The 20S proteasome, a critical component of the ubiquitin-proteasome system, plays a central role in regulating protein degradation in eukaryotic cells. Marizomib (MZB), a natural γ-lactam-β-lactone compound derived from Salinispora tropica, is a potent 20S proteasome covalent inhibitor with demonstrated anticancer properties. Its broad-spectrum inhibition of all three proteasome subunits and ability to cross the blood-brain barrier has made it a promising therapeutic candidate for glioblastoma. Here, we present the cryo-EM structure of the human 20S proteasome in complex with MZB at 2.55 Å resolution. This structure reveals the binding mode of MZB to all six catalytic subunits within the two β-rings of the 20S proteasome, providing a detailed molecular understanding of its irreversible inhibitory mechanism. These findings explain the therapeutic potential of MZB at the molecular level and highlight marine-derived natural products in targeting the proteasome for anticancer treatment.

Center for Discovery and Innovation in Parasitic Diseases Skaggs School of Pharmacy and pharmaceutical Sciences University of California San Diego 9500 Gilman Dr La Jolla CA 92093 USA

Institute of Organic Chemistry and Biochemistry AS CR v v i Flemingovo nam 2 166 10 Prague 6 Czech Republic

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA 92037 USA

Molecules. 2025 Mar 20;30(6):1386. doi: 10.3390/molecules30061386. PubMed

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Feling R. H. et al. Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus salinospora. Angew Chem Int Ed Engl 42, 355–357 (2003). 10.1002/anie.200390115 PubMed DOI

Mincer T. J., Jensen P. R., Kauffman C. A. & Fenical W. Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl Environ Microbiol 68, 5005–5011 (2002). 10.1128/AEM.68.10.5005-5011.2002 PubMed DOI PMC

Roth P. et al. Marizomib for patients with newly diagnosed glioblastoma: A randomized phase 3 trial. Neuro Oncol 26, 1670–1682 (2024). 10.1093/neuonc/noae053 PubMed DOI PMC

Di K. et al. Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brain barrier. Neuro Oncol 18, 840–848 (2016). 10.1093/neuonc/nov299 PubMed DOI PMC

Manton C. A. et al. Induction of cell death by the novel proteasome inhibitor marizomib in glioblastoma in vitro and in vivo. Sci Rep 6, 18953 (2016). 10.1038/srep18953 PubMed DOI PMC

Manasanch E. E. et al. The proteasome: mechanisms of biology and markers of activity and response to treatment in multiple myeloma. Leuk Lymphoma 55, 1707–1714 (2014). 10.3109/10428194.2013.828351 PubMed DOI

Eisenberg-Lerner A. et al. Golgi organization is regulated by proteasomal degradation. Nat Commun 11, 409 (2020). 10.1038/s41467-019-14038-9 PubMed DOI PMC

Tanaka K. The proteasome: overview of structure and functions. Proc Jpn Acad Ser B Phys Biol Sci 85, 12–36 (2009). 10.2183/pjab.85.12 PubMed DOI PMC

Coux O., Tanaka K. & Goldberg A. L. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65, 801–847 (1996). 10.1146/annurev.bi.65.070196.004101 PubMed DOI

Lupas A., Zwickl P., Wenzel T., Seemuller E. & Baumeister W. Structure and function of the 20S proteasome and of its regulatory complexes. Cold Spring Harb Symp Quant Biol 60, 515–524 (1995). 10.1101/sqb.1995.060.01.055 PubMed DOI

Lowe J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539 (1995). 10.1126/science.7725097 PubMed DOI

Orlowski M., Cardozo C. & Michaud C. Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids. Biochemistry 32, 1563–1572 (1993). 10.1021/bi00057a022 PubMed DOI

Mishra R., Upadhyay A., Prajapati V. K. & Mishra A. Proteasome-mediated proteostasis: Novel medicinal and pharmacological strategies for diseases. Med Res Rev 38, 1916–1973 (2018). 10.1002/med.21502 PubMed DOI

Schmidt M. & Finley D. Regulation of proteasome activity in health and disease. Biochim Biophys Acta 1843, 13–25 (2014). 10.1016/j.bbamcr.2013.08.012 PubMed DOI PMC

Fenteany G. et al. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268, 726–731 (1995). 10.1126/science.7732382 PubMed DOI

Groll M. & Potts B. C. Proteasome structure, function, and lessons learned from beta-lactone inhibitors. Curr Top Med Chem 11, 2850–2878 (2011). 10.2174/156802611798281320 PubMed DOI

Macherla V. R. et al. Structure-activity relationship studies of salinosporamide A (NPI-0052), a novel marine derived proteasome inhibitor. J Med Chem 48, 3684–3687 (2005). 10.1021/jm048995+ PubMed DOI

Groll M., Huber R. & Potts B. C. Crystal structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in complex with the 20S proteasome reveal important consequences of beta-lactone ring opening and a mechanism for irreversible binding. J Am Chem Soc 128, 5136–5141 (2006). 10.1021/ja058320b PubMed DOI

Silhan J. et al. Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors. Nature communications 15, 8621 (2024). 10.1038/s41467-024-53022-w PubMed DOI PMC

Prudhomme J. et al. Marine actinomycetes: a new source of compounds against the human malaria parasite. PLoS One 3, e2335 (2008). 10.1371/journal.pone.0002335 PubMed DOI PMC

Robbertse L. et al. Evaluating Antimalarial Proteasome Inhibitors for Efficacy in Babesia Blood Stage Cultures. ACS Omega 9, 44989–44999 (2024). 10.1021/acsomega.4c04564 PubMed DOI PMC

Punjani A., Rubinstein J. L., Fleet D. J. & Brubaker M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature methods 14, 290–296 (2017). 10.1038/nmeth.4169 PubMed DOI

He J., Li T. & Huang S. Y. Improvement of cryo-EM maps by simultaneous local and non-local deep learning. Nature communications 14, 3217 (2023). 10.1038/s41467-023-39031-1 PubMed DOI PMC

Sahu I. et al. The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag. Nat Commun 12, 6173 (2021). 10.1038/s41467-021-26427-0 PubMed DOI PMC

Pettersen E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein science : a publication of the Protein Society 30, 70–82 (2021). 10.1002/pro.3943 PubMed DOI PMC

Afonine P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol 74, 531–544 (2018). 10.1107/S2059798318006551 PubMed DOI PMC

Liebschner D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75, 861–877 (2019). 10.1107/S2059798319011471 PubMed DOI PMC

Emsley P., Lohkamp B., Scott W. G. & Cowtan K. Features and development of Coot. Acta crystallographica. Section D, Biological crystallography 66, 486–501 (2010). 10.1107/S0907444910007493 PubMed DOI PMC

Croll T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr D Struct Biol 74, 519–530 (2018). 10.1107/S2059798318002425 PubMed DOI PMC

Lebedev A. A. et al. JLigand: a graphical tool for the CCP4 template-restraint library. Acta Crystallogr D Biol Crystallogr 68, 431–440 (2012). 10.1107/S090744491200251X PubMed DOI PMC

Williams C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci 27, 293–315 (2018). 10.1002/pro.3330 PubMed DOI PMC

Afonine P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr D Struct Biol 74, 814–840 (2018). 10.1107/S2059798318009324 PubMed DOI PMC

Borissenko L. & Groll M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem Rev 107, 687–717 (2007). 10.1021/cr0502504 PubMed DOI

Adasme M. F. et al. PLIP 2021: expanding the scope of the protein-ligand interaction profiler to DNA and RNA. Nucleic Acids Res 49, W530–W534 (2021). 10.1093/nar/gkab294 PubMed DOI PMC

Fajtova P. et al. Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome. Protein science : a publication of the Protein Society 33, e5225 (2024). 10.1002/pro.5225 PubMed DOI PMC

Ciechanover A. The Ubiquitin-Proteasome Proteolytic Pathway. Cell 79, 13–21 (1994). https://doi.org:Doi 10.1016/0092-8674(94)90396-4 PubMed DOI

Chauhan D. et al. A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib. Cancer cell 8, 407–419 (2005). 10.1016/j.ccr.2005.10.013 PubMed DOI

Nunes A. T. & Annunziata C. M. Proteasome inhibitors: structure and function. Semin Oncol 44, 377–380 (2017). 10.1053/j.seminoncol.2018.01.004 PubMed DOI PMC

Park J. E., Miller Z., Jun Y., Lee W. & Kim K. B. Next-generation proteasome inhibitors for cancer therapy. Translational Research 198, 1–16 (2018). 10.1016/j.trsl.2018.03.002 PubMed DOI PMC

Kegyes D. et al. Proteasome inhibition in combination with immunotherapies: State-of-the-Art in multiple myeloma. Blood Rev 61 (2023). https://doi.org:ARTN 101100 10.1016/j.blre.2023.101100 PubMed DOI

Raninga P. et al. Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition. Theranostics 10, 5259–5275 (2020). 10.7150/thno.42705 PubMed DOI PMC

Zhang Z. et al. Combined treatment of marizomib and cisplatin modulates cervical cancer growth and invasion and enhances antitumor potential in vitro and in vivo. Front Oncol 12, 974573 (2022). 10.3389/fonc.2022.974573 PubMed DOI PMC

Kaplan G. S., Torcun C. C., Grune T., Ozer N. K. & Karademir B. Proteasome inhibitors in cancer therapy: Treatment regimen and peripheral neuropathy as a side effect. Free Radic Biol Med 103, 1–13 (2017). 10.1016/j.freeradbiomed.2016.12.007 PubMed DOI

Eadsforth T. C. et al. Pharmacological and structural understanding of the Trypanosoma cruzi proteasome provides key insights for developing site-specific inhibitors. The Journal of biological chemistry 301, 108049 (2024). 10.1016/j.jbc.2024.108049 PubMed DOI PMC