A Flexible Workflow for Automated Bioluminescent Kinase Selectivity Profiling. (April 2017)

• Record Type: Journal Article
• Authors: Worzella, Tracy; Butzler, Matt; Hennek, Jacquelyn; Hanson, Seth; Simdon, Laura; Goueli, Said; Cowan, Cris; Zegzouti, Hicham
• Is Part Of: SLAS technology. Volume 22:Number 2(2017)
• Page Start: 153
• Page End: 162
• Publication Date: 2017-04
• Subjects: kinase profiling -- bioluminescence -- ADP detection -- selectivity profiles -- liquid handling; Medical laboratory technology -- Periodicals; Laboratories -- Equipment and supplies -- Periodicals; Diagnosis, Laboratory -- Periodicals; 616.075
• DOI: 10.1177/2211068216677248
• Languages: English
• ISSNs: 2472-6303

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.