Validation of putative apicoplast targeting drugs using a chemical supplementation assay in cultured human malaria parasites [PublishAheadOfPrint]

Malaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a non-apicoplast target. Here we use IPP supplementation to discriminate whether 23 purported apicoplast targeting drugs are on or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, clarithromycin), a tRNA synthase inhibitor (mupirocine), and two IPP synthesis pathway inhibitors (fosmidomycin or FR900098) have apicoplast targets. Intriguingly, the latter two drugs leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial post-translation modification does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action.

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