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Jun 1, 2011 (Vol. 31, No. 11)

Kinase Inhibitor Obstacles Falling

Structural Modeling and Improved Assays Propel Advances in Field

  • In-House Screening

    Screening of kinase inhibitors is often outsourced to a CRO due to the number of assays that are involved. Laurie LeBrun, Ph.D., principal scientist, biochemistry, Celgene, described the company’s in-house kinase selectivity strategy.

    “We wanted to develop an internal screening system that had a faster turn-around time than we can get via CROs. The advantage is that the selectivity of our screening hits can be assessed early on in the process. This provides the medicinal chemists additional data to assist in the prioritization of the screening hits and aids in the selection of the best leads.”

    Dr. LeBrun said that the Caliper Life Sciences’ ProfilerPro kinase selectivity assay quickly and easily provided the selectivity snapshots they were seeking. “We could quickly and easily screen early target chemical series and get selectivity results within one day. The 48 kinases included in kits one and two provide good coverage of the kinome.”

    Each plate can profile 12 different compounds against 24 kinases. The enzymes and fluorophore-labeled peptide substrates are predispensed into 384-well microtiter plates. Each profiling kit provides a predictable conversion of substrate (nonphosphopeptide) to product (the phosphopeptide) at the apparent ATP Km, Dr. LeBrun explained. The assays are read using the Lab-on-a-Chip mobility shift assay technology, and she reported that she used the EZ Reader II.

    “When we compared our internal data with that obtained through a CRO, we found a good correlation for the kinase selectivity that we observed internally.”

  • Simple Assay for Optimization

    There are more than 500 protein kinases encoded in the human genome. An inhibitor may target one or more. Thus, delineating activity and potential cross-reactivity can help optimize lead discovery of kinase inhibitors. According to John Moffat, Ph.D., scientist, small molecule biochemical pharmacology at Genentech, there are two key questions that arise after identifying cross-reactivity to other kinases: “Does this activity translate to a functional effect in cells?” and “What is the window (if any) between on-target and off-target functional activity?”

    “High-content screening assays can provide answers to both of these questions,” Dr. Moffat said. “It is a simple way to assess activity in living cells that can complement data obtained from other types of screening assays. We have established a high-content assay that employs 384-well plates screened using only a fluorescent dye for nuclear staining. The images from the fluorescent microscope subsequently are analyzed with software. This gives cell-cycle information similar to flow cytometry, but provides another layer of information such as cell health and nuclear morphology.”

    This approach also allows a better definition of the on-target versus off-target effects of a candidate. “We are working on an oncology program that targets kinases with known or predicted effects on cell-cycle progression. We receive a large number of compounds from our medicinal chemists to profile. For example, we may screen for compounds that are expected to arrest the cell in its G1 cycle. Our screening not only validates this activity, but also helps quantitate the window between on- and off-target effects.”

    Dr. Moffat concluded that “a lot of information can be mined from a relatively simple imaging assay as to the cellular effects of kinase inhibitors that may be missed by other assays. Early identification of the most useful candidates to pursue saves time and money.”

  • Entropy and Selectivity

    Most scientists would understand entropy as a thermodynamic property. However, applying “information entropy” to kinase-inhibitor profiling is a new way to solve the old problem of making sense out of the large amounts of data, according to Joost Uitdehaag, Ph.D., senior research scientist at Merck.

    “Today, researchers are able to identify hits from new libraries of compounds using high-throughput screening. There is a lot of debate as to how to improve selectivity of these hits in the process and to determine when your compound is sufficiently selective. But, it all starts with quantification, with being able to compare actual values. This is what the entropy score for selectivity brings to the field.”

    Dr. Uitdehaag said this process allows one to very quickly choose the best compound to take forward into further testing. “We have proposed a way to calculate a single value from a set of IC50 data to quantify selectivity profiling from panel profiling. It is a powerful way to study molecular mechanisms of kinase inhibitor selectivity.

    “Often, other methods are utilized such as dotting a kinome tree, heat maps or a radius plot, but these only provide a qualitative comparison. For quantitative approaches, others have developed a selectivity score based on kinase-profiling data. But this doesn’t provide sufficient sensitivity. Other common methods include using the so-called Gini score or a partition index. None of these measures are fully adequate.”

    How do these new equations work? They are based on the principle that an inhibitor candidate will assume a Boltzmann distribution across the various targets when confronted by multiple kinases. “This distribution has calculated entropy. If it is, for example, 2.2, which is an average measure of selectivity, the compound has average selectivity. If the calculation ends up to be 1, however, this indicates that the compound is a much more selective inhibitor. One can use this information to quickly select the best candidates after screening. The nice thing is that this method gives consistent values across profiling experiments, so it’s really general.”

    Dr. Uitdehaag also noted that selectivity entropy can be used to study the success of candidates in clinical trials. “We assessed clinically tested inhibitors and determined their selectivity scores. We found that the most successful compounds actually are those with more broad selective profiles. These findings indicate that selective candidates have less of a chance for surviving early clinical trials.”

    Indicating the selectivity of an inhibitor should ultimately be just as commonplace as indicating its IC50, advised Dr. Uitdehaag. “I think, for instance, the selectivity values of inhibitors should be reported when people do biological validation experiments with them. It would make a lot of sense.”

    Although great advances have been made over the last decade in measuring and predicting kinase inhibitor selectivity, a number of issues remain. Advances in structural-guided modeling and in enhancing the selectivity of assays should provide critical improvements for future drug development and therapeutic target expansion.


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