Michel Jung

Effect of α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues

Abstract α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) in vitro , causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.

Inhibition of monoamine synthesis by irreversible blockade of aromatic aminoacid decarboxylase with α-monofluoromethyldopa

Abstract DL-α-monofluoromethyldopa is a potent enzyme-activated irreversible inhibitor of purified aromatic aminoacid decarboxylase. Single doses from 0.25 to 25 mg/kg cause partial to total inhibition of this enzyme in kidney and heart. Inhibition of brain enzyme becomes significant at doses above 2.5 mg/kg and is complete at 100 mg/kg. Enzyme activity begins to return after 24 hr, so that repetition of a dose at 12 hr intervals markedly increases the inhibition. Single doses of 100–250 mg/kg almost completely deplete kidney, heart and brain of endogenous catecholamines by blocking dopa decarboxylation. Serotonin is also decreased, presumbaly by the same mechanism.

FURTHER STUDIES ON THE INHIBITION OF MONOAMINE SYNTHESIS BY MONOFLUOROMETHYLDOPA

1 α‐Monofluoromethyldopa (MFMD, RMI 71963), a potent and selective enzyme‐activated irreversible inhibitor of aromatic l‐amino acid decarboxylase produces a substantial and long‐lasting decrease in the catecholamine content of mouse brain, heart and kidney. 2 Single doses of MFMD reduce the 5‐hydroxytryptamine concentration of mouse brain without altering the tryptophan concentration. 3 In animals treated with MFMD, peripheral but not brain noradrenaline is restored within 1 h to control levels by an intraperitoneal injection of dopamine.

Preparation of 3-chloroacetylpyridine adenine dinucleotide: An alkylating analogue of NAD+

Analogues of coenzymes with reactive groups have been extensively used to detect the presence of functional amino acid residues in the catalytic site of dehydrogenases. Thus, alkylating analogues of NAD+ have been synthesized: the group at C-3 of nicotinamide of NAD+ has been replaced by a diazonium group [ 11, a diazoacetate [2] or a bromoacetyl group, with an aliphatic chain instead of the ribose [3-61 and the adenine has been replaced by a bromoacetylimidazole [6, 71. As basic residues are invoked in the mechanism of the dehydrogenase for the removal of the proton located at the alcohol during the hydrogen transfer [8,9], it was tempting to attack this basic group from the coenzyme side, by the chloroacetyl group, replacing at C-3 the amide group of the pyridinium ring. Our goal was to synthesize the 3-chloroacetylpyridine adenine dinucleotide analogue of NAD+. This is quite similar to 3-acetylpyridine adenine dinucleotide, which is active as hydrogen acceptor with many dehydrogenases. The similarity of the structure may lead to the activity of this analogue as hydrogen acceptor, as well as to alkylation. This activity is therefore a good indication for the alkylation of a residue in the active site. The preparation of this analogue, and of the 3-propionylpyridine adenine dinucleotide are described here and the preliminary results on the alkylation of some dehydrogenases are briefly reported.

Biochemical consequences of reactions catalyzed by GAD and GABA-T

Abstract GABA-T abstracts the pro-4-S proton from GABA. The enzyme abstracts the analogous protons from (+)-γ-acetylenic GABA and (+)-γ-vinyl GABA in each case precipitating its own inactivation. (+)-γ-Acetylenic GABA also irreversibly inhibits GAD indicating a transamination capability of the decarboxylase on this synthetic GABA analogue. γ-Vinyl GABA has no effect on this enzyme in vitro . Administration of either γ-acetylenic or γ-vinyl GABA to rats or mice results in a dose-dependent increase in brain GABA levels. At any given level of GABA-T inhibition the concentrations of GABA are higher after γ-vinyl GABA due to its smaller effect on GAD. Nevertheless, after a dose of γ-vinyl GABA sufficient to raise brain GABA levels to over 300% of control levels for 48 hours, there is a slow decrease in brain GAD activity to 75% and 65% of control levels at 24 and 48 hours respectively. This diminution of GAD activity after administration of γ-vinyl GABA is consistent with a feedback effect of sustained elevated GABA levels on the synthesis of GAD.

α-Fluoromethyl histidine: Inhibition of histidine decarboxylase in pylorus ligated rat

Abstract α-Fluoromethyl histidine is an irreversible inhibitor of histidine decarboxylase. The injection of a single dose to pyloric-ligated rats inhibits gastric mucosal histidine decarboxylase in a dosedependent manner but does not modify histamine content and gastric acid secretion even at the highest dose used. Administration of cimetidine increases histidine decarboxylase activity, decreases histamine level in gastric mucosa and inhibits gastric acid secretion. The co-administration of α-fluoromethyl histidine blocks the augmentation in enzyme activity, maintains lowered histamine level and prolongs the antisecretory action of cimetidine.

Syntheses of DL-2-fluoromethy-p-tyrosine and DL-2-difluoromethyl-p-tyrosine as potential inhibitors of tyrosine hydroxylase

Abstract The syntheses of the title compounds from 3,4-dimethylanisole and ethyl 5-hydroxy-2-methylbenzoate, respectively, are described.

Stereospecific synthesis of (2R,5R)-hept-6-yne-2,5-diamine: a potent and selective enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC)

Hept-6-yne-2,5-diamine (2) and 2-methylhept-6-yne-2,5-diamine (3), while structurally related to the potent ornithine decarboxylase (ODC) inhibitor hex-5-yne-1,4-diamine (1), are stable to in vivo oxidation by monoamine oxidase (MAO). Although the methyl substitution is at a carbon relatively remote from the site of metabolic attack by ornithine decarboxylase (ODC), it has a critical influence on the potencies of these compounds as inhibitors of the enzyme. Of the four stereoisomers, (2R,5R)-(2) is the most active. Unambiguous syntheses of each isomer of (2) from the dianion of 3-trimethylsilyl-N-butoxycarbonylprop-2-ynylamine (5) are presented.

Stereochemistry of the inactivation of 4-aminobutyrate: 2-oxoglutarate aminotransferase and L-glutamate 1-carboxylase by 4-aminohex-5-ynoic acid enantiomers.

Incubation of rat brain or bacterial 4-aminobutyrate aminotransferase (EC 2.6.1.19) with both (S)-(+)- and (R)-(-)-enantiomers of 4- aminohex -5- ynoic acid results in a time-dependent irreversible loss of enzymatic activity. Rat brain glutamate decarboxylase (EC 4.1.1.15) is inactivated by the (S)-(+)-enantiomer while the bacterial glutamate decarboxylase is inactivated by the (R)-(-)-enantiomer. In addition, we demonstrate that (R)-(-)-4- aminohex -5- ynoic acid is a selective and effective inhibitor of rat brain 4-aminobutyrate aminotransferase in vivo.

New fluoroketones as human renin inhibitors

Renin inhibition has been evaluated for a new class of fluorinated ketones, true analogues of peptides that have been retroinverted at the C‐terminal position. The readily formed hydrate of the ketone is proposed to mimic the tetrahedral intermediate that occurs during the enzyme‐catalyzed hydrolysis of amide linkage. From this series of compounds it appears that the number of reverted amide bonds is crucial in terms of activity. Furthermore, a shortening of the C‐terminal part of our peptide analogues and the replacement of the leucine residue in P1 by a cyclohexylalanine leads to the tripeptide analogue 12 a potent renin inhibitor (IC50 = 3.5 × 10−9 M).