Robert X. Xu

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments

SummaryA general approach for assigning the resonances of uniformly 15N- and 13C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The 1H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D 15N-edited 1H, 1H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete 13C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the 1H, 13C, and 15N resonances of the FK506 binding protein denatured in 6.3 M urea.

Side chain and backbone assignments in isotopically labeled proteins from two heteronuclear triple resonance experiments

Two multidimensional heteronuclear NMR experiments are described for assigning the resonances in uniformly 15N‐ and 13C‐labeled proteins. In one experiment (HCNH‐TOCSY), the amide nitrogen and proton are correlated to the side‐chain protons and carbons of the same and preceding residue. In a second triple resonance experiment (HC(CO)NH‐TOCSY), the amide nitrogen and proton of one residue is correlated exclusively with the side‐chain proton and carbon resonances of the preceding residue by transferring magnetization through the intervening carbonyl. The utility of these two experiments for making sequential resonance assignments in proteins is illustrated for [U‐15N, 13C]FKBP (107 residues) complexed to the immunosuppressant, ascomycin.

Crystallization of protein-ligand complexes.

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals.

A 4D HCCH-TOCSY experiment for assigning the side chain1H and13C resonances of proteins

SummaryA 4D HCCH-TOCSY experiment is described for correlating and assigning the1H and13C resonances of protein amino acid side chains that has several advantages over 3D versions of the experiment. In many cases, both the1H and13C chemical shifts can be obtained in the 4D experiment from a simple inspection of the13C(ω3),1H(ω4) planes extracted at the1Hα(ω1)/13Cα(ω2) chemical shifts. Together with the 3D and 4D triple resonance experiments, this allows sequence-specific assignments to be obtained. In addition, the increased resolution of the 4D experiment compared to its 3D counterpart allows. automation of resonance assignments.

Identification and Characterization of 4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist

4-Chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide 3 (GSK3787) was identified as a potent and selective ligand for PPARdelta with good pharmacokinetic properties. A detailed binding study using mass spectral analysis confirmed covalent binding to Cys249 within the PPARdelta binding pocket. Gene expression studies showed that pyridylsulfone 3 antagonized the transcriptional activity of PPARdelta and inhibited basal CPT1a gene transcription. Compound 3 is a PPARdelta antagonist with utility as a tool to elucidate PPARdelta cell biology and pharmacology.

Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).

The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure-activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.

Stereospecific assignments and χ1 rotamers for FKBP when bound to ascomycin from 3JHα,Hβ and 3JN,Hβ coupling constants

3 J Hα,Hβ and 3 J N,Hβ coupling constants were measured for isotopically labeled FKBP when bound to the immunosuppressant, ascomycin, using a 1H‐coupled 3D HCCH‐TOCSY and 15N‐coupled 3D HSQC‐TOCSY experiment, respectively. From an analysis of these two sets of coupling constants, stereospecific β‐proton assignments and χ1 rotamers for FKBP have been obtained. All of the χ1 rotamers were consistent with the χ1 angles measured in the X‐ray crystal structure of the FKBP/FK506 complex, suggesting that the structures of the two complexes are similar.

Discovery of a novel class of PPARδ partial agonists

Anthranilic acid GW9371 was identified as a novel class of PPARdelta partial agonist through high-throughput screening. The design and synthesis of SAR analogues is described. GSK1115 and GSK7227 show potent partial agonism of the PPARdelta target genes CPT1a and PDK4 in skeletal muscle cells.

Discovery of GSK1997132B a novel centrally penetrant benzimidazole PPARγ partial agonist

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor, thought to play a role in energy metabolism, glucose homeostasis and microglia-mediated neuroinflammation. A novel benzimidazole series of centrally penetrant PPARγ partial agonists has been identified. The optimization of PPARγ activity and in vivo pharmacokinetics leading to the identification of GSK1997132B a potent, metabolically stable and centrally penetrant PPARγ partial agonist, is described.