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A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses

Nature Chemical Biology volume 10, pages 298–304 (2014)Cite this article

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Abstract

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1–UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non–small cell lung cancer and osteosarcoma cells. Our findings point to USP1–UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

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Figure 1: ML323 is a potent USP1–UAF1 inhibitor through a mixed inhibition mechanism.
Figure 2: Selective inhibition of USP1 by ML323 revealed by DUB profiling.
Figure 3: ML323 inhibits the cellular activity of USP1–UAF1 and sensitizes cisplatin-resistant cells to cisplatin killing.
Figure 4: The effect of inhibiting USP1–UAF1 by ML323 in the TLS and FA pathways.
Figure 5: Inhibition of USP1–UAF1 by ML323 decreases HR and SCE in U2OS cells.
Figure 6: ML323 inhibits the deubiquitination by USP1–UAF1 in the TLS and FA pathways in response to DNA damage.

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Acknowledgements

We thank M. Cordeiro-Stone (University of North Carolina at Chapel Hill) for xeroderma pigmentosum variant (XPV) human fibroblasts GM02359-hTERT (XP115LO) and Polη-complemented GM02359-hTERT (XPV + Polη) and M. Jasin (Memorial Sloan-Kettering Cancer Center) for DR-GFP U2OS cells. We also thank C. Arrowsmith (University of Toronto and Ontario Cancer Institute) for the USP21 plasmid; A. Tencer for assistance with protein purification; S. Michael and R. Jones for automation support; P. Shinn and D. van Leer for assistance with compound management; and W. Leister, H. Baker, C. Leclair and E. Fernandez for analytical chemistry and compound purification support. This work was supported by US National Institutes of Health (NIH) grant R03DA030552 and in part by NIH grant R01GM097468 to Z.Z. C.A.O. was supported by NIH training grant T32GM008550. Work in the laboratory of Y.W. was supported by NIH grant R01DK082779. T.S.D., A.S.R., D.K.L., A.S., A.J. and D.J.M. were supported by the intramural research program of the National Center for Advancing Translational Sciences and the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research (U54MH084681).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA

    Qin Liang, Ping Zhang, Mark A Villamil, Junjun Chen, Christine A Ott & Zhihao Zhuang

  2. National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA

    Thomas S Dexheimer, Andrew S Rosenthal, Hongmao Sun, Diane K Luci, Anton Simeonov, Ajit Jadhav & David J Maloney

  3. Department of Chemistry, University of California–Riverside, Riverside, California, USA

    Changjun You, Bifeng Yuan & Yinsheng Wang

  4. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi, China

    Qiuting Zhang

  5. Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, New York, USA

    Hui Xiao

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  1. Qin Liang
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Contributions

Q.L., T.S.D. and J.C. designed, performed and analyzed in vitro biochemical assays for DUBs and other proteases. T.S.D. and A.S. designed and performed the HTS assay. Q.L. and P.Z. designed, performed and analyzed cell culture experiments. A.S.R., D.K.L. and D.J.M. performed chemical synthesis and oversaw the chemistry efforts. M.A.V. performed protein purification and native gel analysis. C.Y. and B.Y. performed the cellular DNA replication assay under the guidance of Y.W. Q.Z. and H.X. performed and analyzed HDX experiments. C.A.O. generated substrates for the DUB assay. H.S. and A.J. analyzed HTS data and structure-activity relationship results. Z.Z. and D.J.M. designed the research plan and wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to David J Maloney or Zhihao Zhuang.

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A patent application on the compounds described in the paper as DUB inhibitors has been filed.

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Liang, Q., Dexheimer, T., Zhang, P. et al. A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses. Nat Chem Biol 10, 298–304 (2014). https://doi.org/10.1038/nchembio.1455

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