Description:
Project ID: 2019-47
Background:
Peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that have become significant targets for addressing various metabolic disorders, including lipid dystrophies and diabetes. PPARs are nutrient receptors that have different selective and promiscuous ligands, including fatty acids and eicosanoids. There are three major isoforms of PPARs: PPARα, PPARβ/δ, and PPARγ. PPARα and PPARβ/δ induce genes to regulate lipid uptake and metabolism. Currently, the most commonly used screening methods for PPAR ligands use assays such as LanthaScreen™ TR-FRET PPAR that are GST tagged, or radioactively labeled drug for competition. Conventional methods for screening PPAR ligands also include radioactive assays that use expensive materials and involves safety concerns. One issue with LanthaScreen™ is that all of the drug-protein targets are labeled with GST, which there are several compounds known to interact with this domain. Thus, there is a strong need for cost-effective and safer alternative screening methods.
Invention Description:
Researchers at the University of Toledo have developed a novel binding assay to evaluate the binding capacity of PPAR ligands using the ability of bilirubin (BR) to autofluorescence when bound to the PPAR protein. In comparison with the LanthaScreen™, this novel assay method does not require GST, and non-GST purified PPAR proteins can be utilized. Because bilirubin or biliverdin bind to the PPAR protein and autofluorescence when excited, the displacement of the bilirubin or biliverdin by the test ligand’s binding to the PPAR protein can be determined by measuring a decrease in the fluorescence. The difference in fluorescence is proportional to the extent to which the test ligand binds to the PPAR protein, thereby displacing bilirubin or biliverdin and reducing the fluorescence from the binding between bilirubin or biliverdin and the PPAR protein. This difference is easily measured from the observed fluorescence, and the amount of binding between the test ligand and the PPAR protein can be determined accordingly. The novel assay can be used to screen/discover new ligands for PPAR proteins, allowing for high throughput screening of possible PPAR ligands. The assay also allows for determining specific binding regions of the drug with the receptor by a mutational analysis, which determines amino acids responsible for drug binding in the ligand-binding pocket.
Patent Status: Patent Pending
Applications:
• Competitive binding assays
• Hormone binding
• Drug binding
• Studies for drug-protein interaction
Advantages:
• Significantly cost effective ($100-$200) and fast (1 hour) compared to LanthaScreen™ and radioactive assays.
• The competitive binding assays do not require GST-tagged proteins that is used with LanthaScreen™.
• High throughput screening of PPAR ligands.