Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

[1]  Yuying Guo,et al.  USP21 accelerates the proliferation and glycolysis of esophageal cancer cells by regulating the STAT3/FOXO1 pathway. , 2022, Tissue & cell.

[2]  Xu Yan,et al.  Insights Into the Properties, Biological Functions, and Regulation of USP21 , 2022, Frontiers in Pharmacology.

[3]  Y. Tong,et al.  On the Study of Deubiquitinases: Using the Right Tools for the Job , 2022, Biomolecules.

[4]  C. Arrowsmith,et al.  Validating Small Molecule Chemical Probes for Biological Discovery. , 2022, Annual review of biochemistry.

[5]  Guoxiang Jiang,et al.  USP21 regulates Hippo signaling to promote radioresistance by deubiquitinating FOXM1 in cervical cancer , 2021, Human Cell.

[6]  Laura F. Dagley,et al.  Oligomerization‐driven MLKL ubiquitylation antagonizes necroptosis , 2021, The EMBO journal.

[7]  I. Cornella-Taracido,et al.  Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay. , 2021, ACS medicinal chemistry letters.

[8]  Yong Huang,et al.  Porcine circovirus type 2 infection attenuates the K63-linked ubiquitination of STING to inhibit IFN-β induction via p38-MAPK pathway. , 2021, Veterinary microbiology.

[9]  Qingqu Guo,et al.  De-Ubiquitinating Enzymes USP21 Regulate MAPK1 Expression by Binding to Transcription Factor GATA3 to Regulate Tumor Growth and Cell Stemness of Gastric Cancer , 2021, Frontiers in Cell and Developmental Biology.

[10]  Chiao-Yin Sun,et al.  Disulfiram and 6-Thioguanine synergistically inhibit the enzymatic activities of USP2 and USP21. , 2021, International journal of biological macromolecules.

[11]  Claire Heride,et al.  The deubiquitylase USP9X controls ribosomal stalling , 2020, bioRxiv.

[12]  Sarah A. Boswell,et al.  Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism , 2020, Scientific Reports.

[13]  Michael J. Emanuele,et al.  Dissenting degradation: Deubiquitinases in cell cycle and cancer. , 2020, Seminars in cancer biology.

[14]  Y. Cho,et al.  Ubiquitin-Specific Protease 21 Promotes Colorectal Cancer Metastasis by Acting as a Fra-1 Deubiquitinase , 2020, Cancers.

[15]  Jianbing Huang,et al.  The USP21/YY1/SNHG16 axis contributes to tumor proliferation, migration, and invasion of non-small-cell lung cancer , 2020, Experimental & Molecular Medicine.

[16]  R. DePinho,et al.  USP21 deubiquitinase promotes pancreas cancer cell stemness via Wnt pathway activation , 2019, Genes & development.

[17]  Michael J. Emanuele,et al.  FOXM1 Deubiquitination by USP21 Regulates Cell Cycle Progression and Paclitaxel Sensitivity in Basal-like Breast Cancer , 2019, Cell reports.

[18]  D. Komander,et al.  Breaking the chains: deubiquitylating enzyme specificity begets function , 2019, Nature Reviews Molecular Cell Biology.

[19]  Qiaojun He,et al.  Inhibition of Ubiquitin-Specific Proteases as a Novel Anticancer Therapeutic Strategy , 2018, Front. Pharmacol..

[20]  T. Sixma,et al.  Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins , 2018, EMBO reports.

[21]  B. Lagu,et al.  Novel highly selective inhibitors of ubiquitin specific protease 30 (USP30) accelerate mitophagy. , 2018, Bioorganic & medicinal chemistry letters.

[22]  D. Urban,et al.  A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase , 2018, Scientific Reports.

[23]  E. Prochownik,et al.  The deubiquitinase USP21 stabilizes MEK2 to promote tumor growth , 2018, Cell Death & Disease.

[24]  Scarlett A Dvorkin,et al.  Identification and Structure-Guided Development of Pyrimidinone Based USP7 Inhibitors. , 2018, ACS medicinal chemistry letters.

[25]  Colin R. O'Dowd,et al.  Discovery and characterization of highly potent and selective allosteric USP7 inhibitors. , 2018, Nature chemical biology.

[26]  Gopal Jayaraj,et al.  Pathways of cellular proteostasis in aging and disease , 2018, The Journal of cell biology.

[27]  K. Anderson,et al.  Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7. , 2017, Cell chemical biology.

[28]  Elizabeth C. Townsend,et al.  Molecular basis of USP7 inhibition by selective small-molecule inhibitors , 2017, Nature.

[29]  Florian Gnad,et al.  USP7 small-molecule inhibitors interfere with ubiquitin binding , 2017, Nature.

[30]  S. Kwon,et al.  Ubiquitin-specific protease 21 stabilizes BRCA2 to control DNA repair and tumor growth , 2017, Nature Communications.

[31]  Zhengfan Jiang,et al.  p38 inhibition provides anti–DNA virus immunity by regulation of USP21 phosphorylation and STING activation , 2017, The Journal of experimental medicine.

[32]  Anuradha Singh,et al.  Deubiquitinases and cancer: A snapshot , 2016, Critical Reviews in Oncology/Hematology.

[33]  David Komander,et al.  Ubiquitin modifications , 2016, Cell Research.

[34]  John P. Overington,et al.  The promise and peril of chemical probes. , 2015, Nature chemical biology.

[35]  Anton Simeonov,et al.  A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses. , 2014, Nature chemical biology.

[36]  Pumin Zhang,et al.  USP21 negatively regulates antiviral response by acting as a RIG-I deubiquitinase , 2014, The Journal of experimental medicine.

[37]  Mark E Bunnage,et al.  Target validation using chemical probes. , 2013, Nature chemical biology.

[38]  David Klenerman,et al.  Ubiquitin chain conformation regulates recognition and activity of interacting proteins , 2012, Nature.

[39]  J. Strickler,et al.  Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases. , 2012, Biochimica et biophysica acta.

[40]  Jian Cao,et al.  Histone Ubiquitination and Deubiquitination in Transcription, DNA Damage Response, and Cancer , 2012, Front. Oncol..

[41]  Keith D Wilkinson,et al.  Polyubiquitin binding and cross‐reactivity in the USP domain deubiquitinase USP21 , 2011, EMBO reports.

[42]  Ian Collins,et al.  Probing the Probes: Fitness Factors For Small Molecule Tools , 2010, Chemistry & biology.

[43]  K. Hofmann,et al.  Dissection of USP catalytic domains reveals five common insertion points. , 2009, Molecular bioSystems.

[44]  David Komander,et al.  Breaking the chains: structure and function of the deubiquitinases , 2009, Nature Reviews Molecular Cell Biology.

[45]  E. Trinquet,et al.  HTRF: A Technology Tailored for Drug Discovery –A Review of Theoretical Aspects and Recent Applications , 2009, Current chemical genomics.

[46]  Shao-Cong Sun.  Deubiquitylation and regulation of the immune response , 2008, Nature Reviews Immunology.

[47]  U. Hassiepen,et al.  A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. , 2007, Analytical biochemistry.

[48]  B. Dye,et al.  Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. , 2007, Annual review of biophysics and biomolecular structure.

[49]  René Bernards,et al.  A Genomic and Functional Inventory of Deubiquitinating Enzymes , 2005, Cell.