Benzimidazole analogs inhibit respiratory syncytial virus G protein function

Abstract

Human respiratory syncytial virus (hRSV) is a highly contagious Paramyxovirus that infects most children by age two, generating an estimated 75,000-125,000 hospitalizations in the U.S. annually. hRSV is the most common cause of bronchiolitis and pneumonia among infants and children under 1year of age, with significant mortality among high-risk groups. A regulatory agency-approved vaccine is not available, and existing prophylaxis and therapies are limited to use in high-risk pediatric patients; thus additional therapies are sorely needed. Here, we identify a series of benzimidazole analogs that inhibit hRSV infection in vitro with high potency, using a previously-reported high-throughput screening assay. The lead compound, SRI 29365 (1-[6-(2-furyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]methyl-1H-benzimidazole), has an EC50 of 66μM and a selectivity >50. We identified additional compounds with varying potencies by testing commercially-available chemical analogs. Time-of-addition experiments indicated that SRI 29365 effectively inhibits viral replication only if present during the early stages of viral infection. We isolated a virus with resistance to SRI 29365 and identified mutations in the transmembrane domain of the viral G protein genomic sequence that suggested that the compound inhibits G-protein mediated attachment of hRSV to cells. Additional experiments with multiple cell types indicated that SRI 29365 antiviral activity correlates with the binding of cell surface heparin by full-length G protein. Lastly, SRI 29365 did not reduce hRSV titers or morbidity/mortality in efficacy studies using a cotton rat model. Although SRI 29365 and analogs inhibit hRSV replication in vitro, this work suggests that the G-protein may not be a valid drug target in vivo.

Keywords: Antiviral; G protein; Heparin; SAR; hRSV.

Fig. 1

EC₅₀ and CC₅₀ plots for SRI 29365 in HEp-2 and A549 cells. EC₅₀ and CC₅₀ values were calculated for SRI 29365 against wt hRSV in either (A) HEp-2 cells or (B) A549 cells. In panel C, the concentration-dependent antiviral and cytotoxic responses were calculated for SRI 29365 against wt hRSV in HEp-2 cells. An eight point concentration dose response cytoprotection assay was performed (compound concentration range of 100–0.04 μg/mL) in the presence of hRSV-infected cells and the percent of cell viability (compared to uninfected, undrugged cell controls) was plotted v. test compound concentration (solid line). Each concentration point was performed in triplicate. In parallel, the same concentration range of test compound was added to uninfected cells to determine compound-induced cytotoxicity (dotted line). EC₅₀/CC₅₀ values were converted to Log M (molar) concentrations (x-axis). Dose–response curves were plotted, and EC₅₀ values were calculated using IDBS’ XLfit function 205 [y = A + ((B − A)/(1 + ((C/x)^D)))] with minimum (A) and maximum (B) parameters set at 0 and 100 respectively.

Fig. 2

Time-of-addition assays for SRI 29365. SRI 29365 (5 μM) was added to 96-well microplates containing a confluent monolayer of HEp-2 cells at timepoints of −1, 0, 1, 2, 4, 8 h, post viral infection of the cells with 1 MOI of hRSV Long strain. After a four-day incubation, cell viability was determined (compared to uninfected, undrugged cell controls) (n = 3). The graph shows the change in cell viability as the compound addition timepoint changes, and demonstrates that SRI 29365 did not reduce CPE more than 50% if added at 2 h post-infection.

Fig. 3

An A63V mutation in the hRSV G protein grants resistance to SRI 29365. Panel A shows the hRSV G protein genetic sequence trace of the wt hRSV and Panel B shows the genetic trace of the SRI 29365-resistant mutant hRSV, which shows a C → T mutation at position 500 in the sequence trace. Panel C shows the hRSV G protein domain structure and the location of the translated A63V amino acid mutation at the interface of the transmembrane domain and the virion surface domain. Panel D shows the amino acid sequence conservation of the region of the G protein transmembrane domain flanking residue A63 in RSVA Long (wild-type), RSVA Long (SRI-29365-resistant mutant), RSVA Tracy, and RSVB WI/629-12/06-07.

Fig. 4

SRI 29365 has differential efficacy in HEp-2, A549, and Vero E6 cells. Confluent monolayers of HEp-2, A549, or Vero E6 cells with either the RSVA Long strain wild-type or SRI-29356-resistant mutant in the presence of 5 μM SRI 29365, and the reduction in virus titer (TCID₅₀ reduction) was determined relative to infected (-compound) control cells (n = 4). The compound reduced wt hRSV virus TCID50 values by 300-fold, but only 10-fold in A549 cells, and less than 10-fold in Vero E6 cells. SRI 29365 reduced mutant hRSV virus TCID₅₀ values by less than 10-fold in all cell types.