Limits...
High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

HTS using stably transformed CHO cells.(A) Metabolic basis of the indirect glycolate-induced cytotoxicity. (B) Illustration of the HTS flowchart. The HTS assay parameters were optimized (see Fig. 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037430&req=5

f1: HTS using stably transformed CHO cells.(A) Metabolic basis of the indirect glycolate-induced cytotoxicity. (B) Illustration of the HTS flowchart. The HTS assay parameters were optimized (see Fig. 2).

Mentions: A cell-based model with PH1 disease related metabolic events was reported previously1213. CHO cells are particularly useful for this assay because they do not endogenously express enzymes involved in glyoxylate synthesis (e.g. GO) and detoxification (e.g. AGT). Addition of glycolate to wild type (WT) CHO cells does not confer cytotoxicity because GO is not expressed although LDH is present. This cell-based assay stably expresses GO and/or wild type (WT) or mutant AGT in CHO cells (Fig. 1A). In the transformed CHO-GO cells, glycolate is oxidized to glyoxylate in peroxisomes. Excessive glyoxylate diffuses from the peroxisomes to the cytosol to be further processed into oxalate by endogenously expressed LDH. Both glyoxylate and oxalate are toxic to cells. Addition of glycolate to the CHO-GO cells results in cytotoxicity due to the production of glyoxylate and oxalate. In CHO-GO/AGT cells, expression of WT AGT leads to conversion of toxic glyoxylate into nontoxic glycine which reduces glyoxylate level and thus leads to the decrease of oxalate level. Consequently, co-expression of WT AGT with GO significantly reduces the cytotoxicity induced by addition of glycolate. In contrast, mutated AGT in the CHO-GO/AGT-mutant cells fails to reach the same protective effect as the WT AGT and thus glycolate cytotoxicity is still significantly present.


High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
HTS using stably transformed CHO cells.(A) Metabolic basis of the indirect glycolate-induced cytotoxicity. (B) Illustration of the HTS flowchart. The HTS assay parameters were optimized (see Fig. 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037430&req=5

f1: HTS using stably transformed CHO cells.(A) Metabolic basis of the indirect glycolate-induced cytotoxicity. (B) Illustration of the HTS flowchart. The HTS assay parameters were optimized (see Fig. 2).
Mentions: A cell-based model with PH1 disease related metabolic events was reported previously1213. CHO cells are particularly useful for this assay because they do not endogenously express enzymes involved in glyoxylate synthesis (e.g. GO) and detoxification (e.g. AGT). Addition of glycolate to wild type (WT) CHO cells does not confer cytotoxicity because GO is not expressed although LDH is present. This cell-based assay stably expresses GO and/or wild type (WT) or mutant AGT in CHO cells (Fig. 1A). In the transformed CHO-GO cells, glycolate is oxidized to glyoxylate in peroxisomes. Excessive glyoxylate diffuses from the peroxisomes to the cytosol to be further processed into oxalate by endogenously expressed LDH. Both glyoxylate and oxalate are toxic to cells. Addition of glycolate to the CHO-GO cells results in cytotoxicity due to the production of glyoxylate and oxalate. In CHO-GO/AGT cells, expression of WT AGT leads to conversion of toxic glyoxylate into nontoxic glycine which reduces glyoxylate level and thus leads to the decrease of oxalate level. Consequently, co-expression of WT AGT with GO significantly reduces the cytotoxicity induced by addition of glycolate. In contrast, mutated AGT in the CHO-GO/AGT-mutant cells fails to reach the same protective effect as the WT AGT and thus glycolate cytotoxicity is still significantly present.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus