The Structure of Nitric Oxide Synthase Oxygenase Domain and Inhibitor Complexes

Crane, Brian R.; Arvai, A.S.; Gachhui, Ratan; Wu, Chaoqun; Ghosh, Dipak; Getzoff, Elizabeth D.; Stuehr, Dennis J.; Tainer, John A.

doi:10.1126/science.278.5337.425

Cited by 338 publications

(306 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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“…After the reaction was completed, the solvent was evaporated under reduced pressure to afford a colorless oil (0.465 g, quantatitive yield). 1 H NMR (500 MHz, CD 3 OD) δ (7.657 + 7.642) (d, 2H, J = 7.5Hz), (7.500 + 7.485) (d, 2H, J = 7.5Hz), 7.249 (t, 2H, J = 7.5Hz), 7.170 (t, 2H, J = 7.5Hz), 4 001 mol) dissolved in MeOH (20 mL) at 0 °C was added a 37% formaldehyde solution (0.812 g, 0.745 mL, 0.01 mol), NaBH 3 CN (0.314 g, 0.005 mol) and acetic acid (0.120 g, 0.115 mL, 0.002 mol). The reaction mixture was stirred at room temperature overnight.…”

Section: Experimental General Methods Reagents and Materialsmentioning

confidence: 99%

Conformationally Restricted Dipeptide Amides as Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors

Gómez-Vidal

Martásek

et al. 2006

J. Med. Chem.

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Four new conformationally-restricted analogues of the potent and selective neuronal nitric oxide synthase inhibitor, L-nitroargininyl-L-2,4-diaminobutyramide (1), have been synthesized. N α -Methyl and N α -benzyl derivatives (3 and 4, respectively) of 4N-(L-Arg NO 2 )-trans-4-amino-Lprolineamide (2) are also selective inhibitors, but the potency and selectivity of 3 are weak. Analogue 4 has only one-third the potency and one-half to one-third the selectivity of 2 against iNOS and eNOS, respectively. 3-N-(L-Arg NO 2 )-trans-3-amino-L-prolineamide (6) is as potent an inhibitor of nNOS as is 2; selectivity for nNOS over iNOS is half of that for 2 but the selectivity for nNOS over eNOS is almost double that for 2. The corresponding cis-isomer (5) is a weak inhibitor of nNOS. These results are supported by computer modeling.Nitric oxide (NO) is a ubiquitous biological messenger involved in a variety of physiological processes that acts as a signal transducer but also exerts a variety of regulatory and cytostatic functions. In most instances NO mediates its biological effects by activating guanylate cyclase and increasing cyclic GMP synthesis. However, effects of nitric oxide that are independent of cyclic GMP are also known. 1 Nitric oxide synthase (NOS, EC 1.14.13.39) isoforms 2 are homodimers that catalyze the oxidation of L-arginine to L-citrulline and nitric oxide in a NADPH-and O 2 -dependent process. 3 There are at least three distinct isoforms of NOS. The constitutive endothelial isoform (eNOS) is involved in the regulation of smooth muscle relaxation and blood pressure and in the inhibition of platelet aggregation. A second constitutive isoform is the neuronal NOS (nNOS), which is important for neurotransmission. A third isozyme is the inducible NOS (iNOS), which is located in activated macrophage cells and acts as a cytotoxic agent in normal immune responses. All of the isoforms utilize NADPH, FAD, FMN, tetrahydrobiopterin, and heme as cofactors. The constitutive isoforms also require Ca 2+ and calmodulin for activity, while the inducible isoform has tightly bound Ca 2+ and calmodulin. The crystal structures of the oxygenase domains of murine iNOS monomer, 4 murine and human iNOS dimer, 5,6,7,8 human and bovine endothelial NOS dimers, 9 and rat neuronal NOS dimer 10 indicate a high degree *Address correspondence to this author at the Department of Chemistry Phone: 1−847−491−5653 Email: Agman@chem.northwestern.edu. § Northwestern University ¶ University of Texas † Present address: Departamento de Quimica Farmaceutica y Organica, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain # Developed the eNOS overexpression system in E. coli and the purification of eNOS. ± Developed the overexpression system for nNOS in E. coli and the purification of the nNOS. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of structural similarity within the catalytic center and the dimer interface regions between NOS isoforms. They share only approximately 50% ...

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“…The iNOS isoform has been implicated in the pathogenesis of various diseases; so there is a growing interest in developing potent and highly selective inhibitors 15, 16 . Their targeted design requires detailed insights into the interactions between ligand, substrate and the surrounding protein matrix.…”

Section: Introductionmentioning

confidence: 99%

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

Horn

Nienhaus

2014

F1000Res

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Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOS oxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOS oxy-NO, demonstrating the strong trans effect of the heme-bound NO.

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The Structure of Nitric Oxide Synthase Oxygenase Domain and Inhibitor Complexes

Cited by 338 publications

References 41 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

Conformationally Restricted Dipeptide Amides as Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

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