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

  • Bioorg Med Chem. 2016 Nov 1;24(21):5340-5352. doi: 10.1016/j.bmc.2016.08.050.
Akane Kamiyama  1 ,  Mado Nakajima  1 ,  Liyou Han  1 ,  Kei Wada  2 ,  Masaharu Mizutani  1 ,  Yukiko Tabuchi  1 ,  Akiko Kojima-Yuasa  3 ,  Isao Matsui-Yuasa  3 ,  Hideyuki Suzuki  4 ,  Keiichi Fukuyama  5 ,  Bunta Watanabe  6 ,  Jun Hiratake  7
Affiliations
  • 1. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
  • 2. Organization for Promotion of Tenure Track, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
  • 3. 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.
  • 4. Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
  • 5. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
  • 6. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Electronic address: [email protected].
  • 7. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Electronic address: [email protected].
PMID: 27622749 DOI: 10.1016/j.bmc.2016.08.050
Abstract

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.

Keywords
Active-site residue Lys562; Cytotoxicity; Electrostatic interaction; GGsTop; Mechanism-based inhibitor; Phosphonate diesters; Selectivity; γ-Glutamyl transpeptidase.
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