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.

Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site.

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors.

Inhibition of γ-glutamyl transferase suppresses airway hyperresponsiveness and airway inflammation in a mouse model of steroid resistant asthma exacerbation.

Introduction: Despite recent advances, there are limited treatments available for acute asthma exacerbations. Here, we investigated the therapeutic potential of GGsTop, a γ-glutamyl transferase inhibitor, on the disease with a murine model of asthma exacerbation. Methods: GGsTop was administered to mice that received lipopolysaccharide (LPS) and ovalbumin (OVA) challenges. Airway hyperresponsiveness (AHR), lung histology, mucus hypersecretion, and collagen deposition were analyzed to evaluate the hallmark features of asthma exacerbation. The level of proinflammatory cytokines and glutathione were determined with/without GGsTop. The transcription profiles were also examined. Results: GGsTop attenuates hallmark features of the disease with a murine model of LPS and OVA driven asthma exacerbation. Airway hyperresponsiveness (AHR), mucus hypersecretion, collagen deposition, and expression of inflammatory cytokines were dramatically inhibited by GGsTop treatment. Additionally, GGsTop restored the level of glutathione. Using RNA-sequencing and pathway analysis, we demonstrated that the activation of LPS/NFκB signaling pathway in airway was downregulated by GGsTop. Interestingly, further analysis revealed that GGsTop significantly inhibited not only IFNγ responses but also the expression of glucocorticoid-associated molecules, implicating that GGsTop profoundly attenuates inflammatory pathways. Conclusions: Our study suggests that GGsTop is a viable treatment for asthma exacerbation by broadly inhibiting the activation of multiple inflammatory pathways.

Synthesis and in-vitro antibacterial activity of 7-(3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives.

A series of novel 7-(3-aminopyrrolo[3,4-c]pyrazol-5(2H,4H,6H)-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid derivatives was designed, synthesized and characterized by (1)H-NMR, MS and HRMS. These fluoroquinolones were evaluated for their in-vitro antibacterial activity against representative Gram-positive and Gram-negative strains. Generally, all of the target compounds display rather weak potency against the tested Gram-negative strains, but most of them exhibit good potency in inhibiting the growth of S. aureus including methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis including methicillin-resistant S. epidermidis (MRSE) (MIC: 0.125-8 µg/mL). In particular, the compound 9g is 2 to 32 fold more potent than gemifloxacin (GM), moxifloxacin (MX), gatifloxacin (GT), and levofloxacin (LV) against S. pneumoniae 08-3, K. pneumoniae 09-23, and P. aeruginosa ATCC27853, 4 to 32 fold more potent than MX, GM, and LV against K. pneumoniae 09-21, and more active than or comparable to the four reference drugs against P. aeruginosa 09-32.

Synthesis and in vitro antibacterial activity of fluoroquinolone derivatives containing 3-(N'-alkoxycarbamimidoyl)-4-(alkoxyimino) pyrrolidines.

A series of novel 7-[3-(N'-alkoxycarbamimidoyl)-4-(alkoxyimino)pyrrolidin-1-yl] fluoroquinolone derivatives were designed, synthesized and characterized by (1)H NMR, MS and HRMS. These fluoroquinolones were screened for their in vitro antibacterial activity. Most of them exhibit good potency in inhibiting the growth of Staphylococcus aureus and Staphylococcus epidermidis (MIC: 0.06-4.00 µg/mL). The activity of compounds 33 and 43 against S. aureus including MRSA and S. epidermidis including MRSE (MIC: 0.06-0.125 µg/mL) is more than or comparable to the reference drugs levofloxacin and gemifloxacin. In addition, compound 33 is 32 and 16-32 fold more potent than both the reference drugs against Enterococcus faecium 08-7 and Klebsiella pneumoniae 09-22, respectively.

Design, synthesis, and evaluation of gamma-phosphono diester analogues of glutamate as highly potent inhibitors and active site probes of gamma-glutamyl transpeptidase.

Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the transfer of the gamma-glutamyl group of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and plays a central role in glutathione metabolism. GGT is involved in a number of biological events, such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics and reactive oxygen species through glutathione metabolism, and is also implicated in physiological disorders, such as Parkinson's disease, neurodegerative disease, diabetes, and cardiovascular diseases. In this study, we designed, synthesized, and evaluated a series of gamma-phosphono diester analogues of glutamate as transition-state mimic inhibitors of GGT. The electrophilic phosphonate diesters served as highly potent mechanism-based inhibitors that caused the time-dependent and irreversible inhibition of both the E. coli and human enzymes, probably by phosphonylating the catalytic Thr residue of the enzyme. In particular, one of the inhibitors exhibited more than 6000 times higher activity toward human GGT than acivicin, a classical but nonselective inhibitor of GGT. The dependence of the inactivation rate on the leaving group ability of the phosphonates (Brønsted plot) revealed that the phosphonylation of the catalytic Thr residue proceeded via a dissociative transition-state with substantial bond cleavage between the phosphorus and the leaving group for both E. coli and human GGTs. The binding site of GGT for the Cys-Gly moiety of glutathione or for the acceptor molecules was probed by the phosphonate diesters to reveal a significant difference in the mechanism of substrate recognition between E. coli and human GGT. Thus, in the human enzyme, a specific residue in the Cys-Gly binding site played a critical role in recognizing the Cys-Gly moiety or the acceptor molecules by interacting with the C-terminal carboxy group, whereas the Cys side chain and the Cys-Gly amide bond were not recognized significantly. In contrast, the E. coli enzyme was a nonselective enzyme that accommodated substrates without specifically recognizing the C-terminal carboxy group of the Cys-Gly moiety of gamma-glutamyl compounds or the acceptor molecules. The phosphonate diester-based GGT inhibitors shown here should serve as a blue print for the future design of highly selective GGT inhibitors for use as drug leads and biological probes that gain insight into the hitherto undefined physiological roles of GGT and the relationships between GGT and a variety of diseases.

Gamma-(monophenyl)phosphono glutamate analogues as mechanism-based inhibitors of gamma-glutamyl transpeptidase.

Gamma-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 gamma-(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 betalg = -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.