A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells
Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a β-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 μM, 300 μM, and 100 μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 μM, 6.1 μM, and 3.4 μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.
Additional Information© 2021 Elsevier Masson SAS. Received 22 November 2020, Revised 16 December 2020, Accepted 28 December 2020, Available online 2 January 2021. We thank M. S. Cohen, J. Taunton, and K. Shokat for providing compounds 4,5,6, M. Smythe and C. Crews for YU101, A. M. Weissman for PYR-41, C. C. Wu for β-nitrostyrene analogues (compounds 7, 9,10,11,12), Y. Ye at NIDDK/NIH for providing yeast Cdc48 and hamster NSF plasmids. A.C.J. was supported by NIH Grant F32GM082000; A.F.G.G thanks the Natural Sciences and Engineering Research Council (NSERC) of Canada for a PGS D scholarship; R.J.D. was an HHMI Investigator, and this work was funded in part by HHMI; This work was funded in part by the NIH-NINDS (R01NS102279) to T.F.C. and NIH-NIGMS (R01GM080269) to B.M.S. We thank the anonymous reviewers for constructive criticism. Author contributions: Gang Zhang wrote the manuscript and analyzed the potential interaction between PPA and p97 by molecular docking. Shan Li and Feng Wang performed the anti-proliferative assay and proteomics study. Amanda C. Jones, Alexander F. G. Goldberg, and Scott Virgil synthesized and characterized the target compounds. Tsui-Fen Chou performed experiments in Table 1, Table 2, Table 3, Table 4; Fig. 2, Fig. 4. Amanda C. Jones, Brian M. Stoltz, Raymond J. Deshaies, and Tsui-Fen Chou conceived the project and made the major contribution in the design of the initial work. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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