A chemical rescue screen identifies a Plasmodium falciparum apicoplast inhibitor targeting MEP isoprenoid precursor biosynthesis

Abstract

The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the "Malaria Box" library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

FIG 1

IPP rescue of growth inhibition by MMV-08138. (A) Chemical structure. (B) EC₅₀ curves in the absence and presence of IPP. (C) Time course of growth through the intraerythrocytic cycle. Parasites were treated with drug only, drug plus IPP, or drug plus IPP followed by removal of drug and IPP after the first reinvasion. Parasitemia is normalized to that of an untreated control.

FIG 2

Selection of MMV-08138-resistant parasite populations. (A) Drug selection timeline. (B) EC₅₀ curves of resistant populations.

FIG 3

Identification of IspD mutations in MMV-08138-resistant parasites. (A) Mutations determined by whole-genome and Sanger sequencing of resistant populations 08138R1 (R1), 08138R2 (R2), and 08138R3 (R3). aa, amino acid; nt, nucleotide. (B) Block-level alignment of P. falciparum, A. thaliana, and E. coli IspD, with percent homology of each block. MMV-08138-resistant PfIspD mutation locations are denoted by arrows.

FIG 4

Inhibition of PfIspD enzyme activity by diastereomers of MMV-08138.