Caracterização da ATP difosfohidrolase de Solanum tuberosum como ferramenta biotecnológica no estudo de NTPDases

Abstract

Ectonucleotidases comprise a group of enzime's families, which participate in the hydrolysis of extracelullar nucleotides and may be located on the cell surface, soluble in the extracellular medium or in the interstitial fluids. The NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family widely distributed among organisms and that have as common feature the ability to hydrolyse di- and triphosphate nucleotides (ADP and ATP) in the presence of milimolar concentrations of Ca+2 and Mg+2. Potato apyrase was the first enzyme of the ectonucleotidase family to be purified and its soluble isoform is easily obtained in a high level of yield and purity through conventional purification methods, ensuring high reproducibility in pharmacological and immunological assays. NTPDases are involved in several pathophysiological processes such as immune and inflammatory responses, clotting, tumor formation, neuronal disorders and virulence of microorganisms. Thus, the search for ATP diphosphohydrolases inhibitors has great scientific relevance, since no potent and specific inhibitor for an isoform has been found so far. The aim of this work was to obtain pure Solanum tuberosum apyrase from purification, perform in silico studies to characterize this protein as a biotechnological tool in the study of NTPDases and perform an in vitro screening for compounds and fractions with inhibitory potential against Solanum tuberosum ATP diphosphohydrolase. In the purification process it was possible to evaluate the technique efficiency through identification of potato apyrase by western blotting and mass spectrometry. The protein phosphohidrolictical activity was preserved and verified by calcium phosphate deposit in the same height for ATP and ADP in a non-denaturing gel. The in vitro inhibition evaluation revealed 8 isolated substances (A6, A7, A8, A45, A47, A33, Cu(isapn) e aceturado de diminazeno) and 10 fractions from extracts derived of different plant species (A2, A50, A49, A19, A37, A41, A22, A42, A39 e A30) with promising inhibitory potential against the ATPase activity of potato apyrase. In silico analysis through molecular docking of the isolated substances revealed that all of them have binding spontaneity with the catalytic site of the enzyme, reassuring its inhibitory potential. These results revealed isolated compounds and fractions with promising inhibitory potential on Solanum tuberosum apyrase, reiterating the application of these substances in the treatment of various pathophysiological conditions related to the exacerbation of purinergic signaling. However, further studies are needed to corroborate this hypothesis.