A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins

  • Nat Commun. 2024 Mar 20;15(1):2485. doi: 10.1038/s41467-024-46644-7.
Xiuxiu Lu  1 ,  Monika Chandravanshi  1 ,  Venkata R Sabbasani  2 ,  Snehal Gaikwad  3 ,  V Keith Hughitt  3 ,  Nana Gyabaah-Kessie  3 ,  Bradley T Scroggins  4 ,  Sudipto Das  5 ,  Wazo Myint  6 ,  Michelle E Clapp  7 ,  Charles D Schwieters  8 ,  Marzena A Dyba  9 ,  Derek L Bolhuis  10 ,  Janusz W Koscielniak  11 ,  Thorkell Andresson  5 ,  Michael J Emanuele  12 ,  Nicholas G Brown  12 ,  Hiroshi Matsuo  6 ,  Raj Chari  7 ,  Deborah E Citrin  4 ,  Beverly A Mock  3 ,  Rolf E Swenson  2 ,  Kylie J Walters  13
Affiliations
  • 1. Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
  • 2. Chemistry and Synthesis Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
  • 3. Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD, USA.
  • 4. Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
  • 5. Protein Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, USA.
  • 6. Cancer Innovation Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
  • 7. Genome Modification Core, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
  • 8. Computational Biomolecular Magnetic Resonance Core, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
  • 9. Biophysics Resource, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
  • 10. Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
  • 11. Basic Science Program, Leidos Biomedical Research Inc., NMR Facility for Biological Research, Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
  • 12. Department of Pharmacology and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
  • 13. Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA. [email protected].
PMID: 38509117 DOI: 10.1038/s41467-024-46644-7
Abstract

Proteasome subunit hRpn13 is partially proteolyzed in certain Cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent Apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

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