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Large Kinase Datasets from Fox Chase, Ambit Teams Offer Insights into Inhibitor Selectivity

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By Adam Bonislawski

Two papers published in the current edition of Nature Biotechnology add significantly to the publicly available data on the specificity of kinase inhibitors.

Examining the interactions of thousands of kinase-inhibitor pairs, these studies – one led by scientists at Fox Chase Cancer Center and the other by researchers at Ambit Biosciences – offer information on the specificities of many commercially available inhibitors and provide scientists with a tool to facilitate their work with such reagents.

As key players in cancer and cellular signaling, protein kinases are a major area of interest for both drug developers and basic research scientists, making kinase inhibitors important both as therapeutic agents and research reagents. Because most such agents affect inhibition by binding to the ATP-binding site of their target kinases, which has been well conserved evolutionarily, questions exist about the specificity of these molecules.

The Nature Biotechnology studies found that creating sufficiently specific inhibitors is possible for many protein kinases. However, they also found that many commercially available inhibitors exhibited a wide range of off-target interactions, suggesting the need for caution by scientists using these reagents in their research.

"Using an inhibitor blindly without knowing the selectivity is asking for trouble," Jeffrey Peterson, leader of the Fox Chase Study, told ProteoMonitor. "We found many examples in our study of inhibitors that people assume are selective for their target but that are actually quite non-selective. And that tremendously complicates results from experiments" that have used those inhibitors.

The problem, Peterson noted, is that large-scale screens of kinase inhibitor activity are "quite expensive and require high-throughput technology that every research lab using this type of inhibitor is not going to have." Given that, his team aimed to analyze the specificities of many of the inhibitors currently on the market so as to make that information available to researchers in the field.

The Fox Chase screen was performed by Malvern, Pa.-based assay development firm Reaction Biology, whose director of research services, Sean Deacon, was formerly a post-doc in Peterson's lab. Using HotSpot, a radiometric assay based on conventional filter-binding assays, the researchers screened 178 kinase inhibitors against a panel of 300 recombinant protein kinases.

They then analyzed the results of the screen, using gini scoring to rank the compounds by their selectivity and generating for each inhibitor a uni-specificity score – a measure of the extent to which an inhibitor targeted any single kinase more potently than any other in the panel.

The degree of inhibitor promiscuity the researchers found "came as quite a big surprise," Peterson said. While most compounds that have been approved by the US Food and Drug Administration or are currently in clinical trials had relatively high specificity, many of the less well characterized research reagents screened interacted with a wide range of kinases.

"Selectivity, I think, is generally much better known for clinical candidates, whereas research tool compounds are often used with very little characterization," Peterson said. "And that was what we wanted to shed light on in particular."

People "entrenched in the field are quite aware" of the lack of selectivity of many kinase inhibitors, said Gideon Bollag, senior vice president of research at Berkeley, Calif.-based pharma firm Plexxikon and co-author of a commentary accompanying the two studies. However, he told ProteoMonitor, the research "will probably open some eyes of people who are more peripheral to the field that these compounds are, in fact, not monospecific."

"There are quite a few compounds in [the Fox Chase] dataset that academic labs have used as an inhibitor of kinase X, and you can now look at the data and find out that, indeed, there are many kinases in my experimental system that are inhibited [by this inhibitor], so it makes the conclusions a lot more tenuous," he said. "I think some of the conclusions people have made in past studies need to be re-evaluated."

In addition to prompting re-evaluation of old studies, the two studies offer researchers potential leads for new compounds they might use to inhibit kinases of interest, Bollag noted. "These two studies offer some of the largest [publicly available] collections of data on a large number of kinases versus a large number of compounds" he said.

While the Fox Chase team concentrated on commercially available research tool compounds, the Ambit researchers focused on clinical compounds, said Daniel Treiber, currently senior director of research and development of assay development firm DiscoveRx's KinomeScan division and an author on the Ambit Nature Biotechnology paper.

"We went through the literature for several years tracking compounds that were making their way through the clinic," he told ProteoMonitor. They then paid to have these compounds synthesized for use in their study, which screened 72 inhibitors against 442 kinases.

Calling the two datasets complementary, Treiber noted that while he and his colleagues also found a fair amount of promiscuity in the inhibitors they screened, their work with mature, well-characterized compounds suggests that it should be possible to make dedicated, selective inhibitors to a wide range of protein kinases.

"Our results showed that for the majority of intended targets there is at least one selective inhibitor, with selectivity being defined either as overall kinome selectivity or relative selectivity for the target," he said. "We were actually quite encouraged."

Relative selectivity, Treiber said, means that a compound may have off-target interactions but still affects a particular kinase strongly enough as to effectively function as a specific inhibitor.

"In a one micromolar screen you may identify a lot of off-target hits," he said, "however, you may find that the intended target may have a one nanomolar Kd – so it's very potent – while most of the off-targets are, say, 100 nanomolar or higher."

The Ambit team began its study with an initial screen of the inhibitors at a 10 micromolar concentration, following up hits from that screen with full dose response curves. The Fox Chase study, meantime, analyzed all its kinase-inhibitor interactions at a single 0.5 micromolar concentration.

In the case of both studies, Peterson and Treiber agreed, further investigation is required to determine how well the results of these in vitro screens hold up in vivo.

"Certainly there is not going to be a one-to-one correspondence of the relative potency of an inhibitor in vitro to an inhibitor in vivo," Peterson said. "Nevertheless, in general, I think it's clear that activity in vitro generally correlated with activity in vivo."

"Potency in a biochemical assay is necessary but not sufficient for activity in cellular and in vivo assays," Treiber said. "If you don't have a compound that is potent in a biochemical assay, it's extremely unlikely that you will have activity in a cellular or in vivo milieu. For compounds that are highly potent in a biochemical assay, they generally show activity in a cellular assay."

Datasets from both studies are publicly available – the Ambit set through Nature Biotechnology or by request from Treiber, and the Fox Chase set at that institution's Kinase Inhibitor Resource.

The Ambit and Fox Chase studies follow the recent release of several other large-scale protein kinase datasets, including one released in February by Abbott Laboratories and another released by Bristol-Myers Squibb in December 2010 (PM 3/11/2011).


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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