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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

Optimization of the indirect glycolate cytotoxicity.(A) A collection of eight CHO cell lines were cultured in 384-well plates and assayed for the glycolate toxicity. (B–D) Optimization of assay parameters was carried out in three aspects: seeding CHO cells at different density (B), adding glycolate at a range of concentrations (C), and varying the incubation period (D). Signal-to-basal ratio was evaluated for each parameter. (E) Partial reduction of the indirect glycolate cytotoxicity in the cell-based assay by 2-hydroxy-3-butynoic acid (2H3BA) c, a known GO inhibitor.
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f2: Optimization of the indirect glycolate cytotoxicity.(A) A collection of eight CHO cell lines were cultured in 384-well plates and assayed for the glycolate toxicity. (B–D) Optimization of assay parameters was carried out in three aspects: seeding CHO cells at different density (B), adding glycolate at a range of concentrations (C), and varying the incubation period (D). Signal-to-basal ratio was evaluated for each parameter. (E) Partial reduction of the indirect glycolate cytotoxicity in the cell-based assay by 2-hydroxy-3-butynoic acid (2H3BA) c, a known GO inhibitor.

Mentions: In order to choose the appropriate AGT mutants for compound screening assay, we first evaluated several alleles of AGT mutations (details in the Discussion). We examined six CHO cell lines expressing WT GO and normal or mutated AGT (AGT-MA and AGT-mi; AGT-170, AGT-152, AGT-244, and AGT-41 all in the background of AGT-mi) (Fig. 2A) in a glycolate cytotoxicity assay. Addition of glycolate resulted in cell death in CHO-GO cells but not in untransformed CHO WT cells (Fig. 2A). Co-expression of AGT-MA significantly reduced the cytotoxicity of glycolate, while co-expression of AGT-mi, AGT-170, AGT-152, or AGT-244 partially reduced the cytotoxicity (Fig. 2A). Among these partially functional AGT mutants, AGT-152 gives the largest assay window at a final concentration of 0.25 or 0.5 mM glycolate. In contrast, co-expression of AGT-41 provided little cellular protection, suggesting that this AGT mutant is largely nonfunctional. Therefore, we selected the CHO-GO cell line and CHO-GO/AGT-152 cell line as a model system for respective identification of GO inhibitors and AGT mutant activators.


High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
Optimization of the indirect glycolate cytotoxicity.(A) A collection of eight CHO cell lines were cultured in 384-well plates and assayed for the glycolate toxicity. (B–D) Optimization of assay parameters was carried out in three aspects: seeding CHO cells at different density (B), adding glycolate at a range of concentrations (C), and varying the incubation period (D). Signal-to-basal ratio was evaluated for each parameter. (E) Partial reduction of the indirect glycolate cytotoxicity in the cell-based assay by 2-hydroxy-3-butynoic acid (2H3BA) c, a known GO inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Optimization of the indirect glycolate cytotoxicity.(A) A collection of eight CHO cell lines were cultured in 384-well plates and assayed for the glycolate toxicity. (B–D) Optimization of assay parameters was carried out in three aspects: seeding CHO cells at different density (B), adding glycolate at a range of concentrations (C), and varying the incubation period (D). Signal-to-basal ratio was evaluated for each parameter. (E) Partial reduction of the indirect glycolate cytotoxicity in the cell-based assay by 2-hydroxy-3-butynoic acid (2H3BA) c, a known GO inhibitor.
Mentions: In order to choose the appropriate AGT mutants for compound screening assay, we first evaluated several alleles of AGT mutations (details in the Discussion). We examined six CHO cell lines expressing WT GO and normal or mutated AGT (AGT-MA and AGT-mi; AGT-170, AGT-152, AGT-244, and AGT-41 all in the background of AGT-mi) (Fig. 2A) in a glycolate cytotoxicity assay. Addition of glycolate resulted in cell death in CHO-GO cells but not in untransformed CHO WT cells (Fig. 2A). Co-expression of AGT-MA significantly reduced the cytotoxicity of glycolate, while co-expression of AGT-mi, AGT-170, AGT-152, or AGT-244 partially reduced the cytotoxicity (Fig. 2A). Among these partially functional AGT mutants, AGT-152 gives the largest assay window at a final concentration of 0.25 or 0.5 mM glycolate. In contrast, co-expression of AGT-41 provided little cellular protection, suggesting that this AGT mutant is largely nonfunctional. Therefore, we selected the CHO-GO cell line and CHO-GO/AGT-152 cell line as a model system for respective identification of GO inhibitors and AGT mutant activators.

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