OTIMIZAÇÃO E VALIDAÇÃO DE MÉTODO ENZIMÁTICO SEMIQUANTITATIVO SIMPLES E DE BAIXO CUSTO PARA A BUSCA DE INIBIDORES DE CISTEINO PROTEASES

Abstract

OPTIMIZATION AND VALIDATION OF A SIMPLE AND LOW-COST SEMI-QUANTITATIVE ENZYMATIC METHOD TO SEARCH FOR CISTEINE PROTEASES INHIBITORS. Cysteine proteases are an important class of enzymes involved in several essential biological processes. For this reason, these enzymes are important therapeutic targets, and therefore can be used in enzymatic assays for the search of hits. The most widely used methods in the search of enzyme inhibitors are spectrophotometric, however they have relatively high operational costs. For this reason, simpler, and cheaper methods can be useful in the search for enzyme inhibitors. In this work, a simple and low-cost semi-quantitative method was optimized and validated using papain and skimmed powdered milk (casein) as a substrate. The ideal condition for the method was the one that used powdered milk, 0.250%, papain solution, 9.4 mg mL-1 and incubation time, 18 h. The method validation was performed using the inhibitor E64 and herb extracts, in different concentrations. After carrying out the experiments and statistical analyses, a high reproducibility can be observed, with relative standard deviations below 2.1%. Plant extracts were used to test the method and the results indicated that these extracts are promising for the search of cysteine protease inhibitors. The method developed here has simplicity and reproducibility and has great application, as preliminary study, in the search for inhibitors of cysteine proteases.