Impact of mechanism-based enzyme inactivation on inhibitor potency: implications for rational drug discovery.

Mechanism-based enzyme inactivators (MBEIs) have unique kinetic actions that make predictions of potency, selectivity, and potential for metabolic drug interactions more complex than for competitive antagonists. We have derived a mathematical relationship that links the influence of substrate concentration and binding constant ([S] and K(m), respectively), inhibitor concentration and binding constant ([I] and K(I), respectively), and inactivation rate constant (k(inact)) to enzyme activity (v) and maximal activity (V(max)) at any time (t). The kinetic behavior of this relationship was validated in murine-macrophage cell cultures using MBEIs of nitric oxide synthase (NOS). This initial equation was also used in the derivation of a new relationship that directly links the kinetic parameters of mechanism-based inactivation to inhibitory potency at a particular time (IC((t))(50)). Using this direct relationship, we observed that the predicted rank inhibitory potency of a series of MBEIs was improved over that predicted by the K(I) parameter alone. These relationships offer a fundamental understanding of the kinetics of MBEI action and may be useful in the evaluation of these compounds during the discovery process.

Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta. An additional mechanism for nitroglycerin.

We investigated the involvement of calcitonin gene-related peptide (CGRP) in the vasodilatory mechanism of action of nitric oxide (NO) donors. The functional role of CGRP in NO donor-induced vasodilation of isolated rat aortic rings was determined by incubating these drugs with and without CGRP(8-37), a selective CGRP receptor antagonist. CGRP(8-37) (0.63 microM) induced rightward shifts in the vasodilatory concentration-response curves for nitroglycerin (NTG), Piloty's acid (PA), and SIN-1 (linsidomine). The EC(50) values for NTG, PA, and SIN-1 were increased by 8.3-, 5.2-, and 2.3-fold, respectively (P < 0.05). The release of CGRP from rat aorta in response to NTG and PA was measured specifically by radioimmunoassay. Thirty-minute incubations of NTG or PA with rat aorta induced 189.5 and 214.6% increases, respectively, in CGRP release when compared with the control (P < 0.05). The concentration-response curves of sodium nitroprusside (SNP), S-nitroso-acetylpenicillamine (SNAP), tetranitromethane (TNM), diethylamine NO complex (DEA-NO), and diethylenetriamine/nitric oxide adduct (DETA NONOate) were not inhibited significantly by CGRP(8-37) co-incubation (P 0.05). NO donors also were incubated with aortic strips, and NTG and PA alone induced significant formation of hydroxylamine, a NO(-) metabolite (232.4 and 364.9%, respectively, P < 0.05). These results indicate that only NTG and PA, and to a lesser extent SIN-1, stimulate the release of CGRP from the rat aorta, which subsequently contributes to the vasodilatory activity of these agents. The hydroxylamine formation suggests a possible link between NO(-) generation and CGRP release from the vascular wall.

Differential sensitivity among nitric oxide donors toward ODQ-mediated inhibition of vascular relaxation.

Nitric oxide (NO) donors are believed to exert their vasodilatory action through the activation of soluble guanylate cyclase (sGC), the heme site of which can be specifically inhibited by 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). We examined the vascular relaxation of the rat aorta mediated by eight different NO donors in the presence of ODQ (0.1, 1, or 10 microM), and demonstrated that these NO donors displayed different sensitivities toward ODQ inhibition (ANOVA, P <.05). Among the NO donors studied, S-nitrosothiols such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione exhibited partial resistance toward ODQ inhibition at 0.1 microM ODQ, whereas nitroglycerin (NTG) showed nearly complete inhibition at this concentration of ODQ. Three NO donors representing increasing sensitivity toward ODQ inhibition, SNAP < sodium nitroprusside (SNP) < NTG, were chosen for additional mechanistic studies. ODQ (1 microM) inhibition of vascular relaxation by SNAP and SNP, but not that by NTG, was partially reversed by a sulfhydryl donor, N-acetylpenicillamine (100 microM), and by a phosphodiesterase inhibitor, zaprinast (10 microM), specific for cGMP. Our results strongly indicate that the vascular relaxation mechanism(s) of NO donors is not identical for each. In the rat aorta, NTG appeared to exhibit its vasodilatory effect exclusively through activation of the heme site of sGC. On the other hand, in the intact vascular tissue, SNAP and SNP could bring about vasodilation through a secondary pathway. These results are consistent with the view that SNAP and SNP, but not NTG, can induce vascular relaxation additionally through the activation of the sulfhydryl site of sGC.

Differential interactions of nitric oxide donors with rat oxyhemoglobin.

To estimate the reaction of two primary redox-related species of nitric oxide (i.e. NO+ vs NO*) from a variety of NO donors, we employed the differential interactions of these NO forms with oxyhemoglobin (oxyHb) as a chemical assay. NO+ formation was estimated by the S-nitrosation reaction with oxyHb, and NO* formation via its reaction with the oxygen-heme complex of oxyHb. Under the conditions employed, all NO donors caused concentration-dependent formation of methemoglobin, indicative of NO* liberation. However, the extent of S-nitrosation was substantially different among the NO donors studied. A representative S-nitrosothiol, S-nitroso-N-acetyl-penicillamine, caused significantly more S-nitrosation than nitroglycerin, isobutyl nitrite, sodium nitroprusside, and 3-morpholino-sydnonimine (ANOVA, P < 0.05). These results indicated that NO donors can differ in their interactions with oxyHb, and possibly with other target proteins, in part because they liberate or transfer different ratios of NO redox forms. This difference may contribute, in part, to the diversity of pharmacological effects elicited by NO donors.

In vivo disposition of 3-nitro-L-tyrosine in rats: implications on tracking systemic peroxynitrite exposure.

In many pathological conditions such as inflammatory and neurodegenerative diseases, the in vivo toxicity of nitric oxide has been attributed to the toxic oxidant peroxynitrite. Interaction of peroxynitrite with biological molecules can modify tyrosine residues on the proteins at the ortho position resulting in the formation of the stable end-product, 3-nitro-L-tyrosine (3-NT). Recent investigations indicate that changes in the circulating concentrations of 3-NT in pathological conditions may reflect the extent of nitric oxide-dependent oxidative damage and peroxynitrite toxicity. In the present study, we examined the in vivo disposition characteristics of 3-NT in rats after either a single i.v. bolus dose (10 mg/kg) or a loading and maintenance infusion at 10 or 30 mg/kg. Plasma concentrations of 3-NT were analyzed by a reversed-phase HPLC method. After a single bolus dose of 3-NT at 10 mg/kg, the average half-life of the elimination phase for the drug was 68.5+/-18.4 min (n = 5). Infusions of 3-NT at two different doses (10 and 30 mg/kg) indicated that the pharmacokinetic properties of 3-NT below plasma concentrations of 100 microM were both linear and stationary. Urinary excretion of unchanged 3-NT was minimal, but two distinct metabolites of 3-NT were identified in the urine collected throughout the study. These findings may be useful in the interpretation of the plasma and urine 3-NT concentrations as possible indices of systemic peroxynitrite exposure.

Diethyldithiocarbamate prolongs survival of mice in a lipopolysaccharide-induced endotoxic shock model: evidence for multiple mechanisms.

It is now known that overproduction of nitric oxide (NO) by nitric oxide synthase (NOS) is an important contributing factor for the development of cardiovascular collapse and subsequent death in endotoxic shock. Diethyldithiocarbamate (DETC) is a molecular scavenger of NO and can inhibit overexpression of a number of cytokines during shock through inactivation of transcription factors such as nuclear factor (NF)-kappaB. Thus, DETC may be a useful adjunct in the therapy of endotoxic shock. In our study, we examined the effect of DETC on survival time in a murine model of severe endotoxic shock. Our results indicated that selected in vivo dosage regimens of DETC (intraperitoneal: at -2, -1, 3, 6, and 10 h or at -2, -1, 3, 6, 9, 12, 15, and 18 h relative to lipopolysaccharide administration, 180 mg/kg, at t = 0) in endotoxic mice were effective in increasing survival time when compared with untreated animals and DETC pretreatment was more effective than methylprednisolone (p<.05). DETC was shown to exert multiple beneficial mechanisms, including 1) a decrease in circulating NO, as determined by plasma nitrite/nitrate levels, 2) a reduction in plasma tumor necrosis factor-alpha after lipopolysaccharide induction, and 3) decreased expressions of metalloproteinases such as gelatinase A and B which may be responsible for cellular release of cytokines. These results indicate that DETC and its analogs may be useful in the treatment of endotoxic shock.

Effects of nitro-L-arginine on blood pressure and cardiac index in anesthetized rats: a pharmacokinetic-pharmacodynamic analysis.

PURPOSE: Nitric oxide synthase (NOS) inhibitors such as Nitro-L-arginine (L-NA) are being considered for the management of hypotension observed in septic shock. However, little information is available regarding the pharmacokinetic and pharmacodynamic properties of these agents. Our objective was to examine the relationships between L-NA plasma concentration and various hemodynamic effects such as cardiac index (CI), mean arterial pressure (MAP), and heart rate (HR) elicited by L-NA administration in rats. METHODS: L-NA was infused at doses between 2.5-20 mg/kg/hr in anesthetized rats over one hour. Hemodynamic effects and plasma L-NA levels were determined. RESULTS: Infusion of L-NA resulted in dose-dependent increases in MAP and systemic vascular resistance (SVR), decreases in CI, and minimal change in HR. The relationships between the hemodynamic effects and plasma L-NA levels were not monotonic, and hysteresis was observed. Using nonparametric analysis, the equilibration half-time (t1/2,keo) between plasma L-NA and the hypothetical effect site was determined to be 51.5 +/- 6.6 min, 42.4 +/- 10.1 min, 43.4 +/- 9.0 min for MAP, CI, and SVR, respectively (n = 14). The Emax and EC50 values obtained were + 32.5 +/- 8.4 and 2.6 +/- 1.3 microg/ml for MAP and -52.9 +/- 15.6 and 3.7 +/- 1.8 microg/ml for CI, respectively. CONCLUSIONS: Although L-NA can bring about beneficial elevation of MAP, such effect is always accompanied by a stronger effect on CI depression. Dose escalation of L-NA may bring about detrimental negative inotropic effect and loss of therapeutic efficacy.

Pharmacokinetics and steady-state tissue distribution of L- and D-isomers of nitroarginine in rats.

Nitric oxide synthase (NOS) inhibitors, such as nitro-L-arginine (L-NNA), have been used in vivo as mechanistic probes of the NOS system and as potential therapeutic agents for reversing the hypotension developed in septic shock. Little information is available regarding the pharmacokinetic and biodistribution pattern of these compounds. We have examined the in vivo disposition, as well as steady-state biodistribution, of NNA isomers in rats. Plasma and tissue concentrations of L-NNA were determined by HPLC. After intravenous administration of a bolus dose of 20 mg/kg in rats, plasma concentrations of both L- and D-NNA declined biexponentially, with average half-lives of 12 min and 20 hr for L-NNA, and 15 min and 15 hr for the D-enantiomer, respectively. In contrast to L-NNA, the D-isomer had a higher systemic clearance (170 +/- 20 vs. 70.9 +/- 8.2 ml/hr/kg; p = 0.0004) and shorter mean residence time (17.3 +/- 3.7 vs. 31.7 +/- 3.7 hr; p = 0.0039). Based on these pharmacokinetic characteristics, steady-state plasma concentrations of NNA isomers were achieved within 6-8 hr through the use of a loading dose and maintenance infusion. NNA concentrations achieved in many tissues exceeded plasma concentrations, indicating binding of the drug to tissue components. The tissue-to-plasma distribution coefficient (Kp) for L-NNA was variable among various tissues and ranged from 0.67 for testes to 3.17 for liver. Both kidneys and skeletal muscle had Kp values higher than 2, whereas distribution to the cerebrospinal fluid was minimal (Kp = 0.09). Steady-state distribution of the D-isomer of NNA was similar to that of L-NNA.

Reversed-phase high-performance liquid chromatography method for the analysis of nitro-arginine in rat plasma and urine.

Nitro-L-arginine (L-NNA) is an inhibitor of the enzyme nitric oxide synthase (NOS). We developed a simple, sensitive and reproducible reversed-phase high-performance liquid chromatographic method for detection of nitro-arginine (L- and D-enantiomer) in rat plasma and urine. Samples were treated with perchloric acid, neutralized and eluted through a C8 reversed-phase column with a mobile phase of 18.5 mM heptanesulfonic acid-10% methanol in water, using theophylline as an internal standard. Plasma recovery for both isomers was complete, and the sensitivity limit was 0.5 micrograms/ml. This method may be used for disposition studies of L-NNA in small animals.

Pharmacokinetics, plasma protein binding and urinary excretion of N omega-nitro-L-arginine in rats.

The in vivo disposition of N omega-nitro-L-arginine (L-NOARG) has been examined in rats. Plasma concentrations of L-NOARG following an intravenous dose of 10 mg kg-1 were determined by high-performance liquid chromatography. Plasma L-NOARG concentrations declined biexponentially, with average half-lives of 11 min and 20 h. L-NOARG clearance did not appear to exhibit concentration-dependency below a plasma concentration of 4.56 x 10(-4) M (100 mg l-1). Ultrafiltration studies revealed insignificant binding of L-NOARG to rat plasma proteins. Urinary excretion of unchanged L-NOARG was minimal. These pharmacokinetic information may be useful in the design of in vivo experiments involving L-NOARG, as well as in the interpretation of the pharmacodynamics of this important nitric oxide synthase inhibitor.