Repurposing amide-based drugs unveils inhibition mechanisms of plasmodium falciparum Plasmepsin V enzymatic activity for antimalarial therapy.

Understanding how enzymes are inhibited at the molecular level is essential for rational drug design against malaria parasites. Plasmodium falciparum Plasmepsin V (PfPlmV), an aspartic protease essential for exporting parasite proteins into host red blood cells has emerged as potent target for anti-malarial therapy. In this work, we investigated whether three approved amide-based drugs favipiravir, 5-fluorouracil (5-FU), and saquinavir could inhibit catalytic activity of PfPlmV. All three compounds bound to PfPlmV with affinities of ∼104 M-1, but with distinct binding behaviours. Binding by isothermal titration calorimetry (ITC) showed involvement of both hydrogen bonds and hydrophobic interactions, which stabilized the enzyme-drug complexes. Enzyme kinetics and docking studies demonstrated that the inhibitory effect depends on the degree of catalytic cleft coverage. The results showed that saquinavir interacts with key active-site residues, including the catalytic dyad Asp118 and Asp365, consistent with competitive inhibition having IC50 value of 0.35 ± 0.01 μM and a low Ki value of (0.14 ± 0.02) μM. 5-FU interacts only near Asp118, resulting in mixed-type inhibition with higher IC50 and Ki values. Favipiravir binds at the periphery of the cleft without engaging the catalytic dyad and therefore behaves as a weak, non-inhibitory binder. The insights of interaction-structure-inhibitory activity obtained from this study may guide repurposing strategies to develop selective potent inhibitors/antimalarial agents targeting PfPlmV.