National Center for Advancing Translational Sciences [NCATS]
NIH Chemical Genomics Center [NCGC]
U.S. Environmental Protection Agency [EPA]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

Assay overview:

The Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors of the nuclear receptor superfamily with three distinct subtypes (alpha, garmma and beta/delta). All these subtypes heterodimerize with retinoid X receptor and have different physiological functions. PPARalpha is highly expressed in tissues with high rates of mitochondrial fatty acid oxidation, such as liver, heart, muscle, kidney and cells of the arterial wall. PPARalpha is a major regulator of lipid metabolism and energy homeostasis.

A cell based assay was used to identify activators of PPARalpha. The assay detects binding of a ligand to PPARalpha leading to its activation, translocation and complementation thus forming a functional enzyme capable of hydrolyzing the substrate molecule and generating chemiluminescent signal.

Assay Protocol Summary:

2,000 cells in 6 uL/well were dispensed into white wall/solid bottom 1536-well plates using a Multidrop Combi (Thermo Scientific) dispenser. After incubation 37 C and 5% CO2 overnight, 23 nL compound or DMSO only were transferred by a pin tool. The plates were incubated for 6 hr at 37 C and 5% CO2. 3 ul PathHunter detection reagent (Discoverx) was then added to each well and following 60 min incubation at room temperature, chemiluminescence was quantified on a ViewLux CCD-based plate reader.

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.