Traditionally, progress in the development of protease inhibitors has been hampered to a large degree by their limited selectivity. Therefore, the ability to efficiently profile compounds against a large panel of protease assays is important to successfully discover new generation protease drugs. In order to efficiently profile compounds within and across the various assay panels, we have developed a semi-automated parallel screening technology which is linked to a high capacity data analysis tool.

The compound profiling system as established in the Protease Platform is used to routinely determine the potency and specificity of inhibitors from multiple protease drug discovery projects, and is part of the integrated approach towards protease-directed drug discovery.

Protein kinases are attractive targets for drug discovery programs in many disease areas due to the role they play in mammalian signal transduction. At GlaxoSmithKline we have pursued an integrated screening strategy to identify hits and optimize leads, for potency and selectivity, through to candidate selection. In particular, the selectivity profile of inhibitors is key to understanding their effects on signaling networks and understanding cellular effects. A variety of screening technologies are used offering differing advantages. In addition we characterize in detail the mechanism of inhibition of the hits, leads and candidates we identify.

Different types of kinase assays (e.g. Ser-kinase or Tyr-kinase, primary HTS or selectivity) have different needs and limitations as different assay technologies have their specific strengths and weaknesses. High data quality is crucial for all assays, but throughput and costs are important mainly for HTS assays. For selectivity assays the speed of assay development might be more important. Each assay type can be optimally addressed by a certain assay technology. However, for optimal data comparability it is desirable to use a single assay technology for all kinase assays. Based on our standard kinase assay technology HTRF we have built up a whole platform for the fast and cost-effective development and performance of all kinase assays, primary HTS and compound characterization, in the 5-µL-scale. It comprises tools for the rapid identification of high-quality peptide substrates and suitable corresponding specific antibodies, and standard procedures for effective automated assay development and compound profiling.

HTS using in vitro biochemical assays offer a straightforward approach to discovering small molecule inhibitors. The challenge is choosing a model substrate that both insures the relevance of reactive inhibitors and increases their likelihood of cellular activity. Microfluidic screening of kinase families typically use a peptide substrate whose phosphorylated product can be separated by voltage and pressure. Routine screening of a peptide library revealed multiple substrates for several kinases that have a common motif. These circumstances raise the question as to whether the chosen substrate is valid. Alternatively, a microfluidic-based coupled assay will be described that takes advantage of the linear nature of the kinase pathway. This approach provides a potentially more relevant substrate, offers economic advantages and allows screening of target classes that may not have a model peptide substrate.

We present an application overview of kinase activity profiling using a novel real-time mix-and-read microarray system. This allows enzymatic kinase activity and inhibitor profiling studies on both biochemical single-plex kinase assays as well as cell-lysates using flow-through, PamChip® peptide arrays. Each array contains up to 400 peptides containing phophosites. The system allows Ki, IC50 and MOA to be measured in one array.

Several 96-array microtiterplates can be processed per hour using fully automated, robot compatible, PamStation® array platforms. Each plate can reveal up to 40,000 individual