γ-(Monophenyl)phosphono glutamate analogues as mechanism-based inhibitors of γ-glutamyl transpeptidase
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.
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 2a–d 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 2a–d 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 2a–d
The monophenyl phosphonates 2a–d 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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Human γ-glutamyl transpeptidase 1: Structures of the free enzyme, inhibitor-bound tetrahedral transition states, and glutamate-bound enzyme reveal novel movement within the active site during catalysis
2015, Journal of Biological ChemistryCitation Excerpt :The addition of the methyl group to the phosphate oxygen of the inhibitor creates a diester that reduces the negative charge on the phosphate oxygen, rendering the phosphate more favorable for nucleophilic attack. The second factor proposed by Han and colleagues (31) was that neutral phosphonates would have a higher binding affinity to the active site of hGGT1 than would anionic charged compounds (33). However, the structure of GGsTop-bound hGGT1 revealed that Gly-473 and Gly-474, which interact with the phosphonyl moiety and act as the oxyanion hole, have a partial positive charge.
Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site
2014, Bioorganic and Medicinal ChemistryCitation Excerpt :Therefore the important criteria for GGT inhibitors of practical use both in vitro and in vivo are (1) irreversible inhibition with no regain of enzyme activity until new enzymes are de novo synthesized and (2) high selectivity for GGT without inhibiting glutamine-hydrolyzing biosynthetic enzymes. In view of these criteria, we reported a series of phosphonate esters as active-site directed and transition-state analogue inhibitors of GGT.38,39 The phosphonate esters react in a mechanism-based manner with the N-terminal Thr residue in the small subunit, the catalytic nucleophile of GGT,40,41 to inactivate the enzyme completely.
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Present address: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-cho, Ishikawa 921-8836, Japan.