ja8b07911_si_005.xlsx (154.7 kB)
A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification
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posted on 2018-12-06, 00:00 authored by Christopher
M. Browne, Baishan Jiang, Scott B. Ficarro, Zainab M. Doctor, Jared L. Johnson, Joseph D. Card, Sindhu Carmen Sivakumaren, William M. Alexander, Tomer M. Yaron, Charles J. Murphy, Nicholas P. Kwiatkowski, Tinghu Zhang, Lewis C. Cantley, Nathanael S. Gray, Jarrod A. MartoDespite 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.
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PKN 3 substratescovalent pharmacologic inhibitorsmass spectrometry strategiescovalent inhibitorsoptimized proteomic platformcovalent drug programspharmacologically addressable cysteine-thiolsCDK inhibitor THZ 1acid level readoutstructure-based drug design programsdose-dependent covalent modificationproteome-wide binding profilekinase PKN 3.novel chemoproteomic approachCITe-IdProteome-Wide Covalent Inhibitor Target-Site IdentificationJZ 128
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