Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum

Abstract

The malaria parasite Plasmodium falciparum relies on efficient protein translation. An essential component of translation is the tryptophanyl-tRNA synthetase (TrpRS) that charges tRNAtrp. Here we characterise two isoforms of TrpRS in Plasmodium; one eukaryotic type localises to the cytosol and a bacterial type localises to the remnant plastid (apicoplast). We show that the apicoplast TrpRS aminoacylates bacterial tRNAtrp while the cytosolic TrpRS charges eukaryotic tRNAtrp. An inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits ex vivo Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment ablates apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Apicoplast TrpRS is essential for protein translation and is a promising, specific antimalarial target.

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

“…Although IPP is now used increasingly to discriminate whether or not a drug has a target in the apicoplast, we performed IPP supplementation trials on parasites exposed to two definite nonapicoplast drugs (chloroquine and atovaquone) and two validated apicoplast drugs (azithromycin and fosmidomycin) to establish a comprehensive set of assays focused on apicoplast target validation (Table 1 and Fig. 1) (30,(37)(38)(39)(40)(41)(42). Chloroquine targets the food vacuole in which the parasite digests ingested hemoglobin (43,44), and atovaquone targets the cytochrome bc 1 complex in the mitochondrion (45,46).…”

“…These include isoprenoid precursor biosynthesis, fatty acid biosynthesis, Fe-S cluster assembly, and heme biosynthesis (13)(14)(15)(16). The apicoplast is thus indispensable, and either genetic or pharmaceutical perturbation of its activities kills parasites, making the apicoplast a valid drug target (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”

“…This unique feature allows us to separate out the effect of any drug on the apicoplast from effects on any alternative target during the asexual blood stage of P. falciparum. Here, we used a chemical supplementation approach (37), a technique now routinely in use (30,(38)(39)(40)(41)(42), to test the target of 23 presumed apicoplast drugs, including compounds with confirmed apicoplast targets, confirmed nonapicoplast targets, and compounds with putative apicoplast targets.…”

The Plasmodium falciparum eIK1 kinase (PfeIK1) is central for melatonin synchronization in the human malaria parasite. Melatotosil blocks melatonin action on parasite cell cycle

“…36,45,46 Other indole compound, indolmycin, inhibit the protein translation in P. falciparum apicoplast by inhibiting the tryptophanyl-tRNA synthetase leading to a delayed death of the parasite. 47,48 Since PfeIK1parasites were susceptible to indole derivatives, we also investigated if PfeIK1 knockout would interfere with susceptibility of parasite against classical antimalarials. Zhang et al 49 have reported that parasite treatment with artemisinin resulted in an increase in eIF2 phosphorylation by PK4, and inhibition of this kinase can avoid that parasites enter in latency.…”

Messenger RNAs with large numbers of upstream ORFs are translated via leaky scanning and reinitiation in the asexual stages ofPlasmodium falciparum

“…In this parasite, an essential pathway proposed as a drug target is the protein synthesis machinery (Goodman et al, 2016). P. falciparum employs eukaryotic protein synthesis machinery for translation of nuclear-encoded mRNAs, however, an endosymbiotic organelle, the apicoplast, carries out translation using machinery that resembles prokaryotes (Chaubey et al, 2005;Fichera and Roos, 1997;Roy et al, 1999) and is the target of several anti-malarial drugs (Goodman et al, 2016;Pasaje et al, 2016). Interestingly, the cytosolic translation machinery of P. falciparum also exhibits differences from that of the human host (Jackson et al, 2011;Wong et al, 2014) and hence, has also been proposed to be a drug target (Sheridan et al, 2018;Wong et al, 2017).…”