ABSTRACT
Based on computer modeling of the active site of nitric oxide synthases (NOS), a series of 10 amidine compounds (9-18) was designed including potential inhibitors that involve the coordination of side-chain functional groups with the iron of the heme cofactor. The most potent and selective compound was the methylthio amidine analogue 9, which was more potent than L-nitroarginine with 185-fold selectivity for inhibition of neuronal NOS over endothelial NOS. It also exhibited time-dependent inhibition, but did not involve the mechanism previously proposed for other amidine inhibitors of NOS. None of the compounds, however, exhibited heme-binding characteristics according to absorption spectroscopy.
Subject(s)
Drug Design , Heme/chemistry , Heme/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/pharmacology , Animals , Arginine/analogs & derivatives , Arginine/chemistry , Arginine/pharmacology , Chromatography, High Pressure Liquid , Computer Simulation , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Mice , Molecular Structure , Rats , Spectrum Analysis , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A series of L-nitroarginine-based dipeptide inhibitors are highly selective for neuronal nitric oxide synthase (nNOS) over the endothelial isoform (eNOS). Crystal structures of these dipeptides bound to both isoforms revealed two different conformations, curled in nNOS and extended in eNOS, corresponding to higher and lower binding affinity to the two isoforms, respectively. In previous studies we found that the primary reason for selectivity is that Asp597 in nNOS, which is Asn368 in eNOS, provides greater electrostatic stabilization in the inhibitor complex. While this is the case for smaller dipeptide inhibitors, electrostatic stabilization may no longer be the sole determinant for isoform selectivity with bulkier dipeptide inhibitors. Another residue farther away from the active site, Met336 in nNOS (Val106 in eNOS), is in contact with bulkier dipeptide inhibitors. Double mutants were made to exchange the D597/M336 pair in nNOS with N368/V106 in eNOS. Here we report crystal structures and inhibition constants for bulkier dipeptide inhibitors bound to nNOS and eNOS that illustrate the important role played by residues near the entry to the active site in isoform selective inhibition.
Subject(s)
Guanidines/chemistry , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitro Compounds/chemistry , Protein Conformation , Animals , Cattle , Crystallization , Mannitol/chemistry , Molecular Structure , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type III/genetics , Nitroarginine/chemistry , Point Mutation , Protein Binding , Rats , X-Ray DiffractionABSTRACT
This review includes the non-patent literature up to October 2004 that deals with selective neuronal nitric oxide synthase inhibitors (highest potency is for the neuronal isozyme). Some non-selective inhibitors or selective inducible nitric oxide synthase inhibitors are mentioned if they are related to compounds that are discussed; structures of these compounds generally are not given. In vitro inhibition constants are given either as IC(50) values or as K(i)values. An IC(50) value, the inhibitor concentration that produces 50% inhibition in the presence of a constant concentration of substrate, is obtained by extrapolation of several rate data points to 50% inhibition. K(i) values are derived from several types of plots that relate the concentration of inhibitor with enzyme velocity in the presence of a variety of substrate concentrations [1]. The K(i) value can be estimated from the IC(50) value [2]. Although the two inhibition constants are related, they are not the same; generally, the reported K(i) values tend to be lower than the IC(50) values. If specifics are desired about how the data were collected, then the reader will have to look in the literature cited. No attempt was made to be exhaustive in citing all references related to specific inhibitors; rather, examples of literature references are given for each inhibitor described.
Subject(s)
Enzyme Inhibitors/pharmacology , Nerve Tissue Proteins/antagonists & inhibitors , Nitric Oxide Synthase/antagonists & inhibitors , Animals , Enzyme Inhibitors/classification , Humans , Nitric Oxide Synthase Type IABSTRACT
Three nitric oxide synthase (NOS) isoforms, eNOS, nNOS and iNOS, generate nitric oxide (NO) crucial to the cardiovascular, nervous and host defense systems, respectively. Development of isoform-selective NOS inhibitors is of considerable therapeutic importance. Crystal structures of nNOS-selective dipeptide inhibitors in complex with both nNOS and eNOS were solved and the inhibitors were found to adopt a curled conformation in nNOS but an extended conformation in eNOS. We hypothesized that a single-residue difference in the active site, Asp597 (nNOS) versus Asn368 (eNOS), is responsible for the favored binding in nNOS. In the D597N nNOS mutant crystal structure, a bound inhibitor switches to the extended conformation and its inhibition of nNOS decreases >200-fold. Therefore, a single-residue difference is responsible for more than two orders of magnitude selectivity in inhibition of nNOS over eNOS by L-N(omega)-nitroarginine-containing dipeptide inhibitors.
