Mohamed Boudjelal*, Ana Maria Ruiz-Avendano, Gonzalo Colmenarejo, Sergio A. Senar-Sancho, Ashley Barnes and Stephen A. Harrison* Pages 804 - 819 ( 16 )
Objective: Herein we demonstrate the successful development of a new RORγt-enhanced IL-17F promoter-luciferase reporter assay and its use in a parallel high throughput screening approach, alongside a RORγt TR-FRET assay, to rapidly identify new small molecule RORγt/IL-17 inhibitors and evaluate their mode of action.
Material & Methods: We sought to identify cell-permeable small-molecule inhibitors of RORγt for rapid progression into hit-to-lead chemistry. As such, we developed the IL-17F promoter luciferase reporter assay in a stable human T-cell (Jurkat) line expressing the RORγt receptor and miniaturised it to a final volume of 8 µL in 1536 well plates for HTS use in screening a library of > 350k compounds. In parallel, a RORγt TR-FRET binding assay was employed to cross-screen the same set of compounds. This enabled the rapid identification of a small number of cell permeable RORγt antagonists showing promising activity in both assays and also highlighted a larger group of potentially very interesting hits which inhibited IL-17 reporter activity, but did not appear to modulate RORγt directly.
Result: A rigorous triaging process of the novel non-RORγt IL-17 antagonists was followed, making use of in-silico filtering, historical screening data, selectivity screening using an IL-2 reporter assay with an identical cellular background, and final profiling in a phenotypic PBMC IL-17A production assay. This resulted in the identification of a set of promising small molecule compounds which show IL-17 inhibition via potentially novel pathways.
Conclusion: This technique for the fast identification of cell-permeable IL-17 modulators acting through different mechanisms, highlights the benefits of adopting a parallel approach combining high throughput profiling of hits in multiple assay formats, with robust in-silico triaging.
IL17, RORgamma, high throughput screening, modulators, in-silico, inhibitors.
King Abdullah International Medical Research Centre, Ministry of National Guard Health Affairs, Riyadh, GlaxoSmithKline Screening and Compound Profiling, Tres-Cantos, GlaxoSmithKline Computational Chemistry, Tres-Cantos, GlaxoSmithKline Screening and Compound Profiling, Tres-Cantos, GlaxoSmithKline Biological Sciences, Stevenage, GlaxoSmithKline Clinical Pharmacology Study Science and Operations, Stevenage