Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens

Abstract

The malaria parasite Plasmodium falciparum and related apicomplexan pathogens contain an essential plastid organelle, the apicoplast, which is a key anti-parasitic target. Derived from secondary endosymbiosis, the apicoplast depends on novel, but largely cryptic, mechanisms for protein/lipid import and organelle inheritance during parasite replication. These critical biogenesis pathways present untapped opportunities to discover new parasite-specific drug targets. We used an innovative screen to identify actinonin as having a novel mechanism-of-action inhibiting apicoplast biogenesis. Resistant mutation, chemical-genetic interaction, and biochemical inhibition demonstrate that the unexpected target of actinonin in P. falciparum and Toxoplasma gondii is FtsH1, a homolog of a bacterial membrane AAA+ metalloprotease. PfFtsH1 is the first novel factor required for apicoplast biogenesis identified in a phenotypic screen. Our findings demonstrate that FtsH1 is a novel and, importantly, druggable antimalarial target. Development of FtsH1 inhibitors will have significant advantages with improved drug kinetics and multistage efficacy against multiple human parasites.

Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites

“…Given that actinonin exhibits a different death kinetic to all the established apicoplast housekeeping inhibitors, it seems unlikely that it inhibits apicoplast housekeeping peptide deformylase (67). Rather, recent data demonstrate that actinonin inhibits apicoplast biogenesis in Toxoplasma gondii, and probably P. falciparum, by targeting the apicoplast membrane protein FtsH1 (70), and our results are concordant.…”

“…5). Actinonin resistance in Toxoplasma gondii is conferred by a point mutation in the apicoplast membrane-associated protein FtsH1, and an homologous target is inferred for P. falciparum with protease inhibition rather than posttranslational modification proposed as the mode of action (70).…”

A mevalonate bypass system facilitates elucidation of plastid biology in malaria parasites

“…Prior attempts to investigate the essentiality of apicoplast-specific proteins have been dependent on conditional knockdown approaches and direct supplementation with IPP [15][16][17][18][19]. Using CRISPR-Cas9 in PfMev parasites, we were able to generate the first targeted deletion of a gene encoding an essential apicoplast-specific protein.…”

“…The previously established chemical bypass system has proven to be invaluable to the study of the apicoplast in malaria parasites, having been used to interrogate the essentiality of apicoplast specific genes [15][16][17][18][19], screen for apicoplast-specific drugs [15,[20][21][22][23], and conduct forward genetic screens to elucidate essential apicoplast-specific genes [24]. However, this bypass study are available in GenBank under accession numbers MN822297 and MN822298.…”

An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum

“…Even broader utility of pSN053/054 for enabling in-depth functional studies of diverse parasite genes is underscored by their application in elucidating roles for the claudin-like apicomplexan microneme protein (CLAMP) ligand in red blood cell invasion (Sidik et al, 2016), aspartate proteases Plasmepsin IX and X in red blood cell invasion and egress (Nasamu et al, 2017), and FtsH1 and ATG8 in apicoplast biogenesis/maintenance (Amberg-Johnson et al, 2017; Walczak et al, 2018). In some instances, it can be desirable to achieve even more stringently regulated knockdown to study biological function using a dual aptamer configuration (Ganesan et al, 2016).…”