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

Brindaban C Ranu,Mohammad M Haque,Koustubh Panda,Amit Saha,Nirmalya Mukherjee,Dennis J Stuehr,Arnab Ghosh,Latika Nagpal

doi:10.1074/jbc.m112.446542

Abstract

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.

Highlights

Overproduction of nitric oxide by dimeric inducible nitric-oxide synthase is physiologically harmful
The inhibition of inducible nitric-oxide synthase (iNOS) activity by Pyrimidine imidazole derivative (PID) was twice as fast (0.8 minϪ1 versus 0.4 minϪ1) compared with the parent PIC analog (Fig. 2B). This is consistent with their different rates of binding to the enzyme heme measured in terms of change of absorbance ⌬(460 to 427) nm versus time of binding, which showed PID binding to iNOSfl was 1.9 minϪ1 as compared with 1.4 minϪ1 obtained for PIC (Fig. 2A)
We demonstrate the unique capability of a bulky pyrimidine imidazole derivative, PID, to interact with both the functional entities of the iNOS enzyme, namely the iNOS dimer and monomer to either cause monomerization of already assembled active dimers or prevent dimerization of nascent monomers, through a near-irreversible binding of PID to the iNOS heme moiety

Summary

Background

Overproduction of nitric oxide by dimeric inducible nitric-oxide synthase (iNOS) is physiologically harmful. One of the reasons we decided to revisit the mechanism of inhibition of iNOS by pyrimidine imidazoles, amid several previous reports on the same subject (26 –28), is because we subsequently discovered that these compounds could bind and accomplish inhibition of the dimeric iNOS, the active form of iNOS in a physiological setting, an essential prerequisite for an inhibitor to qualify as a potential candidate for therapeutic intervention of iNOS activity both for the effective clinical control of diseases associated with iNOS overexpression and nitrosative stress as well as a research tool for elucidating the role of this physiologically versatile enzyme in vital life functions in vivo. We found that PID can disrupt the fully assembled, active dimeric form of iNOS in cells, indicating that it has the potential to accomplish complete physiological inhibition of the enzyme and to serve as an in vivo tool for elucidating the role of iNOS in diseases associated with its dysfunctional overexpression as well as a therapeutic inhibitor for clinical management of these diseases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION