Huib32: A Potent and Selective USP32 Inhibitor Modulating Endosomal Processes and Advancing Cell-Permeable USP32 Probes

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Deubiquitinating enzymes (DUBs) are pivotal regulators of ubiquitination, a vital post-translational modification essential for cellular processes. Dysregulated DUB activity disrupts cellular homeostasis, driving diseases like cancer and neurodegeneration. Ubiquitin-specific protease 32 (USP32) has emerged as a promising therapeutic target due to its role in endosomal and autophagosomal dynamics and its association with breast, ovarian, and lung cancers. Here, we describe Huib32 ( H uman de U biquitinase Inhi b itor 32) as a USP32 inhibitor. Cyanimide-containing Huib32 potently and selectively inhibits USP32 by covalently binding to the active site Cys743 in vitro and in cells, enhancing substrate ubiquitination, altering endosomal morphology, and mimicking USP32 depletion. Additionally, we present two activity-based probes (ABPs), Huib32*1 and Huib32*2 , which enable precise detection of USP32 activity and confirm probe selectivity via mass spectrometry. Together, Huib32 and its probes represent a unique approach for targeting USP32, offering new research tools and potential therapeutic avenues for cancer and disorders involving endocytic trafficking.

Authors 12

1. Stephan Scherpe Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

2. Vito Pol Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

3. Raymond Kooij Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

4. Adán Pinto-Fernández

   University of Oxford

   Affiliation as printed

   Chinese Academy for Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom

   Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom

5. Rayman T.N. Tjokrodirijo Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands

6. Darragh Patrick O'Brien

   University of Oxford

   Affiliation as printed

   Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom

7. Iolanda Vendrell

   University of Oxford

   Affiliation as printed

   Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom

8. Dharjath S. Hameed Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

9. Peter A. van Veelen Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands

10. Benedikt Mathias Kessler

    University of Oxford

    Affiliation as printed

    Chinese Academy for Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom

    Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom

11. Paul P. Geurink Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

    Leiden University Medical Center

    Affiliation as printed

    Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

12. Ayşegül Sapmaz Aachen Department of Cell and Chemical Biology Division of Chemical Biology and Drug Discovery

    Leiden University Medical Center

    Affiliation as printed

    Department of Cell and Chemical Biology, Division of Chemical Biology and Drug Discovery, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands