A High Throughput Serum Paraoxonase Assay for Discovery of Small Molecule Modulators of PON1 Activity

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 IC50 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 (IC50 = 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.

Effect of Probucol on Antioxidant Properties of HDL in Patients with Heterozygous Familial Hypercholesterolemia

“…First, we confirmed that treatment with 2-hydroxyquinoline inhibited 90% of PON1 activity in HDL. This inhibition rate was compatible with a previous report 26) . The lag duration time of HDL from probucol-treated patients was reduced by 40% with 2-hydroxyquinoline treatment (Fig.…”

“…A specific inhibitor of PON1 25,26) , 2-hydroxyquinoline (Sigma-Aldrich Corp., St. Louis, MO) was diluted in methanol (10 mM). This stock solution was diluted in PBS.…”

“…This hypothesis was confirmed by the inhibition of PON1 activity. A high throughput serum paraoxonase assay discovered that 2-hydroxyquinoline was the most specific inhibitor of PON1 26) . First, we confirmed that treatment with 2-hydroxyquinoline inhibited 90% of PON1 activity in HDL.…”

The Effects of Anthocyanins and Their Microbial Metabolites on the Expression and Enzyme Activities of Paraoxonase 1, an Important Marker of HDL Function

“…Although the data presented here does not support the notion that direct interactions between anthocyanins and/or their metabolites affect the lactonase or arylesterase activity of PON1, it is not conclusive. The purified enzyme used here is not in its native environment which would include the presence of HDL and the ApoA1 protein, and this may affect the enzyme structural conformation and its stability and therefore its activity [60][61][62][63][64]. In theory it would be possible to use instead serum as a source of PON1.…”

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells

“…Paraoxonase 1 (PON1) was selected in this study for further characterization owing to its primary location of synthesis in the liver and its association with high‐density lipoprotein (HDL) metabolism after secretion into plasma. HDL‐associated PON1 has been reported to reduce oxidative stress in lipoproteins and atherosclerotic lesions . The PON1 gene is activated by PPAR‐γ, which increases the synthesis and release of PON1 enzyme from the liver, thereby reducing atherosclerosis.…”