Suramin Selectivity Inhibits Inducible Nitric Oxide Synthase at the Calmodulin Binding Site
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Excess nitric oxide (NO), synthesized from L-arginine by nitric oxide synthase (NOS), has been linked to a number of pathological conditions. Inhibition of NOS is, therefore, currently a target for drug design. NOS requires binding of calmodulin (CaM), a calcium binding protein ubiquitously expressed in eukaryotes, for activity. The CaM binding site serves as an excellent target for selective inhibition of NOS because studies have demonstrated that the three isoforms of NOS, endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), vary in the orientation and nature by which they bind CaM. Previous studies with wild type CaM showed that suramin, a polysulfonated naphthylurea, inhibits iNOS at lower concentrations than nNOS indicating that it discriminates among calmodulin-binding sites of NOS. This study further investigates the nature of nNOS and iNOS inhibition by suramin by testing it in the presence of CaM with mutations at calcium binding sites 1 and 3 (1Q mutant and 3Q mutant). Kinetic analysis demonstrated that the half-maximal inhibitory constant, IC 50, of suramin for iNOS with wild type CaM and 2.6 µM and 41 µM, in the presence and absence of 1 mM EDTA respectively, and with 1Q mutant CaM, was 6.9 µM and 63 µM. IC 50 of nNOS with wild type CaM was 81 µM and with 3Q mutant CaM, was 48 µM. Moreover, western analyses of nNOS and iNOS in the presence of all three CaM variations indicated that suramin disrupts NOS-CaM binding. These data suggest that suramin selectively inhibits iNOS over nNOS and that inhibition occurs by way of competition for the CaM binding site.