DiscussionKinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activity. However, cost limitations prevent many of these profiling activities from being performed in-house. Our work aims to show that the adoption of ready-to-use kinase enzyme profiling systems allows researchers to quickly and efficiently profile their compounds of interest on-site, generating data on demand and on their timeline. The incorporation of easy to use, affordable liquid handling and detection instrumentation creates a streamlined workflow amenable to even the novice automation user.
Kinase Selectivity Profiling Systems from Promega include kinase and substrate pairs pre-dispensed in 8-tube strip format. Kinases are grouped in single kinase family strips or as a general panel representative of the human kinome. Buffer and ATP are added to the respective strip tubes to create optimized ready-to-assay working solutions. The ADP-Glo™ Kinase Assay technology is used to quantify kinase activity. Programs developed on a Gilson PIPETMAX® liquid handling platform replicate standardized profiling assay plate layouts for single and multi-dose screening. Variables within each automation protocol are used to guide assay assembly, creating a flexible experimental workflow based on user provided parameters such as the number of compounds being profiled, titration increment, and number of kinase families studied. A GloMax® Discover microplate reader preloaded with a SMART Kinase Selectivity Profiling Systems protocol was used for luminescence detection and automatic data analysis. We put the automated profiling workflow into practice by first testing ten compounds in single dose format against the general kinase panel. Compound hits that exhibited < 20% kinase activity were assayed in multi-point dose response format against the respective single kinase families. Compound potencies against the original general kinase panel hit were compared to potencies against other relevant kinases within each family.