Assessing ScMCA-Ia Metacaspase Proteolytic Inhibition by Zinc and Arginine-based Compounds

Metacaspases are cysteine-dependent proteases (analogous to caspases in animal systems) which play a critical role in programmed cell death and stress responses in non-animal organisms. Despite their biological significance, their inhibitory mechanisms remain underexplored. This project investigates inhibition of the Type Ia metacaspase from Schizophyllum commune (ScMCA-Ia) to identify the specific chemical features in synthetic inhibitors that effectively reduce autoproteolysis and enzymatic activity. Building on prior work showing Ca2+-dependent activation of ScMCA-Ia, both qualitative and quantitative assay techniques were used to compare a classical protease inhibitor (Zn2+) with novel synthetic compounds. Purified recombinant ScMCA-Ia was analyzed by SDS-PAGE to visualize autoproteolysis and by kinetic fluorescence assays using the fluorogenic substrate Z-GGR-AMC to quantify activity under different inhibitor concentrations. These complementary approaches allowed inhibition to be assessed both by banding pattern (autoproteolytic cleavage) and by changes in specific activity (substrate cleavage). As expected, Zn2+ inhibits ScMCA-Ia autoproteolysis and reduces enzyme activity, supporting its use as a positive-control. Among the synthetic compounds tested, including variants with different amino-acid side chains (such as lysine- and alanine-based compounds), only the arginine-based compound Z-R-Bz demonstrated dose-dependent inhibition. In contrast, the compound Z-R (lacking the Bz protecting group) did not inhibit ScMCA-Ia across the tested concentrations, which indicates that both the arginine side-chain and the Bz protecting group are important structural features for inhibition. This work provides evidence that specific structural modifications in synthetic compounds can strongly influence metacaspase inhibition and contribute to a broader understanding of fungal metacaspase regulation, supporting the future design of more selective metacaspase-targeting inhibitors.