ATP COMPETITION OF PI3K INHIBITORS USING PHOSPHOCELLULOSE PAPER APPROACH

Author(s): Mahesh Yanamandra, Labanyamoy Kole, Archana Giri and Sayan Mitra

Abstract

Profiling small molecule inhibitors against lipid kinases are challenging owing to fact that lipid substrates/products are difficult to capture on membrane filters unlike protein kinase substrates. We have recently reported the development of an inexpensive & robust methodology for profiling lipid kinase inhibitors in a phosphocellulose plate format. This is a medium throughput 96 well format assay & validated with major PI3K isoforms using specific inhibitors. This unique assay methodology was further extended to inhibitor characterization and evaluated the mechanism of action (MOA) of PI3K inhibitors (LY294002 and TGX-221) with respect to ATP competitiveness against PI3 kinase β. This robust method can be easily adapted in any lipid kinase drug discovery program for compound profiling & in-depth mechanistic applications without having any potential artifacts.

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