Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Garcin, Elsa D.; Arvai, A.S.; Rosenfeld, R.J.; Kroeger, M.D.; Crane, Brian R.; Andersson, Gunilla; Andrews, Glen K.; Hamley, Peter; Mallinder, Philip R.; Nicholls, David J.; St-Gallay, Stephen A.; Tinker, Alan C.; Gensmantel, Nigel P.; Mete, Antonio; Cheshire, David R.; Connolly, Stephen; Stuehr, Dennis J.; Åberg, Anders; Wallace, A.V.; Tainer, John A.; Getzoff, Elizabeth D.

doi:10.1038/nchembio.115

Cited by 215 publications

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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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“…In contrast, known specific inhibitors of iNOS and COX-2 are highly conserved and bind to the active site forming key interactions with Glu377 (iNOS) and Arg513 (COX-2), respectively. 22,23 Our molecular modeling results suggest that the dendrimers of a second generation (G2) and higher are much too large to fit in the active site and would result in highly unfavorable binding interactions (highly solvent exposed) with both iNOS and COX-2 enzymes (Supplemental Figures 9, 10). Our studies suggest that, at most, one arm of the higher generation dendrimer could fit into the active site.…”

Section: ■ Results and Discussionmentioning

confidence: 98%

“…21 Moreover, many well-characterized iNOS inhibitors interact directly with Glu377, obstructing its binding to L-arginine and thus inhibiting iNOS activity. 22 The ligand interaction map of DG1-OH and iNOS reveals that DG1-OH does not form a hydrogen bond with Glu377 (Supplemental Figure 5). Unlike specific inhibitors of iNOS which bind directly to Glu377, DG1-OH makes other interactions including Met374 and Trp372 which help to stabilize DG1-OH at the active site of iNOS, thereby occluding it.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“Click” Dendrimers as Anti-inflammatory Agents: With Insights into Their Binding from Molecular Modeling Studies

et al. 2013

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These studies explore the relationship between the inhibitory actions of low generation dendrimers in stimulated microglia and dendrimer−enzyme interactions using in silico molecular modeling. Low generation (DG0 and DG1) dendrimers with acetylene and hydroxyl terminal groups were tested for their anti-inflammatory activity in microglia stimulated by lipopolysaccharides (LPS), and the results were compared with those from the established anti-inflammatory agents, ibuprofen and celecoxib. We hypothesized that hydroxyl terminal groups of DG0 and DG1 dendrimers could interact with the active sites of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes due to their small size and favorable electrochemical properties. The enzymatic activity of iNOS and COX-2 was determined in the presence of low generation dendrimers using biochemical assays and their values related to dendrimer docking confirmations from in silico molecular modeling. We found that results from the molecular modeling studies correlated well with the in vitro biological data, suggesting that, indeed, hydroxyl terminal groups of low generation dendrimers enable multivalent macromolecular interactions, resulting in the inhibition of both iNOS and COX-2 enzymes.

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“…The iNOS ox Crystal Structure Reveals H 4 B Is a Nitrosation Target-To elucidate the structural effects of nitrosation on the obligate NOS dimer, we grew diffraction quality crystals of iNOS ox in the presence of CysNO and a dimer-stabilizing inhibitor (28,40), and determined the crystal structure (2.2 Å resolution, R free 22.9%, Table 1). We found evidence for two distinct types of NO modifications, both at sites that contribute to the dimer interface: not only S-nitrosylation of the ZnS 4 site, but also N-nitrosation of the pterin cofactors (Fig.…”

Section: Resultsmentioning

confidence: 99%

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

Rosenfeld

Bonaventura

Szymczyna

et al. 2010

Journal of Biological Chemistry

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Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS 4 site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions. Nitric oxide (NO)4 levels are regulated in vivo at the level of synthesis by three closely related forms of nitric-oxide synthase (NOS): inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) (1). All three NOS enzymes are active only as homodimers and contain two modules: the catalytic oxygenase (NOS ox ) and the electron-supplying reductase (NOS red ) (2). The NOS ox dimer contains two catalytic sites at which the substrate L-arginine binds above the heme (3, 4). The symmetrical NOS ox dimer interface is stabilized by a bridging ZnS 4 cluster and two predominantly buried tetrahydrobiopterin (H 4 B or (6R)-5,6,7,8-tetrahydro-L-biopterin) cofactors, each hydrogen-bonded to one heme (4 -7). Critical differences among the three isozymes include location, regulatory mechanisms, dimer stability, and the amount of NO that is produced (8 -10). iNOS rapidly generates large toxic bursts of NO in vivo and has fewer known mechanisms for halting NO production, whereas eNOS and nNOS produce less NO, are calcium-regulated and contain auto-inhibitory elements (9,11,12).Biological signaling by NO is orchestrated by three interconverting redox-active forms: the free radical (NO ⅐ ), the nitroxide anion (NO Ϫ ), and the nitrosonium cation (NO ϩ ) (13). NO ⅐ binds to metal sites in proteins, while NO ϩ reacts with cysteinyl sulfurs forming S-nitrosylated derivatives (14). S-Nitrosylation is a reversible post-translational modification implicated in protein regulation and signaling within intact cells (15-17). As the major...

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Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Cited by 215 publications

References 51 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

“Click” Dendrimers as Anti-inflammatory Agents: With Insights into Their Binding from Molecular Modeling Studies

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

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