In Silico Prediction of Alkaline Phosphatase Interaction with the Natural Inhibitory 5-Azaindoles Guitarrin C and D

The natural 5-azaindoles, marine sponge guitarrin C and D, were observed to exert inhibitory activity against a highly active alkaline phosphatase (ALP) CmAP of the PhoA family from the marine bacterium <i>Cobetia amphilecti</i>, with IC₅₀ values of 8.5 and 110 µM, respectively. The superimposition of CmAP complexes with <i>p</i>-nitrophenyl phosphate (<i>p</i>NPP), a commonly used chromogenic aryl substrate for ALP, and the inhibitory guitarrins C, D, and the non-inhibitory guitarrins A, B, and E revealed that the presence of a carboxyl group at C6 together with a hydroxyl group at C8 is a prerequisite for the inhibitory effect of 5-azaindoles on ALP activity. The 10-fold more active guitarrin C could compete with <i>p</i>NPP for binding sites in the ALP active site due to similarities in size, three-dimensional structure, and the orientation of the COOH group along the phosphate group. However, the inhibition of CmAP and calf intestinal ALP (CIAP) by guitarrin C was observed to occur via a non-competitive mode of action, as evidenced by a twofold decrease in V_max and an unchanged K_m. In contrast, the kinetic model with guitarrin D, with an additional OH group at C7, reflected a mixed type of inhibition, with a decrease in both values. The sensitivity of CIAP to guitarrins C and D was shown to be slightly lower than that of CmAP, with IC₅₀ values of 195 and 230 µM, respectively. Nevertheless, these findings prompted the prediction of complexes of human ALP isoenzymes with guitarrins C and D.