[DOT 2021] Generating Selective Hits for PIM3 Kinase Inhibitors: Example of a Comprehensive Screening Cascade

[DOT 2021] Generating Selective Hits for PIM3 Kinase Inhibitors: Example of a Comprehensive Screening Cascade
File Name/Number:
DOT 2021

Year:
2021


The Proviral Insertion in Murine 3 (PIM3) proto-oncogene is a serine/threonine protein kinase playing critical roles in cancers and energy metabolism. Identification of new PIM kinase inhibitor scaffolds is therefore highly valuable for new therapeutic approaches in oncology and metabolic disorders.

Here we present a comprehensive and robust screening cascade to rapidly identify fully characterized PIM3 inhibitors from Eurofins Discovery’s Compound Libraries.

For this purpose, we produced in-house high-quality recombinant active PIM3 kinase to develop and validate a robust and high-throughput ADP-Glo™ primary assay used for a pilot screen. The high-quality data sets obtained (Z’ = 0.82 ± 0.06, n=44; SNR = 39.6 ± 6.4, n=44; staurosporine (reference compound) on quality control plates pIC50 = 8.39 ± 0.13, n=7) enabled the identification of a first series of active hits , which are in the process to be confirmed, with a PIM3 cellular NanoBRET™ target engagement assay (Z’ > 0.6, SNR 5-6), and mass spectrometry (MS) assay monitoring of site-specific phosphorylation events.

In future, these confirmed hits will be further qualified during a Hit-to-Lead program using Eurofins Discovery’s proprietary KinaseProfiler™ and KINOMEscan® profiling platforms in order to investigate their selectivity against the whole kinome (over 500 kinases), as well as their selectivity against PIM1 and PIM2 kinases (IC50 & KD determination).

In this poster, we describe the comprehensive screening cascade designed for this project together with the results of the pilot studies.