Rapaprotin, an Endopeptidase-Activated Proteasome Inhibitor that Induces 26S Disassembly

Hanjing Peng^#^{1

2}, Zufeng Guo^#^{1

2}, Wei Li Wang^#^{3

4}, Deyao Yin^{3

5

6}, Shitao Zou^{3

5

6}, Thomas Asbell^{1

2}, Brett R Ullman⁷, Maya Thakar⁸, Feiran Zhang^{1

2}, Sam Y Hong⁷, A V Subba Rao^{1

2}, Kunyu Wang^{3

5}, Shuwen Zhang^{3

5

6}, Zhaolong Wu^{3

5}, Xuemei Li³, Aidan A Kendra^{1

2}, Seth S Margolis^{9

10}, William H Matsui⁸, Christian B Gocke⁸, Youdong Mao^{3

5

6

11

12

13}, Jun O Liu^{1

2}

Affiliations

¹ Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
² The SJ Yan and HJ Mao Laboratory of Chemical Biology, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
³ State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, Laboratory of Electron Microscopy, School of Physics, Peking University, Beijing, 100871, China.
⁴ Bioentropy Inc, Cambridge, MA, 02138, USA.
⁵ Peking-Tsinghua Joint Center for Life Sciences, Peking University, Beijing, 100871, China.
⁶ AI for Science (AI4S)-Preferred Program, School of Chemical Biology and Biotechnology, Peking University, Shenzhen, 518055, China.
⁷ Rapafusyn Pharmaceuticals, Baltimore, MD 21205, USA.
⁸ Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, 21287, USA.
⁹ Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
¹⁰ Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
¹¹ National Biomedical Imaging Center, Peking University, Beijing, 100871, China.
¹² Center for Quantitative Biology, Peking University, Beijing, 100871, China.
¹³ AI for Science (AI4S)-Preferred Program, Peking University Shenzhen Graduate School, Shenzhen, 518055, China.

^# Contributed equally.

PMID: 40960209
DOI: 10.1002/anie.202500288

Rapaprotin, an Endopeptidase-Activated Proteasome Inhibitor that Induces 26S Disassembly

Hanjing Peng et al. Angew Chem Int Ed Engl. 2025.

. 2025 Sep 17:e202500288.

doi: 10.1002/anie.202500288. Online ahead of print.

Authors

Affiliations

¹ Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
² The SJ Yan and HJ Mao Laboratory of Chemical Biology, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
³ State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, Laboratory of Electron Microscopy, School of Physics, Peking University, Beijing, 100871, China.
⁴ Bioentropy Inc, Cambridge, MA, 02138, USA.
⁵ Peking-Tsinghua Joint Center for Life Sciences, Peking University, Beijing, 100871, China.
⁶ AI for Science (AI4S)-Preferred Program, School of Chemical Biology and Biotechnology, Peking University, Shenzhen, 518055, China.
⁷ Rapafusyn Pharmaceuticals, Baltimore, MD 21205, USA.
⁸ Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, 21287, USA.
⁹ Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
¹⁰ Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, 21205, USA.
¹¹ National Biomedical Imaging Center, Peking University, Beijing, 100871, China.
¹² Center for Quantitative Biology, Peking University, Beijing, 100871, China.
¹³ AI for Science (AI4S)-Preferred Program, Peking University Shenzhen Graduate School, Shenzhen, 518055, China.

^# Contributed equally.

PMID: 40960209
DOI: 10.1002/anie.202500288

Abstract

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

Keywords: Multiple myeloma; Proteasome disassembly; Proteasome inhibitor; Rapafucin; Rapaprotin.

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References

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Rapaprotin, an Endopeptidase-Activated Proteasome Inhibitor that Induces 26S Disassembly

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Rapaprotin, an Endopeptidase-Activated Proteasome Inhibitor that Induces 26S Disassembly

Authors

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Abstract

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