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.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

The 20S proteasome, a critical component of the ubiquitin-proteasome system, plays a central role in regulating protein degradation in eukaryotic cells. Marizomib (MZB), also known as salinosporamide A, is a natural γ-lactam-β-lactone compound derived from Salinispora tropica and is a potent 20S proteasome covalent inhibitor with demonstrated anticancer properties. Its broad-spectrum inhibition of all three proteasome subunits and its ability to cross the blood-brain barrier has made it a promising therapeutic candidate for glioblastoma. In addition to this, MZB also demonstrates significant inhibition against the 20S proteasome of Trichomonas vaginalis (Tv20S), a protozoan parasite, suggesting its potential for parasitic treatments. 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 enhance the therapeutic potential of MZB for both cancer and parasitic diseases at the molecular level and highlight marine-derived natural products in targeting the proteasome for therapeutic applications.

• Klíčová slova
• 20S, MZB, cryo-EM, marizomib, proteasome,
• MeSH
• elektronová kryomikroskopie MeSH
• inhibitory proteasomu * chemie   farmakologie   MeSH
• laktony * chemie   farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• proteasomový endopeptidasový komplex * chemie   metabolismus   MeSH
• pyrroly * chemie   farmakologie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory proteasomu * MeSH
• laktony * MeSH
• marizomib MeSH Prohlížeč
• proteasomový endopeptidasový komplex * MeSH
• pyrroly * MeSH

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.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH