Metacaspases are novel cysteine proteases found in apicomplexan whose function is poorly understood. Our earlier studies on Plasmodium falciparum metacaspase-2 (PfMCA-2) revealed that the caspase inhibitor, Z-FA-FMK efficiently inhibited PfMCA-2 activity and, expression, and significantly blocked in vitro progression of the parasite developmental cycle via apoptosis-like parasite death. Building on these findings, we synthesized a set of novel inhibitors based on structural modification of Z-FA-FMK with the amides of piperic acid and investigated their effect on PfMCA-2. One of these analogues, SS-5, specifically inhibited the activity and expression of PfMCA-2. The activities of some other known malarial proteases (falcipains, plasmepsins, & vivapain), and human cathepsins-B, D and L, and caspase-3 and -7 were not inhibited by SS-5. SS-5 blocked the development of P. falciparumin vitro (IC50 1µM) and caused prominent morphological distortions. Incubation with SS-5 led to persistent parasite oxidative stress accompanied by depolarization of mitochondrial potential and accumulation of intracellular Ca2+. SS-5 also inhibited the development of P. berghei in a murine model. Our results suggest that the inhibition of PfMCA-2 results in oxidative stress, leading to apoptosis-like parasite death. Thus, SS-5 offers a starting point for optimization of new antimalarials, and PfMCA-2 could be a novel target for antimalarial drug discovery.

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