Peptidomimetic Glutathione Analogues as Novel GT Stable GST Inhibitors Danny Burg, a, * Dmitri V. Filippov, b Ralph Hermanns, a Gijs A. van der Marel, b Jacques H. van Boom b and Gerard J. Mulder a a Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300RA, Leiden, The Netherlands b Division of Bio-Organic Synthesis, Leiden Institute of Chemistry, PO Box 9502, 2300RA, Leiden University, The Netherlands Received 25 May 2001; accepted 18 July 2001 Abstract—Elevated levels of glutathione-S-transferase (GST) isoenzymes are found in many tumor cells and are thought to play a role in the onset of multidrug resistance (MDR). To evaluate the contribution of GST to this process, inhibitors are needed. Glu- tathione (GSH) conjugates, although good GST inhibitors, cannot be used in vivo, because they are eliminated rapidly. In this paper, we describe the synthesis of a series of novel peptidomimetic glutathione analogues that are stabilized against peptidase mediated breakdown. The peptide bonds in GSH were replaced by isosteres, such as the ‘reduced’ amide (which was prepared using a novel method), N-methylamide, urethane, and methylene linkages. The in vitro evaluation of the compounds focuses on GST inhibition and stability towards g-glutamyl-transpeptidase (gGT), the main enzyme involved in GSH breakdown. The compounds were conjugated to the model electrophile ethacrynic acid (EA) to resemble GS-EA, an efficient GST inhibitor. All novel GSH- analogues were shown to inhibit rat liver cytosolic GSTs. Furthermore, peptidomimetic changes of the g-glutamyl-cysteine amide bond greatly improved stability towards gGT. These compounds may therefore be useful in the design of novel in vivo applicable GST inhibitors. # 2001 Elsevier Science Ltd. All rights reserved. Introduction The ubiquitous tripeptide glutathione (GSH), g-l-glu- tamyl-l-cysteinyl-glycine, is the prevalent non-protein thiol in eukaryotic cells. GSH plays a critical role in the cellular protection against potentially harmful electro- philes from xenobiotic sources, or those generated by endogenous oxidative processes. 1 GSH-conjugates are formed by nucleophilic attack of the cysteine sulphydryl on the electrophilic center of a suitable substrate. This process is catalyzed by glutathione-S-transferase (GST). 2 Several members of the GST-isoenzyme family are involved in conjugation of drugs and thereby in drug resistance. Isoenzyme selective GST inhibitors may therefore be used to improve drug response and decrease resistance. GSH-conjugates and their cell- permeable esterified derivatives are effective competitive inhibitors of GST. 3,4 Unfortunately, the inhibitory action of GSH-conjugates is severely limited by their sensitivity towards peptidase-mediated breakdown. Although the g-glutamyl-cysteine peptide bond renders it resistant to most a-peptidases, GSH is rapidly degra- ded by g-glutamyl transpeptidase (gGT). Cleavage by this enzyme, which has particularly high activity in the kidney, prevents the use of GSH conjugates in vivo. Being potential drug candidates, effort has been put into the design of new glutathione analogues as GST inhibi- tors. Several modifications have been made to the GSH peptide backbone, mostly by replacing the amino acid residues by other naturally occurring or synthetic ana- logues. 5 9 Recently, most of the known structural mod- ifications of the glutathione molecule have been reviewed. 10 In recent years, our laboratory has been involved in the development of GSH analogues for the functional mapping and inhibition of GST isoenzymes. This resulted in the development of non-thiol containing S-alkyl-glutathione analogues, in which the cysteinyl- glycine part was replaced by d-aminoadipic acid. These compounds showed improved stability towards g-GT and exhibited in vivo inhibition of some GST iso- enzymes, although inhibition was limited to a and m class GST isoenzymes. 11 15 In this study, new compounds 0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(01)00269-3 Bioorganic & Medicinal Chemistry 10 (2002) 195–205 *Corresponding author. Tel.: +31-71-527-6227; fax; +31-71-527- 6292; e-mail: d.burg@lacdr.leidenuniv.nl