Phosphonate-based irreversible inhibitors of human γ-glutamyl transpeptidase (GGT). GGsTop is a non-toxic and highly selective inhibitor with critical electrostatic interaction with an active-site residue Lys562 for enhanced inhibitory activity.
γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure-activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Akane
A
Kamiyama
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Mado
M
Nakajima
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Liyou
L
Han
Organization for Promotion of Tenure Track, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Kei
K
Wada
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Masaharu
M
Mizutani
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Yukiko
Y
Tabuchi
Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan.
Akiko
A
Kojima-Yuasa
Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan.
Isao
I
Matsui-Yuasa
Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Hideyuki
H
Suzuki
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Keiichi
K
Fukuyama
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Electronic address: bunta@scl.kyoto-u.ac.jp.
Bunta
B
Watanabe
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Electronic address: hiratake@scl.kyoto-u.ac.jp.
Jun
J
Hiratake
D020134:Catalytic Domain / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D008239:Lysine / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D063065:Organophosphonates / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D063065:Organophosphonates; D005723:gamma-Glutamyltransferase; D008239:Lysine
Active-site residue Lys562; Cytotoxicity; Electrostatic interaction; GGsTop; Mechanism-based inhibitor; Phosphonate diesters; Selectivity; γ-Glutamyl transpeptidase