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Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11

Nature Chemical Biology volume 13, pages 486–493 (2017)Cite this article

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Abstract

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

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Figure 1: Identification of a compound that inhibits proteasome function by binding directly to the active site zinc of Rpn11.
Figure 2: Structure–activity relationship optimization yields a potent and selective Rpn11 inhibitor.
Figure 3: Capzimin elicits cellular effects characteristic of proteasome inhibition.
Figure 4: Capzimin blocks cell growth and induces markers of apoptosis.
Figure 5: Capzimin has a global impact on ubiquitination site occupancy.

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Acknowledgements

We thank A. Martin (University of California, Berkeley) for the plasmid that expresses the Rpn11–Rpn8 dimer, E. Zeqiraj (University of Leeds) for providing purified BRISC complex, P. Coffino (The Rockefeller University) for providing the Rpn10-I27V13Pext plasmid, T.M. Kapoor (The Rockefeller University) for providing the RPE1 WT and BTZ-resistant cell lines, National Cancer Institute (Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis) for screening capzimin against 60 human cancer cell lines (http://dtp.cancer.gov), H. Park for testing the cell lines for mycoplasma contamination and Y. Zhang for advice on the competition studies. This work was supported by grants from the Caltech Gates Grubstake Fund and Amgen to R.J.D. and from the NIH (CA164803) to R.J.D. and S.M.C., T.Y. and S.H. were supported by the Gordon and Betty Moore Gordon Foundation, through Grant GBMF775, the Beckman Institute, and the NIH through Grant 1S10RR029594-01A1. R.J.D. is an Investigator of and was supported by the Howard Hughes Medical Institute.

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Author notes

  1. Francesco Parlati & Andrew L Mackinnon

    Present address: Present address: Calithera Biosciences Inc., San Francisco, California, USA.,

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA

    Jing Li, Francesco Parlati, Andrew L Mackinnon & Raymond J Deshaies

  2. Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California, USA

    Tanya Yakushi & Sonja Hess

  3. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA

    Christian Perez, Yuyong Ma & Seth M Cohen

  4. Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA

    Kyle P Carter & Amy E Palmer

  5. Conrad Prebys Center for Chemical Genomics at Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA

    Sharon Colayco, Gavin Magnuson, Brock Brown, Eduard Sergienko, Anthony B Pinkerton, Thomas D Y Chung & Ian Pass

  6. Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute Lake Nona, Orlando, Florida, USA

    Kevin Nguyen, Stefan Vasile, Eigo Suyama & Layton H Smith

  7. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA

    Raymond J Deshaies

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Contributions

J.L. designed, executed, and interpreted all experiments with capzimin, screened the MBP library, and drafted the manuscript. T.Y. executed and interpreted mass spectrometry proteomic analysis with capzimin. F.P. was responsible for the initial development of Rpn11 HTS substrate and fluorescence polarization assay and design and execution of Rpn11 HTS. A.L.M. triaged Rpn11 HTS hits. C.P. executed HDAC6, MMP, hCAII, and GLO1 assay and synthesis of 3021 and 3027 (capzimin). Y.M. synthesized 3027 (capzimin). S.C., G.M., B.B., E. Sergienko, and I.P. developed the HTS assay. K.N., S.V., E. Suyama, and L.H.S. performed the HTS assay. I.P., A.B.P., E. Sergienko, and T.D.Y.C. evaluated and selected hits. K.P.C. designed, executed, and interpreted experiments with zinc reporter. A.E.P. designed and interpreted experiments with zinc reporter. S.H. designed, interpreted and oversaw the mass spectrometric studies with capzimin and revised the manuscript. S.M.C. designed and oversaw capzimin syntheses as well as aided in interpretation of metalloenzyme inhibition studies. R.J.D. designed, interpreted, and oversaw the experiments for the entire study and drafted the manuscript.

Corresponding author

Correspondence to Raymond J Deshaies.

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Competing interests

R.J.D. is a founder, shareholder, consultant, and member of the Scientific Advisory Board of Cleave Biosciences, which is engaged in discovery and development of drugs that target enzymes involved in ubiquitin-dependent protein degradation. S.M.C. is a co-founder, has an equity interest, and receives income as member of the Scientific Advisory Board for Cleave Biosciences and is a co-founder, has an equity interest, and is a member of the Scientific Advisory Board for Forge Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–21 (PDF 2331 kb)

Supplementary Note

Synthesis of 3021 and 3027 (CZM) (PDF 75 kb)

Dataset 1

GlyGly Table information (XLSX 2008 kb)

Dataset 2

Protein Groups with information (XLSX 1594 kb)

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Li, J., Yakushi, T., Parlati, F. et al. Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11. Nat Chem Biol 13, 486–493 (2017). https://doi.org/10.1038/nchembio.2326

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