While kinase assay platforms that probe interactions between inhibitors and a single kinase are sufficient for certain applications, for others, researchers require methods to rapidly and quantitatively characterize hundreds of kinases. With this end in mind, researchers at Ambit Biosciences and DiscoveRx developed a high-throughput method for yielding quantitative ligand-binding data for kinase inhibitors across greater than 80% of the human kinome, which contains more than 500 members.
Once a kinase inhibitor is discovered in initial screening assays, researchers need to determine how those inhibitors interact across the broader kinome, says Daniel Treiber, senior director of research and development at DiscoveRx. A team of Ambit and DiscoveRx scientists, led by Treiber and Patrick Zarrinkar (now at Blueprint Medicines), developed a method known as KINOMEscan and have used this technology to screen the interactions of 72 known, mature kinase inhibitors across 442 kinases in vitro.
KINOMEscan is based on a competitive binding assay that combines affinity chromatography and ultrasensitive protein detection. Researchers immobilize a known kinase inhibitor on a solid support and then introduce a kinase and determine how much kinase is captured in the presence or absence of a test inhibitor.
The kinases, which are labeled with a DNA strand, are then eluted off the solid support, and researchers perform qPCR to quantify the amount of bound kinase. In this way, they determine how effective the test inhibitors are at inhibiting the kinase-ligand interaction and are able to produce full dose-response curves that yield thermodynamic binding constant (Kd) values.
The DiscoveRx team, working closely with epigenetics-based drug discovery pioneer James Bradner of the Dana Farber Cancer Institute, has taken the KINOMEscan technology and extended it to study inhibitors of bromodomains, a class of protein domains that selectively bind to acetylated histone tails and are thought to be disease drivers.
Whether each of the 56 known bromodomains is druggable and can be selectively targeted with an inhibitor remains to be seen, says Treiber, who led a team of DiscoveRx scientists to develop an adapted form of the KINOMEscan methodology to screen broadly for bromodomain inhibitors. So far, Treiber’s team has validated the technology for about 20% of bromodomains and their findings are consistent with and validated by previous literature reports.
When developing an assay to screen for protein inhibitors, researchers often have to compromise quantitative rigor and the number of proteins screened simultaneously to achieve high throughput. According to Treiber, the beauty of the KINOMEscan approach is it has all of these qualities, thanks to the ultrasensitive and quantitative nature of qPCR and the unique semi-automated and generic high-throughput setup.
“The assays are exquisitely sensitive, so it allows us to play games that you can’t play with fluorescence or other traditional technologies,” Treiber says.
Up next, the research team plans to expand upon the bromodomain panel, and then begin to offer it as a service to customers interested in drug screening and optimization.
Characterization of Kinase Inhibitors
Once a drug candidate has been identified and validated in initial screens, researchers need to characterize its effects in cancer cells before progressing into clinical trials. One way to determine if a kinase inhibitor is effective in cancer cells is to determine whether treatment with the inhibitor reduces the phosphorylation of proteins that are downstream of the targeted kinase.
Btk is a known regulator of B-cell receptors and is implicated in malignant B-cell survival. Researchers at Ono Pharmaceuticals wanted to find out how ONO-WG-307, a known Btk kinase inhibitor, affected Btk-dependent signal transduction in two tumor cell lines. In a study performed for Ono Pharmaceuticals, researchers, led by Henrik Daub, Ph.D., svp of science and technology at Evotec, used LC/MS to perform a quantitative phosphoproteomic analysis to help shed light on the cellular mode of action of ONO-WG-307.
The general approach involves incubating cancer cells with isotopically labeled amino acids, which introduces heavier-weight variants into the proteome and enables quantitation, Dr. Daub says. Researchers then incubate the cancer cells in the presence or absence of the compound, lyse the cells, and cleave cellular proteins with a protease.
To isolate the peptide fraction from this extremely complex mixture, researchers use chromatography and then further enrich for phosphorylated peptides. Finally, researchers analyze the enriched phosphopeptides on a LTQ-Orbitrap-Velos mass spectrometer and perform bioinformatic processing to analyze the data. Any changes observed in the intensities of the peptide signals from drug-treated cells, compared to control cells, suggest a change in cellular activity as a result of the drug treatment.
“We typically get a lot of data,” Dr. Daub says, “on the order of up to several hundred thousand peptide identifications initially.” After removing redundant peptide signals, the result is about 10,000 or 15,000 quantifiable signals from distinct phosphorylation sites. Such analyses can yield a wealth of useful information about which downstream signaling pathways are affected by a drug.
In the future, Dr. Daub hopes to see this quantitative phosphoproteomics approach taken to a new level of biological analysis.
“There are quite some interesting developments which can be anticipated,” Dr. Daub explains. For example, Dr. Daub says he hopes to be able to determine not only which phosporylation changes are occurring, but also where in the cell these changes are taking place.
This should be achievable by collecting and analyzing subcellular fractions, Dr. Daub insists. Such analyses done over a time course could also reveal if the process of signaling changes the subcellular location of the proteins.
In addition to shedding light on the cellular mode of action of a kinase inhibitor, quantitative phosphoproteomic analyses can also reveal why a compound may have effects in certain cancer cell lines and not others, and help researchers determine if a promising drug candidate has any off-target effects.
“The strength of doing this in a very broad or more or less global manner is that it’s unbiased,” Daub remarks.
Knowing how a compound works can be just as important in determining that it does work. Quantitative phosphoproteomics makes it possible for researchers “to identify unexpected new things…and get some interesting hints and insights about how and why the kinase inhibitor exerts its therapeutic effects,” Dr. Daub says.