title: Inhibition of glutathione conjugation by glutathione analogues in the perfused rat liver. Effect of esterification on the potency of gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine.
abstract: To assess the role of GST's (glutathione S-transferases) in the (de)toxification of their substrates, an in vivo active inhibitor based on the structure of glutathione (GSH), gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine monoethyl ester (Et-R-Hep), was developed. To increase its effectivity, analogues esterified with alkyl chains of varying lengths and one diesterified derivative (DiEt-R-Hep) were synthesized. The unesterified analogue, R-Hep, was also tested. Their isoenzyme selectivity was characterized using purified rat GST isoenzymes. Furthermore, the extent of inhibition of the GSH conjugation of (RS)-2-bromoisovalerylurea (BIU) was evaluated in rat liver cytosol, isolated hepatocytes, and in liver perfusions. All compounds inhibited Alpha- (1-1 and 2-2) more effectively than Mu (3-3 and 4-4) class GSTs; Pi-(5-5) and Theta (7-7) classes were minimally inhibited. The unesterified R-Hep was the most effective inhibitor towards purified isoenzymes; its Ki value towards GST 3-3 (S-BIU as substrate) was 27 microM. The mono ethyl ester derivative, Et-R-Hep (Ki 270 microM for 3-3), was the most potent inhibitor in hepatocytes and in the perfused liver: 50 microM inhibited the conjugation of (S)-BIU by 50%. Longer ester chains or diesterification did not increase the inhibitory potency; R-Hep had less inhibitory activity. In all systems, only the (S)-enantiomer of BIU, which is conjugated mainly by Alpha class GSTs, was inhibited, confirming Alpha isoenzyme selective inhibition.
authors.affiliation: Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: Ouwerkerk-Mahadevan
authors.forename: R G
authors.initials: RG
authors.lastname: Tirona
authors.forename: R A
authors.initials: RA
authors.lastname: Ripping
authors.forename: J H
authors.initials: JH
authors.lastname: Ploemen
authors.forename: P J
authors.initials: PJ
authors.lastname: van Bladeren
authors.forename: K S
authors.initials: KS
authors.lastname: Pang
authors.forename: J H
authors.initials: JH
authors.lastname: van Boom
authors.forename: G J
authors.initials: GJ
authors.lastname: Mulder
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004951:Esterification; D005973:Glutamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; C108916:glutamyl-(2-aminoadipyl)-N-2-heptylamine; D005973:Glutamine; D005982:Glutathione Transferase; D005978:Glutathione