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

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. Marto

Research output: Contribution to journalArticlepeer-review

56 Scopus citations


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.

Original languageEnglish
Pages (from-to)191-203
Number of pages13
JournalJournal of the American Chemical Society
Issue number1
StatePublished - 9 Jan 2019
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2018 American Chemical Society.


*[email protected] *[email protected] ORCID Christopher M. Browne: 0000-0002-6250-8470 Nathanael S. Gray: 0000-0001-5354-7403 Author Contributions J.A.M. and N.S.G. conceived the concept and research strategy of the project. C.M.B. performed the majority of experiments. Z.M.D, S.B.F, and B.J. contributed equally to the manuscript. Z.M.D. performed functional assays with cell lines and provided expertise with cell culture and genetics. S.B.F. provided critical expertise and advice on mass spectrometry and proteomics. B.J. and T.Z. synthesized all compounds. J.L.J., T.Y, C.J.M., and L.C.C. performed kinase motif analysis and provided biostatistical analysis for it. J.DC.and S.C.S. performed target engagement assays. W.M.A. provided programming expertise. N.P.K. provided expertise and advice toward the entire project and for profiling kinase inhibitors in particular. C.M.B., J.A.M., N.P.K., and N.S.G. cowrote the manuscript. All authors edited the manuscript. Notes L.C.C. is a founder and member of the SAB and holds equity in Agios Pharmaceuticals and Petra Pharmaceuticals. L.C.C. is also a member of the BOD of Agios and an observer on the BOD of Petra. Petra provides partial support for his laboratory. Both Agios and Petra are developing drugs for cancer therapies. Jarrod Marto is a member of the scientific advisory board (SAB) of 908 Devices. N.G. is a founder, SAB member and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Her2llc, Deerfield, and Sanofi. N.G., T.Z., and N.K. are inventors on a patent application covering chemical matter in this publication owned by Dana−Farber Cancer Institute. The authors acknowledge generous support from NIH grants R21 CA178860 (to J.A.M.), R21CA188881 (to J.A.M.), R35 CA197588 (to L.C.C.), R01 CA179483 (to N.S.G.), and U54 HL127365 (to N.S.G.), T32 GM007306 (to Z.D.), the Chleck Foundation (to Z.D.), and by the Dana−Farber Strategic Research Initiative (to J.A.M.).

FundersFunder number
Chleck Foundation
National Institutes of HealthR35 CA197588, R21CA188881, T32 GM007306, R01 CA179483, U54 HL127365
National Cancer InstituteR21CA178860
Astellas Pharma US
Takeda Pharmaceuticals U.S.A.


    Dive into the research topics of 'A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification'. Together they form a unique fingerprint.

    Cite this