Enzyme-activated Irreversible Inhibitors Research Articles

Aromatase inhibition in JAr choriocarcinoma cells by 7alpha-arylaliphatic androgens.

The JAr choriocarcinoma cell cultures have demonstrated high levels of aromatase activity and have been useful for assaying a wide variety of aromatase inhibitors for aromatase inhibition in intact cells. Recently, several 7alpha-arylaliphatic androgens have shown effective inhibition of human placental microsomal aromatase in vitro, with apparent Ki values ranging from 10 to 20 nM. A series of 7alpha-arylaliphatic androst-4-ene-3,17-dione compounds demonstrated potent competitive inhibition, and 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were enzyme-activated irreversible inhibitors. Both series of these potent inhibitors were investigated for the ability to inhibit aromatase activity in JAr cells by measuring the conversion of [1beta-3H]-androstenedione to 3H2O and unlabelled estrone. JAr cell cultures were incubated for 2 h at 37 degrees C with the aromatase inhibitors at concentrations of 10 pM to 10 microM, the percentage of enzyme inhibition was determined, and IC50 values for inhibitors were calculated. Both series of synthetic compounds demonstrated good to excellent aromatase inhibition, and the most effective inhibitors in both series were those compounds with a phenylpropyl substituent at the 7alpha-position of the steroid nucleus. The 7alpha-arylaliphatic androst-4-ene-3,17-diones exhibited inhibition of JAr aromatase activity with IC50 values from 300 to 434 nM. More potent aromatase inhibition was observed with the 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones, which exhibited IC50 values from 64 to 232 nM. Enhanced efficacy of steroidal enzyme-activated irreversible inhibitors compared to competitive inhibitors was observed in these studies and is consistent with previous reports. These results suggest that JAr choriocarcinoma cells with high levels of aromatase activity may be useful in differentiating steroidal aromatase inhibitors exhibiting different mechanisms of enzyme inhibition. In summary, the 7alpha-phenylpropyl androsta-1,4-diene-3,17-dione analogs, which are enzyme-activated irreversible inhibitors, demonstrated the most effective inhibition of aromatase activity present in the JAr cell cultures among the various 7alpha-arylaliphatic androgens.

Biochemistry and Pharmacology of 7α-substituted Androstenediones as Aromatase Inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo . In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K i s ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C 1 C 2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k inact s ranging from 4.4 × 10 −4 to 1.90 x 10 −3 s −1 . The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T 1 2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC 50 values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent Kis ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C1-C2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent kinacts ranging from 4.4 × 10−4 to 1.90 x 10−3 s−1. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T12 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC50 values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.

(Halogenomethyl)phenyl α-D-glucopyranosides as enzyme-activated irreversible inhibitors of yeast α-glucosidase and potential anti-HIV agents

A range of (halogenomethyl)phenyl α-D-glucopyranosides 2–7, prepared from corresponding methylphenyl glucosides by synthetic manipulation of the aglycone moiety, have been investigated as enzyme-activated irreversible inhibitors of yeast α-glucosidase and their anti-HIV activity measured. Compounds 5–7, which also contain a 4- or 6-nitro group in the phenyl ring of the aglycone, are much more effective inhibitors of the enzyme than are compounds 2–4 which lack this feature.

Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors.

Mouse ornithine decarboxylase (ODC) and mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5'-phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent Km values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the kcat/Km (specificity constant) value. These results are consistent with lysine 69 being the residue that forms a Schiff base with the pyridoxal 5'-phosphate co-factor. Mutation C70S did not greatly affect the activity despite its proximity to this lysine but increased the Km about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the Km, suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, alpha-difluoromethylornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5-14C]DFMO was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-delta-methyl-alpha-acetylenicputrescine and was much less sensitive than the wild type enzyme to alpha-monofluoromethyldehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and biochemical studies of 7α-substituted androsta-1,4-diene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

Several 7α-thiosubstituted derivatives of androstenedione have demonstrated effective inhibition of aromatase, the cytochrome P450 enzyme complex responsible for the biosynthesis of estrogens. Introduction of an additional double bond in the A ring resulted in 7α-(4′-amino)phenylthioandrosta-1,4-diene-3,17-dione (7α-APTADD), a potent inhibitor that inactivated aromatase by an enzyme-catalyzed process. Additional 7α-thiosubstituted androsta-1,4-diene-3,17-dione derivatives were designed to further examine enzyme-catalyzed inactivation. Two halogenated and one unsubstituted 7α-phenylthioandrosta-1,4-diene-3,17-diones were synthesized via an acid-catalyzed conjugate Michael addition of substituted thiophenols with androsta-1,4,6-triene-3,17-dione. Two 7α-naphthylthioandrosta-1,4-diene-3,17-diones were synthesized via either acid-catalyzed or based-catalyzed conjugate Michael addition of substituted thionaphthols with androsta-1,4,6-triene-3,17-dione. These agents were evaluated for aromatase inhibitory activity in the human placental microsomal preparation. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent K i's ranging from 12 to 27 nM. Furthermore, these compounds produced time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no aromatase inactivation was observed in the absence of NADPH. This enzyme-activated irreversible inhibition, also referred to as mechanism-based inhibition, can be prevented by the substrate androstenedione. Thus, the apparent K i values for these inhibitors are consistent with earlier studies on 7α-substituted competitive inhibitors that indicate bulky substituents can be accommodated at the 7α-position. On the other hand, differences in the inactivation half-times at infinite inhibitor concentration, in the k app rates of inactivation, and in the apparent K inact's were observed, suggesting that binding of 7α-substituted androsta-1,4-diene-3,17-diones can affect enzyme catalysis and enzyme-mediated inactivation.

Enantiospecific syntheses of alpha-(fluoromethyl)tryptophan analogues: interactions with tryptophan hydroxylase and aromatic L-amino acid decarboxylase.

alpha-Fluoromethyl amino acids are enzyme-activated irreversible inhibitors of amino acid decarboxylases. Aromatic L-amino acid decarboxylase (AADC) is the enzyme responsible for the final step in the biosynthesis of both dopamine and serotonin via decarboxylation of L-dopa and 5-hydroxy-L-tryptophan, respectively. Our goal is to utilize antagonists of the serotonin-producing enzymes (tryptophan hydroxylase and AADC) as the basis for a chemotherapeutic approach to the treatment of carcinoid tumors, a rare tumor type characterized by the overproduction of serotonin. We report here an enantiospecific synthesis of alpha(S)-(fluoromethyl)tryptophan [(S)-11a] and alpha(S)-(fluoromethyl)-5-hydroxytryptophan [(S)-11b], as well as the (R)-enantiomers, based upon recent methodology involving the face-selective alkylation of cyclic tryptophan tautomers. Our synthetic route provided both enantiomers of 11a and 11b with greater than 97% enantiomeric purity based upon evaluation of the NMR spectra of their Mosher's acid derivatives. (S)-11a was evaluated as a substrate for P815 tryptophan hydroxylase and determined to have an apparent Km of 4.31 +/- 1.07 mM, essentially half the value previously reported for the racemic mixture of 11a with rat brain stem tryptophan hydroxylase. (R)-11a was not a substrate for P815 tryptophan hydroxylase. (S)-11b was evaluated as an enzyme-activated irreversible inhibitor of murine liver AADC and determined to have a KI of 24.3 +/- 3.01 microM and a k2 of 2.26 +/- 0.44 min-1. (R)-11b was not an inhibitor of murine liver AADC.

The molecular pharmacology of L-deprenyl

L-Deprenyl, the selective inhibitor of monoamine oxidase type B (MAO-B) has gained wide acceptance as a useful form of adjunct therapeutic drug in the treatment of Parkinson's disease. This review summarizes the molecular pharmacology of L-deprenyl, and the advances in our understanding of its possible mode of action in Parkinson's disease. l-Deprenyl belongs to the class of enzyme-activated irreversible inhibitors also described as ‘suicide’ inhibitors, because the compound acts as a substrate for the target enzyme, whose action on the compound results in irreversible inhibition. l-Deprenyl first of all forms a noncovalent complex with MAO as an initial, reversible step. The subsequent interaction of l-deprenyl with MAO leads to a reduction of the enzyme-bound flavine-adenine dinucleotide (FAD), and concomitant oxidation of the inhibitor. This oxidized inhibitor then reacts with FAD at the N-5-position in a covalent manner. The observed in vitro selectivity of L-deprenyl for MAO-B may be accounted for by differences in the affinities of the two MAO subtypes for reversible interaction with L-deprenyl, differences in the rates of reaction within the noncovalent complexes to form the irreversibly inhibited adduct, or a combination of both these factors. However, if selective inhibition is to be maintained in vivo, correct dosage schedules are critically important, since all selective MAO inhibitors described up to now lack selectivity at high doses. In experimental animals L-deprenyl is protective against the damaging effects of several neurotoxins, including the dopaminergic agents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) and the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Beside MAO-B inhibition, which above all explains the prevention of neurotoxic action of MPTP by preventing its metabolism. L-deprenyl appears to exhibit other mechanisms of action which are independent of its action on MAO-B.

Ortho- and para-(difluoromethyl)aryl-β- d-glucosides: A new class of enzyme-activated irreversible inhibitors of β-glucosidases

New ortho- and para-(difluoromethyl)aryl-β- d-glucosides were stereoselectively prepared in three steps from 1-bromo-2,3,4,6-tetraacetyl glucose, using an appropriate ortho- or para-hydroxybenzaldehyde derivative. The fluorine atoms were introduced by reacting the soformed ortho- or para-O-glucosyl benzaldehyde derivatives with DAST. Title compounds are potent time-dependent irreversible inhibitors of almond β-glucosidase. The inactivation is explained by the enzyme-catalyzed hydrolysis of the glucosidic linkage, releasing an ortho- or para-(difluoromethyl)phenol. The ortho- and para-(difluoromethyl)phenols are assumed to rapidly form fluorinated quinone methides which alkylate a nucleophilic residue of the enzyme active site.

7-Substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3,17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3,17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.

1,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of .alpha.-glucosidases

1,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of .alpha.-glucosidases

The effects of in vivo inactivation of GABA-transaminase and glutamic acid decar☐ylase on levels of GABA in the rat retina

Gabaculine and γ-vinyl GABA (GVG) are specific enzyme-activated irreversible inhibitors of GABA-transaminase (GABA-T). γ-Acetylenic GABA (GAG) irreversibly inhibits both GABA-T and glutamic acid decar☐ylase (GAD). Subcutaneous injection of any of those compounds rapidly elevated levels of GABA in the retinae of rats. After injection of 10 mg/kg gabaculine, levels of retinal GABA climbed 5-fold in 4 h, and peaked 16 h after injection at levels approximately 7 times those from water-injected control rats. They remained significantly elevated compared to control levels for at least 6 days after injection. The postgabaculine increase in levels of retinal GABA was linear with time between 0.5 and 4 h after injection. In contrast, retinal GABA levels peaked at less than 3 times control levels within 8 h of injection of 50 mg/kg GAG and returned to baseline levels within 4 days. GAG, upon coadministration with gabaculine, significantly attenuated the postgabaculine rise in levels of GABA in retinae. Neither the rate of rise, nor the maximum levels, of retinal GABA was so great after injection of GAG plus gabaculine, compared to those after injection of gabaculine alone. The degree to which postgabaculine GABA accumulation was inhibited in the retina by 50 mg/kg GAG closely corresponded with the extent to which that dose of GAG inactivated retinal GAD activity. The results of this study extend previous reports from this laboratory that systematically administered gabaculine, GVG and GAG all inactivate target enzymes more potently in retina than in other brain regions 7. The observation that retinal GABA accumulates linearly with time shortly after subcutaneous injection of gabaculine provides a simple approach to the study of regulation of retinal GABA synthesis in vivo. The correspondence between the inactivation of retinal GAD activity and the attenuation of postgabaculine GABA accumulation in the retina by GAG argues that GABA synthesis in rat retina occurs primarily via the decar☐ylation of l-glutamate catalysed by GAD, and not via other metabolic pathways.

7 α -Substituted androstenediones as effective in vttro and invivo inhibitors of aromatase

Research efforts over the past several years have focused on the synthesis of competitive and irreversible aromatase inhibitors and examination of these inhibitors in microsomal preparations, in cell culture, and in vivo . Several 7α-substituted androstenediones have demonstrated high affinity for placental aromatase, with apparent K i's ranging from 1 to 30 nM. Inactivation of aromatase occurred following incubation with alkylating and enzyme-activated irreversible inhibitors. 7α-(4'-Amino)phenylthio-4-androstene-3,17-dione (7α-APTA) exhibits potent inhibitory activity of aromatase in the MCF-7 human mammary carcinoma cell line with an ED 50 of approximately 25 nM. The inhibitor did not bind to the estrogen receptor of the cells in vitro nor induce levels of progesterone receptors in intact cells. In vivo studies of 7α-APTA in the DMBA-induced rat mammary carcinoma model resulted in 80% of the tumors responding completely or partially at doses of 25 and 50 mg/kg body wt/day. Thus, these 7α-substituted steroidal aromatase inhibitors are effective medicinal agents and may be useful for the treatment of estrogen-dependent breast cancer.

Synthesis of diazomethyl β- d-galactopyranosyl and β- d-glucopyranosyl ketones. Potential affinity-labeling reagents for carbohydrate-binding proteins

3,7-Anhydro-1-deoxy-1-diazo- d- glycero- l- manno-2-octulose ( 6a; diazomethyl β- d-galactopyranosyl ketone) and 3,7-anhydro-1-deoxy-1-diazo- d- glycero- d- gulo-2-octulose ( 6b; diazomethyl β- d-glucopyranosyl ketone) have been prepared. Readily available C-glycosyl compounds possessing the appropriate stereochemistry and hydroxyl-group protection, viz., per- O-acetyl-2,6-anhydroheptononitriles and per- O-acetyl-2,6-anydroheptonamides, were employed as precursors to per- O-acetyl-2,6-anydroheptonic acids. These key intermediates were then converted into mixed carboxylic-carbonic acid anhydrides, and these caused to react with diazomethane, to give the corresponding per- O-acetyl-3,7-anhydro-1-deoxy-1-diazo-2-octuloses. Zemplén deacetylation gave, stereospecifically, the crystalline target-molecules in good overall yield. It is proposed that such C-glycosyl compounds as 6a and 6b, which possess the diazoacetyl functional group as their “aglycon”, will be useful as enzyme-activated irreversible inhibitors (suicide substrates) of glycosidases, and as photoaffinity-labeling reagents and classical affinity-labeling reagents for carbohydrate-binding proteins.

Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationships.

Seventeen 2-aryl-3-haloallylamine derivatives were prepared and evaluated as inhibitors of monoamine oxidase (MAO, EC 1.4.3.4). The synthesis of these compounds was achieved from either alpha-methylstyrene or ring-substituted phenylacetic acid derivatives. With one exception, these 2-arylallylamines were found to be enzyme-activated, irreversible inhibitors of MAO. The most potent inhibitors were ring-substituted derivatives of (E)-2-phenyl-3-fluoroallylamine with IC50 values ranging from 10(-6) to 10(-8) M. Selectivity for the A and B form of MAO was found to depend on the nature of aromatic ring substitution. In general, hydroxyl substitution favored the inactivation of the A form of MAO, while very selective B inhibitors were obtained when the aromatic ring was substituted with a 4-methoxy group. (E)-2-(4-Methoxyphenyl)-3-fluoroallylamine and (E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine proved to be in vitro as selective for the B form of MAO as deprenyl.

Haloenol lactones. Potent enzyme-activated irreversible inhibitors for alpha-chymotrypsin.

Haloenol lactones can act as enzyme-activated irreversible inhibitors for alpha-chymotrypsin: acyl transfer to the active site serine releases a halomethyl ketone that remains tethered in the active site during the lifetime of the acyl enzyme, poised to alkylate an accessible nucleophilic residue. To investigate the structural determinants for chymotrypsin suicide inactivation with haloenol lactones, we prepared a series of nine lactones, differing in ring size (6-membered valerolactones and 5-membered butyrolactones) and in the nature of the aromatic substituent (phenyl and alpha-naphthyl), and the halogen (bromine and iodine). The inactivating behavior of these lactones is characterized by a binding constant (Ki) and three rate constants, for inactivation (k2), catalytic hydrolysis (kc), and spontaneous hydrolysis (kh). The six-membered valerolactones were much more potent inactivators than the butyrolactones, having both higher affinity and more rapid inactivation; the alpha-naphthyl-substituted lactones were also more effective, but the nature of the halogen had relatively little effect. The spontaneous rate of hydrolysis of all of these lactones is low. The turnovers per inactivation of these lactones vary from 91-1.7, with some of the alpha-naphthyl-substituted lactones approaching ideal behavior (stoichiometric inactivation). These studies indicate that several haloenol lactones are effective enzyme-activated irreversible inhibitors of chymotrypsin, and that their potency and efficiency depends markedly upon certain structural features of the lactone system.

A mutant of Chinese hamster ovary cells resistant to alpha-methyl- and alpha-difluoromethylornithine.

We describe a mutant of Chinese hamster ovary cells which is resistant to elevated levels of alpha-methylornithine and alpha-difluoromethylornithine, reversible and enzyme-activated irreversible inhibitors, respectively, of the enzyme ornithine decarboxylase (ODC). The mutant cells have significantly elevated levels of enzyme activity compared to wild-type cells, but several of the physical parameters of the enzyme are completely normal: Michaelis-Menten parameter, Km, affinity for the analog, and half-life. The temporal regulation of this activity in synchronized cells is not perturbed, and the suppression of ODC activity by the addition of putrescine is still observed. Indirect experiments suggest increased concentrations of ODC mRNA in the mutant cells.

A stereochemical concept for the catalytic mechanism of prolylhydroxylase: Applicability to classification and design of inhibitors

The enzyme prolylhydroxylase (E.C.1.14.11.2.) mediates the post-translational conversion of genetically coded prolyl into non-coded trans-4-hydroxyprolyl. It has a key role in the biosynthesis of interstitial collagens and the subcomponent C1q of the first complement component, and its inhibition results in a selective suppression of these proteins. This finding points to the applicability of specific prolylhydroxylase inhibitors in the causal treatment of diseases aggravated by fibrosis or immune complex formation, substances which clearly would be a major breakthrough in clinical pharmacology. The published suggestions on the reaction mechanism of prolylhydroxylase are known to be in conflict with the data on the enzyme, on model hydroxylations and on iron-dioxygen complexes and thus cannot be used as guides for the design of inhibitory structures. The Stereochemical mechanism presented here allows a detailed understanding of the kinetic findings and of the electronic rearrangements during the reaction. The exactly formulated structure-activity relationships invite pinpoint alterations of the enzyme substrates, leading to the design of structures with inhibitory potential. Competitive and enzyme-activated irreversible inhibitors are detailed. The experimental study of some of the delineated substances has begun.

10 beta-propynyl-substituted steroids. Mechanism-based enzyme-activated irreversible inhibitors of estrogen biosynthesis.

A series of 10 beta-propynyl-substituted steroids are reported as mechanism-based enzyme-activated irreversible inhibitors of the human placental aromatase which converts 4-androstene-3,17-dione to 1,3,5(10-estratrien-3-ol-17-one. Thus 10-propargylestr-4-ene-3,17-dione binds to the enzyme with an apparent Ki of 23 nM and causes time-dependent inactivation (pseudo-first order kinact = 1.11 X 10(-3) s-1). The 10-[(1S)-1-hydroxy-3-propynyl]estr-4-ene-3,17-dione analog of the known enzyme-generated intermediate, 4-androsten-19-ol-3,17-dione, behaved similarly and had an apparent Ki of 27 microM and a psuedo-first order kinact of 2.91 X 10(-3) s-1. The stereoisomeric 10-[(1R)-1-hydroxy-2-propynyl]estr-4-ene-3,17-dione did not cause time-dependent inactivation, but bound in a competitive manner with an apparent Ki of 2.5 microM. Finally, 10-(1-oxo-2-propynyl)-estr-4-ene-3,17-dione, the proposed enzyme-generated affinity label and analog of the second intermediate (3,17-dioxoandrost-4-en-19-al) in the enzymatic reaction, bound to the enzyme with an apparent Ki of 12 microM and caused time-dependent inactivation with a pseudo-first order kinact of 5.35 X 10(-4) s-1.

Determination of dopa, dopamine, dopac, epinephrine, norepinephrine, alpha-monofluoromethyldopa and alpha-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection.

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. B using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). Alpha-Difluoromethyldopa (DFMD) and alpha-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.