Donald J. Creighton

Pharmacokinetics and antitumor properties in tumor-bearing mice of an enediol analogue inhibitor of glyoxalase I

Purpose: The enediol analogue S-(N-p-chlorophenyl-N-hydroxycarbamoyl)glutathione (CHG) is a powerful, mechanism-based, competitive inhibitor of the methylglyoxal-detoxifying enzyme glyoxalase I. The [glycyl,glutamyl]diethyl ester prodrug form of this compound (CHG(Et)2) inhibits the growth of different tumor cell lines in vitro, apparently by inducing elevated levels of intracellular methylglyoxal. The purpose of this study was to evaluate the pharmacokinetic properties of CHG(Et)2 in plasma esterase-deficient C57BL/6 (Es-1e) mice after intravenous (i.v.) or intraperitoneal (i.p.) administration of bolus doses of CHG(Et)2. In addition, the in vivo antitumor properties of CHG(Et)2 were evaluated against murine B16 melanoma in these mice, and against androgen-independent human prostate PC3 tumor and human colon HT-29 adenocarcinoma in plasma esterase-deficient nude mice. Methods: Pharmacokinetics were evaluated after either i.v. or i.p. administration of CHG(Et)2 at the maximally tolerated dose of 120u2009mg/kg to both tumor-free male and female mice and male and female mice bearing subcutaneous B16 tumors. Tissue concentrations of CHG(Et)2, CHG and the [glycyl]monoethyl ester CHG(Et) were measured as a function of time by reverse-phase C18 high-performance liquid chromatography of deproteinized tissue samples. The efficacy of CHG(Et)2 in tumor-bearing mice was evaluated after i.v. bolus administration of CHG(Et)2 at 80 or 120u2009mg/kg for 5u2009days each week for 2u2009weeks, or after 14u2009days continuous infusion of CHG(Et)2 using Alzet mini-osmotic pumps. Hydroxypropyl-β-cyclodextrin was used as a vehicle in the efficacy studies. Results: Intravenous administration of CHG(Et)2 resulted in the rapid appearance of CHG(Et)2 in the plasma of tumor-bearing mice with a peak value of 40–60u2009μM, followed by a first-order decrease with a half-life of about 10u2009min. There was a corresponding increase in the concentration of inhibitory CHG in the B16 tumors, with a maximum concentration in the range 30–60u2009μM occurring at 15u2009min, followed by a decrease to a plateau value of about 6u2009μM after 120u2009min. Neither CHG(Et)2 nor its hydrolysis products were detectable in plasma, after i.p. administration of CHG(Et)2 to tumor-free female mice. From the efficacy studies, dosing schedules were identified that resulted in antitumor effects comparable to those observed with the standard antitumor agents Adriamycin (with B16 tumors), cisplatin (with PC3 tumors), and vincristine (with HT-29 tumors). Conclusion: This is the first demonstration that a mechanism-based competitive inhibitor of glyoxalase I effectively inhibits the growth of solid tumors in mice when delivered as the diethyl ester prodrug.

The gene encoding glyoxalase I from Pseudomonas putida: cloning, overexpression, and sequence comparisons with human glyoxalase I

The gene encoding glyoxalase I (GlxI) from Pseudomonas putida has been cloned into the high-expression plasmid pBTacI. In the presence of IPTG, JM109 cells transformed with this vector give expression levels of GlxI 4000-fold higher than wild-type Escherichia coli. Contrary to a previous report, the nucleotide sequence of the gene encodes a 173-amino-acid polypeptide. Edman analysis indicates that the predicted N-terminal methionine is lost post-translationally to yield a 19407-Da protein. Mass spectrometry of the intact protein, and of the peptides generated from treatment with CNBr, does not indicate any additional post-translational modifications of the enzyme. Contrary to previous conclusions, there are no major regions of dissimilarity between the human and bacterial enzymes.

Is the thiolate--imidazolium ion pair the catalytically important form of papain?

A basic step in the catalytic mechanism of papain, as well as other sulfhydryl proteases, involves nucleophilic attack of an active site sulfhydrylgroup on polypeptide substrates resulting in the formation of a thioacyl-enzyme intermediate [ 11. The proximity of an imidazole sidechain to the active site sulfhydryl suggests that this group may play an important role in catalyzing the acylation reaction [2,3]. For the simple system of pH-dependent equilibria shown below, consistent with a bell-shaped pHacylation rate profile, catalytic assistance could occur in either one or both of two extreme tautomeric forms (11,111) envisioned to predominate at the pH-optimum of activity (pH 6.5).

Solvent isotope effects on tautomerization equilibria of papain and model thiolamines

Polgar was the first to argue for the predominance of If in the active site based on the mercaptide ionlike ultraviolet difference absorption spectrum of papain versus carboxymethyland carboxamidomethylpapain near neutral pH 131. Lewis et al. concluded that II is the predominant tautomer (* 90%) based on one interpretation of the results of their titrimetric procedure [4]. On the other hand, all of the observations reported so far regarding the state of the tautomerization equilibrium are best viewed as being in a tentative stage of interpretation (discussion in [.5]). An alternative approach to this problem is reported here which utilizes the difference between the isotopic

Solvent isotope effects on the rates of alkylation of thiolamine models of papain

Kinetic solvent deuterium isotope effects on the rates of alkylation of the active site sulfhydryl group of papain have been used as a probe of the mechanism by which this relatively simple nucleophilic process is catalyzed in the pH range where the enzyme is optimally active with substrates (pH E 5-7). As evidence against general base catalysis, unity isotope effects were reported on the rates of alkylation by methylbromoacetate and by chloroacetamide [ 1,2]. On the other hand, this interpretation is complicated by the reported inverse isotope effects on the rates of alkylation by bromoacetamide (k(H,O)/k(D,O) = 0.74)and by chloroacetate (k(H,O)/k(D,O) = 0.75) [ 1,3]. The possibility has been suggested that observed inverse kinetic isotope effects could arise from preferential reaction of these alkylating agents with the proposed active site thiolate-imidazolium ion pair, formed between the active site sulfhydryl group and a neighboring imidazole function, provided that the tautomerization equilibrium constant (K) controlling the relative concentrations of ion pair (II) and its tautomer (I) is -2 in HzO, eq. (1) [4]:

7 Stereochemistry of Enzyme -Catalyzed Reactions at Carbon

Publisher Summary This chapter discusses the interpretation of enzyme reaction stereochemistry. Examples have been selected from the literature to illustrate broad principles that may be applicable to large classes of enzymes. Particular emphasis is given to enzyme systems for which X-ray crystallographic measurements provide a structural context in which to evaluate possible mechanistic interpretations of stereochemical trends. The chapter uses the Cahn–Ingold–Prelog convention for assigning absolute configuration to chiral centers. Chemically identical substituents at prochiral centers are designated according to Hansons convention. Since 1970, there has been a dramatic increase in the number of enzymes of known stereochemistry, owing to the broad application of numerous elegant methods of stereochemical analysis. An important challenge facing bioorganic chemists is to distinguish between those cases in which the stereochemical features of an enzymic reaction are functionally neutral and the cases that reflect underlying mechanistic advantages unavailable through alternative stereochemical modes.

Caution : the glycylmethyl and glycylethyl esters of glutathione are substrates for glyoxalase I

The glycylmethyl and glycylethyl esters of glutathione have been synthesized and carefully characterized by both 1H-NMR and tandem FAB mass spectrometry. Contrary to previously published studies, these compounds (as their methylglyoxal-thiohemiacetals) do indeed serve as moderately efficient substrates for yeast glyoxalase I, with kcat values that are approx. 3-fold smaller and Km values that are approx. 3-fold larger than those of the thiohemiacetal formed from glutathione. Product inhibition studies show that the glycylmethyl and glycylethyl esters of (S)-D-lactoylglutathione bind approx. 1.4-fold less tightly to the active site than (S)-D-lactoylglutathione. These observations exclude an essential role for the glycyl-CO2- of substrate in active site binding and catalysis.

Synthesis and initial characterization of γ-L-glutamyl-L-thiothreonyl-glycine and γ-L-glutamyl-L-allo-thiothreonylglycine as steric probes of the active site of glyoxalase I

The diastereomeric GSH derivatives gamma-L-Glu-L-allo-thioThr-Gly (6) and gamma-L-Glu-L-thioThr-Gly (6a) have been synthesized as specific probes of the steric environment near the cysteinyl residue of enzyme bound glutathionyl substrates. Experiments with glyoxalase I indicate that while 6a-methylglyoxal thiohemiacetal is a substrate for the enzyme, 6-methylglyoxal thiohemiacetal forms a tight-binding abortive complex with the active site (Ki congruent to 100 microM). Apparently, the small size of the cysteinyl C beta-Hs proton of the normal GSH-methylglyoxal thiohemiacetal substrate for glyoxalase I is a strict requirement for productive substrate binding. These compounds may provide a novel approach to the inhibition of GSH-dependent enzymes.

Enzyme chemistry of dithiohemiacetals : synthesis and characterization of S-D-dithiomandeloylglutathione as an alternate substrate for glyoxalase I

Both the D- and L-forms of S-dithiomandeloylglutathione (1) have been synthesized by a dithioester-interchange reaction between GSH and S-carboxy-methyl(D,L)-dithiomandelate. Kinetic and product analysis studies indicate that yeast glyoxalase I efficiently catalyzes the stereoselective conversion of D-1 to GSH-phenylglyoxal dithiohemiacetal (2), isolated as a disulfide adduct between 2 and a second molecule of GSH. This observation suggests that dithioester substrate analogues should be generally useful as mechanistic probes of enzyme catalyzed reactions involving thiohemiacetal intermediates.

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.