Biomedical subjects
Publications and source records attributed to R B Silverman.
Both 4-(aminomethyl)-1-phenyl-2-pyrrolidinone (4a) and 4-(aminomethyl)-1- (methoxyphenyl)-2-pyrrolidinone (4b) hydrochlorides were synthesized via a six-step sequence, which represents a general approach to 1,4-disubstituted 2-pyrrolidinones. Both of these compounds inactivated monoamine oxidase B and represent the first in a new class of monomamine oxidase inactivators.
A structural similarity of several monobactams (2-4), 3-aminonocardicinic acid (6), 6-aminopenicillanic acid (7), 7-aminocephalosporanic acid (8), and 7-aminodesacetoxycephalosporanic acids (9, 10) to gamma-aminobutyric acid (GABA) and to known inhibitors and substrates of GABA aminotransferase is described. Because of this, the above-mentioned compounds were tested as competitive inhibitors and as inactivators of pig brain GABA aminotransferase. All of the compounds were competitive inhibitors of GABA aminotransferase. On the basis of the inhibitory potency of these conformationally-rigid GABA analogues it is hypothesized that GABA is bound at the active site with its amino and carboxylate groups in a syn orientation. None of the compounds inactivates GABA aminotransferase. These beta-lactam analogues represent the first examples of a new class of inhibitors of GABA aminotransferase.
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On the basis of the mechanism of inactivation of gamma-aminobutyric acid (GABA) aminotransferase by gamma-ethynyl GABA that was reported recently (Burke and Silverman, manuscript submitted), three 4-(oxoalkyl)-substituted GABA analogues, 4-amino-5-oxohexanoic acid (7), 4-amino-5-oxopentanoic acid (8), and 4-amino-6-oxohexanoic acid (9) were synthesized and tested as inactivators. Only 8 was an inactivator of the enzyme. A mechanism for the inactivation by 8 is proposed as well as rationalizations for the lack of inactivation by 7 and 9.
A variety of alkyl-substituted 4-aminobutanoic acid derivatives, including a homologous series of 3-alkyl-4-aminobutanoic acid analogues, 4-methyl isomer analogues, the 3,3-dimethyl analogue, and (E)-4-amino-3-methyl-2-butenoic acid, were synthesized and tested as alternate substrates for purified gamma-aminobutyric acid aminotransferase from pig brain. All of the compounds were substrates, but their activities diminished as the size and bulk of the 3-alkyl substituent increased. Several differences were observed between the alkyl-substituted analogues and the corresponding aryl-substituted compounds previously reported (Silverman, R. B., Invergo, B. J., and Levy, M. A. (1987) J. Biol. Chem. 262, 3192-3195). These findings will be important in future designs of inhibitors of gamma-aminobutyric acid aminotransferase.
(3R,4R),(3S,4S)- and (3R,4S),(3S,4R)-4-amino-5-fluoro-3-phenylpentanoic acid (1a and 1b) were synthesized and studied as selective inactivators of gamma-aminobutyric acid (GABA) aminotransferase. Neither compound caused time-dependent inactivation of the enzyme. Neither compound underwent enzyme-catalyzed transamination nor was fluoride ion eliminated from either compound by the enzyme. No 3-phenyllevulinic acid, the product of elimination of HF followed by enamine hydrolysis, was detected. However, both 1a and 1b were competitive reversible inhibitors of GABA aminotransferase; the Ki for 1a was smaller than the Km for GABA. These results suggest that 1a and 1b bind to the active site of GABA aminotransferase, but gamma-proton removal does not occur. Whereas (S)-4-amino-5-fluoropentanoic acid (AFPA) is a potent inhibitor of L-glutamic acid decarboxylase (GAD), neither 1a nor 1b at concentrations 40 times the Ki of AFPA caused any detectable competitive inhibition of GAD. Therefore, the incorporation of a phenyl substituent at the 3-position of AFPA confirms selective inhibition of GABA aminotransferase over GAD.
2- and 3-substituted vitamin K 2,3-epoxide analogues were synthesized and tested as inactivators, inhibitors, and substrates for beef liver microsomal vitamin K1 epoxide reductase. 2-(X)-3-phytyl-1,4-naphthoquinone 2,3-epoxides, where X is hydroxymethyl, chloromethyl, fluoromethyl, difluoromethyl, and formyl were all competitive inhibitors, but none was an inactivator. Only the 2-hydroxymethyl analogue was reduced to a quinone that was stable enough under the conditions of the experiment to be detected. Vitamin K1 epoxide analogues with modified phytyl chains (1'-hydroxy, 3'-fluoro with isomerized double bond, 1'-hydroxy and 1'-fluoro with saturated double bond, and the corresponding unsubstituted chains) were synthesized. All of the analogues were competitive inhibitors of vitamin K1 epoxide reductase. The nonfluorinated analogues also were shown to be substrates, being reduced to the corresponding quinone without enzyme inactivation. At least one other enzyme besides vitamin K1 epoxide reductase in beef liver microsomes also metabolizes all of these analogues.
There is little difference in the extent of inactivation of beef liver microsomal vitamin K1 epoxide reductase by N-ethylmaleimide (NEM) whether or not the microsomes are pre-treated with dithiothreitol (DTT). The rat liver microsomal enzyme, however, is inactivated by NEM to a much greater extent if the microsomes are pre-treated with DTT. The beef liver enzyme activity is protected from NEM inactivation by the substrate, vitamin K1 epoxide. Ping-pong kinetics are exhibited by the beef liver enzyme. These results support a mechanism for vitamin K1 epoxide reductase in which the function of the required dithiol is to reduce an active site disulfide bond; however, the geometry of the active sites of the enzyme from rat and beef may be different.
It was previously shown (Sayre, L. M., Arora, P. K., Feke, S. C., and Urbach, F. L. (1986) J. Am. Chem. Soc. 108, 2464-2466) that 1,3,3-trimethyl-4-phenyl-2,3-dihydropyridinium salt (the 3,3-dimethyl analogue of 1-methyl-4-phenyl-2,3-dihydropyridinium ion or MPDP+) is a good model for MPDP+ on the basis of its redox potential and was used to show that MPDP+ is unlikely to possess reactivity characteristics which could contribute to the neurotoxicity observed with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ are now shown to interact with monoamine oxidase similar to MPTP and MPDP+, but only in the presence of beta-mercaptoethanol (beta-ME). In the absence of beta-ME, mixed competitive-noncompetitive inhibition kinetics are observed for 3,3-dimethyl-MPTP and 3,3-dimethyl-MPDP+, whereas competitive inhibition kinetics are exhibited by MPTP. In the presence of beta-ME, however, 3,3-dimethyl-MPTP also is a competitive inhibitor. 3,3-Dimethyl-MPTP and 3,3-dimethyl-MPDP+ also are time-dependent inactivators of monoamine oxidase, having identical kinetic constants, as is the case with MPTP and MPDP+. In the presence of beta-ME, but not glutathione, the rate of inactivation increases dramatically. When [beta-ME] and [3,3-dimethyl-MPTP] or [3,3-dimethyl-MPDP+] are varied, there is an optimal concentration of 1.0 mM for all three at which maximal inactivation rates are obtained. Another dramatic effect of the beta-ME is to lower the partition ratio for inactivation from greater than 50 to about one. This suggests that the effect of the beta-ME toward inactivation may be to induce a conformational change in the enzyme, which reorients an active site nucleophile for attack on the activated species. Support for involvement of an active site nucleophile is the finding that inactivation does not lead to a flavin adduct. Three possible mechanisms for inactivation of monoamine oxidase by MPTP and MPDP+ are suggested.
2-(Fluoromethyl)-3-phytyl-1,4-naphthoquinone (7) was synthesized from the known compound 2-bromo-3-methyl-1,4-dimethoxynaphthalene by N-bromosuccinimide bromination of the 3-methyl group, conversion to the corresponding 3-fluoromethyl compound with silver fluoride, attachment of the 3-phytyl substitutent via the lithium diaryl cuprate and phytyl bromide, and then silver oxide oxidation to 7. Epoxidation with basic hydrogen peroxide gave the corresponding 2,3-oxide (1) in a very low yield. Compound 1 was not a time-dependent inhibitor of beef liver microsomal vitamin K epoxide reductase, but it was a competitive, reversible inhibitor. It was not possible to determine if 1 was a substrate for the enzyme because the expected product of reduction, namely 7, rapidly decomposed under the assay conditions.
Because of the importance of the inactivation of GABA aminotransferase to the design of anticonvulsant agents, a seemingly wide variety of inactivators has been investigated; all of the compounds, however, are analogues of GABA, beta-alanine, or delta-aminovaleric acid, which are substrates for the enzyme. Relatively minor modifications in the inactivator structures result in major differences in inactivation mechanisms and enzyme adduct structures. Compounds that inactivate GABA aminotransferase by a Michael addition mechanism, leading to modification of an active-site residue are Class I inactivators. Those that proceed by an enamine mechanism and give ternary adducts are Class II inactivators. Class III inactivators modify only the PLP cofactor; if the inactivation involves aromatization of the inactivator, it is a Class IIIA inactivation, and if no aromatization is involved, then it is a Class IIIB inactivation. The last class of inactivators (Class IV) are not classified on the basis of the mechanism, but, rather, that they require the enzyme to be in the PMP form. There appears to be no trend in partition ratio values when comparing Class I with Class II inactivators. Class III inactivations alter only the cofactor, so it may be possible for these adducts to diffuse slowly out of the active site; reactivation of the apoenzyme would require additional PLP. These inactivators also inactivate a variety of other PLP-dependent enzymes. At this point there does not seem to be a therapeutic advantage of one class of inactivators over another, although the only current example of these inactivators to be useful clinically is gamma-vinyl GABA (vigabatrin), a Class I inactivator recently approved for the drug market in France and the U.K. There is a mechanistic significance, however, for one class over another. If labeling of an active-site amino acid residue is desired, then Class I inactivators should be selected; desire for attachment of the inactivator to both the protein and the cofactor or just to the cofactor would determine whether Class II or Class III inactivators would be chosen. The classification presented here should allow us to think about inactivator structures in terms of their mechanistic potential and, as a result of this, should afford us the opportunity to be able to make predictions regarding inactivation mechanisms for hypothetical new structural classes of inactivators. Since the different mechanistic pathways lead to different types of enzyme adducts, inactivator design may be driven by the class of adduct that is desired.(ABSTRACT TRUNCATED AT 400 WORDS)
Three known mechanism-based inactivators of beef liver mitochondrial monoamine oxidase (MAO) B are tested as inactivators of human placental mitochondrial MAO A. 1-Phenylcyclopropylamine (1-PCPA), 1-benzylcyclopropylamine (1-BCPA), and N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) are time-dependent irreversible inactivators of MAO A. The KI values for 1-PCPA and N-C alpha MBA, analogues of the MAO B substrate benzylamine, are much higher with MAO A than with MAO B. Evidence is presented to show that 1-PCPA inactivates MAO A by attachment to the flavin cofactor, unlike the reaction with MAO B in which 1-PCPA can attach to both a cysteine residue and the flavin [Silverman, R.B., & Zieske, P.A. (1985) Biochemistry 24, 2128-2138]. The reaction of 1-BCPA with MAO A was too slow to study in detail. N-C alpha MBA exhibits the same properties toward inactivation of MAO A that it does for inactivation of MAO B. Attachment in both cases is shown to be to one cysteine residue per enzyme molecule. The results with 1-PCPA indicate that the active site topographies of MAO A and MAO B are different. The ability of N-C alpha MBA to undergo attachment to a cysteine residue in both MAO A and MAO B may lead the way toward peptide mapping of the two isozymes in order to determine differences in their primary structures.
Vitamin K 2,3-epoxide reductase activity from liver microsomes requires only a thiol cofactor, particularly dithiothreitol (DTT). In order to identify a likely physiological cofactor, reduced lipoic acid and reduced thioredoxin were tested as cofactors in beef and rat liver microsomal systems. Reduced lipoic acid is only about one-third as active as DTT in both systems. Thioredoxin, however, is significantly more active than either DTT or reduced lipoic acid in both systems; thioredoxin binds 188 times better than does DTT. The thioredoxin must be in the reduced form since omission of either thioredoxin reductase or NADPH results in complete loss of enzyme activity. The concentration of DTT required to obtain maximal enzyme activity may be as much as 485 times greater than the corresponding concentration of reduced thioredoxin that gives the same enzyme activity.
(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.
Evidence for an enamine mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid is presented. apo-GABA aminotransferase reconstituted with [3H]pyridoxal 5'-phosphate is inactivated by (S,E)-4-amino-5-fluoropent-2-enoic acid and the pH is raised to 12. All of the radioactivity is released from the enzyme as an adduct of the cofactor; no [3H]pyridoxamine 5'-phosphate is generated.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin and also an inactivator of monoamine oxidase (MAO). Since MPTP is a conformationally rigid analogue of N,N-dimethylcinnamylamine, other conformationally rigid analogues of N,N-dimethylcinnamylamine were synthesized and tested as inhibitors and inactivators of MAO. (E)-2-(Phenylmethylene)cyclohexanamine (5a), (E)-N,N-dimethyl-2-(phenylmethylene)cyclohexanamine (5b), 3-phenyl-2-cyclohexen-1-amine (6a), N,N-dimethyl-3-phenyl-2-cyclohexen-1-amine (6b), and (E)- and (Z)-N-methyl-3-(phenylmethylene)piperidine (7 and 8) are all inhibitors and time-dependent inactivators of MAO B, but none is as potent as MPTP. alpha-Methylation and methylation of the amino group in all cases increases the Ki value relative to that for the parent compound. Compounds 5a, 5b, 6a and 6b are highly cytotoxic, but cytotoxicity is not prevented by pretreatment of the cells with pargyline. There does not appear to be a correlation between the configuration of the N,N-dimethylcinnamylamine analogue and its potency as a MAO inactivator.
Mechanism-based enzyme inactivator, alanine racemase, S-adenosylhomocysteine hydrolase, D-amino acid aminotransferase, gamma-aminobutyric acid aminotransferase, arginine decarboxylase, aromatase, L-aromatic amino acid decarboxylase, dihydrofolate reductase, dihydroorotate dehydrogenase DNA polymerase I, dopamine beta-hydroxylase, histidine decarboxylase, beta-lactamase, monoamine oxidase, ornithine decarboxylase, serine proteases, testosterone 5 alpha-reductase, thymidylate synthetase, xanthine oxidase.