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Updated: Jun 30, 2025
Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
Published on: November 11, 2016
A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins
Xiuxiu Lu1, Monika Chandravanshi1, Venkata R Sabbasani2
1Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
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.
Insights
A novel compound, XL44, targets proteasome subunit hRpn13 and related proteins, inducing cancer cell death. This discovery offers new therapeutic strategies by exploiting distinct protein degradation pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Proteasome subunit hRpn13 is proteolyzed in cancer cells, generating hRpn13Pru.
- The DEUBAD domain is degraded, leaving proteasome- and ubiquitin-binding Pru domain intact.
Purpose of the Study:
- To identify and characterize a small molecule binder of hRpn13 Pru.
- To elucidate the mechanism of action of the identified binder.
- To investigate its therapeutic potential in cancer.
Main Methods:
- Structure-guided virtual screening to identify hRpn13 binders.
- Integrated X-ray crystallography and NMR to solve the structure of hRpn13 Pru ligated to XL44.
- TMT-MS (tandem mass tagging mass spectrometry) to identify off-targets.
- Cell viability assays and apoptosis assays.
Main Results:
- XL44 was identified as an hRpn13 binder, with its structure elucidated.
- XL44 treatment depleted hRpn13Pru in myeloma cells.
- Off-targets including PCLAF and RRM2 were identified and also depleted by XL44.
- XL44 induced hRpn13-dependent apoptosis and PCLAF-dependent cell viability restriction.
- PCLAF depletion by XL44 requires a KEN box but not ubiquitination.
Conclusions:
- XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of KEN box proteins.
- XL44 exhibits dual mechanisms for restricting cancer cell viability.
- This study reveals a novel therapeutic strategy targeting distinct protein degradation pathways.
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