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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.


Characterization of the mechanism of novel GO inhibitors.(A,B) For the confirmed GO inhibitors, various doses (1X, 4X, and 8X of IC50) of potassium dichromate (A) or sodium dichromate dihydrate, or various doses (0.5X, 1X, 2X, 4X, and 8X of IC50) of colistimethate sodium (B) were added to purified GO, and the time-series signal of the in vitro GO enzymatic assay was monitored to obtain the reaction velocity for each glycolate concentration tested. For conciseness, data for sodium dichromate dihydrate is not shown since it can be represented by data for potassium dichromate. (C,D) The Lineweaver-Burk curves (also called the double reciprocal curves) were accordingly plotted for potassium dichromate (C) and colistimethate sodium (D).
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f5: Characterization of the mechanism of novel GO inhibitors.(A,B) For the confirmed GO inhibitors, various doses (1X, 4X, and 8X of IC50) of potassium dichromate (A) or sodium dichromate dihydrate, or various doses (0.5X, 1X, 2X, 4X, and 8X of IC50) of colistimethate sodium (B) were added to purified GO, and the time-series signal of the in vitro GO enzymatic assay was monitored to obtain the reaction velocity for each glycolate concentration tested. For conciseness, data for sodium dichromate dihydrate is not shown since it can be represented by data for potassium dichromate. (C,D) The Lineweaver-Burk curves (also called the double reciprocal curves) were accordingly plotted for potassium dichromate (C) and colistimethate sodium (D).

Mentions: To investigate the inhibitory mechanism of confirmed GO inhibitors, we measured the GO enzyme kinetics in the presence of various inhibitor concentrations. These three compounds are actually two types in chemical structures. Potassium dichromate and sodium dichromate dihydrate share the common structure of dichromate and are different salts. Both compounds inhibited GO activity at higher substrate concentrations (Fig. 5A). In comparison, colistimethate sodium inhibited GO activity at all concentrations of substrate (Fig. 5B). The Lineweaver-Burk curve was plotted and the position of the crossing point can be used to suggest the potential inhibitory mechanism for the compounds. Competitive inhibition would have the crossing point on the Y (1/V) axis and non-competitive inhibition would have the crossing point on the X (1/[S]) axis. The crossing points were neither on the Y-axis nor X-axis for all three compounds (Fig. 5C,D), suggesting that the mechanism of action of these compounds is the mixed linear inhibition.


High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
Characterization of the mechanism of novel GO inhibitors.(A,B) For the confirmed GO inhibitors, various doses (1X, 4X, and 8X of IC50) of potassium dichromate (A) or sodium dichromate dihydrate, or various doses (0.5X, 1X, 2X, 4X, and 8X of IC50) of colistimethate sodium (B) were added to purified GO, and the time-series signal of the in vitro GO enzymatic assay was monitored to obtain the reaction velocity for each glycolate concentration tested. For conciseness, data for sodium dichromate dihydrate is not shown since it can be represented by data for potassium dichromate. (C,D) The Lineweaver-Burk curves (also called the double reciprocal curves) were accordingly plotted for potassium dichromate (C) and colistimethate sodium (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Characterization of the mechanism of novel GO inhibitors.(A,B) For the confirmed GO inhibitors, various doses (1X, 4X, and 8X of IC50) of potassium dichromate (A) or sodium dichromate dihydrate, or various doses (0.5X, 1X, 2X, 4X, and 8X of IC50) of colistimethate sodium (B) were added to purified GO, and the time-series signal of the in vitro GO enzymatic assay was monitored to obtain the reaction velocity for each glycolate concentration tested. For conciseness, data for sodium dichromate dihydrate is not shown since it can be represented by data for potassium dichromate. (C,D) The Lineweaver-Burk curves (also called the double reciprocal curves) were accordingly plotted for potassium dichromate (C) and colistimethate sodium (D).
Mentions: To investigate the inhibitory mechanism of confirmed GO inhibitors, we measured the GO enzyme kinetics in the presence of various inhibitor concentrations. These three compounds are actually two types in chemical structures. Potassium dichromate and sodium dichromate dihydrate share the common structure of dichromate and are different salts. Both compounds inhibited GO activity at higher substrate concentrations (Fig. 5A). In comparison, colistimethate sodium inhibited GO activity at all concentrations of substrate (Fig. 5B). The Lineweaver-Burk curve was plotted and the position of the crossing point can be used to suggest the potential inhibitory mechanism for the compounds. Competitive inhibition would have the crossing point on the Y (1/V) axis and non-competitive inhibition would have the crossing point on the X (1/[S]) axis. The crossing points were neither on the Y-axis nor X-axis for all three compounds (Fig. 5C,D), suggesting that the mechanism of action of these compounds is the mixed linear inhibition.

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.