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
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Published:2023-09-26
Issue:19
Volume:28
Page:6812
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ISSN:1420-3049
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Container-title:Molecules
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language:en
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Short-container-title:Molecules
Author:
Potęga Agnieszka1ORCID, Rafalska Dominika1, Kazimierczyk Dawid1, Kosno Michał1ORCID, Pawłowicz Aleksandra2, Andrałojć Witold2ORCID, Paluszkiewicz Ewa1, Laskowski Tomasz1ORCID
Affiliation:
1. Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdańsk University of Technology, Gabriela Narutowicza Str. 11/12, 80-233 Gdańsk, Poland 2. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Zygmunta Noskowskiego Str. 12/14, 61-704 Poznań, Poland
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.
Funder
Dean of Faculty of Chemistry of Gdańsk University of Technology
Subject
Chemistry (miscellaneous),Analytical Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Molecular Medicine,Drug Discovery,Pharmaceutical Science
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