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Synthesis of a high-affinity fluorescent PPARgamma ligand for high-throughput fluorescence polarization assays
Michael J DeGrazia1, Jerry Thompson, John P Vanden Heuvel
1Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
Abstract:
Members of the peroxisome proliferator activated receptor (PPAR) family of transcription factors are under investigation as molecular targets for the treatment of numerous diseases including Alzheimer's, asthma, atherosclerosis, inflammation, multiple sclerosis, cancer, and diabetes. We employed the X-ray crystal structure of the PPARgamma subtype complexed with the potent small molecule agonist GI262570 (farglitazar) to design and synthesize a novel fluorescent and high-affinity probe for homogeneous and high-throughput fluorescent polarization (FP) assays. Examination of this X-ray structure revealed that the phenyl carbon atom meta to the oxazole moiety of GI262570 is exposed to solvent at the bottom of a narrow protein cavity. A derivative of GI262570 was synthesized bearing a linear phenylacetylene-derived side chain comprising propargylamine coupled to fluorescein. This fluorescent analogue was designed to project the fluorophore into the adjacent protein cavity with minimal effects on receptor affinity and maximal effects on fluorescence polarization properties. The recombinant PPARgamma ligand binding domain protein bound tightly and specifically to this probe with Kd=61+/-14 nM as determined by FP measurements. Competition binding assays with known PPARgamma ligands provided Ki values that were highly correlated with analogous values obtained by scintillation proximity (SP) assays. This fluorescent PPARgamma probe enables high-throughput and homogenous FP assays for the identification of novel endogenous and exogenous PPARgamma ligands, and this rational ligand design approach may be applied to other therapeutically important members of the nuclear hormone receptor superfamily.
Insights
Researchers developed a novel fluorescent probe for Peroxisome proliferator activated receptor gamma (PPARγ) to enable high-throughput screening for new drug candidates targeting diseases like diabetes and cancer.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Peroxisome proliferator activated receptor (PPAR) family members are key targets for treating diseases including cancer and diabetes.
- Understanding PPARγ ligand interactions is crucial for drug development.
Purpose of the Study:
- To design and synthesize a novel fluorescent probe for PPARγ.
- To enable homogeneous and high-throughput fluorescent polarization (FP) assays for PPARγ ligand screening.
Main Methods:
- Utilized X-ray crystal structure of PPARγ complexed with GI262570 (farglitazar).
- Synthesized a fluorescent analogue of GI262570 with a fluorescein moiety.
- Determined binding affinity (Kd) using FP assays and validated with competition binding assays.
Main Results:
- The novel fluorescent probe exhibited high affinity for recombinant PPARγ ligand binding domain (Kd=61+/-14 nM).
- FP assay results strongly correlated with scintillation proximity (SP) assays.
- The probe facilitated specific and tight binding to PPARγ.
Conclusions:
- A novel, high-affinity fluorescent probe for PPARγ was successfully developed.
- This probe enables efficient, high-throughput screening for PPARγ ligands.
- The rational design approach can be applied to other nuclear hormone receptors.