July 1, 2006 (Vol. 26, No. 13)

Different Approaches to Identifying and Characterizing Substrates for Kinases

Protein kinases play a central role in many signaling pathways and are key effectors of cellular functions, such as cell proliferation and death. They are implicated in the etiology of many pathological conditions like cancer, central nervous system disorders, and inflammation due to their ability to mediate abnormal protein phosphorylation.

The growing relevance of kinases was highlighted at GTCbio&#8217s recent &#34Protein Kinases in Drug Discovery&#34 meeting.

Identifying Kinase Inhibitors

Roger Bosse, Ph.D., technology and business development leader for discovery and research reagents in molecular medicine at PerkinElmer (www.perkinelmer.com), presented a seminar on PerkinElmer&#8217s kinase solutions. &#34The human genome encodes greater than 500 kinases, and the large majority of them have no known function yet. Hence, major efforts are under way to find a function for each of these kinases.&#34

PerkinElmer&#8217s technologies help to facilitate the identification and characterization of substrates for kinases. Among its portfolio are radiometric (FlashPlates, 33P-ATP radionucleotide) and nonradiometric (AlphaScreen antibody-based and antibody-free, TR-FRET-based LANCE and EasiLyte Kinase for homogenous assays and DELFIA for heterogenous assays) offerings.

The FlashPlate is based on a nonseparation technique and does not require washing or centrifugation, according to Dr. Bosse. This makes it amenable for HTS in an automated format. This technology is antibody-free, and the assays involve the transfer of 33P from 33P-ATP to the kinase substrate resulting in a radioactive output that can be measured.

Dr. Bosse also described the AlphaScreen technology, which is based on energy transfer derived from singlet oxygen produced by AlphaScreen donor beads following excitation at 680 nm. This singlet oxygen reacts with specific dyes embedded within the acceptor bead to generate light detectable between 520&#8211620 nm, when the donor beads are brought into proximity by a molecular interaction between, for example, a kinase product and a specific detection antibody.

Donor and acceptor beads can generate an AlphaScreen signal when they are located within a 200-nm range, which allows one to work with large proteins. AlphaScreen assays generate low background, use low concentrations of antibody and antigen, and can measure a large variety of affinities. Dr. Bosse presented results that demonstrated the use of the AlphaScreen technology in cell-based kinase assays using JNK3 in HepG2 cells, in kinase deorphanization with AlphaScreen Phosphosensor (Lewis metal chelate) beads, and in GPCR deorphanization with SureFire ERK assays.

Nikolai Kley, Ph.D., vp of research at GPC Biotech (www.gpc-biotech.com), discussed the use of chemical dimerizers in his talk on the application of chemical dimerizers to kinase inhibitor target discovery.

The company is building a portfolio of anticancer drugs based on kinase inhibition to address the need for new and improved cancer treatments. The focus of Dr. Kley&#8217s presentation was on the use of chemical dimerizers coupled to a yeast three-hybrid system for kinase inhibitor screens. The concept behind the three-hybrid screen is the generation of a methothrexate-based chemical dimerizer signal as a result of interaction between small molecule drug and a kinase target of interest.

The linker attached to the variable signal domain in this system enables the screening of large libraries of candidate target proteins and provides a means to compare results to other kinase inhibitors in a continuous mode. Dr. Kley shared results that validated the use of this technology in finding compounds against cyclin-dependent kinases and kinases that affect RNA Pol II transcription of the Mcl-1protein.

Said Goueli, Ph.D., research fellow at Promega (www.promega.com), talked about Kinase Glo technology, a tool for discovering a new class of protein kinase inhibitors. This is mainly applicable for developing in vitro biochemical assays during the primary screening stage of kinase inhibitor discovery. It is based on the principle of bioluminescence signal that is generated due to the interaction between ATP and Kinase Glo reagent.

Improved Reagent

The luciferase enzyme in the reagent detects the leftover ATP following a kinase substrate reaction. The enzyme catalyzes the oxygenation of luciferin, which emits the bioluminescent signal. Lumin-escence is inversely proportional to kinase activity. Key advantages of this assay are its sensitivity over conventional fluorescence and its capability to work with a wide range of substrates like peptides, proteins, lipids, and sugars.

Dr. Goueli highlighted results derived from a new form of this reagent, the Kinase Glo Plus, which he noted, can function in a broad linear range of ATP concentrations (1&#8211100 &#181M) with a signal stability of up to four hours. He discussed results that demonstrated robust ATP titration, slow signal decay, strong Z&#180 values, and use of the Kinase Glo Plus reagent in ATP competitive and noncompetitive kinase inhibitor compound screens.

Non-ATP Kinase Inhibitors

&#34As binding of ATP is essential for kinase activity, the discovery of small molecule ligands that compete for the ATP binding site has been the main source for new kinase inhibitors. However, due to the relative structural conservation of ATP binding sites for more than 500 protein kinases in the human genome, molecules that compete with ATP binding often cross-react with many different kinases,&#34 explained Alex Kiselyov, Ph.D., executive vp of R&D at ChemDiv (www.chemdiv.com). On the flip side, kinase inhibitors displaying &#34dual&#34 activity against multiple kinases are of particular interest in oncology due to the synergy potential.

ChemDiv has launched several programs that have yielded non-ATP-competitive kinase antagonists. They have also developed a strategy for identification of non-ATP competitive kinase inhibitors based on the Pocketome algorithm developed by Ruben Abagyan at MolSoft (www.molsoft.com). A focused compound library called the Focused Diversity Set (FDS) has been assembled.

FDS includes a selection of 5,000 fully characterized, chemically distinct compounds annotated against orthogonal biological targets, i.e., targets from different families, pathways, and cellular processes. The company has validated the FDS approach in the identification of novel dual AT1/ETA antagonists, PI3KCA-specific inhibitors, EGFR/KDR, and tubulin modulators for oncological indications, according to Dr. Kiselyov. ChemDiv has also been able to develop non-ATP competitive inhibitors of bcr-Abl kinase that are based on a non-ATP competitive mechanism of enzyme inhibition.

Enrique Michelotti, group leader, medicinal chemistry at Locus Pharmaceuticals (www.locuspharma.com), explained that Locus&#8217 approach to kinase inhibitors is a fragment-based strategy that enables the quick identification of biologically relevant binding sites and novel small molecule drug candidates. The company is focusing its attention on developing therapeutics for the treatment of arthritis/inflammation, cancer, and HIV/AIDS.

Dr. Michelotti discussed and presented results on several of the company&#8217s lead kinase inhibitor compounds. These compounds belong to a new class of p38 inhibitors that is based on allosteric inhibition of a conserved non-ATP kinase site as opposed to the more traditional ATP site-dependent classes.

This new breed of compounds confers high selectivity, potency, and improved safety characteristics. Cell assays demonstrated that the mechanism of action was due to complete inhibition of the p38 specific activation pathway thus validating p38 as the unique intervention point for the LOC allosteric inhibitors, Dr. Michelotti said.

In a seminar on finding hits for kinases, Michael Charlton, Ph.D., group leader of computational chemistry at Evotec (www.evotec.com), explained that his company uses a cheminformatics approach to find kinase inhibitors. Evotec has built a database of over three million compounds derived from various suppliers and categorized them into drug-, lead- and non-druglike compounds based on predetermined criteria and queries.

The company has identified a new way of describing molecules and used a technique known as Kohonen mapping to add several thousand compound candidates to its screening collection. The application of this virtual screening approach to finding kinase inhibitors has been validated by results that demonstrated over 40-fold enrichment compared to a random screening approach.

Finding Kinase Inhibitors

&#34The focus on single purified kinase inhibitor screening has lead to poor efficacy in development, because of the ability of tumor cells to compensate and circumvent a block of a single node in the signaling network. Hence, there is a need for a systems biology approach to assay the activity of the signaling pathways downstream of the kinase targets,&#34 says Steve Horrigan, Ph.D., interim vp of research at Avalon Pharmaceuticals (www.avalonrx.com).

AvalonRx uses this approach for the identification of inhibitory compounds that are relevant in the normal cellular context and tumor function, to screen for the activity of targets that are difficult to isolate or develop assays for, and to identify new druggable nodes for known signaling pathways, explains Dr. Horrigan. Key attributes of AvalonRx include accurate measurements of gene expression and unified data integration.

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