The experiments were performed in duplicate in at least three independent experiments. Determination of the concentration that inhibits 50% of the catalytic activity of the enzyme was carried out by varying the inhibitor concentration with a 1:10 dilution factor. selleck chemicals llc The experiments were performed in duplicate in at least three independent experiments until we obtained a coefficient of non-linear regression R2 ⩾ 0.95. The different concentrations of the inhibitors

were obtained by serial dilution of the compound in water or a suitable solvent. The reactions were performed at pH 9.5 using 50 mM CHES buffer in the presence of 50 mM substrate l-arginine (pH 9.5). The samples were incubated in a water bath at 37 °C for 15 min, and the urea formed was analyzed as described above. We used a mathematical sigmoidal (log IC50) model to determine the IC50, using Origin 8.0 software. All reactions were performed in 50 mM CHES buffer, pH

9.5, containing variable concentrations of the substrate l-arginine (12.5, 25, 50 and 100 mM) at pH 9.5. Inhibitors were used at three different concentrations close to the IC50. The different substrate Duvelisib price and inhibitor concentrations were obtained by serial dilution. A mixture, M1, containing l-arginine (pH 9.5) at double the desirable concentration, and a second mixture, M2, containing the enzyme (2000 units) diluted in 125 mM CHES buffer (pH 9.5), were prepared. The reaction was prepared by mixing 50 μl of M1, 10 μl of inhibitor and 40 μl of M2. The addition of M2 was synchronized every 15 s, followed by immediate incubation in a water bath for 15 min at 37 °C. The urea produced was analyzed as described above. All reactions were performed in duplicate in a minimum of three independent experiments. The constant Ki was determined for inhibitors that showed mechanisms of mixed or competitive inhibition, whereas Ki′ was determined for inhibitors that showed uncompetitive or mixed inhibition (Cornish-Bowden, Celecoxib 1974). Each constant was determined by calculating x for the intersecting points between two lines

obtained by linear regression. For non-competitive inhibition, y = 0 was used for the equation to find the values of the constants Ki and Ki′. Statistical analysis was performed by ANOVA and post hoc Tukey’s tests, using Origin 8.0. For all tests, differences of p < 0.05 were considered significant. Linear regressions were obtained using MS Excel 2010. The target compounds (Table 1) were modeled in silico, and energy minimization was performed over 1000 steps, using the steepest descent method, Gasteiger–Hückel charges, a dielectric constant of 80, and the Tripos force field. The structures were further optimized by the conjugated gradient method. The target enzyme used in this work was a previously constructed comparative model of ARG-L (da Silva, Castilho, Pioker, Silva, & Floeter-Winter, 2002).