Helen Plant1, Choi Lai Tiong-Yip2, Paul Sharpe1, Kirsty Rich1, Elise Gorseth2, Qin Yu2
1Discovery Sciences, AstraZeneca R&D Alderley Park, Macclesfield, Cheshire, UK; 2Infection Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, USA

Respiratory syncytial virus (RSV) infects 99% of children by the age of two and is the leading cause of infant hospitalisation and serious lower respiratory tract infections1. Identification of efficacious RSV therapeutics has been hindered by the lack of robust and convenient primary assays for high-throughput screening (HTS). We have developed a 384-well assay requiring minimal biocontainment, utilising a stable RSV replicon cell line containing a luciferase reporter gene to identify inhibitors of RSV. Using an enriched cryobank of replicon-expressing cells, combined with assay-ready compound plates, we developed a robust, automated HTS assay, with a Z’ of 0.56 + 0.07 across 150 assay plates, and a signal to background ratio >40. The assay was validated with RSV replication tool inhibitors and a compound diversity set in duplicate. The assay was used to screen 0.95 million compounds from the AstraZeneca collection at a single concentration of 10 mM. Actives were confirmed as hits in an lead generation screening cascade, generating concentration response data in the replicon assay, a cytotoxicity assay to remove false positives, and a low throughput RSV live virus ELISA. Hence, the high throughput replicon assay has been shown to identify molecules with convincing RSV inhibition, in doing so enabling true high throughput screening of this target, and increasing the prospects of discovering effective medicines targeting RSV.

Reference
1. Collins, P.L. and Melero, J.A. (2011) Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res., 162, 80-99.