Mechanism of the glutathione transferase-catalyzed conversion of antitumor 2-crotonyloxymethyl-2-cycloalkenones to GSH adducts.

Human glutathione (GSH) transferase (hGSTP1-1) processes with similar kinetic efficiencies the antitumor agents 2-crotonyloxymethyl-2-cyclohexenone (COMC-6), 2-crotonyloxymethyl-2-cycloheptenone (COMC-7), and 2-crotonyloxymethyl-2-cyclopentenone (COMC-5) to 2-glutathionylmethyl-2-cyclohexenone, 2-glutathionylmethyl-3-glutathionyl-2-cycloheptenone, and 2-glutathionylmethyl-2-cyclopentenone, respectively. This process likely involves initial enzyme-catalyzed Michael addition of GSH to the COMC derivative to give a glutathionylated enol(ate), which undergoes nonstereospecific ketonization, either while bound to the active site or free in solution, to a glutathionylated exocyclic enone. Free in solution, GSH reacts at the exomethylene carbon of the exocyclic enone, displacing the first GSH to give the final product.

Molecular basis of the antitumor activities of 2-crotonyloxymethyl-2-cycloalkenones.

The antitumor activity of 2-crotonyloxymethyl-2-cyclohexenone (COMC-6) is not the result of the GSH conjugate (GSMC-6) formed inside tumor cells, as the diethyl ester prodrug form of GSMC-6 displays little antitumor activity with B16 melanotic melanoma in vitro (IC(50) > 460 microM) versus COMC-6 (IC(50) 0.041 microM) and its five- and seven-membered ring homologues. Antitumor activity probably results from a reactive intermediate that forms during conjugation of the COMCs with intracellular GSH.

Glutathionyl transferase catalyzed addition of glutathione to COMC: a new hypothesis for antitumor activity.

[reaction: see text] Data are presented indicating that the potent antitumor activity of 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxy-2-cyclohexenone (COTC) and 2-crotonyloxymethyl-2-cyclohexenone (COMC) is not likely the result of glyoxalase I inhibition, as has long been assumed. An alternative hypothesis is presented, based on the finding that COMC is a substrate for human glutathionyl transferase, which produces a transient, highly electrophilic glutathionylated 2-exomethylenecyclohexanone that can covalently modify proteins and nucleic acids.