Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders
Figure 3
Various cell-free assays to discover PPAR modulator. A. Competition radioreceptor assays are performed by incubating recombinant PPAR protein and radioisotope-labeled ligand in the presence of competitor ligands. Bound ligands are separated from free forms by filtration. The amount of bound radioisotope-labeled ligand is determined by liquid scintillation counting. B. Coactivator-dependent receptor ligand assays (CARLAs) based on the recruitment of a coactivator due to a conformational change of specific ligand-bound PPAR. CARLA is carried out by incubating GST-PPAR and radioisotope-labeled coactivator with a ligand candidate or by incubating radioisotope-labeled PPAR and GST-coactivator with ligand candidates. The amount of ligand-bound PPAR-coactivator complex is determined by pull-down assay. C. In the scintillation proximity assay (SPA), the receptor-ligand complex is bound to the SPA bead through interaction between the biotinylated receptor and the streptavidin moiety located on the surface of the SPA bead. Because no separation step is required, SPA has benefits in its application to HTS. D. In the fluorescence resonance energy transfer (FRET)-based in vitro recruitment assay, GST-PPAR LBD proteins are indirectly linked to EU cryptate, (Eu)K, through (Eu)K-labeled anti-GST antibody, -GST-(Eu)K. Purified recombinant coactivator is biotinylated, and is indirectly linked to XL665, which is produced only when there is a ligand-induced change in receptor conformation that results in binding to the coactivator. The extent of the FRET is measured as a ratio of 665 nm/620 nm X 10,000. E. A simple ELISA based on binding between PPAR and coactivators. The ligand unbound-PPAR weakly binds to the LXXLL motifs of coactivator, whereas ligand loaded-PPAR strongly binds to the LXXLL motifs of coactivator due to the conformational change of PPAR by specific agonists. This binding is detected by a specific anti-PPAR antibody, followed by horseradish peroxidase-conjugated secondary antibody. F. Schematic representation of SPR technology. One of the interacting partners is immobilized on a gold or waveguide layer of the sensor chip using the standard amine-coupling protocol. The other flows over the surface of the sensor chip, allowing interaction with the immobilized interacting partners. The interaction of immobilized partners with interacting molecules gives rise to an increase in mass. The refractive index and the angle of reflected light is thereby changed. As soon as the injection is stopped, the complex is washed with a washing buffer. The interacting molecules are dissociated from the immobilized interacting partner, resulting in a decrease in the signal due to a shift in the angle of the reflected light to its original position.
