Discovery and preliminary evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin receptor antagonists.

The discovery and evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin (IP) receptor antagonists is described. Analogs disclosed showed high affinity for the IP receptor in human platelet membranes with IC50 values of 0.05-0.50 microM, demonstrated functional antagonism by inhibiting cAMP production in HEL cells with IC50 values of 0.016-0.070 microM, and exhibited significant selectivity versus other prostanoid receptors.

1,536-well assay development and screening using whole cell displacement binding and laser scanning imaging.

A screen of a GPCR against Pharmacopeia's combinatorial libraries was performed using 1,536-well plates in a 1.5-microl assay volume with an LSI that was specially modified to enable detection at these volumes. The screen encompassed approximately 4 x 10(6) compounds. The assay uses a CHO cell line that expresses human CXCR1. The plate format chosen was the Corning 1536 low-profile wafer plate. The performance of the screen is evaluated, and the necessity to obtain cytotoxicity data from the same well is described.

Evaluation of compound interference in immobilized metal ion affinity-based fluorescence polarization detection with a four million member compound collection.

IMAP is a non-separation-based, antibody-independent, FP assay that can be applied to many types of protein kinases and phosphatases. This technology is currently being used in many high-throughput screening campaigns throughout the industry. In this technology, a fluorescently labeled peptide substrate is phosphorylated and then captured on immobilized metal (M(III)) nanoparticles, an interaction that is enhanced at low pH (pH 5.5). The binding of the phosphorylated peptide to the nanoparticles is detected using FP. IMAP differs from other FP formats in that the polarization signal is antibody-independent and involves metal coordination complexes detected at low pH. Here, this technology is evaluated against a 4000000-member compound collection using a 1536-well assay design that is devoid of enzymes so that only interference of the compounds with the detection system is measured. Miniaturization of the assay to 1536-well plates is discussed. Compound interference due to inhibition of phosphopeptide binding to the M(III) nanoparticles is not observed. Additionally, it is concluded that the level of fluorescence compound interference is similar to typical FP formats for the majority of the compound collection.