Prediction of Noncompetitive Inhibitor Binding Mode Reveals Promising Site for Allosteric Modulation of Falcipain-2

Abstract

Falcipain-2 (FP-2) is a <i>Plasmodium falciparum</i> cysteine protease that has been extensively targeted to identify novel antimalarials. Remarkably, previous reports have shown that FP-2 can be allosterically modulated and, for a particular noncompetitive chalcone inhibitor, the existing lines of experimental evidence can guide the prediction of its unknown binding mode to the enzyme in a reliable fashion. In this work, we propose a structure of FP-2 in complex with the aforementioned compound that fulfills all of the experimental data, by employing a combination of molecular modeling tools, such as pocket volume measurements, docking, molecular dynamics (MD) simulations, and free energy calculations. Our results show that the studied inhibitor binds a transient pocket occluded in all of the available FP-2 crystal structures and lying in a region previously characterized as a potential allosteric site in related cysteine proteases. In addition, we detected <i>in silico</i> the occurrence of significant community reorganization in FP-2, increased signal transmission between the allosteric pocket and the active site, and change in loop motions and residue p<i>K</i>_a values upon the compound binding, thus providing insight into the uncharacterized allosteric mechanism. Overall, this study yields valuable predictions for the design of novel allosteric inhibitors against FP-2 and other cysteine proteases.