Computational aided search of polyphenols with trypanocidal activity

VALERA-VERA E; SAYE M; REIGADA C; MIRANDA M; PEREIRA

Congreso; DRUG DISCOVERY FOR NEGLECTED DISEASES INTERNATIONAL CONGRESS 2018. 4th Scientific Meeting of the Research Network; 2018

Arginine kinase (AK) is a highly conserved protein of invertebrates that is also found in trypanosomatids. It catalyzes the reversible transference of phosphate between ATP and arginine to help maintaining the energetic balance in conditionswhen more energy is needed; and in Trypanosoma cruzi appears to be a key enzyme for stress response. Different plant extracts have shown inhibition of the enzyme activity in a wide range of organisms including trypanosomatids, andmore precisely some polyphenols have been successfully identified as inhibitors.In this work we used computational techniques to find possible inhibitors of the T. cruzi AK (TcAK) and tested their inhibitory capabilities on the enzyme activity and different life cycle stages of the parasite. With this end, ahomology model of the protein was generated with the iTASSER server to use in molecular docking with a database of 326 polyphenols found in plants, using the software AutoDock 4.5. The compounds predicted to have a strong interaction with the AK active site that also are easily accessible in the market were further tested against recombinant TcAK in inhibition assays in the sense ATP + arginine → ADP + P-Arginine, using coupled reactions to measure NADH consumption. Also, in vitro activity was assayed over epimastigotes, trypomastigotes and Vero cell cultures infected with T. cruzi, counting cells after exposition with different concentration of the compounds. Finally, cytotoxicity over Vero cells was assayed and measured by fixation of the remaining cells and tincture with crystal violet. Capsaicin and delphinidin were the molecules that showed the strongest trypanocidal effect in the nanomolar range on either stage of the parasite life-cycle with high selectivity indexes against Vero cell cultures, but only delphinidin showed to be a strong AK inhibitor, with an IC50 of 7uM. Further metabolic studies and computational analysis will be performed to test if AK is indeed the molecular target of polyphenols in their trypanocidal activity; either way, the high anti-parasitic activity and selectivity of these molecules show the strong potential of these polyphenols as lead compounds in the search for new drugs against Chagas disease.