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Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases

Author:
Stoddard, Ethan G., Killinger, Bryan J., Nair, Reji N., Sadler, Natalie C., Volk, Regan F., Purvine, Samuel O., Shukla, Anil K., Smith, Jordan N., Wright, Aaron T.
Source:
Journal of the American Chemical Society 2017 v.139 no.45 pp. 16032-16035
ISSN:
1520-5126
Subject:
active sites, drugs, glutathione, glutathione transferase, liver, mammals, metabolism, models, moieties, tissues, xenobiotics
Abstract:
Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding “G site” and a substrate binding “H site”. Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the “G site”, and (2) an ABP designed to mimic a substrate molecule and have “H site” activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.
Agid:
5860349