Abstract
Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.
Highlights
Drug development remains an expensive, uncertain and challenging endeavor
The pharmaceutical industry is looking for ways to improve the probability of success for each drug development candidate by selecting the best compounds, and drugspecific biomarkers to precisely measure the level of target inhibition and identify the patients likely to benefit from the drug as early as possible [4]
We reported that treatment of cells with classical, ATP-competitive PDK1 kinase inhibitors do not result in decreased PDK1 T-loop autophosphorylation levels eliminating this phosphorylation site (p-PDK1Ser241) as a candidate target engagement biomarker for PDK1 inhibitor development
Summary
Drug development remains an expensive, uncertain and challenging endeavor. The cost from discovery to regulatory approval of a novel drug averages about $900 million out of pocket, with a capitalized cost of ,$1.78 billion, and the process typically takes up towards 13 years [1,2,3]. The pharmaceutical industry is looking for ways to improve the probability of success for each drug development candidate by selecting the best compounds, and drugspecific biomarkers to precisely measure the level of target inhibition and identify the patients likely to benefit from the drug as early as possible [4]. Some of the guiding principles underlying biomarker-driven drug discovery and development have been conceptualized through a series of sequential questions: Are there active concentrations of drug in plasma, blood and tissue? While pharmacokinetic (PK) measurements of drug concentrations in plasma are routine, establishing whether a drug interacts with its molecular target (i.e. inhibition/activation) sufficiently to obtain a biological response has proven to be a challenging endeavor
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