A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress

Abstract

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre-and post-exocytosis. Here, we report the multi-stage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound, 49c. This scaffold inhibits the pre-exocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PfPMIX) and X (PfPMX), respectively. Conditional excision of PfPMIX revealed its crucial role in invasion, and recombinantly active PfPMIX and PfPMX cleave egress and invasion factors in a 49c sensitive manner. KeywordsMalaria; Plasmodium falciparum; Plasmodium berghei; aspartic protease; invasion; egress; exflagellation; transmission; hydroxyl-ethyl-amine scaffold; peptidomimetic inhibitor; protein maturase Malaria remains a major cause of mortality worldwide, and resistance to existing antimalarials is a growing problem, that requires the development of new drugs urgently. Aspartic proteases are potential targets for chemotherapy (1), and key contributors to Plasmodium falciparum pathogenicity (2, 3). P. falciparum possesses a repertoire of 10 aspartic proteases, named plasmepsins (PMI to X). PMIX and PMX are expressed in mature blood-stage schizonts and invasive merozoites and fulfill indispensable but unknown functions. The activity of several serine and cysteine proteases promotes the destabilization * Corresponding authors: Paco.Pino@unige.ch, Dominique.Soldati-favre@unige.ch.

Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases

“…Incubation of r-PMIXFlag in the presence of 1 lM of 49c leads to an accumulation of the protein, which is not observed, with r-PMIXFlag-F291Y (Appendix Fig S2A). The introduction of the F to Y mutation does not alter the processing of TgROP1 peptide, as both r-PMIXFlag-F291Y and r-PMXFlag-F305Y readily cleave TgROP1 peptide like their WT counterparts, as reported previously (Pino et al, 2017;Appendix Fig S2C and D). The introduction of the F to Y mutation does not alter the processing of TgROP1 peptide, as both r-PMIXFlag-F291Y and r-PMXFlag-F305Y readily cleave TgROP1 peptide like their WT counterparts, as reported previously (Pino et al, 2017;Appendix Fig S2C and D).…”

“…Similarly, the orthologous PfPMIX and PfPMX also cleave rhoptry and microneme proteins, respectively (Nasamu et al, 2017;Pino et al, 2017). Similarly, the orthologous PfPMIX and PfPMX also cleave rhoptry and microneme proteins, respectively (Nasamu et al, 2017;Pino et al, 2017).…”

“…degradation, while the endoplasmic reticulum PfPMV is implicated in the cleavage of proteins destined for export into the host cell. TgASP3 is phylogenetically related to PfPMIX and PfPMX, and members of this cluster act as maturases for microneme and rhoptry proteins, playing a crucial role in invasion and in egress in both parasites, respectively (Dogga et al, 2017;Nasamu et al, 2017;Pino et al, 2017). Both proteases are involved in the export of parasite proteins across the parasitophorous vacuole membrane into the host cell Hammoudi et al, 2015;Bedi et al, 2016).…”

“…Given their broad relevance in infectious diseases and druggability, aspartic proteases have been largely prioritized as targets for chemotherapy (Meyers & Goldberg, 2012;Huizing et al, 2015). Remarkably, 49c blocks invasion and egress of P. falciparum without impacting intraerythrocytic growth (Pino et al, 2017). Recently, a peptidomimetic compound WEHI-842 as well as a hydroxy-ethylamine derivative, compound 1 (Arg-Leu-[Leu-HEA-Ala]-Glu-Ala), has been developed to specifically target PMV, mimicking the PEXEL motif involved in substrate recognition (Gambini et al, 2015;Hodder et al, 2015).…”

“…While inhibitors targeting the HIV aspartic protease are potent components of antiretroviral therapies, the inhibitors directed against the malaria hemoglobin-degrading enzymes became unattractive due to dispensability of these Plasmepsins for parasite survival (Silva et al, 1996;Coombs et al, 2001;Andrews et al, 2006). Moreover, 49c but not 49b specifically inhibits recombinant PfPMIX and PfPMX (r-PMIXFlag and r-PMXFlag), recapitulating the defect in invasion and egress of P. falciparum in the erythrocytic blood stages (Pino et al, 2017). Moreover, among the hydroxy-ethylamine scaffold inhibitors directed against Plasmepsin II (PfPMII; Ciana et al, 2013), a few derivatives including compound 49c showed very potent antimalarial effects only after 72 h of treatment with an IC 50 of 0.6 nM.…”