Inhibiting catalytic activity of Plasmodium falciparum aspartate protease plasmepsin V: A biochemical approach to malaria intervention

Abstract

Plasmepsin V, an aspartate protease, is crucial for the survival of the malarial parasite Plasmodium falciparum, making it a promising target for antimalarial drug development. In this study, the quinoline-based drugs amodiaquine (AQ) and clioquinol (CQ) are repurposed to inhibit the catalytic activity of PfPlmV. Fluorescence quenching assays suggest that both AQ and CQ exhibit moderate binding affinity for PfPlmV. Temperature-dependent thermodynamic analyses indicate the involvement of both H-bonding/electrostatic interactions and hydrophobic interactions in the binding of AQ/CQ with PfPlmV. Isothermal titration calorimetry (ITC) indicates that these interactions are both enthalpically and entropically favourable. Molecular docking studies show that AQ binds directly to the substrate-binding site of PfPlmV, engaging catalytic dyad residues Asp118 and Asp365, whereas CQ binds to PfPlmV but does not directly interact with the catalytic dyad. Molecular dynamics simulations also corroborated the docking results, revealing that AQ forms a more conformationally stable and hydrophobic interactions driven complex with PfPlmV compared to CQ. Further, the enzyme kinetics studies using fluorogenic substrate demonstrated that AQ efficiently inhibits the catalytic activity of PfPlmV with an IC₅₀ value of (0.42 ± 0.04) μM via competitive inhibition mode. In contrast, CQ fails to bind at the catalytic site and does not exhibit any inhibitory effect on PfPlmV activity. These mechanistic insights will lay the foundations for new biochemical approaches to develop targeted therapies aimed at malaria disease.