Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV)

Abstract

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.

Fig. 1.

Identification of 2 series with anti-RSV antiviral effect in a whole-cell high-throughput screening (HTS). (A, Right) Flow chart of the HTS for RSV in HEp-2 cells using cell protection as efficacy determination. (A, Left) Images of healthy HEp-2 cells protected by RSV604 or killed by RSV infection. (B) The isoxazole-pyrazole (6b) and the proline (14b) series identified. (C) Effect of compounds (10 μM) in HEp-2 cells at 96 h postinoculation at an MOI 0.1 measured by plaque assay. (D) Time of addition experiments. Compounds (10 μM) were added at time of addition and 4 h postinoculation to HEp-2 cells at MOI 1. Virus was quantified by qRT-PCR from total cell RNA isolated from infected cells at 12 h postinoculation. (E) Efficacy in HBEC polarized. Virus was measured by qRT-PCR from the apical surface supernatant at days 2, 3, 4, and 5 postinoculation after 5 μM compound addition.

Fig. 2.

Target identification using affinity purification. (A) Chemoproteomics tool compounds for series 1 (6a/6c) and series 2 (14a/14c). (B) The scatter plots highlighting results for 6a/6b (B) and 14b/14c (C) relative to DMSO.

Fig. 3.

ITC data for 6b and huDHODH, with measured K_d = 27 ± 6 nM.

Fig. 4.

Apoptosis in jejunum after administration of brequinar 25 mg/kg per d for 7 d