Human Glyoxalase Research Articles

Glyoxalase-based toolbox for the enantioselective synthesis of α-hydroxy carboxylic acids.

We report highly enantioselective synthesis of L-α-hydroxy carboxylic acids (L-αHCAs) via enzymatic intramolecular Cannizzaro reaction of (hetero)aryl glyoxals in the presence of glutathione-independent human glyoxalase DJ-1. Combined with the optimized synthesis of D-αHCAs using glyoxalases I and II, this approach offers a general, scalable and operationally simple access to both enantiomers of α-hydroxy acids in moderate to excellent yields with uniformly high enantioselectivity.

The Human Glyoxalase Gene Family in Health and Disease.

The glyoxalase gene family consists of six structurally and functionally diverse enzymes with broad roles in metabolism. The common feature that defines this family is based on structural motifs that coordinate divalent cations which are required for activity. These family members have been implicated in a variety of physiological processes, including amino-acid metabolism (4-hydroxyphenylpyruvate dioxygenase; HPD), primary metabolism (methylmalonyl-CoA epimerase; MCEE), and aldehyde detoxication (glyoxalase 1; GLO1) and therefore have significant associations with disease. A central function of this family is the detoxification of reactive dicarbonyls (e.g., methylglyoxal), which react with cellular nucleophiles, resulting in the modification of lipids, proteins, and DNA. These damaging modifications activate canonical stress responses such as heat shock, unfolded protein, antioxidant, and DNA damage responses. Thus, glyoxalases serve an important role in homeostasis, preventing the pathogenesis of metabolic disease states, including obesity, diabetes, cardiovascular disease, renal failure, and aging. This review presents a thorough overview of the literature surrounding this diverse enzyme class. Although extensive literature exists for some members of this family (e.g., GLO1), little is known about the physiological role of glyoxalase domain-containing protein 4 (GLOD4) and 5 (GLOD5), paving the way for exciting avenues for future research.

Crystal structures of human glyoxalase I and its complex with TLSC702 reveal inhibitor binding mode and substrate preference.

Human glyoxalase I (hGLO I) is an enzyme for detoxification of methylglyoxal (MG) and has been considered an attractive target for the development of new anticancer drugs. In our previous report, the GLO I inhibitor TLSC702 induced apoptosis in tumor cells. Here, we determined the crystal structures of hGLO I and its complex with TLSC702. In the complex, the carboxyl O atom of TLSC702 is coordinated to Zn2+ , and TLSC702 mainly shows van der Waals interaction with hydrophobic residues. In the inhibitor-unbound structure, glycerol, which has similar functional groups to MG, was bound to Zn2+ , indicating that GLO I can easily bind to MG. This study provides a structural basis to develop better anticancer drugs.

Salicylate metal-binding isosteres as fragments for metalloenzyme inhibition.

Metalloenzyme inhibitors typically share a common need to possess a metal-binding pharmacophore (MBP) for binding the active site metal ions. However, MBPs can suffer from physicochemical liabilities, impeding the pharmacological properties and drug-likeliness of inhibitors. To circumvent this, problematic features of the MBP can be identified and exchanged with isosteric replacements. Herein, the carboxylic and hydroxyl group of the salicylic acid MBP were replaced and a total of 27 salicylate metal-binding isosteres (MBIs) synthesized. Of these 27 MBIs, at least 12 represent previously unreported compounds, and the metal-binding abilities of >20 of the MBIs have not been previously reported. These salicylate MBIs were examined for their metal-binding features in model complexes, physicochemical properties, and biological activity. It was observed that salicylate MBIs can demonstrate a range of attractive physicochemical properties and bind to the metal in a variety of expected and unexpected binding modes. The biological activity of these novel MBIs was evaluated by measuring inhibition against two Zn2+-dependent metalloenzymes, human glyoxalase 1 (GLO1) and matrix metalloproteinase 3 (MMP-3), as well as a dinuclear Mn2+-dependent metalloenzyme, influenza H1N1 N-terminal endonuclease (PAN). It was observed that salicylate MBIs could maintain or improve enzyme inhibition and selectivity. To probe salicylate MBIs as fragments for fragment-based drug discovery (FBDD), an MBI that showed good inhibitory activity against GLO1 was derivatized and a rudimentary structure–activity relationship was developed. The resulting elaborated fragments showed GLO1 inhibition with low micromolar activity.

Triphenylbismuth dichloride inhibits human glyoxalase I and induces cytotoxicity in cultured cancer cell lines.

Organobismuth compounds, i.e., organic-inorganic hybrid molecules composed of an organic structure and bismuth metal, have been reported to induce cytotoxicity in cancer cells; however, the target proteins associated with this cytotoxicity have not been elucidated. Herein, we investigated the inhibitory effect of five organobismuth compounds on human glyoxalase I (hGLO I), a promising target candidate for cancer therapy. Among these compounds, triphenylbismuth dichloride (Bi-05) exerted a strong inhibitory effect on hGLO I. Indeed, Bi-05 inhibited hGLO I in a dose-dependent manner with an IC50 value of 0.18 µM. Bi-05 also induced cytotoxicity in human leukemia HL-60 cells and human lung cancer NCI-H522 cells, both of which exhibit high expression levels of GLO I. However, the hGLO I-inhibiting and cytotoxic effects of Bi-05 disappeared when the bismuth atom was replaced with an antimony or phosphorus atom. Bismuth(III) nitrate had little inhibitory effect on hGLO I activity and only slightly reduced the viability of cancer cells. In the culture medium of Bi-05-treated HL-60 cells, the concentration of the GLO I substrate methylglyoxal was markedly elevated. In addition, Bi-05 treatment more strongly inhibited human lung cancer NCI-H522 cell (exhibiting high GLO I expression) proliferation than human lung cancer NCI-H460 cell (exhibiting low GLO I expression) proliferation. Furthermore, the cytotoxicity of Bi-05 was significantly decreased by pre- and co-treatment with the methylglyoxal scavengers N-acetyl-L-cysteine and aminoguanidine. Overall, these results suggest that Bi-05 treatment leads to the accumulation of methylglyoxal via GLO I inhibition, resulting in cytotoxic effects in cancer cells.

1,2,4-Triazole-3-thione compounds with a 4-ethyl alkyl/aryl sulfide substituent are broad-spectrum metallo-β-lactamase inhibitors with re-sensitization activity

Metallo-β-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to β-lactam antibiotics. MBLs are highly worrying because of their carbapenemase activity, their rapid spread in major human opportunistic pathogens while no clinically useful inhibitor is available yet. In this context, we are exploring the potential of compounds based on the 1,2,4-triazole-3-thione scaffold as an original ligand of the di-zinc active sites of MBLs, and diversely substituted at its positions 4 and 5. Here, we present a new series of compounds substituted at the 4-position by a thioether-containing alkyl chain with a carboxylic and/or an aryl group at its extremity. Several compounds showed broad-spectrum inhibition with Ki values in the μM to sub-μM range against VIM-type enzymes, NDM-1 and IMP-1. The presence of the sulfur and of the aryl group was important for the inhibitory activity and the binding mode of a few compounds in VIM-2 was revealed by X-ray crystallography. Importantly, in vitro antibacterial susceptibility assays showed that several inhibitors were able to potentiate the activity of meropenem on Klebsiella pneumoniae clinical isolates producing VIM-1 or VIM-4, with a potentiation effect of up to 16-fold. Finally, a selected compound was found to only moderately inhibit the di-zinc human glyoxalase II, and several showed no or only moderate toxicity toward several human cells, thus favourably completing a promising behaviour.

Addition of hydrophobic side chains improve the apoptosis inducibility of the human glyoxalase I inhibitor, TLSC702

Glyoxalase I (GLO I) is a known therapeutic target in cancer. Even though TLSC702, a GLO I inhibitor that we discovered, induces apoptosis in tumor cells, exceptionally higher doses are required compared with those needed to inhibit GLO I activity in vitro. In this work, structure–activity optimization studies were conducted on four sections of the TLSC702 molecule to determine the partial structural features necessary for the inhibition of GLO I. Herein, we found that the carboxy group in TLSC702 was critical for binding with the divalent zinc at the active site of GLO I. In contrast, the side chain substituents in the meta- and para- positions of the benzene ring had little influence on the in vitro inhibition of GLO I. The CLogP values of the TLSC702 derivatives showed a positive correlation with the antiproliferative effects on NCI-H522 cells. Thus, two derivatives of TLSC702, which displayed either high or low lipophilicity due to the types of substituents at the phenyl position, were selected. Even though both derivatives showed comparable inhibitory effects as that of their parent compound, the derivative with the high CLogP value was distinctly more antiproliferative than TLSC702. In contrast, the derivative with the low CLogP value did not decrease cell viability in NCI-H522 and HL-60 cells. These findings suggested that structural improvements, such as the addition of hydrophobic moieties to the phenyl group, enhanced the ability of TLSC702 to induce apoptosis by increasing cell membrane permeability.

4-Amino-1,2,4-triazole-3-thione-derived Schiff bases as metallo-β-lactamase inhibitors.

Resistance to β-lactam antibiotics in Gram-negatives producing metallo-β-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with Ki values in the μM to sub-μM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E.coli clinical strain toward several β-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs.

Effect of piceatannol-rich passion fruit seed extract on human glyoxalase I–mediated cancer cell growth

Passion fruit seed extract (PFSE), a product rich in stilbenes such as piceatannol and scirpusin B, has various physiological effects. It is unclear whether PFSE and its stilbene derivatives inhibit cancer cell proliferation via human glyoxalase I (GLO I), the rate-limiting enzyme for detoxification of methylglyoxal. We examined the anticancer effects of PFSE in two types of human cancer cell lines with different GLO I expression levels, NCI–H522 cells (highly-expressed GLO I) and HCT116 cells (lowly-expressed GLO I). PFSE and its stilbenes inhibited GLO I activity. In addition, PFSE and its stilbenes supressed the cancer cell proliferation of NCI–H522 cells more than HCT116 cells. These observations suggest that PFSE can provide a novel anticancer strategy for prevention and treatment.

Protein-protein interactions of human glyoxalase II: findings of a reliable docking protocol.

Glyoxalase II (GlxII) is an antioxidant glutathione-dependent enzyme, which catalyzes the hydrolysis of S-d-lactoylglutathione to form d-lactic acid and glutathione (GSH). The last product is the most important thiol reducing agent present in all eukaryotic cells that have mitochondria and chloroplasts. It is generally known that GSH plays a crucial role not only in the cellular redox state but also in various cellular processes. One of them is protein S-glutathionylation, a process that can occur through an oxidation reaction of proteins' thiol groups by GSH. Changes in protein S-glutathionylation have been associated with a range of human diseases such as diabetes, cardiovascular and pulmonary diseases, neurodegenerative diseases and cancer. Within a major project aimed at elucidating the role of GlxII in the mechanism of S-glutathionylation, a reliable computational protocol consisting of a protein-protein docking approach followed by atomistic Molecular Dynamics (MD) simulations was developed and it was applied to the prediction of molecular associations between human GlxII (in the presence and absence of GSH) and some proteins that are known to be S-glutathionylated in vitro, such as actin, malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The computational results show a high propensity of GlxII to interact with actin and MDH through its active site and a high stability of the GlxII-protein systems when GSH is present. Moreover, close proximities of GSH with actin and MDH cysteine residues have been found, suggesting that GlxII could be able to perform protein S-glutathionylation by using the GSH molecule present in its catalytic site.

Piceatannol, a natural trans-stilbene compound, inhibits human glyoxalase I

Human glyoxalase I (GLO I), a rate-limiting enzyme for detoxification of methylglyoxal (MG), a by-product of glycolysis, is known to be a potential therapeutic target for cancer. Here, we searched new scaffolds from natural compounds for designing novel GLO I inhibitors and found trans-stilbene scaffold. We examined the inhibitory abilities to human GLO I of commercially available trans-stilbene compounds. Among them, piceatannol was found to have the most potent inhibitory activity against human GLO I. Piceatannol could inhibit the proliferation of human lung cancer NCI-H522 cells, which are dependent on GLO I for survival, in a dose- and time-dependent manner. In addition, piceatannol more significantly inhibited the proliferation of NCI-H522 cells than that of NCI-H460 cells, which are less dependent on GLO I. Importantly, overexpression of GLO I in NCI-H522 cells resulted in less sensitive to the antiproliferative activity of piceatannol. Taken together, this is the first report demonstrating that piceatannol inhibits GLO I activity and the GLO I-dependent proliferation of cancer cells. Furthermore, we determined a pharmacophore for novel inhibitors of human GLO I by computational simulation analyses of the binding mode of piceatannol to the enzyme hot spot in the active site. We suggest that piceatannol is a possible lead compound for the development of novel GLO I inhibitory anticancer drugs.

Crystal structure of Staphylococcus aureus Zn-glyoxalase I: new subfamily of glyoxalase I family

The crystal structures of protein SA0856 from Staphylococcus aureus in its apo-form and in complex with a Zn2+-ion have been presented. The 152 amino acid protein consists of two similar domains with α + β topology. In both crystalline state and in solution, the protein forms a dimer with monomers related by a twofold pseudo-symmetry rotation axis. A sequence homology search identified the protein as a member of the structural family Glyoxalase I. We have shown that the enzyme possesses glyoxalase I activity in the presence of Zn2+, Mg2+, Ni2+, and Co2+, in this order of preference. Sequence and structure comparisons revealed that human glyoxalase I should be assigned to a subfamily A, while S. aureus glyoxalase I represents a new subfamily B, which includes also proteins from other bacteria. Both subfamilies have a similar protein chain fold but rather diverse sequences. The active sites of human and staphylococcus glyoxalases I are also different: the former contains one Zn-ion per chain; the latter incorporates two of these ions. In the active site of SA0856, the first Zn-ion is well coordinated by His58, Glu60 from basic molecule and Glu40*, His44* from adjacent symmetry-related molecule. The second Zn3-ion is coordinated only by residue His143 from protein molecule and one acetate ion. We suggest that only single Zn1-ion plays the role of catalytic center. The newly found differences between the two subfamilies could guide the design of new drugs against S. aureus, an important pathogenic micro-organism.

Design, Synthesis and Biological Evaluation of Potent Human Glyoxalase I Inhibitors.

Several glutathione derivatives bearing the S-(N-aryl-N-hydroxycarbamoyl) or S-(C-aryl-N-hydroxycarbamoyl) moieties (10, 10', 13-15) were synthesized, characterized, and their human glyoxalase I (hGLO1) inhibitory activity was evaluated. Compound 10 was proved to be the effective hGLO1 inhibitor with a Ki value of 1.0 nM and the inhibition effect of compound 10 on hGLO1 was nearly ten-fold higher than that of the strongest inhibitor 2 (Ki=10.0 nM) which has been reported in the field of glutathione-type hGLO1 inhibitors. Its diethyl ester prodrug 10' was able to penetrate cell membrane and had good inhibitory effect on the growth of NCI-H522 cell xenograft tumor model.

Catalytic mechanism of human glyoxalase I studied by quantum-mechanical cluster calculations

Density functional theory has been used to study the mechanism and stereospecificity of the catalytic reaction of human glyoxalase I. We used the quantum mechanical cluster method to model the enzyme active site. Glyoxalase I accepts both enantiomers of the hemithioacetal between methylglyoxal and glutathione and converts them to the S-D enantiomer of lactoylglutathione. We have compared several previously suggested or alternative reaction mechanisms for both substrates on an equal footing. The results show that the coordination shell of the Zn ion in the optimized geometries is more symmetric than in some inhibitor crystal structures, which we assign to differences in the electronic structure and the protonation states of the substrate. The symmetry of the active site model indicates that the enzyme can use the same reaction mechanism for the S and the R enantiomers of the substrate, but with exchanged roles of the two active-site glutamate residues. However, the calculations show some asymmetry (0–4kcalmol−1 differences in reaction energies and activation barriers), caused by the different coordination states of the glutamate residues in the starting crystal structure. Our results indicate that the only possibility for the stereospecificity of glyoxalase I is differences in the electrostatic surroundings and flexibility of the glutamate residues in the active site owing to their neighboring residues in the protein.

TLSC702, a Novel Inhibitor of Human Glyoxalase I, Induces Apoptosis in Tumor Cells.

Human glyoxalase I (hGLO I) is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), which is the side product of tumor-specific aerobic glycolysis. GLO I has been reported to be overexpressed in various types of cancer cells, and has been expected as an attractive target for the development of new anticancer drugs. We previously discovered a novel inhibitor of hGLO I, named TLSC702, by our in silico screening method. Here, we show that TLSC702 inhibits the proliferation of human leukemia HL-60 cells and induces apoptosis in a dose-dependent manner. In addition, TLSC702 more significantly inhibits the proliferation of human lung cancer NCI-H522 cells, which highly express GLO I, than that of GLO I lower-expressing human lung cancer NCI-H460 cells. Furthermore, this antiproliferative effect of TLSC702 on NCI-H522 cells is in a dose- and time-dependent manner. These results suggest that TLSC702 can induce apoptosis in tumor cells by GLO I inhibition, which lead to accumulation of MG. Taken together, TLSC702 could become a unique seed compound for the generation of novel chemotherapeutic drugs targeting GLO I-dependent human tumors.

Benzophenone and its analogs bind to human glyoxalase 1.

Benzophenone is a popular photophore for photoaffinity-labeling. It is also an important framework for drug development; many drugs contain benzophenone or analogous frameworks. The current work reports that benzophenone and its analogs bind to human glyoxalase 1. The binding, however, has little effect on the catalytic activity of this enzyme. The implications of the finding in terms of both drug development and photoaffinity-labeling are discussed.

Novel bivalent inhibitors with sub-nanomolar affinities towards human glyoxalase I

The zinc metalloenzyme glyoxalase I (GlxI) catalyzes the glutathione-dependent inactivation of cytotoxic methylglyoxal. Two competitive bivalent GlxI inhibitors, polyBHG2-62 (Ki=1.0 nM) and polyBHG2-54 (Ki=0.3 nM), were synthesized based on the transition-state analog S-(N-bromophenyl-N-hydroxycarbamoyl) glutathione (BHG). The most effective inhibitor, polyBHG2-54, is the first subnanomolar inhibitor of GlxI, and is over 50-fold more potent than BHG itself.

Inhibition by active site directed covalent modification of human glyoxalase I

The glyoxalase pathway is responsible for conversion of cytotoxic methylglyoxal (MG) to d-lactate. MG toxicity arises from its ability to form advanced glycation end products (AGEs) on proteins, lipids and DNA. Studies have shown that inhibitors of glyoxalase I (GLO1), the first enzyme of this pathway, have chemotherapeutic effects both in vitro and in vivo, presumably by increasing intracellular MG concentrations leading to apoptosis and cell death. Here, we present the first molecular inhibitor, 4-bromoacetoxy-1-(S-glutathionyl)-acetoxy butane (4BAB), able to covalently bind to the free sulfhydryl group of Cys60 in the hydrophobic binding pocket adjacent to the enzyme active site and partially inactivate the enzyme. Our data suggests that partial inactivation of homodimeric GLO1 is due to the modification at only one of the enzymatic active sites. Although this molecule may have limited use pharmacologically, it may serve as an important template for the development of new GLO1 inhibitors that may combine this strategy with ones already reported for high affinity GLO1 inhibitors, potentially improving potency and specificity.

Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory activity on human glyoxalase I

A novel isoflavone, erythgianin A (1), along with nine known compounds 2-10, was isolated from the stem bark of Erythrina poeppigiana (Leguminosae). The unusual isoflavone structure of 1, possessing a highly oxidized 3″,4″-dihydroxy-2″-hydroxymethyl-2″-methyl-2″,3″-dihydropyrano substituent, was determined on the basis of spectroscopic analyses. All of the isolated compounds were evaluated for their in vitro inhibitory activity toward human glyoxalase I. Among the isolates, isolupalbigenin (10) with two prenyl groups showed the highest inhibitory activity.

Zopolrestat as a Human Glyoxalase I Inhibitor and Its Structural Basis

Old drugs new tricks: Zopolrestat, an aldose reductase inhibitor developed by Pfizer for the treatment of diabetic complications, is a potent competition inhibitor of human glyoxalase I (GLOI) in vitro. Crystal structures of GLOI in complex with zopolrestat and indomethacin, a nonsteroidal anti-inflammatory drug and moderate inhibitor of GLOI, provide a structural basis for the development of novel GLOI inhibitors with excellent pharmacokinetics profiles.