L337H Mutant of Rat Neuronal Nitric Oxide Synthase Resembles Human Neuronal Nitric Oxide Synthase toward Inhibitors

Fang, Jianguo; Ji, Haitao; Lawton, Graham R.; Xue, Fengtian; Roman, Linda J.; Silverman, Richard B.

doi:10.1021/jm900380j

Cited by 12 publications

(3 citation statements)

References 34 publications

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“…Apparently, the fluorophenyl tail of the inhibitor may clash with the His342 side chain, which may force the inhibitor to adjust its binding conformation. In the past, without a crystal structure of human nNOS, we used the rat nNOS L337H mutant to explore the binding affinity of inhibitors to the human form (Fang et al, 2009). In the future, the inhibitory assay and structural characterization will be performed with human nNOS samples.…”

Section: The Active Site Of Human Nnosmentioning

confidence: 99%

Structures of human constitutive nitric oxide synthases

Jamal

Plaza

et al. 2014

Acta Cryst D Biol Crystallogr

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Mammals produce three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The overproduction of NO by nNOS is associated with a number of neurodegenerative disorders; therefore, a desirable therapeutic goal is the design of drugs that target nNOS but not the other isoforms. Crystallography, coupled with computational approaches and medicinal chemistry, has played a critical role in developing highly selective nNOS inhibitors that exhibit exceptional neuroprotective properties. For historic reasons, crystallography has focused on rat nNOS and bovine eNOS because these were available in high quality; thus, their structures have been used in structure-activity-relationship studies. Although these constitutive NOSs share more than 90% sequence identity across mammalian species for each NOS isoform, inhibitor-binding studies revealed that subtle differences near the heme active site in the same NOS isoform across species still impact enzyme-inhibitor interactions. Therefore, structures of the human constitutive NOSs are indispensible. Here, the first structure of human neuronal NOS at 2.03 Å resolution is reported and a different crystal form of human endothelial NOS is reported at 1.73 Å resolution.

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Section: The Active Site Of Human Nnosmentioning

confidence: 99%

Structures of human constitutive nitric oxide synthases

Jamal

Plaza

et al. 2014

Acta Cryst D Biol Crystallogr

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“…The 4-methyl group on the aminopyridine ring of 1b was removed in compounds 2a – f because the methyl group of the eutomer of 1b is stabilized at the hydrophobic pocket of rat nNOS lined with Met336, Leu337, and Trp306 (chain B) 23,24. In human NOS, the residue that corresponds to rat nNOS Leu337 is His342, and it is the only residue in the active site that differs between rat nNOS and human nNOS 26. It was hypothesized that demethylated compound 2a – f might fit better in the active site of human nNOS, which does not have this hydrophobic pocket.…”

Section: Introductionmentioning

confidence: 99%

Structure-based design, synthesis, and biological evaluation of lipophilic-tailed monocationic inhibitors of neuronal nitric oxide synthase

Xue¹,

Huang²,

Ji³

et al. 2010

Bioorganic & Medicinal Chemistry

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Selective inhibitors of neuronal nitric oxide synthase (nNOS) have the potential to develop into new neurodegenerative therapeutics. Recently, we described the discovery of novel nNOS inhibitors (1a and 1b) based on a cis-pyrrolidine pharmacophore. These compounds and related ones were found to have poor blood-brain barrier permeability, presumably because of the basic nitrogens in the molecule. Here, a series of monocationic compounds was designed on the basis of docking experiments using the crystal structures of 1a,b bound to nNOS. These compounds were synthesized and evaluated for their ability to inhibit neuronal nitric oxide synthase. Despite the excellent overlap of these compounds with 1a,b bound to nNOS, they exhibited low potency. This is because they bound in the nNOS active site in the normal orientation rather than the expected flipped orientation used in the computer modeling. The biphenyl or phenoxyphenyl tail is disordered and does not form good protein-ligand interactions. These studies demonstrate the importance of the size and rigidity of the side chain tail and the second basic amino group for nNOS binding efficiency and the importance of the hydrophobic tail for conformational orientation in the active site of nNOS.

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“…This pocket is where the second headgroup of this series of double-headed inhibitors fits. The inhibitory potency of 8 for human nNOS is 90 nM, similar to that (70 nM) of compound 1 , 25 which is our most potent human nNOS inhibitor with potential selectivity over human iNOS and eNOS.…”

Section: Results and Discussionmentioning

confidence: 53%

Nitric Oxide Synthase Inhibitors That Interact with Both Heme Propionate and Tetrahydrobiopterin Show High Isoform Selectivity

Kang

Tang

et al. 2014

J. Med. Chem.

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Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of α-amino functionalized aminopyridine derivatives (3–8) were designed to probe the structure–activity relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors.

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L337H Mutant of Rat Neuronal Nitric Oxide Synthase Resembles Human Neuronal Nitric Oxide Synthase toward Inhibitors

Cited by 12 publications

References 34 publications

Structures of human constitutive nitric oxide synthases

Structures of human constitutive nitric oxide synthases

Structure-based design, synthesis, and biological evaluation of lipophilic-tailed monocationic inhibitors of neuronal nitric oxide synthase

Nitric Oxide Synthase Inhibitors That Interact with Both Heme Propionate and Tetrahydrobiopterin Show High Isoform Selectivity

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