplicity of infection [MOI] = 5). Triazole-1, YM53403, and quinoline-2 were added at the indicated times. At 24 h postinfection, viral yield in cell culture supernatant was determined by plaque assays and expressed as log₁₀ PFU/ml. Each data point represents mean ± standard deviation from three replicates. The experiments were repeated in two independent experiments.

FIG 3

Triazole-1 inhibits RSV replication. (A) The effect of triazole-1 on RSV N/P/L/M2-1 protein-driven RSV minigenome transcription. Luciferase assays were performed on HEp-2 cells, which were transfected with a minigenome (Minigenome-Luc) construct or T7 promoter construct (T7pro-Luc) together with pcDNA constructs expressing N, P, L, and M2-1 proteins, and then treated with DMSO or triazole-1 (0.1 μM, 1 μM, and 10 μM). The graph shows the percentage of luciferase activity relative to DMSO (DMSO is set as 100%). Each data point represents mean ± standard deviation from three replicates. The experiments were repeated in two independent experiments. (B) Confocal microscopy of RSV-infected HEp-2 cells that were treated with DMSO or 10 μM triazole-1 and stained for the RSV replication complex (green) and nuclei (blue). The top panel shows the RSV replication foci in DMSO-treated cells, and the bottom panel shows the RSV replication foci in triazole-1-treated cells. Bars, 10 μM.

FIG 4

The effect of purine or pyrimidine on anti-RSV activity of triazole-1. Anti-RSV activity of triazole-1 was evaluated in RSV CPE assays in the presence of 25 μM uridine or adenosine. Anti-RSV activity of the compounds is expressed as a percentage of viable cells compared with DMSO-treated cells. Each data point represents mean ± standard deviation from three replicates. The experiments were repeated in two independent experiments.

FIG 5

Identification of the T1684A mutation on L polymerase in the triazole-1-resistant variant. (A) Triazole-1 and quinoline-2 inhibition of the DMSO-p9 and Triazole-1-p9 viruses. (B) YM-53403 and BI compound D inhibition of the DMSO-p9 and Triazole-1-p9 viruses. Anti-RSV activity of the compounds is expressed as percentage of viable cells compared with DMSO-treated cells. Each data point represents mean ± standard deviation from three replicates. The experiments were repeated in three independent experiments.

FIG 6

Effect of triazole-1 on RSV minigenome transcription mediated by wild-type L protein or the T1684A mutant. Luciferase assays were performed on HEp-2 cells transfected with a minigenome construct (Minigenome-Luc) or T7 promoter construct (T7pro-Luc) together with pcDNA constructs expressing N, P, L (wild-type or T1684A mutant), and M2-1 proteins and treated with 0.316 μM to 10 μM triazole-1. The graph shows the relative luciferase activity compared with DMSO. Each data point represents mean ± standard deviation from three replicates. The experiments were repeated in three independent experiments.