Tuesday, July 14, 2009

Automated Kinase Profiling using a Simple and Robust Automated Platform

Kinases continue to be one of the most important targets in today’s drug discovery efforts. Following the identification of lead compounds through screening efforts, it is important to profile these leads against other kinases within that family, as well as from other families, to ascertain potential off-target effects. Typically profiling efforts include testing multiple concentrations of lead compounds in an IC50 format. Demands on a liquid handler can include performing compound titrations, as well as dispensing multiple kinase/substrate and ATP mixes.

In this application, we used the Precision™ XS Microplate Sample Processor and Synergy MX Monochromator-Based Multi-Mode Microplate Reader, in combination with Promega’s ADP-Glo™ Kinase Assay, to demonstrate automated kinase profiling. The Precision was used to perform compound titrations, as well as dispense all kinase and ADP-Glo reaction components to the wells of the assay plates. Titrations were carried out in 96-well format, with transfer volumes ranging from 20-40 μL. Kinase reactions required three 5 μL dispenses of inhibitor/DMSO, enzyme/substrate mix, and ATP. ADP-Glo reagent dispenses were set at 15 μL, and Kinase Detection Reagent dispenses at 30 μL.

Wortmannin was used as a model lead compound profiled against a series of different kinases. This compound, being a specific inhibitor for PI3 Kinase, generates IC50 values typically in the low nM range for this kinase. At higher concentrations, this compound has also shown inhibitory effects on DNA PK. Results agreed with literature values, with IC50s for PI3 Kinase Alpha and DNA PK being 0.15 and 7.1 nM, respectively. Wortmannin showed little or no effect on the remaining protein kinases.

Wortmannin profiling data

Where do you see kinase profiling demands going in the future? What type of instrumentation do you feel is essential for automated kinase profiling? How can BioTek help you meet your kinase profiling needs?

By: BioTek Instruments

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