Garry J. Southan

Selective pharmacological inhibition of distinct nitric oxide synthase isoforms

Nitric oxide (NO) is produced in physiological and pathophysiological conditions by three distinct isoforms of NO synthase (NOS): endothelial NOS (ecNOS), inducible NOS (iNOS), and brain NOS (bNOS). Selective inhibition of iNOS may be beneficial in various forms of shock and inflammation, whereas inhibition of bNOS may protect against neuroinjury. This article surveys the enzymatic mechanism of NO production, lists the strategies and pharmacological tools for selective inhibition of distinct NOS isoforms, and considers the side-effects of the various approaches. Selective inhibition of NOS isoforms is achieved by: (a) targeting the differential co-factor (calmodulin or tetrahydrobiopterin) requirement of various NOS isoforms, and NOS; (b) targeting the differential substrate requirements of cells expressing various isoforms of NOS (L-arginine uptake blockers or arginase); (c) the use of pharmacological agents that are selectively taken up by cells expressing various isoforms of NOS (7-nitroindazole); or (d) developing pharmacological NOS inhibitors with isoform specificity. The amino acid-based NOS inhibitor, NG-nitro-L-arginine, shows a preference for ecNOS and bNOS over iNOS, whereas L-N6-(1-iminoethyl)lysine is selective for iNOS over bNOS. Certain non-amino acid-based small molecules, such as aminoguanidine and certain S-alkylated isothioureas, also express selectivity towards iNOS and have anti-inflammatory and anti-shock properties. 7-nitroindazole, a bNOS-selective inhibitor, protects in central nervous system injury. Clearly, there are a number of distinct approaches that are worthy of further research efforts in order to achieve even more selective targeting of various NOS isoforms

Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform.

1 The generation of nitric oxide (NO) from L‐arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S‐alkylisothioureas. Unlike most L‐arginine based inhibitors, however, some guanidines and S‐alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6–30 μm (EC50 values for NG‐methyl‐L‐arginine (L‐NMA) and NG‐nitro‐L‐arginine were 159 and > 1000 μm, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L‐arginine in the incubation medium, indicating that these agents may compete with L‐arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50S comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20–100 times weaker than NG‐methyl‐L‐arginine and NG‐nitro‐L‐arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L‐arginine to L‐citrulline in homogenates of bovine endothelial cells and by their pressor effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin‐treated rats caused an increase in MAP and restored 80% of the endotoxin‐induced fall in MAP. 8 High doses of MEG (30–60 mg kg−1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium‐independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2‐aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10 In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS.

The inhibitory effects of mercaptoalkylguanidines on cyclo‐oxygenase activity

1 It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo‐oxygenase (COX‐2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo‐oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX‐1) and the inducible COX (COX‐2) was investigated in cells and in purified enzymes. Aminoguanidine, NG‐methyl‐L‐arginine (l‐NMA) and NG‐nitro‐L‐arginine methyl ester (l‐NAME) were also studied for comparative purposes. 2 Western blot analysis demonstrated a significant COX‐1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10μgml−1) and interferon γ (IFNγ, 100 u ml−1) for 6 h resulted in a significant induction of COX‐2, and a down‐regulation of COX‐1. No COX‐2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX‐1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX‐2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX‐1 and COX‐2 activities was also studied. 3 In experiments designed to measure COX‐1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 μm) for 6 h. This treatment induced a significant production of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α, the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 μm to 3 mm) caused a dose‐dependent inhibition of the accumulation of 6‐keto‐PGF1α, with an IC50 of 20 μm. However, aminoguanidine, L‐NAME or L‐NMA (up to 3 mM) did not affect the production of 6‐keto‐PGFlş in this experimental system. In experiments designed to measure COX‐1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 μm) for 30 min; this also induced a significant production of 6‐keto‐PGF1α, and MEG (1 μm to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L‐NAME nor L‐NMA inhibited the production of prostaglandins. 4 In experiments designed to measure prostaglandin production by COX‐2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX‐2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6‐keto‐PGF1α. Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 μm to 3 mM) caused a dose‐dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS‐stimulated 6‐keto PGFlα production was shown by other mercaptoalkylguanidines (such as N‐methyl‐mercaptoethylguanidine, N,N′‐dimethyl‐mercaptoethylguanidine, S‐methyl‐mercaptoethylguanidine and guanidino‐ethyldisul‐phide), with IC50 values ranging between 34–55 μm. However, aminoguanidine, L‐NAME and L‐NMA (up to 3 mM) did not affect the production of prostaglandins. 5 In comparative experiments indomethacin, a non selective COX inhibitor, and NS‐398, a selective COX‐2 inhibitor, reduced (LPS) stimulated 6‐keto‐PGF1α production in J774 macrophages in a dose‐dependent manner without affecting nitrite release. Indomethacin, but not NS‐398, inhibited 6‐keto‐PGF1α production in the HUVECs. 6 The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX‐1 and COX‐2. MEG dose‐dependently inhibited the purified COX‐1 and COX‐2 activity with IC50 values of 33 μm and 36 μm, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX‐1 metabolites, without affecting COX‐2 activity. High doses of L‐NAME (3 mM) decreased COX‐1 activity only, while L‐NMA (up to 3 mM) had no effect on the activity of either enzyme. 7 These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo‐oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX‐2 are expressed.

Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

1 . Guanidines, amidines, S‐alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L‐arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2 . GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 μm, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L‐arginine and non‐competitive vs. tetrahydrobiopterin. 3 . Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD‐1 cells were stimulated with appropriate mixtures of pro‐inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11,9, 1 and 30 μm, respectively). This suggests that uptake of GED may be cell type‐ and species‐ dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4 . GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 μm). Part of this relaxation was endothelium‐dependent, but was not blocked by methylene blue (100 μm), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine‐induced, endothelium‐dependent relaxations at 10 μm and caused a slight inhibition of the relaxations at 100 μm. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5 . In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg−1 bolus followed by 10 mg kg−1 h−1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15 mg kg−1, i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrenaline of the thoracic aorta ex vivo and protected against the impairment of the endothelium‐dependent relaxations associated with this model of endotoxaemia. The same bolus and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6 . Administration of GED (10 mg kg−1, i.p.) given at 2 h after LPS (120 mg kg−1, i.p.) and every 6 h thereafter caused a significant improvement in the survival rate in a lethal model of endotoxin shock in mice between 12 and 42 h. 7 . In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towards iNOS may lie both at the enzyme level and at the level of cell uptake. GED has beneficial effects in models of endotoxin shock that are driven by iNOS. GED or its derivatives may be useful tools in the experimental therapy of inflammatory conditions associated with NO overproduction due to iNOS expression.

Amidines are potent inhibitors of nitric oxide synthases : preferential inhibition of the inducible isoform

We evaluated the ability of simple alkyl amidines to inhibit the activity of the inducible isoform of nitric oxide (NO) synthase in vitro. In immunostimulated J774 macrophages, 2-iminopiperidine (EC50 = 10 microM) and butyramidine (EC50 = 60 microM) were more potent than NG-methyl-L-arginine (EC50 = 70 microM) in inhibiting nitrite formation. The five amidines tested for their ability to inhibit the conversion of L-arginine to L-citrulline by bovine endothelial cell homogenates (a source of the constitutive, endothelial NO synthase isoform) were less effective than NG-nitro-L-arginine or NG-methyl-L-arginine. The rank-order of the potencies of the amidines against the endothelial NO synthase was, in general, similar to the rank-order of the pressor effects of these agents in anesthetized rats. Thus, certain amidines are potent inhibitors of NO synthase, and are more selective towards the inducible NO synthase than the commonly used L-arginine based NO synthase inhibitors.

Potent inhibition of the inducible isoform of nitric oxide synthase by aminoethylisoselenourea and related compounds

The generation of nitric oxide (NO) by nitric oxide synthase (NOS) can be inhibited by certain guanidines and S-alkylisothioureas. In particular, aminoethylisothiourea (AE-TU) shows selectivity towards the inducible isoform (iNOS) over the endothelial isoform (ecNOS). Here we report on the effects of the selenium analog of AE-TU, aminoethylisoselenourea (AE-SeU), and its homologue, aminopropylisoselenourea (AP-SeU), on the activities of iNOS and ecNOS. AE-SeU and AP-SeU inhibited the conversion of L-arginine to L-citrulline in homogenates of lung taken from endotoxin-treated rats (a model of iNOS acitivity) with potencies (EC50=1.1, and 0.1 microM, respectively) greater than that of N(G)-methyl-L-arginine (L-NMA) (22 microM). In contrast, AE-SeU and AP-SeU were weaker than or similar to L-NMA at inhibiting ecNOS activity in homogenized bovine endothelial cells (EC50 values = 104, 15, and 16 microM, respectively). AE-SeU and AP-SeU potently inhibited nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values of 10 and 4 microM respectively. The corresponding EC50 value for L-NMA was 160 microM. The inhibition was dose-dependently reduced by increasing concentrations of L-arginine in the medium. In vivo, AE-SeU had only modest effects on blood pressure when given as a bolus to anesthetized rats, suggesting only a small effect on ecNOS in vivo, whereas AP-SeU had potent pressor effects similar to those of L-NMA. We found that both AE-SeU and AP-SeU were unstable in aqueous solution at pH values above 6. Their disappearance from solution was accompanied by the appearance of a reductive species, probably free selenol. These findings suggest that AE-SeU and AP-SeU exert their inhibitory effects through intramolecular rearrangement to yield selenoethylguanidine and seleno-propylguanidine. Thus, selenoalkylguanidines are novel inhibitors of iNOS.