title: Isoenzyme selective irreversible inhibition of rat and human glutathione S-transferases by ethacrynic acid and two brominated derivatives.
abstract: In the present study it has been shown that ethacrynic acid can inhibit glutathione S-transferase (GST) of the pi-class irreversibly. [14C]Ethacrynic acid, 0.8 nmol/nmol human P1-1 and 0.8 nmol/nmol rat GST 7-7 could be incorporated, resulting in 65-93% inhibition of the activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Isoenzymes of the alpha- and mu-class also bound [14C]ethacrynic acid, however without loss of catalytic activity. Incorporation ranged from 0.3 to 0.6 and 0.2 nmol/nmol enzyme for the mu- and alpha-class GST isoenzymes, respectively. For all isoenzymes, incorporation of [14C]ethacrynic acid could be prevented by preincubation with tetrachloro-1,4-benzoquinone, suggesting, that a cysteine residue is the target site. Protection of GST P1-1 against inhibition by ethacrynic acid by the substrate analog S-hexylglutathione, indicates an active site-directed modification. The monobromo and dibromo dihydro derivatives of ethacrynic acid were synthesized in an effort to produce more reactive compounds. The monobromo derivative did not exhibit enhanced irreversible inhibitory capacity. However, the dibromo dihydro derivative inhibited both human and rat GST isoenzymes of the pi-class very efficiently, resulting in 90-96% inhibition of the activity towards CDNB. Interestingly, this compound is also a powerful irreversible inhibitor of the mu-class GST isoenzymes, resulting in 52-70% inhibition. The two bromine atoms only marginally affect the strong (reversible) competitive inhibitory capacity of ethacrynic acid, with IC50 (microM) of 0.4-0.6 and 4.6-10 for the mu- and pi-class GST isoenzymes, respectively.
authors.affiliation: Department of Biological Toxicology, TNO Toxicology and Nutrition Institute, Zeist, The Netherlands.
authors.forename: J H
authors.initials: JH
authors.lastname: Ploemen
authors.forename: J J
authors.initials: JJ
authors.lastname: Bogaards
authors.forename: G A
authors.initials: GA
authors.lastname: Veldink
authors.forename: B
authors.initials: B
authors.lastname: van Ommen
authors.forename: D H
authors.initials: DH
authors.lastname: Jansen
authors.forename: P J
authors.initials: PJ
authors.lastname: van Bladeren
mesh_terms: D000818:Animals; D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004976:Ethacrynic Acid / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D051381:Rats
chemical_list: D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D005982:Glutathione Transferase; D005978:Glutathione; D004976:Ethacrynic Acid