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.