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
Ouwerkerk-Mahadevan, S.; Tirona, R.G.; Ripping, R.A.; Ploemen, J.H.; van Bladeren, P.J.; Pang, K.S.; van Boom, J.H.; Mulder, G.J.
Drug Metabolism and Disposition the Biological Fate of Chemicals 25(10): 1137-1143
1997
ISSN/ISBN: 0090-9556 PMID: 9321516 Document Number: 482095
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 K-i value towards GST 3-3 (S-BIU as substrate) was 27 mu-M. The mono ethyl ester derivative, Et-R-HeP (K-i 270 mu-M for 3-3), was the most potent inhibitor in hepatocytes and in the perfused liver 50 mu-M 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.