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 (EC 50 ) 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 (IC 50 ) 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. Despite encouraging progress over the past decade, malaria caused by Plasmodium parasites continues to pose an enormous disease burden (1). New antimalarials with novel mechanisms of action are needed to circumvent existing or emerging drug resistance (2). The apicoplast is a plastid organelle unique to Plasmodium spp. (and other pathogenic Apicomplexa parasites) and is a key target for development of new antimalarials. Due to its prokaryotic origin and evolution as a secondary plastid, it contains pathways that have no counterpart in the human host (3, 4). The apicoplast in Plasmodium is essential for both intraerythrocytic and intrahepatic development in the human host (5, 6).Despite efforts to develop inhibitors of apicoplast function, to date, there have been no primary agents for treatment of acute malaria whose mechanism of action targets this unusual plastid organelle. Antibiotics that inhibit prokaryotic transcription and translation, such as doxycycline and clindamycin, block expression of the apicoplast genome and are active against Plasmodium parasites (5). Unfortunately, these drugs show a "delayed death" phenotype, in which growth inhibition occurs only after 2 replication cycles (96 h). The slow kinetics limit the use of doxycycline and clindamycin to chemoprophylaxis or as partner drugs in combination therapies with faster-acting compounds. Fosmidomycin, which inhibits the enzyme DoxR/IspC for MEP (methyl-D-erythritol-4-phosphate) isoprenoid precursor biosynthesis in the apicoplast, has immediate onset but shows high recrudescence rates clinically when used as monotherapy (7,8). The efficacy of fosmidomycin-based combination therapy is currently being evaluated, with mixe...

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants

“…In data from parasite clone tree culture experiments13, some subclones from HB3-P and W2 contained the premature stop codon allele while others did not. The nonsense mutation in Epac in the Dd2 clone has been previously noted14, as has the separate stop codon in W215. In summary, most of the laboratory-adapted strains have evidently acquired one or more loss-of-function mutations in Epac or in an ApiAP2 gene.…”

The apicoplast: now you see it, now you don’t

“…This simple chemical rescue demonstrated that the only essential metabolic function of the apicoplast in blood-stage Plasmodium is the biosynthesis of isoprenoid precursors. IPP supplementation can apparently rescue parasites from any apicoplast dysfunction, making it a nifty tool to study apicoplast biology including identification of antimalarials with unknown targets as anti-apicoplast agents (Wu et al, 2015), and even perturbations of essential gene products (e.g. expression of a dominant-negative SufC, a member of the iron sulphur complex generating system in the apicoplast) (Gisselberg et al, 2013).…”

Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

“…For example, IPP supplementation has been shown to allow parasite growth during treatment with either the DXR inhibitor, fosmidomycin (FSM), or the IspD inhibitor, 1 R ,3 S -MMV008138202128. In order to determine whether BITZ inhibition was specific to activity against the MEP pathway, we tested for IPP rescue during treatment with compound 8 .…”

“…More recently, a chemical rescue screen identified 4 (1 R ,3 S -MMV008138) from the Medicines for Malaria Venture (MMV) “malaria box” as a potent inhibitor of P. falciparum IspD ( Pf IspD), showing nanomolar inhibition of recombinant enzyme activity and sub-micromolar inhibition of parasite growth (Fig. 2)19202122.…”