Covalent peroxisome proliferator-activated receptor gamma adduction by nitro-fatty acids: selective ligand activity and anti-diabetic signaling actions.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events.

Characterization of a novel non-steroidal glucocorticoid receptor antagonist.

Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K(i)=13.2nM), was identified in a high-throughput screening effort.

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.

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.

An improved beta-lactamase reporter assay: multiplexing with a cytotoxicity readout for enhanced accuracy of hit identification.

A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a beta-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC(50) values of 40 select compounds in a beta-lactamase reporter assay for nuclear factor kappa B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator.

The biosynthesis of GDP-L-colitose: C-3 deoxygenation is catalyzed by a unique coenzyme B6-dependent enzyme.

l-Colitose (1) is a 3,6-dideoxyhexose found in the O-antigen of gram-negative lipopoly-saccharides. While the biosynthesis of many deoxysugars have previously been investigated, l-colitose is distinct in that it originates from GDP-d-mannose. In contrast, other 3,6-dideoxyhexoses arise from CDP-d-glucose.

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.