Inhibition mechanism of Trypanosoma cruzi arginine kinase by delphinidin

VALERA VERA E; MIRANDA MR; MARTÍNEZ SAYÉ M; REIGADA C; PEREIRA CA

Mar del Plata, Buenos Aires

Congreso; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; 2019

Sociedad Argentina de Protozoología

The enzyme arginine kinase from Trypanosoma cruzi (TcAK) catalyzes the interconversion of arginine and phospho-arginine to maintain the ATP/ADP balance, and is involved in the energetic homeostasis of the parasite and stress response. Some polyphenolic compounds have been predicted to inhibit this enzyme activity in virtual screening and further proved to inhibit it in vitro and excreting trypanocidal activity in epimastigotes and trypomastigotes. One of those compounds, delphinidin, had activity in low micromolar concentrations, so we wanted to explore the mechanism of inhibition and produce a model of interaction between delphinidin and TcAK. To this end, TcAK activity was measured in absence or presence of different delphinidin concentrations, varying the arginine concentrations. The resulting data was fitted to the Michaelis-Menten equation to calculate the kinetical parameters Km and Vmax, resulting in a decrease of the Vmax with increasing delphinidin concentrations (p=0.005), while Km had a trend to increase, but with no statistically significant difference (p=0.3). The decrease in Vmax excludes a competitive mode of inhibition, and while further testing is required to reliably state the kind of inhibition, the trend of the km suggests it could be a mixed inhibition with preference towards the TcAK-arginine complex. To produce a model of the interaction we did molecular docking simulations wit AutoDock 4.5 and AutoDock Vina. While some of the binding poses predicted by AutoDock 4.5 overlapped at some extent with the arginine binding site, both programs produced binding poses with good scores located in the ATP/ADP binding site, and poses with lowest scores that binds to a pocket away from the active site. In conclusion, we tested that delphinidin does not compete with arginine when inhibiting TcAK, and computational approaches suggest the binding of delphinidin might be occurring at the ATP/ADP binding site, so further testing of the inhibition mechanism as a function of ATP concentration is needed.