3-Substituted pyrazole derivatives as inhibitors and inactivators of liver alcohol dehydrogenase.

3-Substituted pyrazoles, HOCH2 (1), HOCH2CH2 (2), HOCH2CH2CH2 (3), ClCH2 (4), ClCH2CH2 (5), ClCH2CH2CH2 (6), and CH3CO (7), were synthesized and evaluated in vitro on horse liver alcohol dehydrogenase for their potential as inhibitors of ethanol metabolism. 1 to 6 bound to the enzyme-NAD+ complex with dissociation constants of 40 to 200 microM, much higher than the constants for the corresponding 4-substituted pyrazoles, but with the same absorption maximum at 295 nm. 4 inactivated the enzyme within a few minutes, but NAD+ protected against reaction, and 4 nonspecifically alkylated many sulfur atoms in the protein. The isomer, 4-(chloromethyl)pyrazole, behaved similarly, 5 and 6 strongly inhibited the enzyme in the presence of NAD+, due to formation of the slowly dissociable (10(-3)s-1) enzyme-NAD+-pyrazole complex, but did not irreversibly inactivate the enzyme. 7 inhibits the enzyme weakly (Kp = 5 mM). It appears that the 3-substituted pyrazoles bind to the enzyme-NAD+ complex with the reactive functional group improperly positioned for specific irreversible reaction.

Inactivation of liver alcohol dehydrogenases and inhibition of ethanol metabolism by ambivalent active-site-directed reagents.

Active-site-directed reagents, of the general structure omega-(BrCH2CONH)RCOY, where R = alkyl, aryl, or aralkyl, and Y = OH or NH2, inactivated horse, mouse, rat, and human liver alcohol dehydrogenases at widely different rates, reflecting differences in reagent specificity and in the structures of the enzymes. Treatment of mice and rats with either of two optimally specific reagents, p-(XCH2CONH)C6H4(CH2)3CONH2, where X = Br (7) or CH3SO3 (10), partially (20 to 40%) inactivated alcohol dehydrogenase in liver, inhibited ethanol metabolism, and prolonged the impairment of coordination produced by ethanol in these animals. Although the dose of 7 used (0.13 mmol/kg) approximated the LD50, 10 was effective at a dose of 0.48 mmol/kg that was not acutely toxic.

In vivo evaluation of ambivalent active-site-directed inactivators of liver alcohol dehydrogenase.

In order to decrease the rate of ethanol metabolism for the treatment of acute and chronic alcoholism it would be useful to inhibit liver alcohol dehydrogenase in vivo. Based on a knowledge of the three-dimensional structure of the horse enzyme, we designed active-site-directed inactivators [p-(XCH2CONH)C6H4(CH2)3COHN2] which bind to the enzyme-NAD or enzyme-NADH complex and alkylate methionine residue 306. In vitro, these reagents inactivated mouse, rat, horse and human liver alcohol dehydrogenases faster in the presence than in the absence of NAD or NADH, but with slightly different specificity. Mice and rats pretreated with the reagents eliminated ethanol in blood more slowly than those not treated, and the specific activity of alcohol dehydrogenase in liver homogenates of treated animals was decreased. It appears that the design of active-site-directed reagents is feasible, but these reagents must be improved so that they are more efficacious in vivo.

Glucocorticoid stimulation of blood coagulation factor activities in the fetal lamb.

Blood coagulation factor activities, including factors II, VII, IX, X, XI, and XII, are lower than normal in full-term infants and more severely depressed in premature infants. Previous studies in our laboratory using a fetal lamb model indicated that most coagulation factor activities increase during the latter part of the third trimester of pregnancy. Since glucocorticoid stimulation of the fetus accelerates fetal lung maturation in experimental models and in offsprings of mothers with impending premature labor, we hypothesized that glucocorticoid stimulation might induce increases in blood coagulation factor activities similar to those found near term. Two sets of experiments were carried out in chronically catheterized fetal lambs early in the third trimester (100 to 120 days gestation). In one set of experiments, one twin from each of 10 twin sets was infused with hydrocortisone (2 mg/hr for 48 hr). The twin sibling served as a control. In the other set of experiments, six pregnant ewes were given 15 mg of beta-methasone intramuscularly every 24 hr for two doses. Five pregnant ewes were injected with saline and used as controls. Fetal blood samples were obtained prior to and 48 hr after the start of the hydrocortisone infusions and prior to and 24 hr after the second dose of beta-methasone given to the pregnant ewes. The results of blood coagulation factor activities comparing glucocorticoid-stimulated fetuses with controls demonstrated significant increases in the activities of blood coagulation factors II, V, VII, IX, and X in the stimulated fetuses. There were no significant changes in the activities of factors VIII, XI, or XII. The results confirmed the hypothesis that glucocorticoid stimulation increases many blood coagulation factor activities in the fetus.

Effects of acidosis on fetal and maternal blood coagulation: a fetal lamb model.

The effects of fetal acidosis (mean pH 6.93) on fetal and maternal blood coagulation were measured. Test results from 10 fetal lambs and mother ewes (127 +/- 2 days mean gestation) before and after fetal lactic acid infusions were compared to test results from eight control fetal lambs and mother ewes (127 +/- 3 days mean gestation) before and after control glucose infusion. Significant changes found in acidotic fetal lambs not seen in control fetuses included an increase in the white blood cell count (mean 2800/mm3 before to 3600/mm3 after acidosis; p = 0.0009), a shortening of the thrombin time (mean 17.8 s before to 11.2 s after acidosis; p = 0.0001), and decreases in the activities of factor V (mean 57% before to 37% after acidosis; p = 0.0014) and factor IX (mean 35% before to 29% after acidosis; p = 0.0128). There was also a reduction in the concentration of fibrinogen (mean 147 mg/100 ml before to 125 mg/100 ml after acidosis; p = 0.0492) but no significant changes in the levels of fibrin monomer, fibrinogen/fibrin degradation products, or antithrombin III. In vitro exposure of five different fetal whole blood samples to a pH of 6.9 for 2 h at 37 degrees C did not result in significant changes in any of the coagulation factor activities. A significant decrease in the level of factor V was also found in the mother ewes of the acidotic fetuses (mean 141% before to 113% after acidosis; p = 0.006) and a decrease in the level of maternal factor IX approached significance (mean 119% before to 102% after acidosis; p = 0.0564).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of liver alcohol dehydrogenases by imidoesters generated in solution.

Various omega-halogenated carboxy acids and amides were evaluated as potential active-site-directed reagents for alcohol dehydrogenase. 2-Bromoacetamide and bromoacetic and 3-bromopropionic acids inactivated the enzyme; AMP, NAD+, and NADH markedly decreased the rate of inactivation. Some omega-halogenated carboxyamides, X(CH2)nCONH2, increased the activity of the enzyme with the rate and extent of activation depending on the number of methylene units (n) in the order 3 greater than 4 greater than 2 and on X in the order Br greater than Cl. 4-Chlorobutyramide (0.1 M) activated the horse liver enzyme 20-fold in 24 hr at pH 8.0 and 25 degrees. The activation was not prevented by AMP or 2,2-bipyridine, but was by NADH. The kinetic constants and turnover numbers for human and horse liver alcohol dehydrogenases treated with chlorobutyramide were increased markedly compared to those for native enzymes. Alcohol dehydrogenase treated with chlorobutyramide was not further activated by methyl picolinimidate, an imidoester which activates native enzyme by modifying amino groups in the active sites. Chlorobutyramide does not appear to react directly with the enzyme but cyclizes in the reaction medium to form an intermediate imidoester, 2-iminotetrahydrofuran, which reacts with most of the amino groups of the enzyme.

Characterization and kinetics of native and chemically acitvated human liver alcohol dehydrogenases.

Acetimidylation of the amino groups of alcohol dehydrogenase from human and horse liver yields several modified enzyme forms, which differ in electrophoretic mobility and can be separated by ion exchange chromatography, but which are similar in kinetic characteristics. The acetimidylated, as well as the methylated, enzymes from human livers of the normal phenotype have increased activity and larger Michaelis and inhibition constants. These results suggest that the human enzyme has amino groups at the active sites, as was shown previously for the horse enzyme. The variant subunit occuring in the enzyme isolated from atypical human livers does not seem to be activated by acetimidylation, which may indicate that substitution of proline for Ala-230 or modifiction of Lys-228 is sufficient to fully activate the enzyme. Results of product inhibition studies of native and modified human enzymes are consistent with an Ordered Bi Bi mechanism. However, the major isoenzyme of native human liver alcohol, dehydrogenase exhibits nonlinear kinetics over a wide range of ethanol concentrations. This result may indicate that subunits with different kinetic characteristics are present or that there is negative cooperativity between subunits. After chemical modification, the kinetic patterns become linear, suggesting that the mechanism is altered.

omega-haloalkyl esters of 5'-adenosine monophosphate as potential active-site-directed reagents for dehydrogenases.

A series of esters of adenosine 5'-monophosphate with ethyl, propyl, or hexyl moieties substituted at the omega-position with chlorine or bromine were prepared. The compounds were competitive inhibitors of horse liver alcohol dehydrogenase with respect to coenzyme, NAD+, and had inhibition (dissociation) constants in the range of 40 to 260 microM at pH 8.0, 25 degrees C. The bromoalkyl esters were designed to be active-site-directed inactivators and were chemically reactive as tested with the model compound 4-(p-nitrobenzyl)pyridine. Yeast alcohol dehydrogenase was inactivated by the bromohexyl analog by an active-site-directed mechanism, with a Ki = 1.5 mM and a pseudo-bimolecular rate constant of 0.03 M-1 S-1, which is 150 times larger than the bimolecular rate constant for inactivation by 2-bromoethanol. However, the rates of inactivation of other dehydrogenases treated with 10 mM concentrations of these compounds were generally slower than with the simpler reagent, 2-bromoethanol. Thus, the reactive functional group attached to the AMP moiety may not be properly oriented for affinity labeling of these dehydrogenases. The bromoalkyl esters may be useful for inactivating other enzymes.

Kinetics of native and modified liver alcohol dehydrogenase with coenzyme analogues: isomerization of enzyme-nicotinamide adenine dinucleotide complex.

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.