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Cellzome Uses Proteomic Technology to ID Novel Targets for Gleevec, Two Other Drugs

Cellzome researchers have used the company’s Kinobeads technology to identify novel targets for three drugs, including the blockbuster leukemia drug Gleevec.
While the research may confirm Cellzome’s Kinobeads technology, the findings need further validation. But if the findings hold up, the drugs may have therapeutic utility beyond their current indications.
The study, which appears in the online edition of Nature Biotechnology, describes a “chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins,” the authors wrote.
In short, the study found that the Kinobeads can allow researchers to use mass spectrometry to study the interaction of a drug in a cell or tissue lysate for all possible targets.
While drug makers have paid much attention to kinases and their functions, to date only a small handful of products that inhibit kinases have reached the market. This is due in part to the fact that “kinase inhibitors can be both conformation specific and have multiple targets,” the Cellzome researchers wrote in their paper.
They add that drug compounds directed at the ATP-binding sites of kinases may not specifically bind to a single kinase because humans have more than 500 protein kinases and 2,000 other purine-binding proteins that share similar binding pockets.
In their paper, they describe how they used the Kinobeads technology, a mixed kinase-inhibitor matrix, to develop a new method that captures a substantial portion of the expressed kinome and related proteins. They then use quantitative mass spectrometry to analyze their defined subproteome.
This approach “allows the parallel quantitative determination of protein-affinity profiles of kinase inhibitors in any cell type or primary tissue as well as the differential mapping of drug-induced changes of phosphorylation events on the captured subproteome,” the authors wrote.
“If you expose these Kinobeads to a cell or tissue lysate, [then] kinases and related proteins will bind to these beads, and you can then isolate the beads and map the bound protein by mass spectrometry, [and] identify and quantify the bound kinases,” Gerard Drewes, vice president of discovery research at Cellzome and one of the authors of the Nature Biotechnology paper, told ProteoMonitor recently. Other proteins not related to the kinases will not bind to the beads, he said.
In the presence of a drug, the drug will bind to the target. By doing a differential analysis by mass spectrometry, Drewes said, researchers can determine a quantitative list of the drug’s targets, and deduce affinities for targets in the lysate.
For the Nature Biotechnology paper, Drewes and his colleagues chose three drugs — Gleevec, manufactured by Novartis, Sprycel, developed by Bristol-Myers Squibb; and SKI-606, made by Wyeth and currently undergoing clinical testing — because they inhibit the ABL kinase, a target for chronic myelogenous leukemia.
He also said that for the purposes of the paper, it was important that the company chose at least one drug that has been extensively studied, in this case Gleevec, for which almost 3,500 published studies exist.
“Our thinking was quite simple: That [if] we were able to add something novel for such a well-studied drug as Gleevec, then people would recognize that this technology really has something special to add,” Drewes said.
Because the purpose of the paper was to demonstrate the utility of the Kinobeads technology, which Cellzome believes it has achieved, the company does not plan to do any follow-up and will leave it to the drug manufacturers to do so.
In an e-mail, Novartis said that though there is some evidence that Gleevec may inhibit some processes leading to pulmonary fibrosis, it is “premature to draw any conclusions” from the Cellzome paper. Novartis said it is conducting its own research into Gleevec as a possible treatment for pulmonary fibrosis and scleroderma.
Bristol-Myers Squibb declined to comment, and Wyeth did not respond to requests for comment.

“Our thinking was actually quite simple: That [if] we were able to add something novel for such a well-studied drug as Gleevec, then people would recognize that this technology really has something special to add.”

Unforeseen Targets

For the work described in Nature Biotechnology, the Cellzome scientists chose K562 cells and added Gleevec, Sprycel, and SKI-606 to cell lysates in concentrations ranging from 100 picomoles to 100 micromoles. The lysates were then subjected to Kinobead precipitation.
The kinases and related proteins that bound to the Kinobeads were tryptically digested, and peptides were labeled with an iTRAQ reagent. Drewes and his colleagues then quantified proteins and bound kinases with a Thermo Fisher Scientific LTQ Orbitrap mass spectrometer by “measuring the signal of the iTRAQ reporter ions relative to vehicle-treated lysate,” the researchers said.
“From this data set, dose-response binding profiles were computed for more than 500 proteins in each sample, including approximately 150 kinases,” they wrote.
For Gleevec, they found two targets that had not previously been publicly disclosed, the tyrosine kinase DDR1 and the quinine oxidoreductase NQO2, which they validated using traditional kinase and cell-based assays.
According to Drewes, DDR1 is particularly interesting because it may play a role in diseases whose hallmark is tissue fibrosis. If Cellzome’s findings, which Drewes concedes are still preliminary, hold true, “Gleevec may be a useful therapeutic agent in these diseases, especially since these diseases currently have no good treatment options available.”
For Sprycel and SKI-606, the researchers identified more than 10 novel targets each, some of which were validated with traditional assays. One of the new targets is BTK, an emerging target in inflammatory diseases.
“This would lead us to predict that [they] are fairly immunosuppressive,” Drewes said.
In addition to the work described in the paper, Cellzome uses Kinobeads in-house to screen targets of interest that cannot be detected by more traditional assay methods.

“There are a lot of proteins that are difficult to express or that when expressed are not properly folded, for instance,” Drewes said. “And in those cases, we can circumvent the limitations of those more traditional approaches and we can let the Kinobeads technology screen these proteins right away in a cell or a tissue lysate.”

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