Mechanism of nitric oxide synthase dimerization inhibition by novel pyrimidine imidazoles

Abstract

Over-production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been etiologically linked to several degenerative diseases. Thus its therapeutic control has become clinically imperative. As dimerization of NOS is essential for its activity, several dimerization inhibitors including pyrimidine imidazoles are being evaluated for therapeutic intervention of NOS activity. However, the mechanism of their action is still unclear. Here, we examined the mechanism of inhibition by two novel pyrimidine imidazole derivatives (PIDs) having low toxicity, high affinity and strong selectivity for iNOS using stopped-flow kinetic studies, gel-filtration and spectrophotometric analysis. The binding of our PIDs to iNOS produced an irreversible monomer-inhibitor complex with intact heme, contrary to reported behavior of bulky analogues. Treatment with tetrahydrobiopterin (H 4 B) and l -arginine ( l -Arg) failed to recover any dimer from such monomers, demonstrating the irreversible nature of such inhibition. To elucidate the precise mechanism of action of the PIDs, we engaged two monomeric mutants of the iNOS oxygenase domain (iNOSoxy) whose dimerization capabilities were either recoverable (K82AiNOSoxy) or non-recoverable (D92AiNOSoxy) with H 4 B. We observed that PID bound the H 4 B-pretreated dimeric K82A mutant in a biphasic manner triggering monomerization but with fast monophasic kinetics to the monomeric form of both mutants, disallowing dimer recovery by H 4 B and l -Arg. This indicated that PIDs could not only interact with the iNOS monomer to prevent dimerization but also with the dimer to elicit irreversible monomerization. Overall, this study establishes the strong potential of PIDs as candidates for therapeutic control of diseases related to NO overproduction by iNOS.