Discovery of a Hidden Trypanosoma cruzi Spermidine Synthase Binding Site and Inhibitors through In Silico, In Vitro, and X-ray Crystallography

In drug discovery research, the selection of promisingbindingsites and understanding the binding mode of compounds are crucialfundamental studies. The current understanding of the proteins-ligandbinding model extends beyond the simple lock and key model to includethe induced-fit model, which alters the conformation to match theshape of the ligand, and the pre-existing equilibrium model, selectivelybinding structures with high binding affinity from a diverse ensembleof proteins. Although methods for detecting target protein bindingsites and virtual screening techniques using docking simulation arewell-established, with numerous studies reported, they only considera very limited number of structures in the diverse ensemble of proteins,as these methods are applied to a single structure. Molecular dynamics(MD) simulation is a method for predicting protein dynamics and candetect potential ensembles of protein binding sites and hidden sitesunobservable in a single-point structure. In this study, to demonstratethe utility of virtual screening with protein dynamics, MD simulationswere performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding siteswith a high probability of existence. The structure of the bindingsite obtained through MD simulation revealed pockets in addition tothe active site that was present in the initial structure. Using theobtained binding site structures, virtual screening of 4.8 millioncompounds by docking simulation, in vitro assays,and X-ray analysis was conducted, successfully identifying two hitcompounds.