The glyoxalase enzymatic pathway is highly conserved in nature, and found in the cytoplasm of cells, cell organelles, and mitochondria. This system plays a pivotal role in cell proliferation among all higher eukaryotes, by catalyzing the conversion of cytotoxic methylglyoxal (MEG) to D-lactic acid. Selective inhibition of the first enzyme in this pathway, Glyoxalase I (GxI), results in a buildup of MEG in cancer cells, causing cell death. Generally, cancer cells have higher GxI activity and lower MEG concentrations than normal cells. One hypothesis is that the higher levels of GxI are responsible for unregulated tumor cell proliferation due to removal of MEG as the natural control of abnormal cell growth. If a GxI inhibitor can directly target tumor cells, the levels of MEG will increase and inhibit tumor growth by promoting apoptosis. The primary objective of this research was to evaluate the glutathione 3-methyleneoxindole conjugate (GSMOI) as a potential reversible and/or irreversible inhibitor of human GxI. MOI and GSMOI were synthesized, purified, and characterized by NMR spectrometry. An end point enzymatic assay with yeast GxI was used to determine the concentration of aqueous MEG stock solutions used to form the hemithioacetal substrate with glutathione. Kinetic assays to characterize GxI inhibition were done using UV-Vis spectroscopy by monitoring the rate of conversion of the hemithioacetal substrate to the lactoyl glutathione thioester product at 240 nm in pH 7 KP buffer. Based on initial rate kinetics, we observed a 20% reduction in the rate of hemithioacetal conversion in the presence of 0.1 mM GSMOI. We will report our results to characterize the type of inhibition of GxI, and evidence for irreversible inactivation by GSMOI and MOI.

Exploring the mechanism of glyoxalase I inhibition and inactivation by the glutathione conjugate with 3-methyleneoxindole

255th National Meeting (2018)

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