Structures of Falcipain-2 and Falcipain-3 Bound to Small Molecule Inhibitors: Implications for Substrate Specificity

Abstract

Falcipain-2 and falcipain-3 are critical hemoglobinases of Plasmodium falciparum, the most virulent human malaria parasite. We have determined the 2.9 Å crystal structure of falcipain-2 in complex with the epoxysuccinate E64 and the 2.5 Å crystal structure of falcipain-3 in complex with the aldehyde leupeptin. These complexes represent the first crystal structures of plasmodial cysteine proteases with small molecule inhibitors and the first reported crystal structure of falcipain-3. Our structural analyses indicate that the relative shape and flexibility of the S2 pocket are affected by a number of discrete amino acid substitutions. The cumulative effect of subtle differences, including those at “gatekeeper” positions, may explain the observed kinetic differences between these two closely related enzymes.

Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials

“…Compounds 8 and 9 were evaluated in vitro concerning their ability to inhibit (i) heme polymerization to hemozoin; (ii) falcipain activity; and (iii) development of blood-stage P. falciparum. In order to support observed SAR and to rationalize the activity profile of the novel compounds, we also performed molecular modeling calculations on computational models derived from X-ray structures of FP2 (PDB code: 3BPF) and FP3 (PDB code: 3BWK) co-crystalized with E64 and K11017, respectively [7,16].…”

“…Amongst them, cysteine proteases, especially falcipain-2 (FP2) and falcipain-3 (FP3), are key therapeutic targets [7]. Therefore, compounds able to simultaneously impair hemozoin formation and falcipain proteolytic activity may be potent agents against blood-stage plasmodia with reduced propensity to elicit parasite resistance [11].…”

Molecular docking and 3D-quantitative structure activity relationship analyses of peptidyl vinyl sulfones: Plasmodium Falciparum cysteine proteases inhibitors

“…For this process, we used the molecular docking program GOLD (Genetic Optimization for Ligand Docking) [19,20] from the Cambridge Crystallographic Data Center. The X-ray structure of FP2 with its bound inhibitor E64 (PDB code: 3BPF) [21] was used and prepared for docking with Sybyl X 1.3 software. The original ligand, ions and solvent molecules were removed and the protein was protonated to a pH = 5.5.…”

“…For this validation, we selected the available complex of FP3 with K11017 inhibitor from the PDB databank (PDB code: 3BWK) [24]. The selection of FP3 structure (PDB code: 3BWK) to establish a docking protocol was based on the following reasons: (i) FP2 and FP3 are both cysteine proteases of Plasmodium Falciparum and contribute more or less equally to the digestion of hemoglobin in the food vacuole [21]; (ii) the superimposition of FP2 (3BPF, chainB) and FP3 (3BWK, chainA) using DaliLite server [25], matches 240 a-carbons with an RMSD of 1.1 Å , a Z score of 38.9 and a sequence of identity of 66% and (iii) FP3 X-ray structure (3BWK) give us access to the bioactive conformation of one of the peptidyl vinyl sulfones derivatives of this study (ligand 14). The X-ray FP3 structure presented a missing residue, the C-terminal GLU243, which was added with the module Biopolymer in Sybyl X 1.3 molecular modeling program.…”

Toward the discovery of inhibitors of babesipain-1, a Babesia bigemina cysteine protease: in vitro evaluation, homology modeling and molecular docking studies

“…Hydrophobic residues usually constitute the S2 pocket, but the key residue (residue 205 in papain) present at the bottom of the pocket is not conserved [32]. In this critical position of falcipain-2 and of babesipain-1, we find the polar residue Asp234 [40] and the bulky hydrophobic Phe206 residue, respectively. This difference was suggested to be responsible for a narrower S2 pocket in babesipain-1 compared to that in falcipain-2 and, hence, responsible for the P2 rank ordering observed for babesipain-1, Val [ Leu [ Phe, in contrast with that in falcipain-2 following Leu [ Phe [ Val ordering [6].…”