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A high throughput serum paraoxonase assay for discovery of small molecule modulators of PON1 activity.

Graves TL, Scott JE - Curr Chem Genomics (2008)

Bottom Line: A compound library was screened resulting in no confirmed activators, but 12 confirmed inhibitors.Seven of these hits also inhibited purified human PON1.This compound (IC(50) = 420 nM) may be useful towards a chemical probe for PON1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.

ABSTRACT
PON1 has been demonstrated to be the serum enzyme responsible for detoxifying organophosphate chemical weapons and plays a protective role against atherosclerosis. In order to identify small molecules that modulate PON1 activity in serum, we developed a high throughput kinetic absorbance assay using mouse serum and the organophosphate paraoxon. The IC(50) value obtained for the known PON1 inhibitor, 2-hydroxyquinoline, matched the value reported for purified PON1. A compound library was screened resulting in no confirmed activators, but 12 confirmed inhibitors. Seven of these hits also inhibited purified human PON1. One compound was only two-fold less potent than 2-hydroxyquinoline in the serum assay, but 10-fold more potent against purified PON1. This compound (IC(50) = 420 nM) may be useful towards a chemical probe for PON1. Therefore, this assay has utility as a high throughput assay for discovery of small molecule modulators of PON1 activity that maintain activity in serum.

No MeSH data available.


Related in: MedlinePlus

Reaction time course. Absorbance (optical density) was recorded over time for six replicate reactions initiated at time 0. Serum concentration was 15%. Error bars represent standard deviations. Data are representative of two independent experiments.
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Figure 2: Reaction time course. Absorbance (optical density) was recorded over time for six replicate reactions initiated at time 0. Serum concentration was 15%. Error bars represent standard deviations. Data are representative of two independent experiments.

Mentions: The optimal concentration of serum to use in the serum PON1 assay was determined by varying the percentage of serum in the final reaction (Fig. 1). The rate of the reaction increased in a linear fashion up to 15% serum, after which it became non-linear. The reason for the loss in linearity, i.e. lower than expected velocities at >15% serum concentrations, appears to be due to the high absorbance of the serum; studies in which the reactions were stopped and diluted show a linear increase in product up to the highest concentration of serum tested (60%) (data not shown). The serum concentration that was chosen was 15% and this concentration was used in all subsequent experiments. The linearity of the reaction was determined over extended times to provide information on how quickly the reaction needed to be read in the plate reader after initiation of the reaction with substrate (Fig. 2). The reaction rate was linear for at least 20 minutes indicating that there is ample time to read the plate after addition of paraoxon.


A high throughput serum paraoxonase assay for discovery of small molecule modulators of PON1 activity.

Graves TL, Scott JE - Curr Chem Genomics (2008)

Reaction time course. Absorbance (optical density) was recorded over time for six replicate reactions initiated at time 0. Serum concentration was 15%. Error bars represent standard deviations. Data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803440&req=5

Figure 2: Reaction time course. Absorbance (optical density) was recorded over time for six replicate reactions initiated at time 0. Serum concentration was 15%. Error bars represent standard deviations. Data are representative of two independent experiments.
Mentions: The optimal concentration of serum to use in the serum PON1 assay was determined by varying the percentage of serum in the final reaction (Fig. 1). The rate of the reaction increased in a linear fashion up to 15% serum, after which it became non-linear. The reason for the loss in linearity, i.e. lower than expected velocities at >15% serum concentrations, appears to be due to the high absorbance of the serum; studies in which the reactions were stopped and diluted show a linear increase in product up to the highest concentration of serum tested (60%) (data not shown). The serum concentration that was chosen was 15% and this concentration was used in all subsequent experiments. The linearity of the reaction was determined over extended times to provide information on how quickly the reaction needed to be read in the plate reader after initiation of the reaction with substrate (Fig. 2). The reaction rate was linear for at least 20 minutes indicating that there is ample time to read the plate after addition of paraoxon.

Bottom Line: A compound library was screened resulting in no confirmed activators, but 12 confirmed inhibitors.Seven of these hits also inhibited purified human PON1.This compound (IC(50) = 420 nM) may be useful towards a chemical probe for PON1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707, USA.

ABSTRACT
PON1 has been demonstrated to be the serum enzyme responsible for detoxifying organophosphate chemical weapons and plays a protective role against atherosclerosis. In order to identify small molecules that modulate PON1 activity in serum, we developed a high throughput kinetic absorbance assay using mouse serum and the organophosphate paraoxon. The IC(50) value obtained for the known PON1 inhibitor, 2-hydroxyquinoline, matched the value reported for purified PON1. A compound library was screened resulting in no confirmed activators, but 12 confirmed inhibitors. Seven of these hits also inhibited purified human PON1. One compound was only two-fold less potent than 2-hydroxyquinoline in the serum assay, but 10-fold more potent against purified PON1. This compound (IC(50) = 420 nM) may be useful towards a chemical probe for PON1. Therefore, this assay has utility as a high throughput assay for discovery of small molecule modulators of PON1 activity that maintain activity in serum.

No MeSH data available.


Related in: MedlinePlus