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

Xiuxiu Lu; Monika Chandravanshi; Venkata R Sabbasani; Snehal Gaikwad; V Keith Hughitt; Nana Gyabaah-Kessie; Bradley T Scroggins; Sudipto Das; Wazo Myint; Michelle E Clapp; Charles D Schwieters; Marzena A Dyba; Derek L Bolhuis; Janusz W Koscielniak; Thorkell Andresson; Michael J Emanuele; Nicholas G Brown; Hiroshi Matsuo; Raj Chari; Deborah E Citrin; Beverly A Mock; Rolf E Swenson; Kylie J Walters

doi:10.1038/s41467-024-46644-7

A structure-based designed small molecule depletes hRpn13^Pru and a select group of KEN box proteins

Nat Commun. 2024 Mar 20;15(1):2485. doi: 10.1038/s41467-024-46644-7.

Authors

Xiuxiu Lu¹, Monika Chandravanshi¹, Venkata R Sabbasani², Snehal Gaikwad³, V Keith Hughitt³, Nana Gyabaah-Kessie³, Bradley T Scroggins⁴, Sudipto Das⁵, Wazo Myint⁶, Michelle E Clapp⁷, Charles D Schwieters⁸, Marzena A Dyba⁹, Derek L Bolhuis¹⁰, Janusz W Koscielniak¹¹, Thorkell Andresson⁵, Michael J Emanuele¹², Nicholas G Brown¹², Hiroshi Matsuo⁶, Raj Chari⁷, Deborah E Citrin⁴, Beverly A Mock³, Rolf E Swenson², Kylie J Walters¹³

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].

Abstract

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13^Pru 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, hRpn13^Pru 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 hRpn13^Pru and ubiquitin-independent loss of select KEN box containing proteins.

MeSH terms

Cytoplasm / metabolism
Intracellular Signaling Peptides and Proteins
Membrane Glycoproteins* / metabolism
Proteasome Endopeptidase Complex* / metabolism
Transcription Factors
Ubiquitin / metabolism

Substances

Proteasome Endopeptidase Complex
Membrane Glycoproteins
Intracellular Signaling Peptides and Proteins
Ubiquitin
Transcription Factors

Abstract

MeSH terms

Substances

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