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

The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria.

Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target.

As resistance to current therapies spreads, novel antimalarials are urgently needed. In this work, we examine the potential for therapeutic intervention via the targeting of Plasmodium IspD (2-C-methyl-D-erythritol 4-phosphate cytidyltransferase), the second dedicated enzyme of the essential methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. Enzymes of this pathway represent promising therapeutic targets because the pathway is not present in humans. The Malaria Box compound, MMV008138, inhibits Plasmodium falciparum growth, and PfIspD has been proposed as a candidate intracellular target. We find that PfIspD is the sole intracellular target of MMV008138 and characterize the mode of inhibition and target-based resistance, providing chemical validation of this target. Additionally, we find that the Pf ISPD genetic locus is refractory to disruption in malaria parasites, providing independent genetic validation for efforts targeting this enzyme. This work provides compelling support for IspD as a druggable target for the development of additional, much-needed antimalarial agents.