A high-capacity assay for PPARgamma ligand regulation of endogenous aP2 expression in 3T3-L1 cells

A novel class of insulin-sensitizing agents, the thiazolidinedines (TZDs), has proven effective in the treatment of type 2 diabetes. These compounds, as well as a subclass of non-TZD insulin-sensitizing agents, have been shown to be peroxisome proliferator-activated receptor (PPAR) gamma agonists. PPARgamma plays a critical role in adipogenesis and PPARgamma agonists have been shown to induce adipocyte differentiation. Here, PPARgamma ligand activity has been assessed in murine 3T3-L1 cells, a commonly used in vitro model of adipogenesis, by measuring their ability to induce adipocyte fatty acid-binding protein (aP2) mRNA expression. In order to perform this task, we have developed a novel, multiwell assay for the direct detection of aP2 mRNA in cell lysates that is based on hybridization of mRNA to target-specific oligonucleotides. These oligonucleotide probes are conjugated to enzymes that efficiently process unique chemical substrates into robust fluorescent products. Ribosomal protein 36B4 mRNA, a gene whose expression is unaffected by adipogenesis, serves as the control in the assay. Two assay formats have been developed, a single analyte assay in which aP2 and 36B4 mRNA expression are assayed in separate lysate aliquots and a dual analyte assay which can measure aP2 and 36B4 mRNA simultaneously. Both forms of the assay have been used to quantify attomole levels of aP2 and 36B4 mRNAs in differentiating 3T3-L1 preadipocytes treated with PPARgamma agonists. The potencies of PPARgamma agonists determined by this novel methodology showed good correlation with those derived from aP2 mRNA slot-blot analysis and PPARgamma transactivation assays. We conclude that the aP2 single and dual analyte assays both provide specific and sensitive measurements of endogenous aP2 mRNA levels that can be used to assess the activity of PPARgamma ligands in 3T3-L1 cells. Since the assay obviates the need for RNA isolation and is performed in an automatable multiwell format, it can serve as a high-throughput, cell-based screen for the identification and characterization of PPARgamma modulators.     

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.     

Design,synthesis, and evaluation of simple phenol amides as ERRγ agonists

Herein we report the design and synthesis of a series of simple phenol amide ERRγ agonists based on a hydrazone lead molecule. Our structure activity relationship studies in this series revealed the phenol portion of the molecule to be required for activity. Attempts to replace the hydrazone with more suitable chemotypes led to a simple amide as a viable alternative. Differential hydrogen-deuterium exchange experiments were used to help understand the structural basis for binding to ERRγ and aid in the development of more potent ligands.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

牛红细胞超氧化物歧化酶活性中心的紫外光谱研究

应用差示分光光度法研究了牛红细胞Ｃｕ２Ｚｎ２ＳＯＤ的紫外光谱,归属和讨论了酶活性中心金属离子与配体间全部电荷转移谱带,给出了相应的配体轨道光学电负性,特别研究了涉及Ｚｎ２＋的电荷转移谱带．     

Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor

Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms.     

The azatryptophan-based fluorescent platform for in vitro rapid screening of inhibitors disrupting IKKβ-NEMO interaction

The nuclear factor-κB (NF-κB) plays an important role in inflammatory and immune responses. Aberrant NF-κB signaling is implicated in multiple disorders, including cancer. Targeting the regulatory scaffold subunit IκB kinase γ (IKKγ/NEMO) as therapeutic interventions could be promising due to its specific involvement in canonical NF-κB activation without interfering with non-canonical signaling. In this study, the use of unnatural amino acid substituted IKKβ with unique photophysical activity to sense water environment changes upon interaction with NEMO provides a powerful in vitro screening platform that would greatly facilitate the identification of compounds having the potential to disrupt IKKβ-NEMO interaction, and thus specifically modulate the canonical NF-κB pathway. We then utilized a competitive binding platform to screen the binding ability of a number of potential molecules being synthesized. Our results suggest that a lead compound (−)-PDC-099 is a potent agent with ascertained potency to disrupt IKKβ-NEMO complex for modulating NF-κB canonical pathway.     

Solubilization, identification, and localization of vitellogenin-binding sites in follicles of the cockroach, Nauphoeta cinerea

Binding sites for vitellogenin were solubilized and analyzed either with a filter assay or with ligand blotting. We tested sodium dodecylsulfate (SDS), Chaps, octyl-beta-D-glucoside, and sodium deoxycholate and found SDS and sodium deoxycholate to be most effective in solubilizing both high and low molecular weight binding sites. In the filter assay the sodium deoxycholate extracts, but not the SDS extracts, maintained binding activity after dilution of the solubilizer below its critical micellar concentration. In ligand blotting we consistently observed, in vitellogenic follicles, binding sites with an apparent Mr of approximately 200,000, 35,000, and three closely spaced bands between 14,000 and 20,000. Binding of vitellogenin to all binding sites was suppressed in the presence of the drug suramin. Analysis of corpora lutea and oothecae as well as of ovariole sheath, follicle cell/basal lamina, and oocyte plasma membrane preparations showed that the 35 and 14-20 kDa binding sites are located in the outer follicle compartments, and the 200 kDa binding site in the oocyte plasma membrane. In the latter we occasionally also observed binding sites with an apparent Mr of approximately 150,000, 95,000, 67,000 and 30,000, particularly at stages after ovulation. The 35 and 14-20 kDa binding sites, as visualized in stained gels and in ligand blotting, are rather abundant and were also seen in several other male and female tissues of Nauphoeta and even in other species. They also bound other 14C-labeled hemolymph proteins and thus appear to be rather unspecific. As binding analysis with nonsolubilized and sodium deoxycholate-solubilized membranes revealed that the quantity of vitellogenin bound by binding sites of the outer follicle compartments was low, it is conceivable that the abundance of the 14-20 kDa and 35 kDa binding sites in ligand blotting is merely an effect of SDS and does not reflect the in vivo situation. We suppose that the 200 kDa binding site of the oocyte plasma membrane represents the vitellogenin receptor involved in endocytosis.     

Immobilized ligands of high stability: Hydroxyalkyl methacrylate gel conjugates with oligo- and polypeptides

The long-time stability of conjugates prepared from epoxy derivatives of hydroxyalkyl methacrylate gels and peptides or proteins has been investigated at various pH values. These conjugates were found to be extremely stable. The observed slow release of nitrogen is due mainly to a splitting of the peptide bond adjacent to the covalent bond anchoring the peptide to the matrix. This peptide bond is partially labilized by microenvironmental influences of the matrix. Since in all experiments only a fraction of the immobilized ligand became detached, it is suggested that there is a subpopulation of fixed ligands which are susceptible to a matrix-induced bond splitting.