Tuesday, December 11, 2018

A Chemoproteomic Strategy for Direct and Proteome-wide Covalent Inhibitor Target-site Identification

Christopher Michael BrowneBaishan JiangScott B FicarroZainab M DoctorJared Lee JohnsonJoseph D CardSindhu Carmen SivakumarenWilliam M AlexanderTomer YaronCharles Joseph MurphyNicholas P KwiatkowskiTinghu ZhangLewis C. CantleyNathanael S Gray, and Jarrod A. Marto

Journal of the American Chemical Society 2018
DOI: 10.1021/jacs.8b07911

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.

Oncogenic KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

Minh V. Huynh, Derek Parsonage, Tom E. Forshaw, Venkat R. Chirasani, G. Aaron Hobbs, Hanzhi Wu, Jingyun Lee, Cristina M. Furdui, Leslie B. P...