Glycolate oxidase (GO) and
alanine:glyoxylate aminotransferase (AGT) are both involved in the peroxisomal
glyoxylate pathway. Deficiency in AGT function causes the accumulation of intracellular
oxalate and the
primary hyperoxaluria type 1 (PH1). AGT enhancers or GO inhibitors may restore the abnormal peroxisomal
glyoxylate pathway in PH1 patients. With stably transformed cells which mimic the
glyoxylate metabolic pathway, we developed an indirect
glycolate cytotoxicity assay in a 1,536-well plate format for high throughput screening. This assay can be used to identify compounds that reduce indirect
glycolate-induced cytotoxicity by either enhancing AGT activity or inhibiting GO. A pilot screen of 4,096 known compounds identified two membrane permeable GO inhibitors: dichromate
salt and
colistimethate. We also developed a GO
enzyme assay using the
hydrogen peroxide-
Amplex red reporter system. The IC50 values of
potassium dichromate,
sodium dichromate, and
colistimethate sodium were 0.096, 0.108, and 2.3 μM in the GO
enzyme assay, respectively. Further
enzyme kinetic study revealed that both types of compounds inhibit GO activity by the mixed linear inhibition. Our results demonstrate that the cell-based assay and GO
enzyme assay developed in this study are useful for further screening of large compound libraries for
drug development to treat PH1.