Tracy Worzella1, Gediminas Vidugiris1, Jolanta Vidugiriene1, Jacquelyn Turri1, Hicham Zegzouti1, Lyndsey Helley1, Jessica Merlino2, Michael Reitman2
1Promega Corporation, Madison WI; 2Tecan Group AG, Männedorf, Switzerland
Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activity. While automation is necessary to screen and identify putative inhibitors, it can also provide benefit for followup characterization studies where throughput is not a primary concern. Smaller, more cost-effective platforms are now available that offer the user programming flexibility and ease of use to carry out the tasks needed to profile their compounds of interest in-house. Combined with sensitive reagents, these instruments comprise a semi-automated workflow for lower throughput kinase profiling applications.
We explored this concept by combining complete kinase enzyme systems with smaller footprint laboratory instrumentation platforms for non-contact compound delivery, assay setup, and signal detection. Kinase enzyme systems (KES) from Promega were used as the source of enzymes for this project. KES are comprised of kinases, substrates, buffers and co-factors supplied with optimized reaction conditions for maximum performance out-of-the box. Kinase activity was measured using the ADP-Glo™ Assay, a luminescent assay where light production is directly proportional to ADP produced in the kinase reaction. The HP D300 Digital Dispenser, available from Tecan, was used to generate assay-ready-plates of titrated compounds dispensed in volume ranges from 300nL down to 13pL. A Hudson SOLO™ multichannel pipettor was used to assemble kinase reactions in standard and low-volume 384-well microplates, as well as perform the ADP-Glo™ Assay. A Promega GloMax® or Tecan Safire2 multimode plate reader was used to quantify luminescence.
Comparative studies were performed to assess the impact of compound replicate number on IC50 determination. Inhibitor titrations were performed with the HP D300 using from one to four replicates per concentration. Results show potencies that are in close agreement between the different titration methods. Small-scale proof-of-concept profiling studies were then conducted to assess selective and off-target inhibitory effects of compounds against small panels of kinases that were assembled together in an easy to use 8 well-strip format, including receptor tyrosine kinase and lipid kinase families.