0 in, 2009). A group of 13 flavonoids (Table 1) was selected to determine a structure–activity relationship using ARG-L as the drug target. The compounds were screened at 125 μM concentrations in the presence of 50 mM substrate l-arginine at pH 9.5, the
optimal pH of the enzyme. Under these conditions, only three compounds, apigenin, isovitexin and vitexin, inhibited less than 50% of the enzyme activity. Galangin and quercitrin achieved 50–70% inhibition, whereas isoquercitrin, isoorientin and orientin achieved 70–75% inhibition. The best inhibitors were fisetin (87%), luteolin (83%), quercetin (83%) and 7,8-dihydroxyflavone Wnt inhibitor (80%). Using the same conditions, these compounds did not significantly inhibit ARG-1 from the rat, which was used as a model for the mammalian enzyme. At a concentration of 1 mM, all of the tested compounds inhibited ARG-1 by <50%. Based on results from this study, the flavonoids showed specific inhibition of ARG-L, and did not act through the ARG-1 route. The interaction of fisetin with ARG-1 was less stable than that with ARG-L, confirming the selectivity of fisetin for the
parasite enzyme. The energy value found for the interaction between fisetin and ARG-1 was −62.5 kcal/mol, compared to −85.8 kcal/mol with the parasite ARG-L. Fisetin docking (Fig. 3) shows a rotation of 180° in the position of interactions with ARG-1 and ARG-L. There MAPK Inhibitor Library is an inversion Rho of fisetin interaction with the distinct enzyme when it looks for Ser150 and Asp245 in ARG-L, and equivalent amino acids Ser137 and Asp234 in ARG-1: the catechol group from fisetin donates a hydrogen bond (H-bond) to Ser150 in ARG-L, while, in ARG-1, the
hydroxyl group at position 7 on the flavone group donates an H-bond to Ser137, which is the position equivalent to Ser150 in ARG-L. This inversion allows for a close hydrophobic interaction of His154 and His139 with the double ring of the flavone group of fisetin, and enhances the stability of this inhibitor with ARG-L. The constants Ki and Ki′ refer to the equilibrium established between the enzyme (E) and substrate (S) in the presence of an inhibitor (I). The inhibition constant Ki refers to the dissociation constant of the complex EI, while Ki′ refers to the dissociation of the EIS ( Cornish-Bowden, 1974). Eight compounds, with an IC50 of less than 20 μM, were selected for analysis of the mechanism of enzyme inhibition. The aglycone compounds, such as quercetin, luteolin and fisetin, exhibited mixed inhibition, while the glycoside flavonoids, such as orientin and isoorientin, showed uncompetitive inhibition. The compounds quercitrin, isoquercitrin and 7,8-dihydroxyflavone showed non-competitive inhibition. Table 1 summarizes the kinetic data obtained with the Dixon and Cornish-Bowden plots that were used to calculate the constants Ki and Ki′ (Fig. 1).