High Throughput Screening Assay to Identify Modulators of IL-17 Expression.

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.

Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors.

NR1/NR2A is a subtype of N-methyl-d-aspartate receptors (NMDARs), which are glutamate and glycine-gated Ca(2+)-permeable channels highly expressed in the central nervous system. A high-throughput screening (HTS) campaign using human osteosarcoma (U-2 OS) cells transiently transduced with NR1/NR2A NMDAR subunits, tested in a specifically designed fluorometric imaging plate reader (FLIPR)/Ca(2+) assay, identified sulfonamide derivative series, exemplified by 3-chloro-4-fluoro-N-[(4-{[2-(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide (compound 1) and thiodiazole derivative N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (compound 13) as novel NR1/NR2A receptor antagonists. Compounds 1 and 13 displayed submicromolar and micromolar potency at NR1/NR2A receptor, respectively, although they did not show activity at NR2B-containing receptor up to 50 µM concentration. Addition of 1 mM glycine, but not 1 mM l-glutamate, was able to surmount compound 1 and 13 inhibitory effects in FLIPR NR1/NR2A assay. However, compounds 1 and 13 displaced a glutamate site antagonist [(3)H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid ([(3)H]CGP 39653) to a greater extent than the glycine site antagonist [(3)H]3-[(E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid ([(3)H]MDL 105,519), in rat brain cortex binding assay. Results of FLIPR cell-based, electrophysiological, and biochemical binding assays suggest that compounds 1 and 13 are the prototypes of novel classes of NMDAR ligands, which to the best of our knowledge are the first selective antagonists at NR1/NR2A over NR1/NR2B receptor, and might constitute useful tools able to elucidate the relative role of the NR2A subunit in physiological and pathological conditions.