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

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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.


(A) Concentration-response of chromium(II) chloride (450782, Sigma-Aldrich) determined in the GO enzymatic assay. (B) Immunofluorescence images of GO localized in peroxisomes. CHO GO cells were treated with 100 μM potassium dichromate, sodium dichromate dihydrate, or colistimethate sodium for 2 h, then immunostained for GO, the peroxisomes (catalase) and the nucleus (DAPI), and finally analyzed by fluorescence microscope (Scale bar, 30 μm). Compound treatment did not significantly change the localization and amount of GO in peroxisome.
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f6: (A) Concentration-response of chromium(II) chloride (450782, Sigma-Aldrich) determined in the GO enzymatic assay. (B) Immunofluorescence images of GO localized in peroxisomes. CHO GO cells were treated with 100 μM potassium dichromate, sodium dichromate dihydrate, or colistimethate sodium for 2 h, then immunostained for GO, the peroxisomes (catalase) and the nucleus (DAPI), and finally analyzed by fluorescence microscope (Scale bar, 30 μm). Compound treatment did not significantly change the localization and amount of GO in peroxisome.

Mentions: Both potassium dichromate and sodium dichromate dihydrate share a functional group in structure. In comparison to the dichromate (Cr2O72−) group, chromium(II) chloride, which has Cr2+, does not inhibit GO enzymatic activity (Fig. 6A). The result suggests unique structural and chemical features of the dichromate group in this function. In addition, GO is specifically targeted to the peroxisomes. Consequently, authentic GO inhibitors should inhibit GO functional activity but should not change its cellular localization. We found that GO were physiologically present in the peroxisomes of CHO-GO cells without significant change after treatment with the 100 μM compound which produced the maximal inhibition of the glycolate toxicity (Fig. 6B), further demonstrating the direct functional inhibition of GO by these three compounds.


High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
(A) Concentration-response of chromium(II) chloride (450782, Sigma-Aldrich) determined in the GO enzymatic assay. (B) Immunofluorescence images of GO localized in peroxisomes. CHO GO cells were treated with 100 μM potassium dichromate, sodium dichromate dihydrate, or colistimethate sodium for 2 h, then immunostained for GO, the peroxisomes (catalase) and the nucleus (DAPI), and finally analyzed by fluorescence microscope (Scale bar, 30 μm). Compound treatment did not significantly change the localization and amount of GO in peroxisome.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: (A) Concentration-response of chromium(II) chloride (450782, Sigma-Aldrich) determined in the GO enzymatic assay. (B) Immunofluorescence images of GO localized in peroxisomes. CHO GO cells were treated with 100 μM potassium dichromate, sodium dichromate dihydrate, or colistimethate sodium for 2 h, then immunostained for GO, the peroxisomes (catalase) and the nucleus (DAPI), and finally analyzed by fluorescence microscope (Scale bar, 30 μm). Compound treatment did not significantly change the localization and amount of GO in peroxisome.
Mentions: Both potassium dichromate and sodium dichromate dihydrate share a functional group in structure. In comparison to the dichromate (Cr2O72−) group, chromium(II) chloride, which has Cr2+, does not inhibit GO enzymatic activity (Fig. 6A). The result suggests unique structural and chemical features of the dichromate group in this function. In addition, GO is specifically targeted to the peroxisomes. Consequently, authentic GO inhibitors should inhibit GO functional activity but should not change its cellular localization. We found that GO were physiologically present in the peroxisomes of CHO-GO cells without significant change after treatment with the 100 μM compound which produced the maximal inhibition of the glycolate toxicity (Fig. 6B), further demonstrating the direct functional inhibition of GO by these three compounds.

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.