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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.


Confirmation of HTS hit compounds.(A) Mechanism of the in vitro GO enzymatic assay. Recombinant GO oxidizes glycolate to glyoxylate, and hydrogen peroxide is a co-product. Amplex red reagent reacts with hydrogen peroxide in a 1:1 stoichiometry and produces fluorescent resorufin. The resulting fluorescent signal is an accurate linear reflection of the initially available H2O2, which is itself a linear reflection of the in vitro GO enzymatic activity. (B) The Michaelis-Menten plot for the in vitro GO enzymatic assay. (C) Six HTS hit compounds were tested in the GO enzymatic assay. Compounds were added at a series of doses to purified GO for 10 min followed by the addition of glycolate to start the reaction. After another 10 min, Amplex red reagent was added and the wells were evaluated for fluorescence signal. Signal of GO without addition of compound was defined as 100% response while signal of GO without addition of glycolate was defined as 0% response.
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f4: Confirmation of HTS hit compounds.(A) Mechanism of the in vitro GO enzymatic assay. Recombinant GO oxidizes glycolate to glyoxylate, and hydrogen peroxide is a co-product. Amplex red reagent reacts with hydrogen peroxide in a 1:1 stoichiometry and produces fluorescent resorufin. The resulting fluorescent signal is an accurate linear reflection of the initially available H2O2, which is itself a linear reflection of the in vitro GO enzymatic activity. (B) The Michaelis-Menten plot for the in vitro GO enzymatic assay. (C) Six HTS hit compounds were tested in the GO enzymatic assay. Compounds were added at a series of doses to purified GO for 10 min followed by the addition of glycolate to start the reaction. After another 10 min, Amplex red reagent was added and the wells were evaluated for fluorescence signal. Signal of GO without addition of compound was defined as 100% response while signal of GO without addition of glycolate was defined as 0% response.

Mentions: To investigate if the six hit compounds directly inhibit GO, we developed a GO enzymatic assay using the hydrogen peroxide-Amplex red reporter system that is described in Fig. 4A. The Km value of glycolate for GO in this enzyme assay was 74.5 μM (Fig. 4B). To determine the inhibitory mechanism of action for these compounds, a series of compound concentration was added into the GO enzyme kinetic assay (Fig. 4C). The GO enzymatic assay confirmed three compounds that directly and efficiently inhibit GO enzymatic activity including potassium dichromate (IC50 = 96.6 nM), sodium dichromate dihydrate (IC50 = 108.0 nM), and colistimethate sodium (IC50 = 2.3 μM). All three compounds produced sigmoidal dose-response curves. Similar to their weak effect as observed in CHO-cell-based assay, the other two compounds of SB 222200 and telmisartan only partially inhibit GO enzymatic activity (less than 50% inhibition at 100 μM). The 6th compound syrosingopine did not inhibit GO enzymatic activity in this assay. We then observed this compound has self-fluorescence that could be the reason as a false positive compound. Together, three compounds (potassium dichromate, sodium dichromate dihydrate, and colistimethate sodium) identified from the primary screen were confirmed as GO inhibitors in this GO enzymatic assay.


High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1
Confirmation of HTS hit compounds.(A) Mechanism of the in vitro GO enzymatic assay. Recombinant GO oxidizes glycolate to glyoxylate, and hydrogen peroxide is a co-product. Amplex red reagent reacts with hydrogen peroxide in a 1:1 stoichiometry and produces fluorescent resorufin. The resulting fluorescent signal is an accurate linear reflection of the initially available H2O2, which is itself a linear reflection of the in vitro GO enzymatic activity. (B) The Michaelis-Menten plot for the in vitro GO enzymatic assay. (C) Six HTS hit compounds were tested in the GO enzymatic assay. Compounds were added at a series of doses to purified GO for 10 min followed by the addition of glycolate to start the reaction. After another 10 min, Amplex red reagent was added and the wells were evaluated for fluorescence signal. Signal of GO without addition of compound was defined as 100% response while signal of GO without addition of glycolate was defined as 0% response.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Confirmation of HTS hit compounds.(A) Mechanism of the in vitro GO enzymatic assay. Recombinant GO oxidizes glycolate to glyoxylate, and hydrogen peroxide is a co-product. Amplex red reagent reacts with hydrogen peroxide in a 1:1 stoichiometry and produces fluorescent resorufin. The resulting fluorescent signal is an accurate linear reflection of the initially available H2O2, which is itself a linear reflection of the in vitro GO enzymatic activity. (B) The Michaelis-Menten plot for the in vitro GO enzymatic assay. (C) Six HTS hit compounds were tested in the GO enzymatic assay. Compounds were added at a series of doses to purified GO for 10 min followed by the addition of glycolate to start the reaction. After another 10 min, Amplex red reagent was added and the wells were evaluated for fluorescence signal. Signal of GO without addition of compound was defined as 100% response while signal of GO without addition of glycolate was defined as 0% response.
Mentions: To investigate if the six hit compounds directly inhibit GO, we developed a GO enzymatic assay using the hydrogen peroxide-Amplex red reporter system that is described in Fig. 4A. The Km value of glycolate for GO in this enzyme assay was 74.5 μM (Fig. 4B). To determine the inhibitory mechanism of action for these compounds, a series of compound concentration was added into the GO enzyme kinetic assay (Fig. 4C). The GO enzymatic assay confirmed three compounds that directly and efficiently inhibit GO enzymatic activity including potassium dichromate (IC50 = 96.6 nM), sodium dichromate dihydrate (IC50 = 108.0 nM), and colistimethate sodium (IC50 = 2.3 μM). All three compounds produced sigmoidal dose-response curves. Similar to their weak effect as observed in CHO-cell-based assay, the other two compounds of SB 222200 and telmisartan only partially inhibit GO enzymatic activity (less than 50% inhibition at 100 μM). The 6th compound syrosingopine did not inhibit GO enzymatic activity in this assay. We then observed this compound has self-fluorescence that could be the reason as a false positive compound. Together, three compounds (potassium dichromate, sodium dichromate dihydrate, and colistimethate sodium) identified from the primary screen were confirmed as GO inhibitors in this GO enzymatic assay.

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.