A reinvestigation of inhibitors of glyoxalase I

It has been reported earlier that nucleotides, nucleosides and a series of structurally related compounds as well as compounds based on transition state analogy inhibit yeast glyoxalase I. In our study on the metabolic regulation of glyoxalase I, we have found that nucleotides such as ATP, GTP and different classes of other reagents based on transition state analogy (D-isoascorbate, dihydroxyfumaric acid, rhodizonic acid) do not inhibit yeast or goat liver glyoxalase I. The reported inhibition of glyoxalase I by these compounds has been found to be due to the interference of these compounds with the absorbancy at 240 nm of S-D-lactoylglutathione formed by the glyoxalase I reaction. Glyoxalase I from goat liver has been found to be strongly and competitively inhibited by lactaldehyde. But, lactaldehyde has very little inhibitory effect on yeast glyoxalase I. Lactaldehyde is formed from methylglyoxal, the substrate for glyoxalase I by the enzyme methylglyoxal reductase. D-Lactaldehyde inhibits the liver enzyme more strongly than L-lactaldehyde.     

Recent advances in the discovery and development of glyoxalase I inhibitors

Glyoxalase I (GLO1) is a homodimeric Zn²⁺-metalloenzyme that catalyses the transformation of methylglyoxal (MG) to d-lacate through the intermediate S-d-lactoylglutathione. Growing evidence indicates that GLO1 has been identified as a potential target for the treatment cancer and other diseases. Various inhibitors of GLO1 have been discovered or developed over the past several decades including natural or natural product-based inhibitors, GSH-based inhibitors, non-GSH-based inhibitors, etc. The aim of this review is to summarize recent achievements of concerning discovery, design strategies, as well as pharmacological aspects of GLO1 inhibitors with the target of promoting their development toward clinical application.     

Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) of 5.4+/-0.6 microM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.     

Inhibitors of glyoxalase I in vitro

Inhibition in vitro of human red blood cell glyoxalase I activity was measured by the decrease in the rate of formation of S-D-lactoyl-glutathione as determined by the change in absorbance at 240 nm. The percentage activity remaining was determined after addition of various potential inhibitor compounds and the concentration for 50% activity was obtained by graphical interpolation. The inhibitors were selected on the basis of their similarity to a possible transition-state enediol intermediate of methylglyoxal. The most effective inhibitors were dihydroxycoumarins with a 50% inhibition of 0.03 mM. Inhibition of methylglyoxal catabolism suggests possible application as chemotherapeutic agents based on the inhibitor characteristics of methylglyoxal.     

Assay of glyoxalase I in blood

Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) was assayed using alcoholic, acidic 2,4-dinitrophenylhydrazine to follow the disappearance of methylglyoxal over time, with the absorbance of formed methylglyoxal bis-hydrazone measured at 432 nm. Erythrocyte glyoxalase I activities were found to be 64, 41, and 18 mumole of S-lactoyl glutathione formed min-1 X ml-1 of red blood cells in rat, human, and rabbit blood and 174 mumole X min-1 X mg-1 of protein for yeast. The Km values found in millimolar hemimercaptal were about 0.5. Glyoxalase I activity can be determined in crude tissue preparations without interference from biological materials.     

Nonstereospecific substrate usage by glyoxalase I

Glyoxalase I operates on a mixture of rapidly interconverting diasteriomeric thiohemiacetals, formed in a preequilibrium step between glutathione and alpha-ketoaldehyde. That both diasteriomers are directly used as substrates by the enzyme from yeast and from porcine erythrocytes is an outcome of a series of isotope-trapping experiments in which pulse solutions composed of the two diasteriomeric thiohemiacetals, due to [3H]glutathione and phenylglyoxal, are rapidly mixed with chase solutions containing excess unlabeled glutathione and successively increasing concentrations of glyoxalase I. As the enzyme approaches infinite concentration in the chase solution, the radioactivity incorporated into the S-mandeloylglutathione product approaches 100% of the total radioactivity due to both diasteriomers from the pulse solution. The special properties of the active site that allow the enzyme to accommodate both diasteriomeric substrate forms may also account for the fact that the cis and the trans isomers of various para-substituted S-(phenylethenyl)glutathione derivatives are both strong competitive inhibitors of the enzyme. A catalytic mechanism is proposed for glyoxalase I involving catalyzed interconversion of the bound diasteriomeric thiohemiacetals before transformation to final product.     

Human red cell glyoxalase I polymorphism

Human erythrocyte glyoxalase I has been subjected to starch gel electrophoresis, and its isoenzymatic forms have been visualized by a new positive staining procedure. The enzyme exhibits polymorphism and holds promise as a useful new genetic marker.     

Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.     

Polymorphism of glyoxalase I in Vienna.

Phenotype and gene frequencies of the GLO I polymorphism in Vienna are given. No exception to the postulated rule of inheritance could be found in 23 families with 51 children and 132 mother-child pairs. Linkage with the HLA system is confirmed, but no linkage disequilibrium between GLO alleles and HLA-A, B, C genes was detected. The use of the GLO I polymorphism in paternity cases is discussed.     

Relative distribution of glutathione transferase, glyoxalase I and glyoxalase II in helminths

Glutathione transferase, glyoxalase I and glyoxalase II activities were not evenly distributed among the major helminth groups. Intestinal cestodes and digeneans had higher glutathione transferase activity than parasitic nematodes. High glyoxalase II activity was found in cestodes and digeneans but no glyoxalase I was detectable. Glyoxalase I and II were both detected in nematodes. These results are discussed in relation to the enzymes' suggested role in protection against secondary lipid peroxidation products.     

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae.

The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program HYPERFIT. Glyoxalase I exhibited saturation kinetics on 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters Km 0.53 +/- 0.07 mM and V (3.18 +/- 0.16) x 10(-2) mM.min(-1). Glyoxalase II also showed saturation kinetics in the SD-lactoylglutathione concentration range of 0.15-3 mM and Km 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10(-3) mM.min(-1) were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program GEPASI. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis-Menten model. The kinetic parameters obtained, Km 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis-Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.     

Design,synthesis and biological evaluation of novel glyoxalase I inhibitors possessing diazenylbenzenesulfonamide moiety as potential anticancer agents

The enzyme glyoxalase-I (Glo-I) is an essential therapeutic target in cancer treatment. Significant efforts have been made to discover competitive inhibitors of Glo-I as potential anticancer agents. Herein, we report the synthesis of a series of diazenylbenzenesulfonamide derivatives, their in vitro evaluation against Glo-I and the resulting structure–activity relationships. Among the compounds tested, compounds 9h and 9j exhibited the highest activity with IC₅₀ 1.28 µM and 1.13 µM, respectively. Docking studies to explore the binding mode of the compounds identified key moieties that may contribute to the observed activities. The active compounds will serve as suitable leads for further chemical optimization.     

Cloning and characterization of glyoxalase I from soybean

Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No. X68819) resembling a GST being described as a glyoxalase I. To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures. When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or gamma-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean. When the clone X68819 was expressed in E. coli, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3. GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzymes are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%).     

Isolation and sequencing of a gene coding for glyoxalase I activity from Salmonella typhimurium and comparison with other glyoxalase I sequences

The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn²⁺ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.     

Induction of mouse liver glyoxalase I by hypobaric hypoxia

Hypobaric hypoxia at 0.45 atm induced a reversible increase of mouse liver glyoxalase I. The levels of this enzyme increased after an exposure of 20 h and 20 + 20 h, whereas the activity decreased to the control values after 20 h at room pressure. Before the treatment, some animals received tritiated leucine (i.p.). Glyoxalase I was purified to homogeneity. The pure enzyme from the treated animals showed 20-times more radioactivity than the controls. Thus, the increase in specific activity is due to new protein synthesized in response to the treatment at 0.45 atm. The activities of glyoxalase II and glutathione S-transferase were not affected by the treatment.     

Purification and characterization of glyoxalase I from Hansenula mrakii

Glyoxalase I was purified from Hansenula mrakii IFO 0895 which was resistant to 25 mM methylglyoxal. The molecular weight of the purified enzyme was calculated to be 38,000 by both gel-filtration of Sephadex G-150 and SDS-PAGE. The enzyme was almost specific to methylglyoxal (K_m = 0.91 mM). The activity of the enzyme was not inhibited by metal ion chelators such as EDTA, which is a potent inhibitor for glyoxalase Is from other sources.     

Nitric oxide inactivates glyoxalase I in cooperation with glutathione

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.     

Purification and characterization of glyoxalase I from Pseudomonas putida

Glyoxalase I was purified to apparent homogeneity from Pseudomonas putida. The enzyme was a monomer with a molecular weight of 20,000. The enzyme was most active at pH 8.0. The Km values for methylglyoxal and 4,5-dioxovale-rate are 3.5 mM and 1.2 mM, respectively. Contrary to the case of eukaryotic enzymes, chelating agents showed little inhibitory effects on the enzyme activity. Among the metal ions tested, Zn++ specifically and completely inhibited the activity of the enzyme at a millimolar level. The properties of bacterial glyoxalase I were quite different from mammalian and yeast enzymes.     

The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid

Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl X coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in which L-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid, L-alanine, glutathione, and purified L-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These 'metal-free' homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO4 greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.