Antifungal imidazoles block assembly of inducible NO synthase into an active dimer

Cytokine-inducible nitric oxide synthase (iNOS) is a homodimeric enzyme that generates nitric oxide (NO) and L-citrulline from L-arginine (L-Arg) and O2. The N-terminal oxygenase domain (amino acids 1-498; iNOSox) in each subunit binds heme, L-Arg, and tetrahydrobiopterin (H4B), is the site of NO synthesis, and is responsible for the dimeric interaction, which must occur to synthesize NO. In both cells and purified systems, iNOS dimer assembly is promoted by H4B, L-Arg, and L-Arg analogs. We examined the ability of imidazole and N-substituted imidazoles to promote or inhibit dimerization of heme-containing iNOSox monomers, or to affect iNOS dimerization in cells. Imidazole, 1-phenylimidazole, clotrimazole, and miconazole all bound to the iNOSox monomer heme iron. Imidazole and 1-phenylimidazole promoted iNOSox dimerization, whereas clotrimazole (30 microM) and miconazole (15 microM) did not, and instead inhibited dimerization normally promoted by L-Arg and H4B. Clotrimazole also bound to iNOSox dimers in the absence of L-Arg and H4B and caused their dissociation. When added to cells expressing iNOS, clotrimazole (50 microM) had no effect on iNOS protein expression but almost completely inhibited its dimerization and consequent NO synthesis over an 8-h culture period, without affecting calmodulin interaction with iNOS. Thus, imidazoles can promote or inhibit dimerization of iNOS both in vitro and in cells, depending on their structure. Bulky imidazoles like clotrimazole block NO synthesis by inhibiting assembly of the iNOS dimer, revealing a new means to control cellular NO synthesis.