Abstract

Introducing FLIPR Tetra as a Novel Application for Capturing High-Throughput Biochemical Kinetic Data
Lisa McWilliams and Puneet Khurana
Mechanistic Biology & Profiling, Discovery Sciences, R&D, Astrazeneca, Cambridge, UK


Within the Mechanistic Biology and Profiling (MBP) Department of AstraZeneca’s Discovery Sciences organization, we recognize the importance of kinetic biochemical data even at the early phase of drug discovery. Traditionally, post high throughput screening, Structure Activity Relationship (SAR) data has been generated by measuring IC50 values as a measure of biochemical efficacy. We believe that solely using potency to drive SAR leads to excluding structures that could be developed and would have better overall drug properties. Understanding the kinetics of how compounds interact with the biological target is critical information to guide selection of the best leads from a campaign.

The challenge in earlier phases of a Drug Discovery is that there are compounds from multiple series requiring profiling, however, generating kinetic data on large numbers of compounds is extremely challenging using standard photomultiplier tube (PMT) readers. PMT readers typically read only a small number of wells on the plate at a time then move on to the next wells, this means that for kinetic studies it usually takes around 1.5 minutes to read the whole 384 well plate before starting again on the next timepoint. The problem is even greater for higher density microtiter plates, a full 1536 plate would take roughly 6 mins per cycle on standard readers. Biochemical kinetic characterization of inhibitors requires quantitative measure of enzyme activity at very small-time intervals, and therefore standard readers are limited to low throughput kinetic studies.

In contrast, the CCD camera within FLIPR captures whole plate level data per read which reduces the time interval to as low as 1 sec per read irrespective of the plate type, 384 or 1536. Using a tyrosine kinase as a model system, we have validated a novel application of FLIPR for delivering 384-plate based biochemical kinetic assays offering many folds increase in data throughput. The throughput is compatible with Structure Kinetic Relationship based progress during Hit to Lead stage, facilitating mechanistically driven medicinal chemistry along with traditional SAR based approach.