title: A Flexible Workflow for Automated Bioluminescent Kinase Selectivity Profiling.
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.
authors.affiliation: 1 Promega Corporation, R&D Department, Madison, WI, USA.
authors.forename: Tracy
authors.initials: T
authors.lastname: Worzella
authors.affiliation: 1 Promega Corporation, R&D Department, Madison, WI, USA.
authors.forename: Matt
authors.initials: M
authors.lastname: Butzler
authors.affiliation: 1 Promega Corporation, R&D Department, Madison, WI, USA.|3 Exact Sciences Corporation, Madison, WI, USA.
authors.forename: Jacquelyn
authors.initials: J
authors.lastname: Hennek
authors.affiliation: 2 Gilson, Inc., Middleton, WI, USA.
authors.forename: Seth
authors.initials: S
authors.lastname: Hanson
authors.affiliation: 2 Gilson, Inc., Middleton, WI, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Simdon
authors.affiliation: 1 Promega Corporation, R&D Department, Madison, WI, USA.
authors.forename: Said
authors.initials: S
authors.lastname: Goueli
authors.affiliation: 1 Promega Corporation, R&D Department, Madison, WI, USA.
authors.forename: Cris
authors.initials: C
authors.lastname: Cowan
authors.affiliation: 1 Promega Corporation, R&D Department, Madison, WI, USA.
authors.forename: Hicham
authors.initials: H
authors.lastname: Zegzouti
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D010770:Phosphotransferases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D057188:Workflow
chemical_list: D004791:Enzyme Inhibitors; D010770:Phosphotransferases
keywords: ADP detection; bioluminescence; kinase profiling; liquid handling; selectivity profiles