High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors

Abstract

Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

Immunobiotic Lactobacillus administered post-exposure averts the lethal sequelae of respiratory virus infection

“…A number of drugs are currently in development for the treatment of RSV disease, including antivirals based on siRNA (DeVincenzo et al, 2010) and the cell fusion inhibitor GS-5806 (DeVincenzo et al, 2014). Just recently, Plant et al (2015) have described a high-throughput screening assay designed to identify agents that inhibit RSV replication. Our groups have focused on the role of the inflammation and its contribution to acute respiratory virus infection, and we have documented the efficacy of combined antiviral/anti-inflammatory approaches in studies carried out using in vivo models (Rosenberg and Domachowske, 2012; Bonville et al, 2003; Bonville et al, 2004).…”

Current Landscape of Antiviral Drug Discovery

“…Inhibition of the L-protein, the RNA-dependent RNA-polymerase of RSV, seems an ideal target given its critical function in viral replication; however, little success has been realized prior to the 2014 disclosure of the leading L-protein inhibitor AL-8176 ( vide infra ). The recent disclosure of an RSV replicon assay 22 and a screening cascade to identify RSV inhibitors in a target-agnostic fashion 23 demonstrate the continued desire both in academia and in the pharmaceutical industry to discover novel mechanisms of action for the treatment of RSV.…”

Priming of the Respiratory Tract with ImmunobioticLactobacillus plantarumLimits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM)

“…The antiviral agent ribavirin is currently recommended only for severely ill and immunocompromised children (4, 5). New antiviral agents that focus specifically on hRSV are in development (6)(7)(8), and the inflammatory responses characteristic of severe hRSV disease are also recognized as targets for therapeutic intervention (9, 10).…”

“…The antiviral agent ribavirin is currently recommended only for severely ill and immunocompromised children (4, 5). New antiviral agents that focus specifically on hRSV are in development (6)(7)(8), and the inflammatory responses characteristic of severe hRSV disease are also recognized as targets for therapeutic intervention (9, 10).Pneumonia virus of mice (PVM) is a rodent pathogen of the same family and genus as hRSV. PVM infection in inbred strains of mice reproduces many of the clinical and pathological features of the more severe forms of hRSV disease, which has facilitated the exploration of new therapeutic strategies in vivo (11,12).…”