Protein kinases serve as critical mediators of signal transduction in human cells and impact virtually all aspects of cellular physiology, from the coordination of the cell cycle and cell division to apoptosis. The deregulation of kinase mediated signaling is now known to be involved in a variety of human diseases that include, diabetes, inflammation, cardiovascular diseases, tumor cell proliferation and metastasis making them a target for drug development. Structurally, kinases share a very similar architecture at the active-site (ATP-binding domain), making selectivity an issue in drug discovery and development. Thus, the development of rapid and economic methods for profiling drug candidates against a large panel of kinases in whole cells will not only aid in anticipating potential long term toxicit, but also help in understanding kinase targeted polypharmacology and aid in identifying new targets for old compounds. In this Phase I application, we will develop a split-luciferase based luminescent assay for kinase profiling in a cellular setting. These cell-based assays will meet a critical need for directly monitoring the effect of compounds on a target kinase not only to ascertain cell-permeability and cellular toxicity, but also to establish efficacy in the cellular mlieu in the presence of adaptor and regulatory proteins. We will test the generality of the approach for kinases across various groups and evaluate the feasibility of our assay for high throughput screening using a panel of 80 known kinase inhibitors. Our goal is to make 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 n many more opportunities for success.
Our genome encodes 518 different protein kinases that serve as traffic signals for the many pathways that regulate the physiology of a normal the cell. The activities of 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, whic 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 work inside cells for advancing the discovery of new and effective therapies for human diseases.