High-throughput biochemical kinase selectivity assays: panel development and screening applications.

Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles.

Identification and SAR around N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, a selective alpha2C adrenergic receptor antagonist.

The discovery of the CNS-penetrant and selective alpha(2C) adrenergic receptor antagonist N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, 13 is described. Structure-activity studies demonstrate the structural requirements for binding affinity, functional activity, and selectivity over other alpha(2)-AR subtypes.

Multiplexed G-protein-coupled receptor Ca2+ flux assays for high-throughput screening.

An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined as the screening of multiple targets within the same experiment, is an example of this strategy. Here, the authors describe a technique that allows multiplexing of a common assay type used to study G-protein-coupled receptors: changes in intracellular Ca2+ levels as measured by Molecular Device's fluorometric imaging plate reader (FLIPR). The multiplexed FLIPR assays showed the expected pharmacological properties of single assays, with good reproducibility and Z* factors. The authors used them to screen large compound libraries in 2 multiplexed assay designs. The 1st used a single-cell line expressing 2 different receptors and the 2nd a mixture of 2 cell lines of the same type each expressing distinct receptors. Screening using these multiplexed assays produced significant savings in reagents, time, and human resources and allowed the authors to quickly identify specific and selective hits.

Monitoring changes in gene expression in renal ischemia-reperfusion in the rat.

BACKGROUND: Although acute renal failure (ARF) is a relatively common disorder with major morbidity and mortality, its molecular basis remains incompletely defined. The present study examined global gene expression in the well-characterized ischemia-reperfusion model of ARF using DNA microarray technology. METHODS: Male Wistar rats underwent bilateral renal ischemia (30 min) or sham operation, followed by reperfusion for 1, 2, 3 or 4 days. Plasma creatinine increased approximately fivefold over baseline, peaking on day 1. Renal total RNA was used to probe cDNA microarrays. RESULTS: Alterations in expression of 18 genes were identified by microarray analysis. Nine genes were up-regulated (ADAM2, HO-1, UCP-2, and thymosin beta4 in the early phase and clusterin, vanin1, fibronectin, heat-responsive protein 12 and FK506 binding protein in the established phase), whereas another nine were down-regulated (glutamine synthetase, cytochrome p450 IId6, and cyp 2d9 in the early phase and cyp 4a14, Xist gene, PPARgamma, alpha-albumin, uromodulin, and ADH B2 in the established phase). The identities of these 18 genes were sequence-verified. Changes in gene expression of ADAM2, cyp2d6, fibronectin, HO-1 and PPARgamma were confirmed by quantitative real-time polymerase chain reaction (PCR). ADAM2, cyp2d6, and PPARgamma have not previously been known to be involved in ARF. CONCLUSION: Using DNA microarray technology, we identified changes in expression of 18 genes during renal ischemia-reperfusion injury in the rat. We confirmed changes in five genes (fibronectin, ADAM2, cyp 2d6, HO-1 and PPARgamma) by quantitative real-time PCR. Several genes, not previously been identified as playing a role in ischemic ARF, may have importance in this disease.