Editorial Article
Monday February 01, 2010
by Caitlin Smith
Kinases are finally getting the recognition they deserve. Yes, there are a lot of them—and they are essential for normal cellular functioning. However, there remains a relative dearth of information about their seemingly endless activities. “Over 400 human diseases have been linked with defects in kinase signalling pathways,” says Steven Pelech, president and CSO at Kinexus, “and only about 15% of the human protein kinases have been seriously pursued as drug targets. At the end of the last millennium, very few major companies were seriously pursuing kinases as drug targets. Today, at least a third of human clinical trials are based on the use of compounds that are kinase inhibitors. We suspect that before the end of this decade, more than half of the annual spending in the pharmaceutical industry will be deployed on the development of kinase-based therapeutics.” Researchers have many investigative tools at their disposal, including analyzing kinase activity, the binding of kinase peptide fragments to small molecules, and the interaction of kinases with kinase inhibitors.
Profiling by enzyme activity
Much of the kinase profiling performed today is based on kinase activity, such as Caliper Life Sciences’ ProfilerPro Kinase Profiling kits. This series of eight kits (each containing a different panel of kinases) allows you to use 24 enzymes per kit to profile up to 12 compounds. “Caliper’s microfluidic lab-on-a-chip technology directly measures substrate and product to provide a more precise measurement of enzyme activity, free of typical assay artifacts,” says Seth Cohen, senior director of microfluidic applications and support at Caliper Life Sciences. “Our ProfilerPro Kinase Profiling kits are designed for ‘in-house’ use with simple add, mix, incubate and read format. The selectivity profile of a compound can be determined the same day the compound is available.”
Enzyme activity assays may also be facilitated by instruments such as Bio-Rad’s new BioPlex 2200 System, a fully automated, random access, multiplex testing platform. The BioPlex is designed to save labor and reagent costs, while speeding assays with multiplexing and automation.
Profiling by peptide binding
For high-throughput, peptide-based kinase profiling using time-resolved fluorescence resonance energy transfer (FRET), PerkinElmer’s LANCE®Ultra platform offers validation for more than 300 kinase targets. PerkinElmer also offers cell-based assays using full-length proteins. “Interesting cases are described when drug candidates exhibit a different profile on peptides versus a full-length protein,” says Martina Bielefeld-Sevigny, VP and general manager of drug discovery and research reagents at PerkinElmer. “This can be seen as a problem by one group and as an interesting observation on mechanism of action by another. Here our Alpha technologies come into play, perfectly suited to work with large proteins and cell extracts.” Their AlphaScreen®SureFire® range of kits covers most targets, screens, and pathways, and are optimized for their automated liquid handling platform, including the 1536-well Alpha Plates.
Profiling by inhibitor binding
Exploring how kinases interact with kinase inhibitors is a common method used in drug discovery. “Kinexus offers a variety of proteomics services that permit our clients to track the actions of kinase inhibitor drug candidates on treated cells in culture and animals to uncover their mechanisms of action and potential side effects,” says Pelech. “This includes the application of our Kinex™ 800 Antibody Microarray and our Kinetworks™ Multi-immunoblotting services. These approaches utilize hundreds of antibodies that target specific protein kinases and key regulatory phosphorylation sites in their substrates. These services are very useful for in vivo profiling of the actions of kinase inhibitors, but they are also helpful for identifying appropriate kinase targets for drug development.”
The physiological substrates for most kinases are still unknown. “One of the new services provided by Kinexus allows for the discovery of physiological substrates of up to 380 known protein kinases, along with identification of the actual phosphorylated site on the substrate, and a phospho-site-specific antibody reagent that can be used to monitor its phosphorylation in future assays,” says Pelech. “This novel method involves the use of our Kinex™ 800 Antibody Microarray in combination with proprietary information on the consensus substrate specificity information that Kinexus has developed for most of the known human protein kinases.”
Kinexus recently launched a new microarray-based kinase inhibitor profiling service that Pelech hopes will reduce screening costs. “Our method is based on the ability of a candidate drug to interfere with the binding of an ATP analogue to more than 150 kinases that are individually spotted in triplicate in the grid of a glass slide,” he says. “With four grids per chip that allows for the testing of three compounds, Kinexus can offer counter screening assays for about $1.50 per compound per kinase with triplicate measurements. This promises to markedly reduce the costs of kinase drug screening, and presents the exciting possibility that it may be feasible to identify even more potent kinase targets for existing drugs that can increase their therapeutic applications. The miniaturization of the compound screening process and expansion of test kinases with the Kinex™ Protein Kinase Microarray can significantly enhance the ability of pharmaceutical industry to provide better quality drugs for a wider range of disease indications.”
Ambit Biosciences also uses binding assays to learn about kinases. They measure active site directed binding, which interferes with the kinase binding to a known inhibitor. Their results are very similar to those reporting activity assays. “But they give you some advantage over activity assays because you don’t need to have active enzymes, for one thing, which means we can work with activated as well as unactivated states and look at the difference in binding affinity of compounds to the two states, which is becoming more and more important in the field,” says Patrick Zarrinkar, VP of technology development at Ambit Biosciences. “And it allows us to look at catalytically inactive kinase domains. The other distinguishing feature is simply the breadth of coverage that these assays have allowed us to build for kinase profiling. Our panel covers more than 80% of the canonical human protein kinases.”
Zarrinkar believes that the field may be moving away from the one-assay-per-kinase system. “Kinase inhibitors interact differently with different activation states of their targets,” he says. “In fact, most kinases target the unactivated state. And there are multiple activation states for kinases. Or autoinhibited states ... so really what you need is multiple assays for each kinase that represent the different activation states to really understand how a compound behaves.” Zarrinkar says that this year Ambit will introduce assays for autoinhibited and non-autoinhibited versions of the class III receptor tyrosine kinases. He also notes that in the future, much can be gained from analyzing compounds across a range of kinase targets. “That will allow you to understand why compounds have the profiles they do, which can then feed back into medicinal chemistry and help chemists fine tune those profiles,” he says. “People always say they have a kinase inhibitor that’s better than others because it hits [for example] kinase A, C, and F, but that’s usually serendipitous—it was not by design. So if one understands the rules of the game better, the design of dialing in and out specific activities will become more feasible in the future.”