Abstract

Kinase discovery efforts are particularly challenging due to the conservation of active sites and high structural homology between kinases, leading to safety liabilities and sometimes suboptimal success rates in high-throughput screening (HTS) campaigns. Access to a highly purified source of active recombinant kinases and a full range of technologies amenable to automation and high throughput facilitate the design of rapid and efficient discovery cascade of kinase inhibitor compounds. In addition, the design and execution of today's screening cascades requires coordinated management of chemical libraries, as well as optimized project and resource management, to meet the current standards of modern hit finding programs to be delivered on time. We present here a comprehensive and robust hit identification cascade fully designed by our HTS and biophysics experts to rapidly (< 6 months) identify selective PIM3 kinase inhibitors from our proprietary libraries. This cascade integrates all phases of assay development for primary screening (ADP-Glo® assays on a recombinant active PIM3 Kinase produced by Eurofins DiscoverX™) as well as the confirmation and ranking of the identified PIM3 kinase hits with mass spectrometry-based (RF-MS) and cellular NanoBRET™ target engagement (TE) based assays. Hit confirmation will use our biophysical platform employing the most recent technologies: SPR, MST Spectral Shift, ITC & TSA. From our proprietary libraries of 71 000 compounds, there were 946 confirmed hits through HTS, and from these, 140 compounds were selected for their potential medicinal chemistry properties. These were tested for selectivity against PIM1 and PIM2 to reach the 16 confirmed hits distributed in 6 clusters. The binding of the 16 compounds will be confirmed by SPR (Biacore 8K+) and MST Spectral Shift (Dianthus) to give access to kinetic parameters (Kon, Koff, residence time), binding affinity (KD) and mechanisms of actions. Overall, a robust screening cascade was successfully implemented to obtain highly selective and tractable kinase inhibitors within weeks. In doing so, this ensures that subsequent Hit-to-Lead and Lead Optimization programs can rapidly and efficiently generate safe pre-clinical candidates.