Involvement of human glutathione S-transferase M1-1 in the glutathione conjugation of the antitumor unsymmetrical bisacridine derivative C-2028.

Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. The aim of this study was to investigate the possible role of human glutathione S-transferase M1-1 (hGSTM1-1) in the glutathione (GSH) conjugation of a representative UA, C‑2028.
0.05 mg/mL recombinant hGSTM1-1 (expressed in Escherichia coli, obtained from Sigma-Aldrich, MO, USA), 0.01 mM C-2028, and 5 mM reduced L-glutathione (GSH) in 0.1 M Hepes buffer solution (pH 6.5) were incubated at 37 °C for 0 and 1 hrs in a total volume of 50 µL. MgCl2 solution (2 mM) was added for enzyme stimulation. The incubations were terminated by adding ice-cold 1 M HCl to the incubation mixtures (10:90, v/v). The samples were vortexed, placed in ice for 10 min, and centrifuged at 10000 x g for 10 min. Aliquots of the supernatants (50 µL) in triplicate were then analyzed directly by reversed-phase liquid chromatography (RP-LC) with UV-vis detection and/or diode array and multiple wavelength detection, and monitored by MS.