The Plasmodium falciparum 20S proteasome (Pf20S) has emerged as a promising antimalarial target. Development of therapeutics to this target 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. Proteasome assembly was carried out in insect cells by co-expressing all fourteen subunits along with the essential 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 the propeptides of the β2 and β5 subunits 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 inhibited Pf20S β5 with an IC50 of 22.4 nM and 90-fold selectivity over human β5. Structural studies using cryo-EM elucidated the basis for the selective binding of J-80. 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

Center for Malaria Therapeutics and Antimicrobial Resistance Department of Medicine Columbia University Medical Center New York New York 10032 United States

Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography 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

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Ciechanover A. & Schwartz A. L. The ubiquitin-proteasome pathway: the complexity and myriad functions of proteins death. Proc Natl Acad Sci U S A 95, 2727–2730 (1998). 10.1073/pnas.95.6.2727 PubMed DOI PMC

Jung T. & Grune T. The proteasome and the degradation of oxidized proteins: Part I-structure of proteasomes. Redox Biol 1, 178–182 (2013). 10.1016/j.redox.2013.01.004 PubMed DOI PMC

Moreau P. et al. Proteasome inhibitors in multiple myeloma: 10 years later. Blood 120, 947–959 (2012). 10.1182/blood-2012-04-403733 PubMed DOI 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 110, 3281–3290 (2007). 10.1182/blood-2007-01-065888 PubMed DOI PMC

Groen K., van de Donk N., Stege C., Zweegman S. & Nijhof I. S. Carfilzomib for relapsed and refractory multiple myeloma. Cancer Manag Res 11, 2663–2675 (2019). 10.2147/CMAR.S150653 PubMed DOI PMC

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

Li H. et al. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature 530, 233–236 (2016). 10.1038/nature16936 PubMed DOI PMC

Xie S. C. et al. The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis. Nat Microbiol 4, 1990–2000 (2019). 10.1038/s41564-019-0524-4 PubMed DOI

Almaliti J. et al. Development of Potent and Highly Selective Epoxyketone-Based Plasmodium Proteasome Inhibitors. Chemistry 29, e202203958 (2023). 10.1002/chem.202203958 PubMed DOI PMC

Sulzen H., Fajtova P., O’Donoghue A. J., Silhan J. & Boura E. Structural Insights into Salinosporamide a Mediated Inhibition of the Human 20S Proteasome. Molecules 30 (2025). 10.3390/molecules30061386 PubMed DOI PMC

Khare S. et al. Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature 537, 229–233 (2016). 10.1038/nature19339 PubMed DOI 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 116, 9318–9323 (2019). 10.1073/pnas.1820175116 PubMed DOI PMC

Gantt S. M. et al. Proteasome inhibitors block development of Plasmodium spp. Antimicrob Agents Chemother 42, 2731–2738 (1998). 10.1128/AAC.42.10.2731 PubMed DOI PMC

Reynolds J. M., El Bissati K., Brandenburg J., Gunzl A. & Mamoun C. B. Antimalarial activity of the anticancer and proteasome inhibitor bortezomib and its analog ZL3B. BMC Clin Pharmacol 7, 13 (2007). 10.1186/1472-6904-7-13 PubMed DOI 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 19, 1535–1545 (2012). 10.1016/j.chembiol.2012.09.019 PubMed DOI PMC

Li H. et al. Identification of potent and selective non-covalent inhibitors of the Plasmodium falciparum proteasome. J Am Chem Soc 136, 13562–13565 (2014). 10.1021/ja507692y PubMed DOI PMC

Xie S. C. et al. Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome. J Med Chem 61, 10053–10066 (2018). 10.1021/acs.jmedchem.8b01161 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

LaMonte G. M. et al. Development of a Potent Inhibitor of the Plasmodium Proteasome with Reduced Mammalian Toxicity. J Med Chem 60, 6721–6732 (2017). 10.1021/acs.jmedchem.7b00671 PubMed DOI 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 115, E6863–E6870 (2018). 10.1073/pnas.1806109115 PubMed DOI PMC

Dogovski C. et al. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance. PLoS Biol 13, e1002132 (2015). 10.1371/journal.pbio.1002132 PubMed DOI PMC

Stokes B. H. et al. Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness. Elife 10 (2021). 10.7554/eLife.66277 PubMed DOI PMC

Martinez-Vega R. et al. Regional action needed to halt antimalarial drug resistance in Africa. Lancet 405, 7–10 (2025). 10.1016/S0140-6736(24)02706-5 PubMed DOI PMC

Xie S. C., Dick L. R., Gould A., Brand S. & Tilley L. The proteasome as a target for protozoan parasites. Expert Opin Ther Targets 23, 903–914 (2019). 10.1080/14728222.2019.1685981 PubMed DOI

Hsu H. C. et al. Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors. Nat Commun 14, 8302 (2023). 10.1038/s41467-023-44077-2 PubMed DOI 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 301, 108049 (2025). 10.1016/j.jbc.2024.108049 PubMed DOI PMC

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

Toste Rego A. & da Fonseca P. C. A. Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes. Mol Cell 76, 138–147 e135 (2019). 10.1016/j.molcel.2019.07.014 PubMed DOI 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 20 (2019). 10.3390/ijms20092231 PubMed DOI PMC

Murata S., Yashiroda H. & Tanaka K. Molecular mechanisms of proteasome assembly. Nat Rev Mol Cell Biol 10, 104–115 (2009). 10.1038/nrm2630 PubMed DOI

Mischlinger J., Agnandji S. T. & Ramharter M. Single dose treatment of malaria - current status and perspectives. Expert Rev Anti Infect Ther 14, 669–678 (2016). 10.1080/14787210.2016.1192462 PubMed DOI

Zhang H. et al. Structure-Activity Relationship Studies of Antimalarial Plasmodium Proteasome Inhibitors horizontal line Part II. J Med Chem 66, 1484–1508 (2023). 10.1021/acs.jmedchem.2c01651 PubMed DOI 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 277, 29698–29709 (2002). 10.1074/jbc.M203088200 PubMed DOI

Shenai B. R., Sijwali P. S., Singh A. & Rosenthal P. J. Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum. J Biol Chem 275, 29000–29010 (2000). 10.1074/jbc.M004459200 PubMed DOI

Eakin A. E., McGrath M. E., McKerrow J. H., Fletterick R. J. & Craik C. S. Production of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi. J Biol Chem 268, 6115–6118 (1993). PubMed

Budenholzer L., Cheng C. L., Li Y. & Hochstrasser M. Proteasome Structure and Assembly. J Mol Biol 429, 3500–3524 (2017). 10.1016/j.jmb.2017.05.027 PubMed DOI PMC

Hirano Y. et al. Dissecting beta-ring assembly pathway of the mammalian 20S proteasome. EMBO J 27, 2204–2213 (2008). 10.1038/emboj.2008.148 PubMed DOI PMC

Li X., Kusmierczyk A. R., Wong P., Emili A. & Hochstrasser M. beta-Subunit appendages promote 20S proteasome assembly by overcoming an Ump1-dependent checkpoint. EMBO J 26, 2339–2349 (2007). 10.1038/sj.emboj.7601681 PubMed DOI PMC

Chen P. & Hochstrasser M. Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly. Cell 86, 961–972 (1996). 10.1016/s0092-8674(00)80171-3 PubMed DOI

Adolf F. et al. Visualizing chaperone-mediated multistep assembly of the human 20S proteasome. Nat Struct Mol Biol 31, 1176–1188 (2024). 10.1038/s41594-024-01268-9 PubMed DOI PMC

Zhang H., Zhou C., Mohammad Z. & Zhao J. Structural basis of human 20S proteasome biogenesis. Nat Commun 15, 8184 (2024). 10.1038/s41467-024-52513-0 PubMed DOI PMC

Zhao J. et al. Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins. Proc Natl Acad Sci U S A 119, e2207200119 (2022). 10.1073/pnas.2207200119 PubMed DOI PMC

Han Y., Han Q., Tang Q., Zhang Y. & Liu K. Molecular basis for the stepwise and faithful maturation of the 20S proteasome. Sci Adv 11, eadr7943 (2025). 10.1126/sciadv.adr7943 PubMed DOI PMC

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

Silva E. B. et al. Enhancing schistosomiasis drug discovery approaches with optimized proteasome substrates. Protein Sci 34, e70180 (2025). 10.1002/pro.70180 PubMed DOI PMC

Deni I. et al. Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome. Cell Chem Biol 30, 470–485 e476 (2023). 10.1016/j.chembiol.2023.03.002 PubMed DOI 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 60, 9279–9283 (2021). 10.1002/anie.202015845 PubMed DOI PMC

Bennett J. M. et al. Covalent Macrocyclic Proteasome Inhibitors Mitigate Resistance in Plasmodium falciparum. ACS Infect Dis 9, 2036–2047 (2023). 10.1021/acsinfecdis.3c00310 PubMed DOI PMC

Yoo E. et al. Defining the Determinants of Specificity of Plasmodium Proteasome Inhibitors. J Am Chem Soc 140, 11424–11437 (2018). 10.1021/jacs.8b06656 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

Shevchenko A., Tomas H., Havlis J., Olsen J. V. & Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1, 2856–2860 (2006). 10.1038/nprot.2006.468 PubMed DOI

Mastronarde D. N. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36–51 (2005). 10.1016/j.jsb.2005.07.007 PubMed DOI

Li X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10, 584–590 (2013). 10.1038/nmeth.2472 PubMed DOI PMC

Zivanov J., Nakane T. & Scheres S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020). 10.1107/S2052252520000081 PubMed DOI PMC

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

Pettersen E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605–1612 (2004). 10.1002/jcc.20084 PubMed DOI

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

Emsley P. & Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132 (2004). 10.1107/S0907444904019158 PubMed DOI

Adams P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213–221 (2010). 10.1107/S0907444909052925 PubMed DOI PMC

Adams P. D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011). 10.1016/j.ymeth.2011.07.005 PubMed DOI PMC

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

Perez-Riverol Y. et al. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res 53, D543–D553 (2025). 10.1093/nar/gkae1011 PubMed DOI PMC