Theodore W. C. Lo

The reaction of methylglyoxal with aminoguanidine under physiological conditions and prevention of methylglyoxal binding to plasma proteins

Increased formation of methylglyoxal in clinical diabetes mellitus and metabolism by the glyoxalase system has been linked to the development of clinical complications of diabetes: retinopathy, neuropathy and nephropathy. Aminoguanidine has been proposed as a prophylactic agent for preventive therapy of diabetic complications. Methylglyoxal reacted with aminoguanidine under physiological conditions to form two isomeric triazines, 3-amino-5-methyl-1,2,4-triazine and 3-amino-6-methyl-1,2,4-triazine. The mean second order rate constant for the reaction of methylglyoxal with aminoguanidine, kMG.AG = 0.39 +/- 0.06 M-1 sec-1 at pH 7.4 and 37 degrees. Under these conditions, no methylglyoxal bisguanylhydrazone was detected. Aminoguanidine prevented the irreversible modification of human plasma protein by a physiological concentration of methylglyoxal (1 microM); the median inhibitory concentration IC50 value of aminoguanidine was 203 +/- 16 microM (N = 28). The scavenging of methylglyoxal by aminoguanidine may contribute to the beneficial effects of aminoguanidine in the prevention of vascular pathogenesis in diabetes.

Inhibition of proliferation of human leukaemia 60 cells by diethyl esters of glyoxalase inhibitors in vitro

Diethyl esters of the glutathione S-conjugate S-p-bromobenzylglutathione, an inhibitor of glyoxalase I, and S-p-nitrobenzoxycarbonylglutathione, an inhibitor of glyoxalase II, induced growth arrest and toxicity in human leukaemia 60 cells in culture. The median growth inhibitory concentration IC50 values were 8.3 microM (95% C.I. 7.0-9.9 microM) for S-p-bromobenzylglutathione diethyl ester and 56 microM (95% C.I. 36-86 microM) for p-nitrobenzoxycarbonylglutathione. Monoethyl ester and unesterified derivatives were inactive. The diethyl ester derivatives were also toxic to mature human neutrophils under the same culture conditions where the respective median toxic concentration IC50 values were 39.7 (95% C.I. 35.4-44.5 microM) and 127 (95% C.I. 123-132 microM) microM. Diester derivatives may be of future interest in studying the cytotoxicity of glutathione S-conjugates and for the development of cytotoxic anti-tumour agents.

A simplified method for the purification of human red blood cell glyoxalase. I. Characteristics, immunoblotting, and inhibitor studies.

Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. TheKM value for methylglyoxal-glutathione hemithioacetal was 192±8 µM and thekcat value was 10.9±0.2 × 104 min−1 (N = 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had aKi value of 0.16±0.04 µM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with aKi value of 3.12±0.88 µM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with aKi value of 18±8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations.

γ-L-Glutamyl-O-acyl-L-serylglycine derivatives: synthesis, purification and evaluation as inhibitors of glyoxalases

Oxygen ester analogues of S-D-lactoylglutathione, γ-L-glutamyl-O-acyl-L-serylglycine derivatives, were synthesised and evaluated as inhibitors of glyoxalase II. They were competitive inhibitors where the inhibition constant Ki, decreased with increase in acyl chain length. γ-L-Glutamyl-O-acyl-L-serylglycine derivatives also inhibit glyoxalase I. These compounds provide a novel route to glyoxalase II inhibitors for cancer chemotherapy.