Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione: Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Abstract

Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. A significant route of phase II drug metabolism is conjugation with glutathione (GSH), which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes. The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA, C-2028. GSH-supplemented incubations of C-2028 with rat, but not with human, liver cytosol led to the formation of a single GSH-related metabolite. Interestingly, it was also revealed with rat liver microsomes. Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole. Therefore, the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate. In turn, incubations of C-2028 and GSH with human recombinant glutathione S-transferase (GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form, respectively. These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule. Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction. Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry. Mechanisms for their formation were proposed. The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important. This may affect the patient's drug clearance due to GST activity, loss of GSH, or the interactions with GSH-conjugated drugs. Moreover, GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Naturally Derived Phenethyl Isothiocyanate Modulates Induction of Oxidative Stress via Its N-Acetylated Cysteine Conjugated form in Malignant Melanoma

“…Last, GSH levels were minimally affected (at any time of exposure), suggesting prevention of GSH conjugation (Figure 5C). In addition, a reversible inhibitor of GST (ethacrynic acid) was utilized (Figure 5A) as previously described by others [35][36][37]. Finally, cells pre-treated for 4 h to 30 µM of ethacrynic acid, followed by exposure to PhEF extract (0.75-2.5% v/v) for another 0.5-48 h, provided optimized experimental conditions in the context of not being associated with the induction of any cytotoxicity (Figure S5A, Table S3).…”

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

“…GAP can remove and detoxify carcinogens, but high expression of GAP may protect cancer cells by conferring resistance to some drugs [ 21 , 22 ]. Targeting GAP is a potential strategy to induce cancer cell death [ 22 , 23 ]. Therefore, monitoring the metabolite profiling of GAP is of great importance and contributes to research on drugs and diseases.…”

In Vitro Enzyme Kinetics and NMR-Based Product Elucidation for Glutathione S-Conjugation of the Anticancer Unsymmetrical Bisacridine C-2028 in Liver Microsomes and Cytosol: Major Role of Glutathione S-Transferase M1-1 Isoenzyme

“…Further, we showed that C-2028 was rapidly conjugated with glutathione (GSH) to only one main product (GSH S-conjugate) via the catalytic action of rat microsomal and cytosolic GSH S-transferases (GSTs) [8]. No kinetic data for this reaction have been assessed yet.…”