Abstract
Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activities. However, cost and logistical limitations prevent profiling activities from being performed in-house. We describe the development of an automated and flexible kinase profiling workflow that combines ready-to-use kinase enzymes and substrates in convenient eight-tube strips, a bench-top liquid handling device, ADP-Glo Kinase Assay (Promega, Madison, WI) technology to quantify enzyme activity, and a multimode detection instrument. Automated methods were developed for kinase reactions and quantification reactions to be assembled on a Gilson (Middleton, WI) PIPETMAX, following standardized plate layouts for single- and multidose compound profiling. Pipetting protocols were customized at runtime based on user-provided information, including compound number, increment for compound titrations, and number of kinase families to use. After the automated liquid handling procedures, a GloMax Discover (Promega) microplate reader preloaded with SMART protocols was used for luminescence detection and automatic data analysis. The functionality of the automated workflow was evaluated with several compound-kinase combinations in single-dose or dose-response profiling formats. Known target-specific inhibitions were confirmed. Novel small molecule-kinase interactions, including off-target inhibitions, were identified and confirmed in secondary studies. By adopting this streamlined profiling process, researchers can quickly and efficiently profile compounds of interest on site.