The Plasmodium falciparum 20S proteasome (Pf20S) is a promising antimalarial target. Therapeutic development has previously relied on native purifications of Pf20S, which is challenging and has limited the scope of previous efforts. Here, we report an effective recombinant Pf20S platform to facilitate drug discovery. Complex assembly is carried out in insect cells by co-expressing all fourteen subunits along with the essential Pf chaperone homolog, Ump1. Unexpectedly, the isolated proteins consisted of both a mature and an immature complex. Cryo-EM analysis of the immature complexes revealed structural insights detailing how Ump1 and β2/β5 pro-peptides coordinate β-ring assembly, which differ from human and yeast homologs. Biochemical validation confirmed that β1, β2, and β5 subunits of the mature proteasome were catalytically active. Clinical proteasome inhibitors, bortezomib, carfilzomib and marizomib were potent but lacked Pf20S selectivity. However, the tripeptide-epoxyketone J-80 potently and selectively inhibited Pf20S β5 (IC50= 22.4 (1.3) nM, 90-fold over human β5), with cryo-EM elucidating the structural basis for its specific binding. Further evaluation of novel Pf20S-selective inhibitors such as the reversible TDI-8304 and irreversible analogs 8304-vinyl sulfone and 8304-epoxyketone confirmed their potency and selectivity over the human constitutive proteasome. This recombinant Pf20S platform facilitates detailed biochemical and structural studies, accelerating the development of selective antimalarial therapeutics.

Cancer Metabolism and Microenvironment Program Sanford Burnham Prebys Medical Discovery Institute La Jolla 92037 USA

Center for Discovery and Innovation in Parasitic Diseases Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA USA

Department of Microbiology and Immunology Columbia University Medical Center New York New York 10032 United States

Department of Microbiology and Immunology Weill Cornell Medicine New York New York 10065 United States

Department of Pathology Stanford University School of Medicine Stanford California 94305 United States

Department Pharmaceutical Sciences College of Pharmacy The University of Jordan Amman Jordan

Institute of Organic Chemistry and Biochemistry AS CR v v i Prague Czech Republic

Pharmaceutical Synthesis Group Universidade Federal do Rio Grande do Sul Porto Alegre RS Brazil

Scripps Institution of Oceanography University of California San Diego La Jolla CA USA

See more in PubMed

Ciechanover A. and Schwartz A.L., The ubiquitin-proteasome pathway: the complexity and myriad functions of proteins death. Proc Natl Acad Sci U S A, 1998. 95(6): p. 2727–30. PubMed PMC

Jung T. and Grune T., The proteasome and the degradation of oxidized proteins: Part I-structure of proteasomes. Redox Biol, 2013. 1(1): p. 178–82. PubMed PMC

Moreau P., et al. , Proteasome inhibitors in multiple myeloma: 10 years later. Blood, 2012. 120(5): p. 947–59. PubMed PMC

Kuhn D.J., et al. , Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood, 2007. 110(9): p. 3281–90. PubMed PMC

Groen K., et al. , Carfilzomib for relapsed and refractory multiple myeloma. Cancer Manag Res, 2019. 11: p. 2663–2675. PubMed PMC

Ramirez K.G., Ixazomib: An Oral Proteasome Inhibitor for the Treatment of Multiple Myeloma. J Adv Pract Oncol, 2017. 8(4): p. 401–405. PubMed PMC

Li H., et al. , Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature, 2016. 530(7589): p. 233–6. PubMed PMC

Xie S.C., et al. , The structure of the PA28–20S proteasome complex from Plasmodium falciparum and implications for proteostasis. Nat Microbiol, 2019. 4(11): p. 1990–2000. PubMed

Almaliti J., et al. , Development of Potent and Highly Selective Epoxyketone-Based Plasmodium Proteasome Inhibitors. Chemistry, 2023. 29(20): p. e202203958. PubMed PMC

Sulzen H., et al. , Structural Insights into Salinosporamide a Mediated Inhibition of the Human 20S Proteasome. Molecules, 2025. 30(6). PubMed PMC

Khare S., et al. , Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature, 2016. 537(7619): p. 229–233. PubMed PMC

Wyllie S., et al. , Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition. Proc Natl Acad Sci U S A, 2019. 116(19): p. 9318–9323. PubMed PMC

Gantt S.M., et al. , Proteasome inhibitors block development of Plasmodium spp. Antimicrob Agents Chemother, 1998. 42(10): p. 2731–8. PubMed PMC

Reynolds J.M., et al. , Antimalarial activity of the anticancer and proteasome inhibitor bortezomib and its analog ZL3B. BMC Clin Pharmacol, 2007. 7: p. 13. PubMed PMC

Li H., et al. , Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity. Chem Biol, 2012. 19(12): p. 1535–45. PubMed PMC

Li H., et al. , Identification of potent and selective non-covalent inhibitors of the Plasmodium falciparum proteasome. J Am Chem Soc, 2014. 136(39): p. 13562–5. PubMed PMC

Xie S.C., et al. , Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome. J Med Chem, 2018. 61(22): p. 10053–10066. PubMed PMC

Prudhomme J., et al. , Marine actinomycetes: a new source of compounds against the human malaria parasite. PLoS One, 2008. 3(6): p. e2335. PubMed PMC

LaMonte G.M., et al. , Development of a Potent Inhibitor of the Plasmodium Proteasome with Reduced Mammalian Toxicity. J Med Chem, 2017. 60(15): p. 6721–6732. PubMed PMC

Kirkman L.A., et al. , Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance. Proc Natl Acad Sci U S A, 2018. 115(29): p. E6863–E6870. PubMed PMC

Hsu H.C., et al. , Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors. Nat Commun, 2023. 14(1): p. 8302. PubMed PMC

Eadsforth T.C., et al. , Pharmacological and structural understanding of the Trypanosoma cruzi proteasome provides key insights for developing site-specific inhibitors. J Biol Chem, 2025. 301(1): p. 108049. PubMed PMC

Silhan J., et al. , Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors. Nat Commun, 2024. 15(1): p. 8621. PubMed PMC

Toste Rego A. and da Fonseca P.C.A., Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes. Mol Cell, 2019. 76(1): p. 138–147 e5. PubMed PMC

Satoh T., et al. , Molecular and Structural Basis of the Proteasome alpha Subunit Assembly Mechanism Mediated by the Proteasome-Assembling Chaperone PAC3-PAC4 Heterodimer. Int J Mol Sci, 2019. 20(9). PubMed PMC

Murata S., Yashiroda H., and Tanaka K., Molecular mechanisms of proteasome assembly. Nat Rev Mol Cell Biol, 2009. 10(2): p. 104–15. PubMed

Mischlinger J., Agnandji S.T., and Ramharter M., Single dose treatment of malaria - current status and perspectives. Expert Rev Anti Infect Ther, 2016. 14(7): p. 669–78. PubMed

Zhang H., et al. , Structure-Activity Relationship Studies of Antimalarial Plasmodium Proteasome Inhibitors horizontal line Part II. J Med Chem, 2023. 66(2): p. 1484–1508. PubMed PMC

Withers-Martinez C., et al. , Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells. Characterization, comparison with the parasite protease, and homology modeling. J Biol Chem, 2002. 277(33): p. 29698–709. PubMed

Shenai B.R., et al. , Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum. J Biol Chem, 2000. 275(37): p. 29000–10. PubMed

Eakin A.E., et al. , Production of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi. J Biol Chem, 1993. 268(9): p. 6115–8. PubMed

Han Y., et al. , Molecular basis for the stepwise and faithful maturation of the 20S proteasome. Sci Adv, 2025. 11(2): p. eadr7943. PubMed PMC

Adolf F., et al. , Visualizing chaperone-mediated multistep assembly of the human 20S proteasome. Nat Struct Mol Biol, 2024. 31(8): p. 1176–1188. PubMed PMC

Almaliti J., et al. , Development of Potent and Highly Selective Epoxyketone-Based Plasmodium Proteasome Inhibitors. Chemistry–A European Journal, 2023. 29(20): p. e202203958. PubMed PMC

Fajtova P., et al. , Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome. Protein Sci, 2024. 33(12): p. e5225. PubMed PMC

Silva E.B., et al. , Enhancing schistosomiasis drug discovery approaches with optimized proteasome substrates. Protein Sci, 2025. 34(6): p. e70180. PubMed PMC

Schrader J., et al. , The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design. Science, 2016. 353(6299): p. 594–8. PubMed

Deni I., et al. , Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome. Cell Chem Biol, 2023. 30(5): p. 470–485 e6. PubMed PMC

Zhan W., et al. , Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice. Angew Chem Int Ed Engl, 2021. 60(17): p. 9279–9283. PubMed PMC

Bennett J.M., et al. , Covalent Macrocyclic Proteasome Inhibitors Mitigate Resistance in Plasmodium falciparum. ACS Infect Dis, 2023. 9(10): p. 2036–2047. PubMed PMC

Yoo E., et al. , Defining the Determinants of Specificity of Plasmodium Proteasome Inhibitors. J Am Chem Soc, 2018. 140(36): p. 11424–11437. PubMed PMC

Robbertse L., et al. , Evaluating Antimalarial Proteasome Inhibitors for Efficacy in Babesia Blood Stage Cultures. ACS Omega, 2024. 9(45): p. 44989–44999. PubMed PMC

Shevchenko A., et al. , In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc, 2006. 1(6): p. 2856–60. PubMed

Mastronarde D.N., Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol, 2005. 152(1): p. 36–51. PubMed

Li X., et al. , Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods, 2013. 10(6): p. 584–90. PubMed PMC

Zivanov J., Nakane T., and Scheres S.H.W., Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ, 2020. 7(Pt 2): p. 253–267. PubMed PMC

Punjani A., et al. , cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods, 2017. 14(3): p. 290–296. PubMed

Pettersen E.F., et al. , UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem, 2004. 25(13): p. 1605–12. PubMed

Emsley P., et al. , Features and development of Coot. Acta Crystallogr D Biol Crystallogr, 2010. 66(Pt 4): p. 486–501. PubMed PMC

Emsley P. and Cowtan K., Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr, 2004. 60(Pt 12 Pt 1): p. 2126–32. PubMed

Adams P.D., et al. , PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr, 2010. 66(Pt 2): p. 213–21. PubMed PMC

Adams P.D., et al. , The Phenix software for automated determination of macromolecular structures. Methods, 2011. 55(1): p. 94–106. PubMed PMC

Pettersen E.F., et al. , UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci, 2021. 30(1): p. 70–82. PubMed PMC