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

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.