A new class of peroxisome proliferator-activated receptor agonists with a novel binding epitope shows antidiabetic effects.

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the NR1 subfamily of nuclear receptors. The PPARs play key roles in the control of glucose and lipid homeostasis, and the synthetic isoform-specific PPAR agonists are used clinically to improve insulin sensitivity and to lower serum triglyceride levels. All of the previously reported PPAR agonists form the same characteristic interactions with the receptor, which have been postulated to be important for the induction of agonistic activity. Here we describe a new class of PPARalpha/gamma modulators, the 5-substituted 2-benzoylaminobenzoic acids (2-BABAs). As shown by x-ray crystallography, the representative compounds BVT.13, BVT.762, and BVT.763, utilize a novel binding epitope and lack the agonist-characteristic interactions. Despite this, some compounds within the 2-BABA family are potent agonists in a cell-based reporter gene assay. Furthermore, BVT.13 displays antidiabetic effects in ob/ob mice. We concluded that the 2-BABA binding mode can be used to design isoform-specific PPAR modulators with biological activity in vivo.

Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation.

The nuclear receptor heterodimers of liver X receptor (LXR) and retinoid X receptor (RXR) are key transcriptional regulators of genes involved in lipid homeostasis and inflammation. We report the crystal structure of the ligand-binding domains (LBDs) of LXRalpha and RXRbeta complexed to the synthetic LXR agonist T-0901317 and the RXR agonist methoprene acid (Protein Data Base entry 1UHL). Both LBDs are in agonist conformation with GRIP-1 peptides bound at the coactivator binding sites. T-0901317 occupies the center of the LXR ligand-binding pocket and its hydroxyl head group interacts with H421 and W443, residues identified by mutational analysis as critical for ligand-induced transcriptional activation by T-0901317 and various endogenous oxysterols. The topography of the pocket suggests a common anchoring of these oxysterols via their 22-, 24- or 27-hydroxyl group to H421 and W443. Polyunsaturated fatty acids act as LXR antagonists and an E267A mutation was found to enhance their transcriptional inhibition. The present structure provides a powerful tool for the design of novel modulators that can be used to characterize further the physiological functions of the LXR-RXR heterodimer.

A triple mutant of the Drosophila ERR confers ligand-induced suppression of activity.

The steroid hormone (NR3) subfamily of nuclear receptors was until recently believed to be restricted to deuterostomes. However, a novel nuclear receptor belonging to the NR3 subfamily was recently identified in the Drosophila melanogaster genome, indicating the existence of an ancestor before the evolutionary split of deuterostomes and protostomes. This receptor, termed the Drosophila estrogen-related receptor (dERR), most closely resembles the human and mouse estrogen-related receptors (ERRs) in both the DNA binding domain (DBD) (approximately 85% identical) and the ligand binding domain (LBD) (approximately 35% identical). Here we describe the functional analysis and rational design of ligand responsive dERR mutants created by protein engineering of the LBD. On the basis of homology modeling, three amino acid residues in the LBD were identified and mutated to enable ligand-dependent suppression of transcriptional activity. Our results show that the Y295A/T333I/Y365L triple mutant is significantly suppressed by the known ERR inverse agonists 4-hydroxytamoxifen (OHT) and diethylstilbestrol (DES), in comparison to the wild-type dERR receptor, which was inefficiently suppressed by these substances. The coactivator mGRIP-1 (mouse glucocorticoid receptor interacting protein 1) was shown to significantly increase the activity of the triple mutant in transfection experiments, and the addition of OHT resulted in an efficient suppression of the activity. Accordingly, the ability to functionally interact with a coactivator is still maintained by the Y295A/T333I/Y365L mutant. These findings demonstrate the potential of using rational design and engineering of the LBD to study the function of a nuclear receptor lacking identified ligands.

Synthesis and pharmacological evaluation of a new class of peroxisome proliferator-activated receptor modulators.

A series of 5-substituted 2-benzoylaminobenzoic acids has been synthesized and assayed for PPARalpha/gamma activity. Both dual activators and selective PPARgamma agonists have been identified. This class of compounds was shown to activate the PPARgamma receptor through interaction with a novel binding site.

Identification of residues in the PXR ligand binding domain critical for species specific and constitutive activation.

The cytochrome P450 family of enzymes has long been known to metabolize a wide range of compounds, including many of today's most common drugs. A novel nuclear receptor called PXR has been established as an activator of several of the cytochrome P450 genes, including CYP3A4. This enzyme is believed to account for the metabolism of more than 50% of all prescription drugs. PXR is therefore used as a negative selector target and discriminatory filter in preclinical drug development. In this paper we describe the design, construction and characterization by transient transfection of mutant receptors of the human and mouse PXR ligand binding domains. By modeling the human PXR ligand binding domain we have identified and mutated two polar residues in the putative ligand binding pocket which differ between the human and the mouse receptor. The first residue (Q285 in human/I282 in mouse) was mutated between the two species with the corresponding amino acids. These mutants showed that this residue is important for the species specific activation of PXR by the ligand pregnenolone-16alpha-carbonitrile (PCN), while having a less pronounced role in receptor activation by rifampicin. The second residue to be mutated (H407 in human/Q404 in mouse) unexpectedly proved to be important for the basal level of activation of PXR. The H407A mutant of the human receptor showed a high level of constitutive activity, while the Q404H mutant of the mouse receptor demonstrated a sharply decreased basal activity compared to wild-type.