Elsevier

Bioorganic & Medicinal Chemistry

Volume 14, Issue 17, 1 September 2006, Pages 6043-6054
Bioorganic & Medicinal Chemistry

γ-(Monophenyl)phosphono glutamate analogues as mechanism-based inhibitors of γ-glutamyl transpeptidase

https://doi.org/10.1016/j.bmc.2006.05.008Get rights and content

Abstract

γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione and plays a central role in glutathione homeostasis. We report here the synthesis and evaluation of a series of hydrolytically stable γ-(monophenyl)phosphono glutamate analogues with varying electron-withdrawing para substituents on the leaving group phenols as mechanism-based and transition-state analogue inhibitors of Escherichia coli and human GGTs. The monophenyl phosphonates caused time-dependent and irreversible inhibition of both the E. coli and human enzymes probably by phosphonylating the catalytic Thr residue of the enzyme. The inactivation rate of E. coli GGT was highly dependent on the leaving group ability of phenols with electron-withdrawing groups substantially accelerating the rate (Brønsted βlg = −1.4), whereas the inactivation of human GGT was rather slow and almost independent on the nature of the leaving group. The inhibition potency and profiles of the phosphonate analogues were compared to those of acivicin, a classical inhibitor of GGT, suggesting that the phosphonate-based glutamate analogues served as a promising candidate for potent and selective GGT inhibitors.

Graphical abstract

A series of hydrolytically stable monophenyl phosphonoates has been synthesized and found to serve as irreversible and mechanism-based inhibitors of Escherichia coli and human γ-glutamyl transpeptidase.

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Introduction

γ-Glutamyl transpeptidase (GGT; EC 2.3.2.2) is a heterodimeric enzyme found widely among organisms from bacteria to mammals and catalyzes the first step in glutathione (GSH) metabolism.1, 2, 3, 4 In mammals, GGT is bound to the external surface of plasma membrane and is expressed in high concentrations in kidney tubules, biliary epithelium, and brain capillaries.1, 4, 5 This enzyme plays critical roles in GSH homeostasis by breaking down extracellular GSH to provide cells with secondary source of cysteine, the rate-limiting substrate for intracellular GSH biosynthesis,6, 7, 8, 9 and in detoxification of electrophilic/oxidative xenobiotics through the metabolism of GSH conjugates to confer resistance against oxidative stress and anti-tumor drugs such as cisplatin.10, 11 The expression of GGT is often increased significantly in human tumors, and its roles in tumor progression12 and the expression of malignant phenotypes of cancer cells such as drug resistance10, 11, 13, 14 and metastasis15, 16, 17 have been repeatedly suggested.18 GGT is also implicated in many physiological disorders such as neurodegenerative disease,19, 20 diabetes,21, 22 and cardiovascular disease23, 24 in relation to oxidative stress and GSH homeostasis.25 Therefore, the important roles played by GGT in GSH-mediated detoxification and cellular response to oxidative stress strongly suggest that GGT is an attractive pharmaceutical target for cancer chemotherapy and a vast array of physiological disorders related to oxidative stress caused by reactive oxygen species.

GGT catalyzes the cleavage of the γ-glutamyl bond of GSH, its S-conjugates, and structurally diverse γ-glutamyl amides to transfer the γ-glutamyl group to water (hydrolysis) or to a variety of amino acids and peptides (transpeptidation) through a modified ping–pong mechanism involving a γ-glutamyl enzyme intermediate (Scheme 1).26, 27 Although a number of inhibitors have been reported to date, little compounds appear to serve as potent and specific inhibitors of GGT. (αS,5S)-α-Amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125), a natural product derived from Streptomyces sviceus,28 is a classical and most widely used irreversible inhibitor of GGT29, 30, 31, but is a strong inhibitor of glutamine-dependent amidotransferases32, 33 and inactivates many physiologically important enzymes involved in purine and pyrimidine biosynthesis to exert potent cytotoxicity.34, 35 Other glutamine antagonists such as l-azaserine and 6-diazo-5-oxo-l-norleucine (DON) inhibit GGT,36 but these compounds also serve as potent inhibitors of glutamine-dependent amidotransferases.34 The reversible and weak inhibition of GGT by l-serine–borate complex37 lead to the development of L-2-amino-4-boronobutanoic acid (γ-boroGlu), a γ-boronic acid analogue of glutamic acid, as a potent GGT inhibitor with an inhibition constant (Ki) of a nanomolar range.38, 39 This compound is believed to form a covalent bond with a hydroxy residue in the active site to mimic the transition-state of GGT catalysis, but the inhibition is reversible, and the inactivated enzyme regained activity rapidly.38

We previously reported that 2-amino-4-(fluorophosphono)butanoic acid (1), a γ-tmonofluorophosphono derivative of glutamic acid, served as an extremely effective mechanism-based affinity labeling agent that inactivated Escherichia coli GGT with a second-order rate constant for enzyme inactivation (kon) of 48,000 M−1 s−1.40 This compound was used successfully for the identification of the catalytic nucleophile of E. coli GGT as Thr-391,40 the N-terminal residue of the small subunit, suggesting that GGT is a member of the N-terminal nucleophile hydrolase family.41, 42 Compound 1 is a promising lead for potent and selective inhibitors of GGT, because it reacts with the catalytic residue of GGT in a mechanism-based manner to form a stable monophosphonate bond with its catalytic Thr to inactivate the enzyme. However, the monofluorophosphonate 1 is highly reactive and is hydrolytically unstable in alkaline to neutral media.40 Herein we report the synthesis and evaluation of a series of hydrolytically stable γ-(monophenyl)phosphono glutamate analogues 2ad with varying electron-withdrawing groups at the para-position of the leaving group phenols as mechanism-based inhibitors of E. coli and human GGTs (Scheme 2). The monophenyl phosphonates 2ad served as irreversible inhibitors that caused time-dependent inhibition of both the E. coli and human enzymes, but with significantly higher activity toward E. coli GGT. The dependence of the inactivation rates on the leaving group ability suggested significant differences in the inactivation transition state between the E. coli and human enzymes. The phosphonate-based glutamate analogues are compared with acivicin in terms of the potency and the mechanism of inactivation.

Section snippets

Synthesis and stability of monophenyl phosphonates 2ad

The monophenyl phosphonates 2ad were synthesized as shown in Scheme 3. The amino group of 2-amino-4-phosphonobutanoic acid (APBA) was protected with 4-nitrobenzyloxycarbonyl [p(NO2)Z] group, and the α-carboxy group was esterified selectively under acidic conditions to afford the methyl ester 4 or the ethyl ester 4′ with a free γ-phosphono group. The methyl ester 4, however, was partially hydrolyzed (22%) to the acid 3 during purification on an ODS column (MeOH–H2O), probably because the

Materials and methods

All chemicals were obtained commercially and used without further purification unless otherwise noted. 7-(γ-l-Glutamylamino)-4-methylcoumarin (γ-Glu-AMC) and (αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) were purchased from Sigma. Racemic 2-amino-4-phosphonobutanoic acid (APBA) was synthesized by the reported procedure.58, 59 Racemic 2-amino-4-(fluorophosphono)butanoic acid (1) was synthesized previously.40, 44 Dry toluene and dry CH2Cl2 were prepared by distillation

Acknowledgments

We thank Asahi Kasei Corporation for a generous gift of human γ-glutamyl transpeptidase HC-GTP (T-72). This study was supported in part by a Grant-in-Aid for Scientific Research from Japan for the Promotion of Science for J.H. (contract No. 16310152).

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    Present address: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-cho, Ishikawa 921-8836, Japan.

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