Comprehensive and Fast High Throughput Screening Cascade to Generate Highly Selective Hits for PIM3 Kinase

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 & confirmation cascade fully designed by our HTS and project management experts to rapidly ( < 6 months) identify selective PIM3 kinase inhibitors from our proprietary libraries (70K+ compounds sub-set, nanodispensed assay ready plates, Echo®). This cascade integrates all phases of assay development for primary screening (ADP-Glo® assays on a recombinant active PIM3 Kinase produced by Eurofins DiscoverX™, screening performed on PHERAstar® FSX) as well as the confirmation and ranking of the identified PIM3 kinase hits with mass spectrometry-based (RapidFire-Q-TRAP 5500 mass spectrometer (RF-MS)) and cellular NanoBRET™ target engagement (TE) based assays.

Recombinant active PIM3 kinase was produced in-house to develop a robust high throughput ADP-Glo primary assay used to screen Eurofins Discovery’s Diversity Focused and Hinge Binder libraries (70K+ compounds). High-quality data sets were obtained (Z’ = 0.86 ± 0.05, SNR = 35.1 ± 2.7) with a hit rate of 1.54% enabling the selection of 1,000 hits (946 confirmed in ADP-Glo dose response curves, exhibiting pIC₅₀ from 3.42 to 7.72). From the 946 confirmed hits, 140 compounds were selected and confirmed in both PIM3 cellular NanoBRET TE assay (Z’ = 0.83 ± 0.06), and a mass spectrometry assay monitoring of site-specific phosphorylation events (Z’ > 0.6); leading to the confirmation of 67 hits in NanoBRET assay (pIC₅₀ from 5.55 to 6.71). Further triaging will take place employing Eurofins Discovery's proprietary KinaseProfiler™ kinase activity and KINOMEscan® active site-directed competition binding assay platforms, together with RF-MS assay, to investigate their selectivity against PIM1 & PIM2 kinases (IC₅₀ & KD) as well as the whole kinome.

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. This study also demonstrates the importance of having access to high-quality sources of compound libraries, purified proteins, various technologies amenable to miniaturization and HTS, which, combined with project management and scientific skills, lead to successful high performance hit finding campaigns.