The human genome encodes 518 protein kinases that catalyze the phosphorylation of client proteins. Signal transduction mediated by protein impacts virtually all aspects of cellular physiology, from the coordination of the cell cycle and cll division to apoptosis. Not surprisingly, the deregulation of kinases is implicated in many diseases including diabetes, inflammation, cardiovascular diseases, tumor cell proliferation and metastasis, making them a target for drug development. Small molecule drug discovery targeting protein kinases has seen enormous success in the past decade with over 25 drugs approved for oncology and most recently rheumatoid arthritis with many more in the pipeline for diverse indications. However, the similarity of the ATP binding active site in kinases targeted by small molecules continues to make selectivity a significant concern in drug discovery and development, since a promiscuous drug is expected to give rise to undesired adverse-effects, especially for long term therapy. In this Phase II application, we will develop a split-luciferase based luminescent assay for kinome-wide screening and profiling of inhibitors in a cellular setting. Currently, there are several kinome scale in vitro biochemical assays that are used for screening and identification of kinase inhibitors, but these assays do not provide any information on the ability of a molecule to cross cell membranes or engage its desired target in a cell. In addition, most biochemical assays use the catalytic kinase domain, thereby limiting the identification of potentially more selective allosteric inhibitors that bind sites distal to the AT-binding site. We hypothesize that the development of kinome wide cell-based assays, which are in its infancy, will meet a critical need. The cell-based kinome assays proposed herein will allow for directly monitoring the effect of compounds on a target kinase to not only to ascertain cell-permeability and cellular toxicity, but also to establish efficacy in the cellular milieu in the presence of other ATP binding proteins. Our goal is to render cellular kinase profiling assays both easily available and affordable, so that compound profiling in a cellular or native context can be done earlier and thereby lead to early identification of failures, resulting in many more opportunities for success and new and safer clinical candidates.
The activities of the 518 human protein kinases are tightly regulated inside cells and their dysregulation has been implicated in many diseases, validating them as therapeutic targets. The challenge in designing drugs against kinases stems from their cross-reactivity, which arises due to similar architecture of many kinases at the conserved ATP-binding pocket. Screening compounds against a large number of kinases in their native cellular context can help develop a selectivity fingerprint, which can be used to make critical decisions for advancing a compound into the clinic and provide efficacious drugs. Our application will seek to develop low-cost, sensitive, luminescence based kinase assays that measure direct binding of the target kinase by compounds inside cells and can be used for advancing the discovery of new and effective therapies for human diseases.