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

J Am Chem Soc. 2019 Jan 9;141(1):191-203. doi: 10.1021/jacs.8b07911. Epub 2018 Dec 20.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Cyclin-Dependent Kinase-Activating Kinase
  • Cyclin-Dependent Kinases / antagonists & inhibitors
  • Cyclin-Dependent Kinases / chemistry
  • Dose-Response Relationship, Drug
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Proteomics*

Substances

  • Protein Kinase Inhibitors
  • protein kinase N
  • Protein Kinase C
  • Cyclin-Dependent Kinases
  • Cyclin-Dependent Kinase-Activating Kinase