Kenneth T. Douglas

Inhibition of glyoxalase I: a possible transition-state analogue inhibitor approach to potential antineoplastic agents?

The glyoxalase system contains two enzymes. Glyoxalase I requires glutathione (GSH) as coenzyme and catalyses the disproportionation of methylglyoxal into the thiol ester of GSH and lactic acid. Glyoxalase II then hydrolyses this ester to free GSH and lactate PI. There have been suggestions that the ubiquitous [2] glyoxalase enzyme system may aid in cell growth regulation by controlling the level of methylglyoxal [2-41. Indeed, the proposal [3,5 ] that selective inhibition of glyoxalase I may provide carcinostatic activity by preventing metabolism of cw-ketoaldehydes in tumour cells has considerable experimental support. The carcinostatic activity of a-ketoaldehydes [6] is well-known, but their chemotherapeutic use is obviated by rapid metabolism to cw-hydroxyacids (by the glyoxalase system). Based on the above, approaches to antineoplastic agents have included preparation and screening of substituted-glutathione analogues [5,7,8]. For example, & GSR (R = -(CH& CO.Ar, -CHsAr, -(CHs), H) are powerful competitive inhibitors of glyoxalase I, [3,9,10] and are cytotoxic to L1210 leukaemia and KB cells in tissue culture [5]. In addition, several Sand Nsubstituted cysteinylglycines have been evaluated as glyoxalase inhibitors [8]. However, rapid metabolism rendered such compounds inactive in vivo [7,8,11] and attempts have been made to find degradation-resistant versions [7,8]. o-Hydroxythiolesters (product-like inhibitors) have no significant antileukaemic activity [ 121.

Partial transition-state inhibitors of glyoxalase I from human erythrocytes, yeast and rat liver.

Glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) converts the hemithiolacetal of glutathione and an alpha-ketoaldehyde to S-D-lactoylglutathione which is hydrolysed under the catalytic influence of glyoxalase II to produce D-lactate and regenerate glutathione. There is much evidence that glyoxalase I operates via an enediol intermediate, and in this study a number of inhibitors are described which were designed based on the enediol moiety of this reactive intermediate. These enediol and paene-enediol moieties were combined with groups designed to make use of an adjacent hydrophobic site and can be described as partial transition-state analogues. Derivatives of lapachol and kojic acid were good competitive inhibitors of glyoxalase I from various sources unless the free hydroxy group was blocked or replaced. Flavones with strong inhibitors of glyoxalase I and gallocyanine (a dye) showed spectral changes on binding to glyoxalase I indicative of binding to a metal-ion site (probably Zn2+ or Mg2+). The use of the enediol-binding determinant to produce glyoxalase I inhibitors is discussed as a route to potential antitumour derivatives.

Glutathione derivatives as inhibitors of glutaredoxin and ribonucleotides reductase from Escherichia coli

Ribonucleotide reductase requires a dithiol as hydrogen donor for reduction of ribonucleotides to the corresponding deoxyribonucleotides [ 1,2]. Originally, thioredoxin, a small dithiol protein, was found to be the hydrogen donor in the reaction [I]. The oxidized thioredoxin (thioredoxin&) is reduced by the specific enzyme NADPH-thioredoxin reductase. In [3], another system was found in an Escherichia coli mutant lacking detectable thioredoxin. The monothiol, glutathione, is hydrogen donor in the pres ence of glutaredoxin, a novel small protein that thus couples the oxidation of glutathione to the reduction of a ribonucleotide [3]. To study the mechanism of action of glutaredoxin we have used the glutathione analogues l-5 shown below in scheme 1. Our results provide evidence for a glutathione-binding site on ribonucleotide reductase.

Inhibition of mammalian glyoxalase I (lactoylglutathione lyase) by N-acylated S-blocked glutathione derivatives as a probe for the role of the N-site of glutathione in glyoxalase I mechanism.

A series of twelve S-blocked and N,S-blocked glutathione derivatives has been studied as inhibitors of glyoxalase I [R)-S-lactoylglutathione methylglyoxal-lyase (isomerising), EC 4.4.1.5) from human erythrocytes. A number of new N,S-blocked glutathiones have been synthesised. Inhibition at pH 7.0, 25 degrees C was linear-competitive in all cases and the Ki values were interpreted in terms of the absence of a specific binding interaction for the N-site of the inhibitor and the absence of coupling between binding processes at N- and S-sites (the regions around the NH2 and HS groups, respectively, of GSH analogues bound to enzyme). These observations are in strong contrast to previous results with the yeast enzyme. Some Ki values were measured for yeast glyoxalase I. A special binding interaction of the phenyl groups with enzyme from both species was found for glutathione derivatives with N-acyl groups of structure -NH X CO X X X Y X Ph but not for -NH X COPh, where X and Y were variously -CH2-, -NH- and -O-. Studies were made of the range of stability of human erythrocyte glyoxalase I to pH. The pH profiles for the Ki values of S-p-bromobenzyl)glutathione and N-acetyl-S-(p-bromobenzyl)glutathione indicated no pH dependence for the latter and little, if any, for the former inhibitor. The mean Ki over the pH range 5-8.5 for S-(p-bromobenzyl)glutathione was 1.21 +/- 0.37 microM and for N-acetyl-S-(p-bromobenzyl)glutathione in the same pH range, Ki decreased from 1.45 +/- 0.26 microM to 0.88 +/- 0.11 M.

Inhibition by glutathione derivatives of bovine liver glyoxalase II (hydroxyacylglutathione hydrolase) as a probe of the N- and S-sites for substrate binding

The nature of the binding determinants used in the interaction of glutathione-based derivatives and bovine liver glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) has been investigated. Linear competitive inhibition was observed for S-blocked and S,N-blocked glutathiones with bovine liver glyoxalase II (molecular weight 22 500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis; pI = 7.48 by analytical isoelectric focussing). There is a significant hydrophobic region on the enzyme to bind substituents around the sulphydryl-derived moiety of the substrate--a hydrophobic S-site. However, there is no evidence for binding of the N-site of the substrate (or inhibitor) to glyoxalase II. In contrast to glyoxalase I, there is no linkage between binding forces used at the S- and N-sites. Binding of S,N-dicarbobenzoxyglutathione is pH-dependent, showing dependence on an ionisation with pKapp approximately equal to 7.2 (binding more tightly at higher pH), as is the kcat value (pKapp approximately equal to 7.8) for S-D-lactoylglutathione.

Photolability of bleomycin and its complexes

Bleomycin has been found to be very sensitive to photolysis and on irradiation with the full spectral range of a medium-pressure mercury lamp undergoes a number of photo-induced reactions; there is a process in which the absorbance at 310 nm decreases, and a slower process in which it increases with photolysis time. The faster process can be studied by irradiation at approximately 300-350 nm, is complete in approximately 8 min, obeys first order kinetics, but is itself biphasic. The Fe(II) and Cu(II) complexes of bleomycin are also photolabile, as is the bleomycin-DNA complex.

Photo-induced covalent labelling of malate dehydrogenase by quercetin.

Abstract Quercetin powerfully inhibits malate dehydrogenase reversibly and cooperatively with 50% inhibition at 2.5μM at pH 7.50. Irradiation with light of wavelengths ≳350nm, of a mixture of malate dehydrogenase and quercetin leads to covalent inhibition whose extent is directly related to quercetin concentration, inversely related to enzyme concentration, and partially protected against by NADH. Prephotolysis of quercetin followed by incubation (in the dark) with malate dehydrogenase led to a time-dependent covalent inhibition.

Nucleophilic versus general base catalysis in phosphyl (PV) group transfer: application to α-chymotrypsin action

The hydrolysis of aryl dimethylphosphinates is shown to be catalysed via a nucleophilic pathway by a series of bases including imidazole which is less efficient than phosphate dianion. The more reactive nucleophilic pathway is allowed because dimethylphosphinate is less sterically hindered than diphenyl phosphinate where general base-catalysis predominates. Acylation of α-chymotrypsin by 4-nitrophenyl diphenylphosphinate, a bona fide general base mechanism, has a low solvent deuterium oxide isotope effect not characteristic of such a mechanism.

A photoaffinity label derivative of glutathione and its inhibition of glyoxalase I

Glutathione (GSH) plays many roles in biochemistry. In addition to its coenzyme functions (e.g., with glyoxalase) it is involved in the detoxification of xenobiotics, maintenance of -SH levels of proteins, disulphide exchange processes, removal of hydrogen peroxide, organic peroxides and free radicals and possibly in amino acid transport across membranes, as well as other phenomena [ 1,2]. With this involvement of GSH in such a myriad of biological activities in mind, we have synthesised a photoaffinity analogue of glutathione (1) based on the azidophenacyl group, introduced in [3].

Yeast glyoxalase I. Circular dichroic spectra and pH effects.

Large scale isolation and physicochemical characterisation of yeast glyoxalase I showed that this enzyme contained small amounts of carbohydrates. Circular dichroic spectra of the enzyme measured in the presence and absence of S-(p-bromobenzyl)glutathione indicated perturbation of a tyrosine on binding of this competitive inhibitor. Values of Ki for competitive inhibitors were pH invariant over the accessible pH range.