Richard G. Knowles

Nitric oxide synthases: structure, function and inhibition

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

1400W Is a Slow, Tight Binding, and Highly Selective Inhibitor of Inducible Nitric-oxide Synthase in Vitro and in Vivo

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric- oxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s−1 and a binding constant of 2.0 μM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 μM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value ≤ 7 nM. In contrast, inhibition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with Ki values of 2 μM and 50 μM, respectively. Thus, 1400W was at least 5000-fold selective for iNOS versus eNOS. This selectivity was similar to that observed in rat aortic rings, in which 1400W was greater than 1000-fold more potent against rat iNOS than eNOS. Finally, 1400W was greater than 50-fold more potent against iNOS than eNOS in a rat model of endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greater than of any previously described iNOS inhibitor.

Anti-inflammatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver and aorta of the rat

The induction by endotoxin of Ca2(+)-independent nitric oxide (NO) synthase in the lung and liver of the rat was prevented by the glucocorticoids dexamethasone and cortisol but not by progesterone. The activity of the constitutive Ca2(+)-dependent NO synthase in the brain and the aorta was not affected by treatment with either endotoxin or glucocorticoids. In the aorta a Ca2(+)-independent NO synthase was also found following endotoxin treatment of rats, and this induction was likewise prevented by dexamethasone. The Ca2(+)-dependent NO synthase in the aorta was located in the vascular endothelium, whereas the Ca2(+)-independent enzyme was predominantly located in the vascular smooth muscle layer. Inhibition of induction of the Ca2(+)-independent NO synthase in vivo may underlie some of the physiological and pharmacological effects of the anti-inflammatory glucocorticoids.

Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands.

The formation of nitric oxide (NO) by an L-arginine:NO synthase and its stimulation of the soluble guanylate cyclase was studied in rat whole adrenal and bovine cortex and medulla cytosol. In the presence of L-arginine, the stimulation of soluble guanylate cyclase was accompanied by the formation of citrulline and NO2-, formed from NO. The NO synthase was NADPH- and Ca(2+)-dependent and was inhibited by several L-arginine analogues. These results indicate that rat and bovine adrenal cytosol contains an L-arginine:NO synthase.

The formation of nitric oxide donors from peroxynitrite

1 Administration of peroxynitrite (ONOO−, 30–300 μM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half‐life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO− which have half‐lives in the order of 1–2 s under these conditions. However the relaxation was inhibited by oxyhemoglobin, suggesting that the compound could be converted to NO in the vascular tissues or in the superfusate. 2 The products of the reaction between ONOO− and Krebs buffer containing 11 mM glucose, but not glucose‐free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO− with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO− in order to characterize their pharmacological actions both in the cascade bioassay and on platelets and to study their ability to release NO. 3 These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D‐glucose, were equally effective if L‐glucose was used as a reactant and were reversed by oxyhaemoglobin. 4 The products of the reaction between ONOO− and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+ and L‐cysteine. 5 These results indicate that ONOO− reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donor(s) with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO− in vivo.

GW274150, a novel and highly selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), shows analgesic effects in rat models of inflammatory and neuropathic pain.

Abstract Nitric oxide (NO), synthesised by different isoforms of nitric oxide synthase (NOS), has been linked with the development and maintenance of nociception. We studied the role of the inducible isoform, iNOS, in two different rat pain models with an inflammatory component. iNOS was immunohistochemically detected locally in the paw 6 h after Freunds Complete Adjuvant (FCA) injection, showing a plateau at 24–72 h and falling slowly in the following weeks. This correlated with the late phase of the hypersensitivity to pain revealed in the behavioural tests. A highly selective iNOS inhibitor GW274150 (1–30 mg/kg orally, 24 h after FCA) suppressed the accumulation of nitrite in the inflamed paw indicating substantial iNOS inhibition. At the same time it partially reversed FCA‐induced hypersensitivity to pain and edema in a dose‐dependent manner. After Chronic Constriction Injury (CCI) surgery to the sciatic nerve, iNOS presence was only detected locally in the region of the nerve (inflammatory cells). GW274150 (3–30 mg/kg orally, 21 days after surgery) also reversed significantly the CCI‐associated hypersensitivity to pain. No iNOS was detectable in dorsal root ganglia, spinal cord or brain in either model. This study demonstrates a role for peripherally‐expressed iNOS in pain conditions with an inflammatory component and the potential value of iNOS inhibitors in such conditions.

Synthesis of nitric oxide in the bovine retina

In the absence of light, high concentrations of cGMP open ion channels in the plasma membranes of rod outer segments. The source of stimulation of retinal guanylate cyclase is not known. Nitric oxide is a potent stimulator of guanylate cyclase in other cell systems. The present data demonstrate that nitric oxide synthase, an enzyme responsible for the production of nitric oxide, is present in retina, and specifically in the rod outer segments. This enzyme uses L-arginine as a substrate and is NADPH- and calcium- dependent. L-arginine-derived nitric oxide may be a source of activation of guanylate cyclase in the retina.

The L-arginine : nitric oxide pathway

The L-arginine: nitric oxide pathway is widely recognized as an important regulator of cell function and communication in a variety of physiological and pathophysiological situations. Recent advances in the biochemistry and molecular biology of nitric oxide synthases have contributed significantly to our understanding of the regulation of nitric oxide synthesis in health and disease. This pathway has been implicated in the pathogenesis of septic shock, hypertension, and atherosclerosis as well as in the antihypertensive action of converting enzyme inhibitors. Progress in this field, which spans the cardiovascular, immune, and nervous systems, has been rapid, and its full potential is yet to be realized.

In vitro pharmacological characterization of vilanterol, a novel long-acting β2-adrenoceptor agonist with 24-hour duration of action.

Vilanterol trifenatate (vilanterol) is a novel, long-acting β2-adrenoceptor (β2-AR) agonist with 24 h activity. In this study, we describe the preclinical pharmacological profile of vilanterol using radioligand binding and cAMP studies in recombinant assays as well as human and guinea pig tissue systems to characterize β2-AR binding and functional properties. Vilanterol displayed a subnanomolar affinity for the β2-AR that was comparable with that of salmeterol but higher than olodaterol, formoterol, and indacaterol. In cAMP functional activity studies, vilanterol demonstrated similar selectivity as salmeterol for β2- over β1-AR and β3-AR, but a significantly improved selectivity profile than formoterol and indacaterol. Vilanterol also showed a level of intrinsic efficacy that was comparable to indacaterol but significantly greater than that of salmeterol. In cellular cAMP production and tissue-based studies measuring persistence and reassertion, vilanterol had a persistence of action comparable with indacaterol and longer than formoterol. In addition, vilanterol demonstrated reassertion activity in both cell and tissue systems that was comparable with salmeterol and indacaterol but longer than formoterol. In human airways, vilanterol was shown to have a faster onset and longer duration of action than salmeterol, exhibiting a significant level of bronchodilation 22 h after treatment. From these investigations, the data for vilanterol are consistent, showing that it is a novel, potent, and selective β2-AR receptor agonist with a long duration of action. This pharmacological profile combined with clinical data is consistent with once a day dosing of vilanterol in the treatment of both asthma and chronic obstructive pulmonary disease (COPD).

GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo

1 GW274150 ([2‐[(1‐iminoethyl) amino]ethyl]‐L‐homocysteine) and GW273629 (3‐[[2‐[(1‐iminoethyl)amino]ethyl]sulphonyl]‐L‐alanine) are potent, time‐dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100‐fold) or neuronal NOS (nNOS) (>80‐fold). GW274150 and GW273629 are arginine competitive, NADPH‐dependent inhibitors of human iNOS with steady state Kd values of <40 and <90 nM, respectively. 2 GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time‐dependent manner, reaching IC50 values of 0.2±0.04 and 1.3±0.16 μM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260‐fold and 219‐fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150‐fold and 365‐fold selective for iNOS against eNOS and nNOS, respectively. 3 The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half‐life of ∼6 h. That of GW273629 was also biphasic in rats, producing a terminal half‐life of ∼3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (∼10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice. 4 GW274150 was effective in inhibiting LPS‐induced plasma NOx levels in mice with an ED50 of 3.2±0.7 mg kg−1 after 14 h intraperitoneally (i.p.) and 3.8±1.5 mg kg−1 after 14 h when administered orally. GW273629 showed shorter‐lived effects on plasma NOx and an ED50 of 9±2 mg kg−1 after 2 h when administered i.p. 5 The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice.