title: 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.
authors: Discovery Sciences, AstraZeneca, Macclesfield, Cheshire, UK helen.plant@astrazeneca.com.
authors: Helen
authors: H
authors: Plant
authors: Discovery Sciences, AstraZeneca, Macclesfield, Cheshire, UK.
authors: Clare
authors: C
authors: Stacey
authors: Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors: Choi-Lai
authors: CL
authors: Tiong-Yip
authors: Discovery Sciences, AstraZeneca, Macclesfield, Cheshire, UK.
authors: Jarrod
authors: J
authors: Walsh
authors: Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA.
authors: Qin
authors: Q
authors: Yu
authors: Discovery Sciences, AstraZeneca, Macclesfield, Cheshire, UK.
authors: Kirsty
authors: K
authors: Rich
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D055808:Drug Discovery / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008826:Microbial Sensitivity Tests; D012136:Respiratory Syncytial Viruses / Q000187:drug effects* / Q000502:physiology; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents
keywords: antiviral drugs; cell-based assays; respiratory diseases; ultra-high-throughput screening