Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles

• Published in: The Journal of biological chemistry Vol. 288; no. 27; pp. 19685 - 19697
• Main Authors: Nagpal, Latika, Haque, Mohammad M., Saha, Amit, Mukherjee, Nirmalya, Ghosh, Arnab, Ranu, Brindaban C., Stuehr, Dennis J., Panda, Koustubh
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.07.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Arginine - chemistry; Arginine - pharmacology; Biopterins - analogs & derivatives; Biopterins - chemistry; Biopterins - pharmacology; Cell Line; Dimerization Inhibitors; Enzyme Inhibitors; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Enzyme Kinetics; Enzymology; Heme - chemistry; Heme - metabolism; Imidazoles - chemistry; Imidazoles - pharmacokinetics; Mice; Mutation, Missense; Nitric Oxide; Nitric Oxide Synthase Type II - antagonists & inhibitors; Nitric Oxide Synthase Type II - chemistry; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Nitric-oxide Synthase; Nitrosative Stress; Protein Multimerization - drug effects; Protein Multimerization - genetics; Pyrimidine Imidazoles; Pyrimidines - chemistry; Pyrimidines - pharmacology; Enzyme Inhibitors; Dimerization Inhibitors; Pyrimidine Imidazoles; Nitric-oxide Synthase; Nitrosative Stress; Enzyme Kinetics; Nitric Oxide
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.446542

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases. Background: Overproduction of nitric oxide by dimeric inducible nitric-oxide synthase (iNOS) is physiologically harmful. Results: Pyrimidine imidazole derivative (PID) binds to both the iNOS dimer and monomer causing irreversible monomerization and inhibition of dimerization, respectively. Conclusion: PID can physiologically inhibit iNOS both during and after its assembly into active enzyme. Significance: Our study reveals PID's dual ability to inhibit iNOS as well as their kinetic mechanisms.