Effect of Esterification on the Potency of γ-L-Glutamyl-α-(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), γ-L-glutamyl-α-(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 μM. The mono ethyl ester derivative, Et-R-Hep (Ki 270 μM for 3–3), was the most potent inhibitor in hepatocytes and in the perfused liver: 50 μM 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.
Footnotes
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Send reprint requests to: G. J. Mulder, Ph.D., Division of Toxicology, LACDR, Leiden University, P. O. Box 9503, 2300 RA Leiden, The Netherlands.
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The work was supported by a grant from the Netherlands Organization for Scientific Research (NWO; reg. no. 900–21-142).
- Abbreviations used are::
- GST
- glutathioneS-transferase
- GSH
- glutathione
- Et-R-Hep
- (R)-5-ethyloxycarbonyl-2-γ-(S)-glutamylamino-N-2-heptylpentamide
- d-Aad
- d-α-aminoapidic acid
- BIU
- 2-bromoisovalerylurea
- (R)- and (S)-IU-G
- GSH conjugates of (S) and (R)-BIU, respectively
- R-Hep
- (R)-5-carboxy-2-γ-(S)-glutamylamino-N-2-heptylpentamide
- Glu-Oct-Hep
- 2-γ-(S)-glutamylamino-N-2-heptyloctamide
- BSA
- bovine serum albumin
- CDNB
- 1-chloro-2,4-dinitrobenzene
- EPNP
- 1,2-epoxy-3-(-p-nitrophenoxy)propane
- Do-R-Hep
- (R)-5-dodecyloxycarbonyl-2-γ-(S)-glutamylamino-N-2-heptylpentamide
- Pt-R-Hep
- (R)-5-pentyloxycarbonyl-2-γ-(S)-glutamylamino-N-2-heptylpentamide
- DiEt-R-Hep
- (R)-5-ethyloxycarbonyl-2-γ-ethyloxycarbonyl-(S)-glutamylamino-N-2-heptylpentamide
- (S)-BI
- (S)-bromoisovaleric acid
- (R)-I-G
- GSH conjugate of (S)-BI
- The American Society for Pharmacology and Experimental Therapeutics
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