DiscussionThe goal of treating diabetes is to restore regulated insulin secretion. Accili et al have demonstrated that ablation, or inhibition, of FoxO1 results in generation of functional insulin-producing cells in the mouse gut and in human iPS cells-derived gut organoid cultures [1,2]. Our work focuses on the identification of small molecule inhibitors of FoxO1 activity. To achieve this, we developed reporter gene assays using large scale transiently transfected assay-ready cells. A limited 130k compound screen was conducted resulting in the identification of several chemotypes with low uM potency and ≥ 10-fold selectivity against key selectivity target FoxoA2 but with limited SAR. Building on this success, a 1 million compound HTS was initiated to increase chemical diversity. To achieve this scale of screening, minor refinements in the cell generation process and assay were initiated resulting in reduced reagent generation costs and improved assay performance. We were able to generate 3.7E+10 assay-ready cells in 7 weeks and complete the primary screen in 5 weeks demonstrating the feasibility of using transiently transfected assay-ready cells in a large HTS for a novel and unprecedented target. More importantly, these efforts have identified new chemical series that have the potential to benefit the project.