Discovering a hidden binding site of spermidine synthase inhibitors for Chagas disease by combining molecular simulations and X-ray crystallography

Background Chagas disease is caused by the parasite Trypanosoma cruzi and is one of the neglected tropical diseases. Although two types of drugs are currently available, new drugs are still required because they have serious side effects. To develop a therapeutic agent for trypanosomiasis, we focused on spermidine synthase (SpdSyn) as the target protein and determined the hidden binding site which was not identified in the X-ray structure for obtaining seed compounds using a computational simulation. Methodology/Principal Findings Molecular dynamics (MD) simulation was performed for TcSpdSyn to predict new binding sites. These results indicated that the highly druggable binding site was discovered around Glu22. We also conducted docking simulation for the new binding site and in vitro assay to determine half-maximal inhibitory concentration (IC50) value. Furthermore, to confirm ligand of binding site and pose, we conducted X-ray crystallographic studies. As a result, two compounds were discovered as inhibitors of TcSpdSyn with IC50 values of 82.27 and 43.41 μM, respectively. X-ray crystallographic analysis shows that two inhibitors are bound to the hidden binding site which is detected by computational simulation. Conclusions/Significance MD simulation revealed that there are new sites in the TcSpdSyn that are not an active site. This site exists near Glu22 and Asp77, and crystal structures revealed that compounds 1 and 2 are bound to the hidden binding site, as predicted by MD simulations, and interacts with Glu22 and Asp77 through hydrogen bonds. 4MCHA which has been reported as known inhibitor binds to the TcSpdSyn active site while interacting with Asp171. Therefore, these inhibitors we discovered differs in binding mode from a known inhibitor and this new binding site is useful for antitrypanosomiasis target.