Abstract 4953: Identification of direct target engagement biomarkers for kinase drug discovery using quantitative mass spectrometry: PDK1 case study

Abstract

Abstract The human genome encodes some 500 protein kinases and an estimated twenty percent of drugs currently in development target this enzyme class. Because kinase inhibitors frequently bind to the conserved ATP-binding pocket, in vitro kinase selectivity profiling of drug candidates are an integral part of the small-molecule drug discovery process. Likewise, the co-development of pharmacodynamic (PD) biomarkers is critical to drug development as they link drug-target inhibition in cells with biological activity. As such PD biomarkers are essential for the interpretation of in vivo PK/PD/efficacy studies, which ultimately inform medicinal chemistry. Here, we present a universal mass-spectrometry-based approached to discover kinase drug-target engagement biomarkers using the phosphoinositide dependent protein kinase 1 (PDK1) as an example. Western blot analysis using a commercial available phosphosite-specific PDK1 antibody (Ser241) revealed that classical, ATP-competitive PDK1 inhibitors do not dynamically impact PDK1 T-loop phosphorylation in cells, despite cell biochemical inhibition of phospho-AKT(Thr308), a bona fide PDK1 substrate phosphorylation site. However, because receptor tyrosine kinases (RTKs), lipid kinases and other pathways often signal via AKT, the monitoring of phospho-AKT as a PD biomarker for PDK1 is not sufficient for delineating the specificity of small-molecule inhibitors and ascertain the degree of PDK1 target engagement in cells. Here, we used stable isotope labeling by amino acids (SILAC) in cell culture combined with immunoprecipitation of PDK1 protein from cells for quantitative mass-spectrometry-based identification of phosphorylation sites modulated by PDK1 inhibitor treatment. We mapped 10 Serine/Threonine phosphorylation sites on PDK1 including several novel phosphorylation sites. Of these, two PDK1 phosphorylation sites (Ser410 and Thr513) were modulated by pharmacological PDK1 inhibition and the degree of dephosphorylation correlated with inhibitor potency in PDK1 enzymatic assays. By contrast, pharmacological inhibition of PDK1 did not quantitatively modulate PDK1 T-loop phosphorylation (Ser241) consistent with the initial western blot analysis. Altogether, our study provides proof-of-concept for identifying novel kinase target engagement PD biomarkers for PDK1, complementing pathway biomarkers such p-AKT and p-S6RP, which are modulated broadly by PI3K-pathway targeting agents including inhibitors of PI3K/mTOR and RTKs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4953.