Conformationally constrained farnesoid X receptor (FXR) agonists: alternative replacements of the stilbene.

To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.

Conformationally constrained farnesoid X receptor (FXR) agonists: heteroaryl replacements of the naphthalene.

To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.

GSK4112, a small molecule chemical probe for the cell biology of the nuclear heme receptor Rev-erbα.

The identification of nonporphyrin ligands for the orphan nuclear receptor Rev-erbα will enable studies of its role as a heme sensor and regulator of metabolic and circadian signaling. We describe the development of a biochemical assay measuring the interaction between Rev-erbα and a peptide from the nuclear receptor corepressor-1 (NCoR). The assay was utilized to identify a small molecule ligand for Rev-erbα, GSK4112 (1), that was competitive with heme. In cells, 1 profiled as a Rev-erbα agonist in cells to inhibit expression of the circadian target gene bmal1. In addition, 1 repressed the expression of gluconeogenic genes in liver cells and reduced glucose output in primary hepatocytes. Therefore, 1 is useful as a chemical tool to probe the function of Rev-erbα in transcriptional repression, regulation of circadian biology, and metabolic pathways. Additionally, 1 may serve as a starting point for design of Rev-erbα chemical probes with in vivo pharmacological activity.

Substituted isoxazole analogs of farnesoid X receptor (FXR) agonist GW4064.

Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.

Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064.

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.

Automated in-line gel filtration for native state mass spectrometry.

Characterization of protein-ligand complexes by nondenaturing mass spectrometry provides direct evidence of drug-like molecules binding with potential therapeutic targets. Typically, protein-ligand complexes to be analyzed contain buffer salts, detergents, and other additives to enhance protein solubility, all of which make the sample unable to be analyzed directly by electrospray ionization mass spectrometry. This work describes an in-line gel-filtration method that has been automated and optimized. Automation was achieved using commercial HPLC equipment. Gel column parameters that were optimized include: column dimensions, flow rate, packing material type, particle size, and molecular weight cut-off. Under optimal conditions, desalted protein ions are detected 4 min after injection and the analysis is completed in 20 min. The gel column retains good performance even after >200 injections. A demonstration for using the in-line gel-filtration system is shown for monitoring the exchange of fatty acids from the pocket of a nuclear hormone receptor, peroxisome proliferator activator-delta (PPARdelta) with a tool compound. Additional utilities of in-line gel-filtration mass spectrometry system will also be discussed.

Rev-erbalpha, a heme sensor that coordinates metabolic and circadian pathways.

The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. We show that heme binds reversibly to the orphan nuclear receptor Rev-erbalpha, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbalpha-mediated gene repression. Thus, Rev-erbalpha serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism.

Crystallization of protein-ligand complexes.

Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

Human PXR forms a tryptophan zipper-mediated homodimer.

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal beta-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface beta-strands, generating a 10-stranded intermolecular beta-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR's ability to interact with DNA, RXRalpha, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor's recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor.

Structural disorder in the complex of human pregnane X receptor and the macrolide antibiotic rifampicin.

The human nuclear xenobiotic receptor, pregnane X receptor (PXR), detects a variety of structurally distinct endogenous and xenobiotic compounds and controls expression of genes central to drug and cholesterol metabolism. The macrolide antibiotic rifampicin, a front-line treatment for tuberculosis, is an established PXR agonist and, at 823 Da, is one of the largest known ligands for the receptor. We present the 2.8 A crystal structure of the ligand-binding domain of human PXR in complex with rifampicin. We also use structural and mutagenesis data to examine the origins of the directed promiscuity exhibited by the PXRs across species. Three structurally flexible loops adjacent to the ligand-binding pocket of PXR are disordered in this crystal structure, including the 200-210 region that is part of a sequence insert novel to the promiscuous PXRs relative to other members of the nuclear receptor superfamily. The 4-methyl-1-piperazinyl ring of rifampicin, which would lie adjacent to the disordered protein regions, is also disordered and not observed in the structure. Taken together, our results indicate that one wall of the PXR ligand-binding cavity can remain flexible even when the receptor is in complex with an activating ligand. These observations highlight the key role that structural flexibility plays in PXR's promiscuous response to xenobiotics.

Helix 1/8 interactions influence the activity of nuclear receptor ligand-binding domains.

The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.

2.1 A crystal structure of human PXR in complex with the St. John's wort compound hyperforin.

The nuclear xenobiotic receptor PXR is activated by a wide variety of clinically used drugs and serves as a master regulator of drug metabolism and excretion gene expression in mammals. St. John's wort is used widely in Europe and the United States to treat depression. This unregulated herbal remedy leads to dangerous drug-drug interactions, however, in patients taking oral contraceptives, antivirals, or immunosuppressants. Such interactions are caused by the activation of the human PXR by hyperforin, the psychoactive agent in St. John's wort. In this study, we show that hyperforin induces the expression of numerous drug metabolism and excretion genes in primary human hepatocytes. We present the 2.1 A crystal structure of hyperforin in complex with the ligand binding domain of human PXR. Hyperforin induces conformational changes in PXR's ligand binding pocket relative to structures of human PXR elucidated previously and increases the size of the pocket by 250 A(3). We find that the mutation of individual aromatic residues within the ligand binding cavity changes PXR's response to particular ligands. Taken together, these results demonstrate that PXR employs structural flexibility to expand the chemical space it samples and that the mutation of specific residues within the ligand binding pocket of PXR tunes the receptor's response to ligands.

A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.

A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.

Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids.

The 2.7 A X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interacted with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, which was reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids did not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. Our results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids.