Internationale Zeitschrift für biomedizinisches Data Mining
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Abstrakt

A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification

Jarrod A Marto

Abstract

Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a serious hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide an immediate, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a completely unique chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents together with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as replacement selective covalent inhibitor of PKN3. Using JZ128 as a search compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a strong complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.