Tina M. Hallis

Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening

The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug-drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z-factors of >or=0.5. Seven- to 15-point concentration-response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format.

Insights into the Branched‐Chain Formation of Mycarose: Methylation Catalyzed by an (S)‐Adenosylmethionine‐Dependent Methyltransferase

A C-methyltransferase involved in methyl-branch formation in sugars has been characterized for the first time. TylC3, an (S)-adenosylmethylthionine(AdoMet)-dependent enzyme, catalyzes the attachment of a methyl branch [Eq. (1)] in the biosynthesis of L-mycarose, an unusual sugar found in tylosin and as its O-3-methyl derivative in erythromycin. The C-3 methylation proceeds with inversion of configuration and does not require the assistance of any cofactors. The turnover rate is 1.4±0.1 min-1 . TDP=thymidine-5-dihydrogenphosphate.

Fluorescent cascade and direct assays for characterization of RAF signaling pathway inhibitors.

RAF kinases are part of a conserved signaling pathway that impacts cell growth, differentiation, and survival, and RAF pathway dysregulation is an attractive target for therapeutic intervention. We describe two homogeneous fluorescent formats that distinguish RAF pathway inhibitors from direct RAF kinase inhibitors, using B-RAF, B-RAF V599E, and C-RAF. A Förster-resonance energy transfer (FRET) based method was used to develop RAF and MEK cascade assays as well as a direct ERK kinase assay. This method uses a peptide substrate, that is terminally labeled with a FRET-pair of fluorophores, and that is more sensitive to proteolysis relative to the phosphorylated peptide. A second time-resolved FRET-based assay using fluorescently labeled MEK substrate was used to detect direct inhibitors of RAF kinase activity. The cascade assays detect compounds that interact with activated and unactivated kinases within the recapitulated RAF pathway, and the direct assays isolate the point of action for an inhibitor.

Characterisation of a sugar epimerase enzyme involved in the biosynthesis of a vancomycin-group antibiotic

An enzyme involved in the biosynthesis of the n4-epi-vancosamine substituents of a vancomycin group antibiotic nhas been expressed and its role as a nTDP-4-keto-6-deoxyglucose-3,5-epimerase demonstrated.

Stereospecificity of hydride transfer for the catalytically recycled NAD+ in CDP-d-glucose 4,6-dehydratase

Abstract CDP-D-glucose 4,6-dehydratase (E od ), found in the biosynthetic pathway of 3,6-dideoxysugars, contains a tightly bound NAD + that is recycled during catalysis. The stereochemical preference of the hydride transfer to and from the coenzyme in E od was determined to be pro-S by analyzing the NAD + produced when the apoenzyme was incubated with stereospecifically labeled NADH and its product, CDP-6-deoxy-D- glycero -L- threo -4-hexulose.

Development and Validation of a Cell-Based Assay for the Nuclear Receptor Retinoid-Related Orphan Receptor Gamma

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at μM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.