Development and Application of a High-Throughput Fluorescence Polarization Assay to Target Pim Kinases.

Pim proteins consisting of three isoforms (Pim-1, Pim-2, and Pim-3) are a family of serine/threonine kinases that regulate fundamental cellular responses such as cell growth, differentiation, and apoptosis. Overexpression of the Pim kinases has been linked to a wide variety of hematological and solid tumors. Thus, all three Pim kinases have been studied as promising targets for anticancer therapy. Here, we report on the development and optimization of an immobilized metal ion affinity partitioning (IMAP) fluorescence polarization (FP) method for Pim kinases. In this homogeneous 384-well assay method, fluorescein-labeled phosphopeptides are captured on cationic nanoparticles through interactions with immobilized trivalent metals, resulting in high polarization values. The apparent Km values for adenosine triphosphate (ATP) were determined to be 45 ± 7, 6.4 ± 2, and 29 ± 5 μM for Pim-1, Pim-2, and Pim-3, respectively. The assay yielded robustness with Z'-factors of >0.75 and low day-to-day variability (CV <5%) for all three Pim kinases. The IMAP FP assay was further validated by determining IC50 values for staurosporine and a known Pim inhibitor. We have also used an IMAP FP assay to examine whether compound 1, an ATP mimetic inhibitor designed through structure-based drug design, is indeed an ATP-competitive inhibitor of Pim kinases. Kinetic analysis based on Lineweaver-Burk plots showed that the inhibition mechanism of compound 1 is ATP competitive against all three Pim isoforms. The optimized IMAP assay for Pim kinases not only allows for high-throughput screening but also facilitates the characterization of novel Pim inhibitors for drug development.