Inhibition mechanism of Trypanosoma cruzi arginine kinase by delphinidin

VALERA VERA EA; REIGADA C; SAYÉ M; MIRANDA MR; PEREIRA CA

Mar del Plata, Buenos Aires

Congreso; Reunión Anual de Sociedades de Biociencia. SAIC. SAFE. SAB. SAP. AACYTAL. NANOMED-ar. HCS; 2019

The enzyme arginine kinase from Trypanosoma cruzi Tc AK) 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 act ivity in virtual screening and further proved to inhibit it in vitro andexcreting trypanocidal activity in epimastigotes and trypomastigotes. One of those compounds, delphinidin, had activity in low micromolar concentrations, so we wanted to explore the m echanism of inhibition and produce a model of interaction between delphinidin and Tc AK. To this end, Tc AK activity was measured in absence or presence of different delphinidin concentrations, varying the arginine concentrations. The resulting data was fitt ed 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 statistical ly significant differe nce (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 Tc AK argini ne 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 someextent with the arginin e binding site, both programs pro duced 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 Tc AK, and comp utational approaches suggest the binding of delphinidin might beoccurring at the ATP/ADP binding site, so further testing of the inhibition mechanism as a function of ATP concentration is needed.