Development and evaluation of a boronate inhibitor of gamma-glutamyl transpeptidase.
Gamma-glutamyl transpeptidase (gamma-GT) plays a central role in the metabolism of glutathione and is also a marker for neoplasia and cell transformation. We have investigated the compound L-2-amino-4-boronobutanoic acid (ABBA) as a structural analog of the putative ternary complex formed by the enzyme, L-serine, and borate, proposed to function as a transition state analog inhibitor. ABBA was found to be a potent inhibitor of the enzyme, with Ki = 17 nM using typical assay conditions (pH 8, gamma-glutamyl-p-nitroanilide substrate, 20 mM glycyl-glycine acceptor). ABBA is a stable amino acid analog with pK values determined from 13C and 11B NMR to be 2.3, 11.0 (amino titration), and 7.9 (boronate titration). The structural similarity to glutamate suggested that it might function as a glutamate analog for some glutamate-dependent enzymes or receptors. Transamination of pyruvate by ABBA to yield alanine in the presence of glutamic pyruvic transaminase was demonstrated by 13C NMR. The 2-keto-4-boronobutanoic acid transamination product is apparently fairly labile to hydrolysis, leading to formation of 2-ketobutanoic acid plus borate. The latter is also subsequently transaminated to yield 2-aminobutanoic acid. Both of these metabolites were observed in the 13C NMR spectrum. However, the corresponding transamination of oxaloacetate by ABBA in the presence of glutamic oxaloacetic transaminase was not observed. Effects of ABBA on the growth of cultured rat liver cell lines ARL-15C1 (nontumorigenic, low gamma-GT activity) and ARL-16T2 (tumorigenic, high gamma-GT activity) were also investigated, both in standard Williams Media as well as in a low cysteine growth medium. A high concentration (1 mM) of ABBA inhibited the growth of both cell lines in both media, with the degree of inhibition greater in the low cysteine medium. Alternatively, growth inhibition by 10 microM ABBA could be observed only in the low cysteine media. In general, there were no significant differences between the two cell lines in terms of sensitivity to ABBA.
London
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Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. london@niehs.nih.gov
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D000409:Alanine / Q000378:metabolism; D000410:Alanine Transaminase / Q000378:metabolism; D000613:Aminobutyrates / Q000378:metabolism; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001665:Binding Sites; D001896:Boron Compounds / Q000737:chemistry / Q000494:pharmacology*; D002247:Carbon Isotopes; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D005973:Glutamine / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver; D009682:Magnetic Resonance Spectroscopy; D062907:Oxaloacetic Acid / Q000378:metabolism; D011522:Protons; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism
C426904:2-amino-4-boronobutanoic acid; D000613:Aminobutyrates; D001896:Boron Compounds; D002247:Carbon Isotopes; D003470:Culture Media; D011522:Protons; D005973:Glutamine; D062907:Oxaloacetic Acid; C001300:gamma-glutamine-4-nitroanilide; C012223:alpha-aminobutyric acid; D019289:Pyruvic Acid; D005723:gamma-Glutamyltransferase; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D003545:Cysteine; D000409:Alanine