May 15, 2007 (Vol. 27, No. 10)

Regulating Fundamental Cellular Processes Such as Proliferation, Differentiation, and Apoptosis

Kinases are enzymes that catalyze the transfer of phosphate groups from high-energy donor molecules to specific target molecules. These intracellular phosphorylation cascades initiated by protein kinases are the most important signaling events in eukaryotic cells, says Wen-Chieh Laio, Ph.D., research scientist in the intracellular signaling department of R&D Systems (

The importance of kinases and the keen interest in their therapeutic potential is underscored by a pair of upcoming conferences, both in Boston in late May and early June. The first will be GTC Bio’s “Protein Kinases in Drug Discovery,” which will be followed by Cambridge Healthtech’s “Protein Kinase Targets Conference.”

Kinases can regulate almost all fundamental cellular processes, such as proliferation, differentiation, apoptosis, and cell cycle. More important, a number of diseases, including cancer, diabetes, and chronic inflammatory diseases such as rheumatoid arthritis, are caused by the abnormality of kinase-mediated cell signaling pathways.

Currently, in the pharmaceutical industry, according to Dr. Laio, GPCRs and kinases are the two largest classes of targets for the development of pharmacological agents to treat and prevent disease.

Kinases have turned out to be one of the most targeted molecules for oncology drug discovery and development. Several recently FDA-approved anticancer drugs are kinase inhibitors. For example, Gleevec, an inhibitor of Abl kinase, is used for treatment of chronic myeloid leukemia, and gefitinib is an EGFR tyrosine kinase inhibitor used to treat non-small-cell lung cancer.

Many methods are used to measure the activation and inhibition of kinases. Generally speaking, they can be categorized into two classes. First, kinase activity can be measured in the enzyme assay using either a peptide or protein as the substrate. Many different detection systems have been developed, including the radioactive assay, Western blotting, gel-based assay, the colorimetric ELISA-based assay, the FRET assay, and other formats. However, these assays can only measure kinase activity in vitro.

Kinase phosphorylation detected by phospho-specific antibodies has recently emerged to measure kinase activation and inhibition. When kinases become activated, they increase their phosphorylation status. The phospho-specific antibodies recognize the phosphorylated forms of kinases, but not the unphosphorylated kinases. By using these phospho-specific antibodies, numerous commonly used kinase assays have been developed, which include cell-based assays.

Small Molecule Therapeutics

Scheduled to speak at the GTC Bio event on kinase inhibitors as therapeutics for chronic inflammatory diseases, Maria Webb, Ph.D., vp, preclinical research, biological and pharmacological sciences at Pharmacopeia (, notes that, up to now, most kinase-based drugs have been targeted for oncology, where therapeutics have “targeted more than a single kinase, thus are not selective.” But because of different mechanism of action and risk-benefit issues, kinase drugs for chronic inflammatory diseases are likely to be “selective for the kinase targeted in the disease process.”

Pharmacopeia is focused on small molecule therapeutics and recently entered into an agreement with Wyeth under which the latter will market drugs for immunological and inflammatory diseases. Pharmacopeia retains full commercialization rights for topical treatment of skin and eye diseases that result from the collaboration.

Dr. Webb notes that her group runs both cell-free assays that measure the enzymatic activity of kinases as well as numerous cell-based assays that are carefully developed for the kinase of interest—Jak 3, for example.

“To be certain that we are as thorough as possible in assessing kinase selectivity,” Dr. Webb says, “when we run a Jak 3 assay we also look at Jak 2, Jak 1, and Tyk 2. An enzyme assay alone doesn’t tell you that you’re inhibiting activity in the cell. One must set up functional measures at related kinases such as other members of the Jak family and look downstream for inhibition of cell signaling, stat5 phosphorylation for example, and read-outs of T-cell activity.” Additionally, activity at unrelated kinases is also assessed in broad or specifically chosen kinase panels. To accomplish this selectivity assessment, Pharmacopeia maintains an in-house kinase panel that includes near and distant members of the kinome and uses several outside services to run broad kinase panels.

Reinforcing the need for selectivity, Mark McCoy, research fellow at the Schering-Plough Research Institute (, will report on NMR-guided design of protein kinase inhibitors at the GTC Bio conference. His group uses NMR methods to search for low molecular weight hits that can either be assembled into leads using a fragment-based approach or serve as scaffolds for chemical exploration that is guided by SAR from NMR data.

While the reliable evaluation of weak hits poses a formidable challenge for bioassay-based screening, NMR methods are well-suited to provide accurate data on low affinity binders.

“When we’re looking at the binding of low molecular weight compounds, they generally have low affinities, but generate strong NMR signals even at micro-molar levels,” Dr. McCoy states.

Ligand-detected NMR methods such as ATP-STD NMR are used to rapidly and accurately determine µM–mM Ki’s of ATP-competitive inhibitors. Binding of non-ATP competitive compounds can also be detected, Dr. McCoy notes. Both active and inactive kinases can be screened.

Concepts such as ligand binding efficiency are used to compare the affinities of scaffolds with different molecular weights and to monitor the effectiveness of substitutions to the scaffold. The high reliability NMR data for weak hits can also impact the evaluation of existing kinase screening libraries. In one example, the Schering-Plough group used SAR from NMR data to identify the correct binding mode of a series of low-affinity screening hits that, through structure-guided optimization, ultimately lead to a potent series of Akt inhibitors.

“Although Akt is implicated in tumor development,” Dr. McCoy states, “it might not be desirable to inhibit all three members of the Akt family.”

STD NMR is also useful for looking at the NMR signal of ATP after it has bound to kinase. Dr. McCoy notes that NMR detects atomic properties of the whole molecule, can reliably distinguish ATP from ADP, and determines that the kinase is whole and functional.

Cellular Assays

Speaking at Cambridge Healthtech’s conference, Josephine Atienza, Ph.D., senior scientist, cell biology and assay development at ACEA Biosciences (, will report on “a novel, quantitative, real-time, and label-free cellular assay” for identification of small molecule inhibitors of EGFR (epidermal growth factor receptor). Noting that there are different ways to identify chemical entities that inhibit kinases, and there are advantages and disadvantages to each, Dr. Atienza adds that “A label-free cell based assay has the advantage of being unbiased. If an assay relies on labeling, then you are looking for a specific type of inhibition, whereas the ACEA technology provides an unrestricted and unbiased query.”

The label-free nature of this assay allows for screening and possibly discovery of new classes of inhibitor compounds that have novel mechanisms of action against inhibitor-resistant forms of kinases, which are an increasing concern in cancer treatment, and which express a strong selection for proliferation and survival.

“Cells are plated in special microtiter plates called E-plates®. They are pretreated with compounds, followed by EGF ligand, and inhibition of EGFR and EGF-mediated cellular changes are monitored using RT-CES®. Sensors in the bottom of the wells detect and quantify cellular changes. The specificity of the assay to EGFR stems from the use of a model cell system that only expresses EGFR1.”

The system is called RT-CES for real-time cell electronic sensing. It measures changes in impedance as a result of ligand-dependent cellular changes such as morphology, adhesion, and cell number. “The unbiased nature of this screen can lead to classes of compounds that have novel mechanism of inhibition and utility against inhibitor-resistant forms of kinases,” Dr. Atienza asserts.

ACEA has established itself as a key player in label-free technology, mostly in secondary screening to validate gene targets and compounds using validated applications such as cell-, compound-, and viral-mediated cytotoxicity to name a few. A number of big pharmas and biotechs have adopted ACEA’s technology for a range of cell-based assays, the company claims.

R&D Systems is also actively involved in cell-based assay development. “We have developed an ELISA assay using fluorogenic substrates to measure kinase phosphorylation in whole cells,” Dr. Laio notes. “The assay eliminates the need to prepare cell lysates and can be used to investigate both kinase pathways and the effects of kinase inhibitors. Cells are grown in 96-well plates and, following treatment, are fixed and permeabilized in the wells. The target protein phosphorylation is measured using a double immunoenzymatic labeling procedure. The cells are simultaneously incubated with two primary antibodies derived from different species: a phospho-specific antibody and a normalization antibody that recognizes the total protein regardless of phosphorylation status.

“Species-specific secondary antibodies labeled with either horseradish peroxidase (HRP) or alkaline phosphatase (AP), and spectrally distinct fluorogenic substrates for either HRP or AP are used for detection. The fluorescence of phosphorylated protein is normalized to that of the total-protein in each well to correct for well-to-well variations. This simple and efficient cell-based ELISA is amenable to high-throughput screening of kinase inhibitors.”

Enzyme Separation

Also appearing at the Cambridge Healthtech conference, Caliper Life Sciences’ ( Seth Cohen, Ph.D., will present a paper describing the utility of the company’s microfluidic lab-on-a-chip technology for the discovery and development of small molecule enzyme inhibitors. The common assay format for these enzyme assays—the mobility shift assay—provides for an integrated separation step on the chip, enabling the detection and quantitation of both substrate and product without the use of radioactivity. The ability to perform these assays in kinetic mode allows for more rapid assay development (i.e., Km determination) as well as greatly reduced material and wells required to study compound mechanism of action.

“In fact, because only nanoliters of sample are utilized,” Dr. Cohen notes, “you can sample out of the same well many times.” The technology is routinely used for lead optimization, secondary screening, and kinase selectivity analysis. Two assay platforms support the mobility shift assay format—the LabChip 3000 and the DeskTop Profiler. The DeskTop Profiler and corresponding ProfilerPro Assay Plates were designed for in-house kinase profiling, Caliper says. The assay plates contain all reagents (including enzymes, substrates, and buffers) needed to profile up to 12 compounds across 48 different kinase enzymes at the benchtop, according to the company.

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