Kinase Library Screening
The kinome is a major focus in drug discovery and, once again, the approach of using whole cells to evaluate large compound libraries is a primary strategy. The term kinome refers to the 500 or so protein kinases in the human proteome that are known to be responsible for a complex array of overlapping cellular regulatory functions. As such, they represent appealing targets for drugs that may hold therapeutic potential in the treatment of cancer, cardiovascular disease, and a host of other disorders. In fact, a number of drugs already on the market work through inhibition of protein kinases such as Gleevec, so there is a demand for rapid screening systems for kinase inhibitors.
Whole cells are a much more physiologically relevant model than traditional cell-free approaches, which cannot provide information on specificity of compounds, membrane permeability, or their cellular toxicity. TGR BioSciences’ (www.tgr-biosciences.com.au) has developed an approach that combines its SureFire™ kinase assay kits with PerkinElmer’s (www.perkinelmer.com) AlphaScreen® assay technology.
The new technology allows the high-throughput detection of phosphorylated cellular proteins in a homogeneous assay format. One such product, the SureFire ERK kit, measures phosphorylation of the extracellular signal-related kinases or EKR protein, enabling researchers to easily screen GPCR activation in whole cells.
The AlphaScreen SureFire ERK assay offers a method for primary and secondary screening of GPCR targets, including those not optimally coupled through the calcium or cAMP pathways. Unlike other proximity-based technologies, AlphaScreen beads can be up to 200 nm apart, allowing for the detection of simple to complex biological interactions and substrates of almost any size.
Like all proximity-based assays, AlphaScreen depends on bringing together two molecules (in this case two distinct types of beads) whose proximity causes the production of a signal. The SureFire assay employs phospho- and non-phospho antibodies, specific to the protein of interest, which coat the beads. Only the phosphorylated protein interacts with both antibodies and brings the two sets of beads together, thus producing a response.
Michael Crouch, Ph.D., director of screening technologies at TGR, discussed the use of AlphaScreen technology as it applied to several projects. Dr. Crouch and his associates have demonstrated that the SureFire system is an effective screening tool for libraries of potential inhibitors of inflammatory mediators such as tumor necrosis factor. “We developed the SureFire technology and then partnered with PerkinElmer,” Dr. Crouch explained. “At present, ours is the only homogeneous high-throughput system for detecting cell-based phosphorylation.”
As Dr. Couch explained, TGR produces 12 kits for detection of inhibitors of various kinase pathways, and another dozen are in the works for release in the coming year. This will enable the screening of hundreds of thousands of drug candidates, and the use of the whole-cell assay allows compounds with toxic side effects to be eliminated early on in the screening process.
“Previously, screening was truly laborious, involving immunoblotting, ELISA, flow cytometry, or cellular imaging, technologies that did not measure the entry of the candidate into cells,” Dr. Crouch continued. “Now drug companies can narrow down a million compounds to find the one satisfactory contender.”