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. 2023 Sep 26;28(19):6812.
doi: 10.3390/molecules28196812.

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

Agnieszka Potęga  1 , Dominika Rafalska  1 , Dawid Kazimierczyk  1 , Michał Kosno  1 , Aleksandra Pawłowicz  2 , Witold Andrałojć  2 , Ewa Paluszkiewicz  1 , Tomasz Laskowski  1
Affiliations

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

Agnieszka Potęga et al. Molecules. .

Abstract

This work is the next step in studying the interplay between C-2028 (anticancer-active unsymmetrical bisacridine developed in our group) and the glutathione S-transferase/glutathione (GST/GSH) system. Here, we analyzed the concentration- and pH-dependent GSH conjugation of C-2028 in rat liver microsomes and cytosol. We also applied three recombinant human GST isoenzymes, which altered expression was found in various tumors. The formation of GSH S-conjugate of C-2028 in liver subfractions followed Michaelis-Menten kinetics. We found that C-2028 was conjugated with GSH preferentially by GSTM1-1, revealing a sigmoidal kinetic model. Using a colorimetric assay (MTT test), we initially assessed the cellular GST/GSH-dependent biotransformation of C-2028 in relation to cytotoxicity against Du-145 human prostate cancer cells in the presence or absence of the modulator of GSH biosynthesis. Pretreatment of cells with buthionine sulfoximine resulted in a cytotoxicity decrease, suggesting a possible GSH-mediated bioactivation process. Altogether, our results confirmed the importance of GSH conjugation in C-2028 metabolism, which humans must consider when planning a treatment strategy. Finally, nuclear magnetic resonance spectroscopy elucidated the structure of the GSH-derived product of C-2028. Hence, synthesizing the compound standard necessary for further advanced biological and bioanalytical investigations will be achievable.

Keywords: GSTM; NMR-based product elucidation; anticancer unsymmetrical bisacridine; enzyme kinetics; glutathione S-conjugate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative HPLC chromatograms of unsymmetrical bisacridine C-2028 and/or its GSH S-conjugate (GS-C-2028) in rat liver microsomes (RLMs) and cytosol (RCyt) as well as with recombinant human GSTs (A1-1, M1-1, and P1-1) after the indicated incubation time. For experimental details, see the Materials and Methods section (Section 4). The scheme of possible GST/GSH-mediated conjugation reaction of C-2028 was shown at the top [8].
Figure 2
Figure 2
Influence of the pH of the 0.1 M Hepes buffer (reaction buffer) on the GST-dependent formation of GSH S-conjugate of C-2028 in rat liver microsomes (RLMs) and cytosol (RCyt). For experimental details, see the Materials and Methods section (Section 4). Data are expressed as means ± SD (standard deviation) of three independent determinations. Statistically significant differences were assessed using two-way ANOVA with Bonferroni multiple comparisons (* p < 0.05, **** p < 0.0001).
Figure 3
Figure 3
Area under the GSH S-conjugate of C-2028 peak against different concentrations of recombinant human GSTM1-1 as a function of reaction time. For experimental details, see the Materials and Methods section (Section 4). Data are expressed as means ± SD of two independent determinations.
Figure 4
Figure 4
Kinetics of the GST-dependent formation of GSH S-conjugate of C-2028 in (a) rat liver microsomes (RLMs), (b) rat liver cytosol (RCyt), and (c) with recombinant human GSTM1-1 normalized to protein content as a function of C-2028 concentration. Eadie-Hofstee plots were shown as insets on the graphs. For experimental details, see the Materials and Methods section (Section 4). Data are expressed as means ± SD of three independent determinations.
Figure 5
Figure 5
NMR-derived structure of the resulting GSH S-conjugate of C-2028. Panels (a–c) show fragments of the 1H-13C HMBC spectrum (recorded at 55 °C), which contain diagnostic proton-carbon correlations enabling unambiguous localization of the peptide moiety, according to the bidirectional arrows displayed at the GSH S-conjugate’s structure.
See this image and copyright information in PMC

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