Methyl-methoxylpyrrolinone and flavinium nucleus binding signatures on falcipain-2 active site.

Following the increasing reports of human toxicity and plasmodium resistance to artemisinin and its derivatives, falcipain-2 (FP-2) is now emerging as the choice antimalarial drug target. Coincidentally, FP-2 is the in vivo target of naturally occurring, therapeutically safe flavonoids (stenopalustroside, myricetin, and fisetin) and symplostatin (symplostatin 4) compounds known to exhibit potent in vitro and in vivo antiplasmodial actions. Here, the structural bases for their inhibitory actions have been studied using molecular dynamics simulation. Myricetin and fisetin act as proton transfer tunnel breakers by inserting between His174 and Cys42, which are key active site residues of FP-2, stenopalustroside inhibits the polarization of His174 by Asn173; a major preparatory step for Cys42/His174 proton transfer process. The roles of flavonoids are favored by T-shaped pi-pi interactions with His174. Symplostatin 4 inserts its methyl-methoxylpyrrolinone moiety into the active site where its proton acceptor function prepares Cys42 for nucleophilic attack on the Michael α,β-unsaturated bonds on its 4(S)-amino-2(E)-pentenoate moiety. Further analyses of the structures identified a unique bridge formed on FP-2 active site groove by stenopalustroside and symplostatin 4 during interaction with the sub-site I of FP-2, whereas fisetin preferentially interacts with sub-site II and myricetin interacts with sub-site III residues. Ultimately, symplostatin-4, myricetin, and fisetin were better than stenopalustroside at trapping FP-2 in its inactive state as revealed by comparative RSMD plots with X-ray structures of FP-2 co-crystallized with inhibitors. Comparative estimates of free energy of binding using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) method suggested that His174 protonation may further enhance stenopalustroside-FP-2 interaction. The unique binding signatures of the ligands within the FP-2 active site groove and its sub-sites may explain the subtle differences in their IC50 values and their mechanism of inhibition.