Structure- and function-based design of Plasmodium-selective proteasome inhibitors

Abstract

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next generation anti-malarial agents.

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…A separate approach to directly overcoming ART resistance has come from the discovery that inhibitors of the P. falciparum 20S proteasome can synergize with ART in drug-sensitive or drug-resistant parasites 96,97 . Encouragingly, one parasite-specific inhibitor showed antimalarial efficacy in a rodent in vivo model of malaria.…”

Activity‐based probes for the multicatalytic proteasome

“…While b5 inhibition is able to effectively block parasite replication, b2 inhibitors are less effective as a single agent but synergize with the antimalarial drug dihydroartemisinin. A combined b2/ b5 inhibitor showed single-agent efficacy in a rodent model of malaria [31,60].…”

“…Recent work has identified parasite proteasomes as novel targets in infectious disease, including malaria, leishmaniasis, Chagas disease, and trypanosomiasis [60,69]. The mycobacterial proteasome has also been investigated as a therapeutic target, and inhibitors selective for the Mycobacterium tuberculosis proteasome over the human proteasome have been developed [70].…”

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

“…With these improvements allowing higher resolution structures to be obtained, there have already been a number of structures published in complex with ligands and inhibitors (Fig. 3), from large soluble complexes such as the proteasome and -galactosidase to smaller membrane proteins including TRP channels and gamma secretase (Bartesaghi et al, 2014;Bai et al, 2015;Paulsen et al, 2015;Gao et al, 2016;Li et al, 2016).…”

“…(a) Resiniferatoxin ligand density from TRPV1 at 2.95 Å resolution (EMDB entry 8117, PDB entry 5irx; Gao et al, 2016). (b) 3.6 Å resolution proteasome EM density showing bound inhibitor (EMBD entry 3231, PDB entry 5fmg; Li et al, 2016). For both panels the EM density map is shown in mesh format and side chains and bound inhibitor are shown in stick format and are coloured light blue, dark blue, red and yellow for carbon, nitrogen, oxygen and sulfur, respectively.…”