Crystal Structure of Constitutive Endothelial Nitric Oxide Synthase

Raman, C.S.; Li, Huiying; Martásek, Pavel; Král, Vladimı́r; Masters, Bettie Sue Siler; Poulos, T.L.

doi:10.1016/s0092-8674(00)81718-3

Cited by 604 publications

(496 citation statements)

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“…5 Investigation into the synthesis and chemistry of novel isoform-selective NOS inhibitors has been an ongoing challenge, even though the general pharmacophore requirements are well established. [6][7][8][9][10][11][12] Synthesis of substrate (L-arginine) based peptidomimetic non-selective as well as selective nNOS inhibitors have been extensively reported in the literature. 13 In an effort to improve PK/PD properties by decreasing their peptidic nature, various small molecule selective nNOS inhibitors have also been reported.…”

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confidence: 99%

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

Renton¹,

Speed²,

Maddaford³

et al. 2011

Bioorganic & Medicinal Chemistry Letters

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A series of 1,6-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). By varying the basic amine side chain at the 1-position of the indole ring, several potent and selective inhibitors of human neuronal NOS were identified. In general compounds with bulkier side chains displayed increased selectivity for nNOS over eNOS and iNOS isoforms. One of the compounds, (R)-8 was shown to reduce tactile hyperesthesia (allodynia) after oral administration (30 mg/kg) in an in vivo rat model of dural inflammation relevant to migraine pain. Keywords1,6-Disubstitued indole derivatives; Nitric oxide; Nitric oxide synthase; Nitric oxide synthase inhibitors; Selective neuronal nitric oxide synthase inhibitors Nitric oxide (NO) is an inorganic free radical that has diverse roles both in normal and pathological processes, including the regulation of blood pressure, neurotransmission and macrophage defense systems. 1 NO is synthesized from the enzyme catalysis of L-arginine to L-citrulline by three isoforms of nitric oxide synthase (NOS): two constitutive forms in neuronal cells (nNOS) and endothelial cells (eNOS), and an inducible form in macrophage cells (iNOS). Overstimulation or overproduction of NO by nNOS and iNOS has been shown to play a key role in several disorders, including septic shock, arthritis, diabetes, ischemiareperfusion injury, pain and various neurodegenerative diseases. 2 However, any inhibitors to treat these conditions must avoid eNOS inhibition as this will lead to unwanted effects such as enhanced white cell and platelet activation, hypertension and atherogenesis. 3 Therefore, the development of selective NOS inhibitors is of considerable interest, both from a therapeutic perspective and also as specific pharmacological tools. 4 Although there is low homology among the three NOS primary sequences (~50%), the active sites of the enzymes appears to be relatively conserved with 16 out of 18 residues within 6 Å being identical, presumably explains the difficulty obtaining selective NOS The pharmacophore model we adopted for the arginine binding site of the NOS enzyme includes a guanidine isosteric group (amidine group) and a basic amine group, both attached to a central aryl scaffold (indole core) as shown in Figure 1. 4,15 The amidine group makes an important bidentate interaction with the conserved glutamic acid residue to achieve the necessary potency; whereas the basic amine is assumed to provide the nNOS isoform selectivity. 15 Our design strategy is based on an indole core as an aryl scaffold and exploring various basic amine side chains for achieving the NOS isoform selectivity. As part of our ongoing efforts to find small molecule selective nNOS inhibitors for treating CNS disorders, herein we report the synthesis and biological activity evaluations of a series of 1,6-disubstituted indole derivatives and in vivo activity of (R)-8 in a rat model relevant to migraine pain. 15 Two general approaches were undertaken for the prepa...

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confidence: 99%

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

Renton¹,

Speed²,

Maddaford³

et al. 2011

Bioorganic & Medicinal Chemistry Letters

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“…The degree of hydrophobicity of a particular non-covalent interaction was rarely discussed, perhaps because it is not fully known, or due to the fact that, upon transfer to the gas phase, hydrophobic stabilization effects diminish, while electrostatic effects are generally enhanced. It has been reported that, in the endothelial NOS isotype, 55% of the dimer interface consists of hydrophobic residues, thereby showing that dimer stability relies heavily on entropic effects of the surrounding aqueous solvent [29]. Due to the high degree of homology between the NOS isoforms, it may be inferred that hydrophobic effects likely play a significant role in the dimerization of both the inducible and neuronal isoforms as well.…”

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confidence: 99%

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Smith

Siu

Rafferty

2004

J. Am. Soc. Mass Spectrom.

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The investigation of protein quaternary structure, protein-cofactor, and protein-ligand interactions by mass spectrometry is often limited by the fragility of such interactions under experimental conditions. To develop more gentle conditions of perhaps general use, we used as a model for study the oxygenase domain of murine inducible nitric oxide synthase (iNOS), which is homodimeric, binds heme and tetrahydrobiopterin H 4 B cofactors, and the substrate L-arginine. The energetics of the collisions in q2 and in the lens region of the mass spectrometer were manipulated for varying the degree of solvation around the non-covalently bound ions. Furthermore, the number of low-energy collisions in the collision cell of the instrument was varied, focusing and dampening the ion beam. Under gentle source collision conditions, and using multiple low-energy collisions in the collision cell of the mass spectrometer, dimers of the iNOS oxygenase domain containing heme, H 4 B, and arginine were observed intact after electrospraying at pH values near neutrality; a mutant of this protein (Trp188 3 Phe) was monomeric and did not bind cofactors. The pH dependence of the iNOS oxygenase domain under acidic conditions was also studied; while heme remained bound to the protein between pH 2.5 and 4.0, the dimeric structure was disrupted. Our findings confirm that non-covalently bound macromolecular complexes are retained and observable using electrospray mass spectrometry under the appropriate experimental conditions. (J Am Soc Mass Spectrom 2004, 15, 629 -638)

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“…Consistent with this was the observation that radioligand binding with [$H]H % Bip and [$H]NO # Arg revealed a dual allosteric interaction between the H % Bip-binding site and -arginine-binding site of NOS-I [35], whereas for NOS-II this has still remained controversial [37,38]. However, at the crystal level H % Bip was not found to affect substrate binding [34], in direct disagreement with an earlier report [16]. Moreover, in enzyme kinetic studies, H % Bip did not alter the IC &!…”

Section: Discussionmentioning

confidence: 69%

“…Interestingly, the stabilizing effects of H % Bip might be isoform-dependent. For NOS-I, H % Bip is required for dimer stabilization [17,24,25] but not for subunit assembly [26,27] ; for NOS-II and III, the current evidence that supports a role for H % Bip in subunit assembly is inconsistent [23,[28][29][30][31][32][33] even when the enzymes have been solved at the crystal level [16,34].…”

Section: Introductionmentioning

confidence: 99%

“…In a radioligand binding study, Mayer and co-workers demonstrated a 6-fold increase in the affinity of NOS-I for [$H]H % Bip in the presence of -arginine [35]. For NOS-II, both positive [37] and negative [38] [34] as had previously been demonstrated [16]. Consequently, it remains to be established whether this binding-site co-operativity is a general phenomenon for all NOS isoforms and of relevance during catalysis.…”

Section: Introductionmentioning

confidence: 99%

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Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

Kotsonis

Fröhlich

Shutenko

et al. 2000

Biochemical Journal

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The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H4Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H4Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H4Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H4Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H4Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H4Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor NΩ-nitro-L-arginine. Importantly, H4Bip was found to react chemically with superoxide (O2-−) and enzyme-bound H4Bip was consumed under O2-−-generating conditions in the absence of substrate. These results suggest that H4Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O2-−.

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Crystal Structure of Constitutive Endothelial Nitric Oxide Synthase

Cited by 604 publications

References 71 publications

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

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