Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models*

  1. Anton Simeonov‡4
  1. From the ‡NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20892,
  2. the §Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02215, and
  3. the Center for Structural Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294
  1. ↵3 To whom correspondence may be addressed: NCATS, National Institutes of Health, 9800 Medical Center Dr., Rockville, MD 20850. Tel.: 301-217-4021; Fax: 301-217-5736; E-mail: boxerm{at}mail.nih.gov.
  2. ↵4 To whom correspondence may be addressed: NCATS, National Institutes of Health, 9800 Medical Center Dr., Rockville, MD 20850. Tel.: 301-217-4021; Fax: 301-217-5736; E-mail: asimeono{at}mail.nih.gov.

  1. ↵1 Both authors contributed equally to this work.

  2. Edited by George DeMartino

  • ↵2 Present address: Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA 02139.

Abstract

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Footnotes

  • ↵* This work was supported by the NCATS Intramural Research Program, Molecular Libraries Initiative of the National Institutes of Health Common Fund, National Institutes of Health Grant U54 MH 084681, Alabama Drug Discovery Alliance, National Institutes of Health/NCI Grant 5P30CA13148-37, and Translational Research Grant from the Leukemia & Lymphoma Society Grant 6237-13. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • Received May 16, 2016.
  • Revision received September 4, 2016.
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  1. First Published on September 28, 2016 doi: 10.1074/jbc.M116.738567 The Journal of Biological Chemistry 291, 24628-24640.
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