Biochemistry of protozoan Metacaspases: Structure, processing, and inhibition

Metacaspases are calcium-dependent cysteine proteases (clan CD, family C14) present in fungi, protozoa, and plants but absent from mammals. Although structurally related to metazoan caspases, they differ in substrate specificity (cleaving after arginine or lysine), calcium-dependent activation, and monomeric architecture. In protozoan parasites such as Trypanosoma brucei, T. cruzi, Leishmania spp., Plasmodium spp., and Toxoplasma gondii, metacaspases regulate programmed cell death, cell cycle progression, protein quality control, differentiation, and virulence. Their absence in humans and essentiality in multiple parasitic life stages make them attractive drug targets for neglected tropical diseases. This review integrates structural, biochemical, functional, and pharmacological data on protozoan metacaspases. We examine calcium-dependent activation, the role of calcium homeostasis, functional diversification across parasite lineages, and the involvement of metacaspases in apoptotic and non-apoptotic pathways. Recent advances in small-molecule inhibitor discovery are covered, including oxime-based compounds, chalcones, flavonoids, peptidomimetics, and non-peptidyl scaffolds from virtual screening and rational design. A comparative biochemical analysis highlights species-specific differences in calcium dependence, processing, and regulation. Inhibitors are systematically classified by kinetic mechanism, offering a framework for understanding their mode of action and guiding structure-based drug design. We address selectivity challenges arising from metacaspase conservation across eukaryotes, distinguish experimentally validated binding sites from computationally predicted ones, and critically evaluate current in vivo and in vitro evidence for inhibitors in disease models. Finally, we discuss isoform redundancy, species-specific regulation, and translational development challenges, along with future research directions. Abreviations: YCA1, metacaspase deSaccharomyces cerevisiae; CaMCA-Ia, metacaspase deCandida albicans; TbMCA, metacaspase deTrypanosoma brucei; TcMCA, metacaspase deTrypanosoma cruzi; PCD, morte celular programada; TcoMCA5, metacaspase deTrypanosoma congolense; LmjMCA, metacaspase deLeishmania major; PfMCA, metacaspase de Plasmodium falciparum; TgMCA, metacaspase de Toxoplasma gondii; ADME, absorption, distribution, metabolism, excretion; FRET, fluorescence resonance energy transfer; HAT, human African trypanosomiasis; IMC, inner membrane complex; MCA, metacaspase; PDB, Protein Data Bank; RNAi, RNA interference; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.