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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.


Scatter plots of DMSO screening plates.The signal-to-basal ratio (S/B), coefficient of variation (CV), and Z’ factor were calculated and displayed. The wells in column 3 contained untransformed CHO WT cells while the wells in column 1 and 2 contained CHO-GO cells. GO DMSO assay plate contained CHO-GO cells in columns 4–48 (A), and GO/AGT-152 DMSO assay plate contained CHO-GO/AGT-152 cells in columns 4–48 (B). All wells were added with 23 nl DMSO for 1 h followed by glycolate addition (final glycolate concentration of 0.5 mM and DMSO concentration of 0.57%).
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f3: Scatter plots of DMSO screening plates.The signal-to-basal ratio (S/B), coefficient of variation (CV), and Z’ factor were calculated and displayed. The wells in column 3 contained untransformed CHO WT cells while the wells in column 1 and 2 contained CHO-GO cells. GO DMSO assay plate contained CHO-GO cells in columns 4–48 (A), and GO/AGT-152 DMSO assay plate contained CHO-GO/AGT-152 cells in columns 4–48 (B). All wells were added with 23 nl DMSO for 1 h followed by glycolate addition (final glycolate concentration of 0.5 mM and DMSO concentration of 0.57%).

Mentions: First we tested the assay performance under a compound screening condition. After cell seeding and culture in 1,536-well plates, compounds were added 1 h before glycolate addition to allow compound diffusion into cells. The compound screening protocol included four cell/reagent addition steps and four incubation periods without any plate-washing steps (Table 1). Two types of cells were used in HTS including CHO-GO cells for identification and confirmation of GO inhibitors and CHO-GO/AGT-152 cells for discovering AGT-152 activators. A DMSO plate (without compounds) was tested to assess the parameters of assay performance. For CHO-GO cells, the S/B ratio was 5.4-fold, the coefficient of variation (CV) was 12.8%, and Z´ factor was 0.78 determined in the DMSO plate (Fig. 3A). The S/B ratio was 4.0-fold, the CV was 13.7%, and Z´ factor was 0.70 as obtained in the DMSO plate of CHO-GO/AGT-152 cells (Fig. 3B). Thus, both cell lines produced robust assay windows and Z’ factors which are suitable for HTS.


High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
Scatter plots of DMSO screening plates.The signal-to-basal ratio (S/B), coefficient of variation (CV), and Z’ factor were calculated and displayed. The wells in column 3 contained untransformed CHO WT cells while the wells in column 1 and 2 contained CHO-GO cells. GO DMSO assay plate contained CHO-GO cells in columns 4–48 (A), and GO/AGT-152 DMSO assay plate contained CHO-GO/AGT-152 cells in columns 4–48 (B). All wells were added with 23 nl DMSO for 1 h followed by glycolate addition (final glycolate concentration of 0.5 mM and DMSO concentration of 0.57%).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Scatter plots of DMSO screening plates.The signal-to-basal ratio (S/B), coefficient of variation (CV), and Z’ factor were calculated and displayed. The wells in column 3 contained untransformed CHO WT cells while the wells in column 1 and 2 contained CHO-GO cells. GO DMSO assay plate contained CHO-GO cells in columns 4–48 (A), and GO/AGT-152 DMSO assay plate contained CHO-GO/AGT-152 cells in columns 4–48 (B). All wells were added with 23 nl DMSO for 1 h followed by glycolate addition (final glycolate concentration of 0.5 mM and DMSO concentration of 0.57%).
Mentions: First we tested the assay performance under a compound screening condition. After cell seeding and culture in 1,536-well plates, compounds were added 1 h before glycolate addition to allow compound diffusion into cells. The compound screening protocol included four cell/reagent addition steps and four incubation periods without any plate-washing steps (Table 1). Two types of cells were used in HTS including CHO-GO cells for identification and confirmation of GO inhibitors and CHO-GO/AGT-152 cells for discovering AGT-152 activators. A DMSO plate (without compounds) was tested to assess the parameters of assay performance. For CHO-GO cells, the S/B ratio was 5.4-fold, the coefficient of variation (CV) was 12.8%, and Z´ factor was 0.78 determined in the DMSO plate (Fig. 3A). The S/B ratio was 4.0-fold, the CV was 13.7%, and Z´ factor was 0.70 as obtained in the DMSO plate of CHO-GO/AGT-152 cells (Fig. 3B). Thus, both cell lines produced robust assay windows and Z’ factors which are suitable for HTS.

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.