Crystal Structures of the Human Peroxisome Proliferator-Activated Receptor (PPAR)α Ligand-Binding Domain in Complexes with a Series of Phenylpropanoic Acid Derivatives Generated by a Ligand-Exchange Soaking Method.

Peroxisome proliferator-activated receptor (PPAR)α, a member of the nuclear receptor family, is a transcription factor that regulates the expression of genes related to lipid metabolism in a ligand-dependent manner, and has attracted attention as a target for hypolipidemic drugs. We have been developing phenylpropaonic acid derivatives as PPARα-targeted drug candidates for the treatment of metabolic diseases. Recently, we have developed the "ligand-exchange soaking method," which crystallizes the recombinant PPARα ligand-binding domain (LBD) as a complex with intrinsic fatty acids derived from an expression host Escherichia (E.) coli and thereafter replaces them with other higher-affinity ligands by soaking. Here we applied this method for preparation of cocrystals of PPARα LBD with its ligands that have not been obtained with the conventional cocrystallization method. We revealed the high-resolution structures of the cocrystals of PPARα LBD and the three synthetic phenylpropaonic acid derivatives: TIPP-703, APHM19, and YN4pai, the latter two of which are the first observations. The overall structures of cocrystals obtained from the two methods are identical and illustrate the close interaction between these ligands and the surrounding amino acid residues of PPARα LBD. This ligand-exchange soaking method could be applicable to high throughput preparations of co-crystals with another subtype PPARδ LBD for high resolution X-ray crystallography, because it also crystallizes in complex with intrinsic fatty acid(s) while not in the apo-form.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

Expression and purification of the ligand-binding domain of peroxisome proliferator-activated receptor alpha (PPARalpha).

We describe the expression and purification of the C-terminally His-tagged ligand-binding domain of the peroxisome proliferator-activated receptor alpha (PPARalpha-LBD). Using a modified expression and purification strategy a five times higher protein yield was achieved compared to existing protocols. In summary, a modified Escherichia coli strain was used, which allows rare codons to be expressed more efficiently, and moreover, conditions for cell growth and cell lysis were improved. Protein purification was achieved in a two-step approach using nickel affinity chromatography followed by anion exchange chromatography. The identity of PPARalpha-LBD was confirmed by online-nano-high performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS).