%0 Generic %A Browne, Christopher M. %A Jiang, Baishan %A Ficarro, Scott B. %A Doctor, Zainab M. %A Johnson, Jared L. %A Card, Joseph D. %A Sivakumaren, Sindhu Carmen %A Alexander, William M. %A Yaron, Tomer M. %A Murphy, Charles J. %A Kwiatkowski, Nicholas P. %A Zhang, Tinghu %A Cantley, Lewis C. %A Gray, Nathanael S. %A Marto, Jarrod A. %D 2018 %T A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification %U https://acs.figshare.com/articles/dataset/A_Chemoproteomic_Strategy_for_Direct_and_Proteome-Wide_Covalent_Inhibitor_Target-Site_Identification/7495793 %R 10.1021/jacs.8b07911.s006 %2 https://acs.figshare.com/ndownloader/files/13889801 %K PKN 3 substrates %K covalent pharmacologic inhibitors %K mass spectrometry strategies %K covalent inhibitors %K optimized proteomic platform %K covalent drug programs %K pharmacologically addressable cysteine-thiols %K CDK inhibitor THZ 1 %K acid level readout %K structure-based drug design programs %K dose-dependent covalent modification %K proteome-wide binding profile %K kinase PKN 3. %K novel chemoproteomic approach %K CITe-Id %K Proteome-Wide Covalent Inhibitor Target-Site Identification %K JZ 128 %X Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major 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 a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination 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 a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful 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. %I ACS Publications