Kinomics is illuminating drug development with an ever-growing toolbox of screening assays.

If the recent spate of publications and presentations at meetings is any indication, kinomics is emerging as the premier field for innovative drug development. The "kinome"refers to the entirety of the genes and their products involved in protein phosphorylation, a signaling activity of vital importance in the regulation of numerous disease processes.

In the mid-1990s, the sequencing of the human genome spilled out a wealth of new information; the most significant being the numbering of the totality of kinases at approximately 500. This was less than originally predicted, but at roughly 1 percent of the total genome, not a trifling number.

The study of the kinome demonstrates in a dramatic fashion the intersection of fundamental bioscience and applied biotechnology, as the relevance of cellular regulatory events to disease has become clear. Many protein kinases play a role in malignant transformation and were discovered years ago as oncogenes.

But this is just one feature of these critical regulatory molecules. In recent years, the role of kinases in phosphorylation of proteins pivotal in immune dysfunction, aging processes, and cardiovascular disorders has been described. As researchers scramble to expand basic science investigations, the understanding of the role of protein kinases in cancer has been translated into the drugs Iressa (AstraZeneca) and Gleevec (Novartis).

Can investigators move beyond these studies on the molecular roles of the kinases and develop new drug candidates?

False starts
In the early days of omics investigations, many pharma companies embarked upon broad screening programs, searching for drug candidates that would inhibit critical kinases involved in cancer and other disease processes. These studies were not fruitful, failing to reveal useful therapeutics, in large part because the candidates lacked specificity. Researchers now understand that the inability to target an individual kinase is because most small molecule kinase inhibitors are ATP-competitive. Throughout the protein kinase family, the ATP-binding pocket is well-conserved, so inhibitors that occupy this space will often cross-react with unintended targets. By profiling against a large and diverse panel of protein kinases, many putative therapeutics can be screened and eliminated early in the drug discovery process. This detailed understanding of possible agents ensures that there will be no ugly surprises along the path of clinical trials.

On the other hand, there is a burden of redundancy within kinase regulatory pathways, so inhibiting a single kinase target with great specificity might leave other enzymes in the same complex unscathed. The outcome could be complete inhibition of one critical kinase, but a disease continuing unimpeded through the pathway of another homologue. This biological reality provides all the more reason for a diverse and extensive panel of kinases, and multiple screening options, including cell-based assays.

High-throughput screening
Several companies offer high-throughput screening technology and contract services focused on kinases (see related article). "Selection of a particular kinome technology is largely a matter of personal preferences,"says Steve Davies, PhD, director of R&D at the Millipore Corporation's Bioscience Division in Dundee, Scotland. "We believe our direct measurement of phosphorylation using radioactivity avoids some of the uncertainties of techniques that use indirect approaches such as antibodies."

The use of radioisotopes is much more convenient than in the past; Millipore has switched to the use of the safer ³³P isotope, environmentally-friendly scintillation fluid and much smaller quantities of reagents per assay.

Millipore provides a KinaseProfiler Service for screening through the use of a large kinase library. While the human kinome contains approximately 518 genes for protein kinases, the kinome offers many more actual drug targets, including disease-relevant mutants, drug-resistant mutants, such as the Gleevec-resistant Abl mutants, and phosphorylation states.

For those who prefer a non-radioactive alternative, Millipore has also developed a rapid screening method in collaboration with CisBio International, Bagnols-sur-Ceze, France, for high-throughput, large-scale kinase profiling of chemical libraries. This service, KinaseProfiler HTRF, takes advantage of the homogeneous time-resolved fluorescent platform offered through CisBio.

The screening technology is the HTRF version of Millipore's KinEASE fluorescence polarization homogeneous assay, in which a phosphorylated peptide is generated as a kinase reaction product. The peptide contains a fluorescein molecule attached to it, and when phosphorylated will bind a specific antibody. The antibody-peptide complex will rotate only slowly and will retain a polarization signal, unlike the much smaller, freely rotating peptide. Thus the fluorescence signal is a measure of the amount of kinase active in the sample.

Screening technology
CisBio International also offers a homogeneous assay based on time-resolved fluorescence resonance energy transfer (FRET) and the use of fluorophores with long

click to enlarge  HTRF KinEASE STK kits use a proprietary anti-phosphoserine specific monoclonal antibody labeled with Eu3+ -Cryptate and a proprietary biotinylated kinase substrate detected using XL665-labeled streptavidin; the basic assay involves a first step for enzymatic reaction using a generic peptide supplied with the kits and then a second step for HTRF detection. (Source: CisBio Inc.)

emission half-lives. FRET is a technology employing two different fluors, a donor and an acceptor, which, when in close proximity, will generate a unique fluorescence signal. In order to develop the reaction, a specific protease is added, which cleaves the non-phosphorylated peptides and eliminates the FRET signal. The uncleaved, phosphorylated peptide maintains the signal. The assay figures prominently in screening for inhibitors of the kinases.

"This platform has features that separate it from other TR-FRET products,"according to Michel Fink, PhD, head of biology at CisBio. These include the use of Europium, a lanthanide fluor with an extremely long half-life. The CisBio product is conjugated to Eu3+ Cryptate, a compound conferring increased assay stability in conjunction with a patented ratiometric measurement that allows correction for quenching and sample interferences the assay.

The product is used to build a technology for measuring kinase inhibition, referred to as the HTRF KinEASE STK kit (see figure below). It employs a proprietary anti-phosphoserine specific monoclonal antibody-labeled with europium (Eu3+)-cryptate and a biotinylated kinase substrate detected using XL665-labeled streptavidin. The basic assay involves incubation of the kinase in the presence or absence of compounds and the appropriate substrate. ATP is added to start the reaction, followed by the Eu3+ Cryptate and XL665 conjugates and the EDTA-containing detection buffer bring it to a grinding halt. The two detection reagents can be pre-mixed, allowing for a single step addition.

Fink states, "We have validated this assay on more than 60 tyrosine kinases, either cytosolic or receptor. A specific kinase reaction buffer was optimized in order to enable the assessment of all TKs. Signal stability, S/B and Z' factor can be easily determined for the most favorable assay conditions. DMSO influence was also characterized."

Alternative kinome technologies
Other firms offer kinase screening services similar to those of Millipore. "We've assembled a collection of more than 280 human kinases,"says Chris Armstrong, PhD, director of discovery collaboration at Invitrogen Discovery Sciences, Invitrogen Corporation, Madison, Wisc. The company's SelectScreen kinase profiling service is also based on a FRET assay system and the company's own kinase library for evaluating drug candidates. The Z'-LYTE assay uses a synthetic peptide substrate, which upon receiving a kinase-transferred phosphate group from ATP, is converted to a FRET pair.

The kinase drug discovery technology has been extensively evaluated by Invitrogen scientists and their pharmaceutical and biotech partners. When the Z'-LYTE assay was compared with a classical radiometric assay measuring inhibition of one of the kinases with the compound typhosin AG1478, the two assays were comparable.

"Another major focus of our kinase platform is the build-out of mutant forms of kinases found in disease,"Armstrong states. There are an increasing number of genetic variant kinases including an emerging set of drug-resistant mutations arising in patients treated with kinase-targeted drugs such as Gleevec and Iressa. In order to address evolution in kinase inhibitor design, Invitrogen has added a collection of mutant kinases to their SelectScreen library.

According to Armstrong, companies are no longer just going after the ATP-binding site with classical Type I inhibitors. "They are also examining a new spectrum of inhibitors that bind to allosteric sites that may be distant from the ATP pocket or that preferentially bind to inactive forms of kinases referred to as types II and III."These alternative approaches are being developed to overcome challenges of selectivity and to investigate drug-resistant mutations in the clinic.

Smaller is better
FRET-based technologies may be popular tools for kinome studies, but they aren't the only possible screening platforms, nor are discovery of drug targets by compound screening of large and random libraries the only avenue to finding new drug candidates. A kinase drug discovery program at Roche, Basel, Switzerland, explores the use of focused libraries containing substances that are selected based on target or compound specific properties and delineated using surface plasmon resonance.

"Sufficient quantity and quality of protein for this work is our greatest bottleneck,"says Michael Hennig, PhD, section head of molecular structure research at Roche. Hennig recounted his experiences with fragment-based

click to enlarge  This circular diagram represents an artistic interpretation of the phylogenetic relationships between all members of the human kinase family. Kinases are organized into nine major groups (TK, TKL, STE, CK1, AGC, CMGC, CAMK, Other and Atypical), based on similarity between amino acid sequences in enzyme catalytic domains with two exceptions. The three-dimensional structure in the middle of the diagram is a model based on cAMP-dependent protein kinase. (Source: Invitrogen Corporation)

focused screening, in which thousands of candidates having molecular weights less than 300-kD were examined. The selection of the two thousand compounds making up the fragment library is highly rational, based on preselected criteria. Because of their small size, interaction with targets will display low affinity. And for this reason, Hennig needed a very sensitive assay system.

In addition to the molecular weight criterion, Hennig and his colleagues placed a number of other restrictions on the fragments, including easy access, high solubility, availability of the compound in powder form, ease of synthesis and excellent compound purity. Moreover, crystal structure analysis to define molecular interactions is required to aid in rapidly generating compounds with optimized biophysical and binding properties. Due to the use of biophysical assays including surface plasmon resonance (SPR) and protein crystallography, the application of this method is restricted to proteins that can be investigated in solution and produced in milligram quantities.

The screening uses the SPR system available through the Biacore A100 instrument (GE Healthcare), which has the capability to screen about 1,000 compounds per day. The principle is based on detecting changes in mass of molecules in the aqueous layer bathing a gold sensor chip surface by measuring changes in refractive index. Thus the binding of a ligand to a protein molecule can be followed without labeling and accurate "on"and "off"constants are readily obtained. The Biacore system is applied as a filter to select compounds for follow-up investigations including X-ray structural analysis of the protein-ligand interaction.

Hennig described Roche's program to screen compounds binding to the beta-amyloid- cleaving enzyme (BACE), a target in Alzheimer's disease. It is believed that release of a portion of the beta-amyloid protein from the cell surface produces the raw material that will congregate in the plaques that clog neurons and prevent the proper transmission of signals in the brain. According to this hypothesis, drugs that block the asp-protease BACE might prevent the initiation and progression of Alzheimer's disease. In addition, a viable candidate drug would have to cross the blood-brain barrier, in order to have access to the cells of the central nervous system.

Hennig and his colleagues have screened their fragment library against BACE and have identified a number of candidates which bound to the enzyme. All but one of the positive fragments bound to the S1 pocket of the enzyme which appears to be a key site for ligand-binding. Based on the screening results and structural information, the tyramine fragment binding to the S1 pocket was selected for chemistry efforts and ortho-substituted molecules were synthesized exhibiting greatly improved Kd. Hennig cautions against irrational exuberance. "Library fragment screening is one important step in drug development,"he says, emphasizing that the overall process results in substances that can be followed by chemists for further optimization. But, he says, "at this point, we still have a long way to go to build the ideal drug candidate."

About the Author
K. John Morrow, Jr. is a biotechnology writer and consultant based in Newport, Ky.

This article was published in G&P magazine: Vol. 7, No. 7, July, 2007, pp. G2-G6.