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.

A multi-year longitudinal study of water quality parameters in four salmon-bearing and recreational streams on mount hood, Oregon.

Four streams-Clear Fork, Lost Creek, Camp Creek and Still Creek-in northwestern Oregon's Sandy River Basin were monitored for temperature, dissolved oxygen levels, and fecal bacterial concentrations in a multi-year analysis examining stream health for recreational users and anchor habitat for Pacific Salmon. Temperatures were recorded using micro -T temperature loggers at 15 locations, during 22 July - 5 September 2006, 2 July - 4 September 2007, 20 June - 7 September 2008, 23 June - 9 September 2009, and 2 July -9 September 2010. The Seven-Day Average Maximum water temperature (7-DAM) of 13°C was used as a reference value for the biological limit governing suitable salmonid spawning and egg incubation conditions. The maximum 7-DAM temperatures occurred on different dates and all streams neared or exceeded the 13°C standard at least once each summer. Dissolved oxygen levels were measured at weekly or longer intervals in 2006, 2007, 2008, and 2009. Dissolved oxygen levels fell below the 9.0 ppm standard for Clear Fork on almost half the sampling dates in 2006, 2007, and 2009. Concentrations of the bacterial genus Enterococcus were measured as an indicator of fecal contamination. Samples were collected at 15 sites along the four streams. Weekly samples were collected during a 9 week period from July - September 2007, an 11 week period from June - September 2008, and an 11 week period from June - September 2009. Enterococcus counts exceeded the federal recommended national criterion value of 61 colony forming units (CFU) per 100 mL every year in Camp Creek and occasionally elsewhere, with exceedances trending towards late summer.

HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.