Incubation of rat brain 4-aminobutyrate aminotransferase with 4-amino-hex-5-enoic acid, a substrate analog of 4-aminobutyric acid, results in a time-dependent irreversible loss of enzymatic activity. In the presence of 0.1 mM inhibitor the half-life of the inactivation process is approximately 6 min. Low concentrations of L-glutamic acid or 4-aminobutyric acid protect against this inactivation, while 2-oxoglutarate prevents this protection, suggesting that only the pyridoxal form of the enzyme is susceptible to inhibition by 4-amino-hex-5-enoic acid.The irreversible inhibition of mammalian 4-aminobutyrate aminotransferase by 4-amino-hex-5-enoic acid is selective. There is no inhibition of this enzyme from Pseudomonas.fZuorescens with the inhibitor at mM concentrations. Even at 10 mM there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited over 500 times more slowly than rat brain 4-aminobutyrate transaminase.4-Aminobutyric acid is believed to be an important inhibitory neurotransmitter in mammalian brain [l]. The major pathway for its degradation is via transamination with 2-oxoglutarate. Fowler and John [2] have described the specific irreversible inhibition of brain aminobutyrate transaminase by ethanolamine-0-sulphate, a substrate analog of 4-aminobutyric acid, which was effective in vivo when given intracisternally. Recently, 4-amino-hex-5-enoic acid (4-acetylene derivative of aminobutyric acid) was shown to be a potent catalytic inhibitor of aminobutyrate transaminase from Pseudomonas fluovescens [3,4]. In addition, it is effective against mammalian brain aminobutyrate transaminase in vitvo [5] and in vivo [5,6] when administered peripherally (P.o., i.p., i.v.).The concept of catalytic inhibition of enzymes as an approach toward the rational design of specific, irreversible, active-site-directed inhibitors of enzymes has attracted much attention, and has recently been Ahhrevintiom. Pyridoxal-P, pyridoxal phosphate. Ei7:yrnes.
␣v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with ArgGly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some ␣v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of ␣v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for ␣v3 and ␣v5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. IntroductionThe formation of new blood vessels from existing vasculature, or angiogenesis, is essential for successful tumor growth and for the development of metastases. Previous work has suggested that certain endothelial cell integrins, including ␣v3 and ␣v5, actively contribute to the angiogenic process. 1,2 More recently, endogenous inhibitors of angiogenesis have been shown to target these integrins. 3 Integrins are transmembrane receptors for extracellular matrix (ECM) and basement membrane proteins that are composed of 2 noncovalently associated subunits, ␣ and . 4 To date, 18 ␣ and 8  subunits have been identified in mammals, and their association in various combinations leads to the formation of at least 24 receptors with distinct ligand specificity. Besides mediating stable adhesion, integrins transmit signals that regulate cell survival, growth, motility, and remodeling of their extracellular environment. [4][5][6] The integrins ␣v3 and ␣v5 bind to ECM molecules through an Arg-Gly-Asp (RGD)-binding site. Based on the concept that they are proangiogenic receptors, specific inhibitors, including blocking monoclonal antibodies, RGD peptides, and RGD peptidomimetics, have been developed and evaluated in vivo. 7 Indeed, pharmacologic agents targeted to ␣v3, ␣v5, or both, have been reported to block tumor and retinal angiogenesis. [8][9][10][11][12][13][14] Some of these angiogenesis inhibitors, including a humanized monoclonal anti␣v3 (Vitaxin; MedImmune, Gaithersburg, MD) and an ␣v3/ ␣v5-selective RGD-based cyclic peptide (cilengitide), have entered clinical trials. 15,16 Early experiments show that a monoclonal antibody directed against ␣v3 inhibits angiogenesis by inducing the apoptosis of angiogenic blood vessels. 17...
A structural analog, 5'-{[(Z)4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (MDL 73811), of decarboxy S-adenosyl-L-methionine, the product of the reaction catalyzed by S-adenosyl-L-methionine (AdoMet) decarboxylase (DC), was found to inhibit Trypanosoma brucei brucei AdoMet DC. The inhibition was time dependent (T.s, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 ,uM), and was apparently irreversible.The natural substrate of the reaction, AdoMet, protected the enzyme from inactivation, suggesting that MDL 73811 was directed at the enzyme active site and was probably catalytically activated. Administration of MDL 73811 to T. b. brucei-infected rats resulted in rapid inhibition of AdoMet DC activity, a decrease in spermidine, and an increase in putrescine in the trypanosomes isolated from treated rats. Treatment of T. b. brucei-infected mice with MDL 73811 (20 mg/kg of body weight intraperitoneally twice daily for 4 days) resulted in cures of the trypanosome infections. Additionally, drug-resistant T. brucei rhodesiense infections in mice were cured by either a combination of MDL 73811 (50 mg/kg intraperitoneally three times per day for 5 days) and relatively low oral doses of a-difluoromethylornithine or MDL 73811 (50 mg/kg per day for 7 days) administered alone in implanted miniosmotic pumps. These data suggest that MDL 73811 and, perhaps, other inhibitors of AdoMet DC have potential for therapeutic use in various forms of African trypanosomiasis.
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