May 15, 2010 (Vol. 30, No. 10)

With Over 500 Inhibitors in Various Stages of Development, Effective Assays Are Critical

Currently, there are only eight small molecule kinase inhibitors approved and on the market; however, researchers have approximately 500 kinase inhibitors working their way through discovery and preclinical phases, thus making protein kinase inhibitors a force to be reckoned with. “Speed, cost, accuracy, and quantitation will determine success in this space,” observes Michael Crouch, Ph.D., CSO at TGR Biosciences.

Protein kinases play a major role in multiple cellular processes and are thus important drug targets, explains Said Goueli, research fellow and technological development team leader at Promega.

“The successful introduction in the last ten years of several kinase inhibitors as drugs for treatment of cancer, metabolic diseases, and autoimmune diseases, for example, is driving research forward.”

Interest in protein kinases is burgeoning, and the upcoming “Protein Kinases in Drug Discovery” meeting will enable researchers to discuss technological advances, as well as some of their product-development successes.

TGR Biosciences started out developing  molecules and looking for new receptor modulators, notes Dr. Crouch. “We needed readout of cell-based activity, and there was nothing on the market that did that, so we developed technology to measure changes in cell behavior as well as protein phosphorylation of cells.”

Techniques for measurement of kinase activity in cell-based assays in high throughput, which allows a more physiological view of test compound action on signal transduction pathways when testing large compound libraries, is a recent development and one that has taken the research community some time to get used to, observes Dr. Crouch.

“We needed to convince people that this was advantageous; once you measure phosphorylation in cells, you can look at cell behavior. This is a capability they could really exploit.”

TGR’s SureFire allows the analysis of cellular kinase activity in both cell lines and primary cells, including growth arrested cells, since no cellular transfection is required. Dr. Crouch notes that the assays, which use PerkinElmer’s AlphaScreen® technology, are homogeneous, and the lack of assay washing steps means the whole assay procedure can be automated with liquid handlers, providing for large sample throughput.

“Assays for over 40 distinct targets are now available and cover most of the major cellular signal-transduction pathways,” he adds. “Thus, the same lab infrastructure can address compound specificity, as well as pathway point of action.”

Because of the small amount of cellular extract that is used per assay point, Dr. Crouch explains that multiple replicates of a cell lysate can be analyzed using AlphaScreen SureFire for different targets, with the versatility of the user defining which proteins are to be tested. This allows for signal pathway profiling in a single experiment, and up to 20 different analytes can be quantitated at one time. Speed and accessibility in the lab are two key drivers.

There is much work to be done, not only in developing screening for the next generation of kinase inhibitors, but also in demonstrating their selectivity across the kinome. “Technology needs to be universal, HTS-formatted, sensitive, and robust,” says Dr. Goueli.

“Universal platforms are highly desirable so kinase profiling can be applied to the various kinases across the kinome regardless of their substrate. These include serine threonine kinases, protein tyrosine kinases, lipid kinases, sphingosine kinases, and they are not limited to a subset of kinases.”

Promega’s ADP-Glo™ Kinase Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.

It can be used to monitor the activity of virtually any ADP-generating enzyme, using up to 1mM ATP, Dr. Goueli says. The company addresses the issue of enzyme quality by providing ADP Glo in a format that includes the kinase, substrate, and buffer in one system—a complete solution to assure that the assay and substrate are performing according to expectations. “You will then be able to do, not only kinase screening, but also kinase profiling based on inhibitor selectivity.”

The ADP-Glo Kinase Assay is performed in a multiwell plate and can detect kinase activity in a reaction volume as low as 5 µL. The assay is performed in two steps—first, after the kinase reaction is completed, an equal volume of ADP-Glo reagent is added to terminate the kinase reaction and deplete the remaining ATP. The kinase detection reagent is then added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction.

The light generated is measured using a luminometer. Luminescence can be correlated to ADP concentrations by using an ATP-to-ADP conversion curve.

B-Cell Mediated Diseases

Avila Therapeutics engineers molecules that it says are able to selectively silence drug targets. “We generate covalent drugs that strongly and resiliently bond to specific disease-causing proteins, thereby silencing them and eradicating the corresponding disease,” explains Juswinder Singh, Ph.D., CSO and co-founder.

Avila is focused on Bruton’s tyrosine kinase (Btk), which is expressed in B cells and thus “represents an exciting target for the treatment of autoimmune diseases and hematological cancers.” This has been a difficult target for pharma to create selective inhibitors. “We designed AVL-292, a highly selective, covalent Btk inhibitor that potently inhibits Btk enzymatic activity in vitro.”

According to Dr. Singh, AVL-292 demonstrates impressive efficacy in several rodent arthritis disease models. “And, because AVL-292 binds to Btk irreversibly, we have developed a covalent probe assay that enables direct measurement of Btk occupancy and can correlate target occupancy with pharmacodynamic response.”

Erythropoietin-producing hepatocellular carcinoma (Eph) receptors are highly conserved transmembrane tyrosine kinases made up of multiple domains that participate in an array of complex cell signaling pathways. Marcie Glicksman, Ph.D., co-director of the laboratory for drug discovery at the Harvard NeuroDiscovery Center, and her group are studying the EphB3 receptor subtype, which is expressed during embryonic development and in discrete areas of the adult brain, including the cerebellum and hippocampus. It co-localizes to brain regions with high levels of ephrin B ligand expression.

“We identified this as a kinase for stroke, and it is an important kinase for neuroprotection,” she says. “It’s an interesting case of kinase and ligand bi-directional signaling. A lot of the biology is not yet well understood.

“Figuring out cell selectivity is a challenge, more selective is better. People do these large panels, but you don’t know what’s going to be relevant in the cell and in the animal. Kinase activity in the cell and animal is sometimes more relevant than at the isolated protein.”

Binding mode of Avila Therapeutics’ Btk inhibitor (blue) to the Btk kinase catalytic domain (green): The company’s drug binds specifically to the Btk catalytic binding site then binds uniquely with a Cys (yellow) in the binding site, leading to selective silencing.

RTK Inhibitor Discovery

Zhao Ren, M.D., Ph.D., staff scientist at Elan Pharmaceuticals, and his co-workers developed a novel cellular assay in-house. Receptor tyrosine kinases (RTKs) “are an important class of drug targets because of the critical roles they play in cell proliferation and differentiation,” notes Dr. Ren. RTK inhibitors have been aggressively pursued in many different therapeutic areas, including oncology and inflammatory diseases, “and some of these are potential drug candidates.”

According to Dr. Ren, homogenous biochemical assays using recombinant kinase domains of RTKs have been widely adopted to drive inhibitor discovery and structure-activity relationship (SAR) studies. However, there is a lack of robust cellular assays that can measure RTK activity in a high-throughput fashion.

“Interestingly, people examine this class of target using biochemical assays, expressing the kinase domains in bacteria and insect cells, purifying them. We, however, want to explore a different strategy. Cellular assays that can measure RTK activity in their natural environment might be a better approach. A much higher hurdle is developing an assay that works in cells.”

The challenges here are both conceptual and technical, Dr. Ren explains. What happens to a complex receptor is not a single event but a cascade of activities. “We need to figure out what is the best step to monitor receptor activation and what is the best assay technology available.

“We ultimately decided to use AlphaScreen, a luminscent homogeneous immunoassay. This is a simple but effective method to monitor molecular interactions. When two biomolecules form a complex, they also bring a pair of donor and acceptor beads to close proximity. Which, in turn, generates luminescent signals when excited with a laser. That’s how you measure activities in this type of assay.”

Using PerkinElmer’s AlphaScreen technology, Dr. Ren developed several robust cellular RTK assays measuring receptor autophosphorylation. The high assay sensitivity enables miniaturization, and the homogenous nature of these assays renders efficient automation with liquid handlers and robotics.

“We need a lot of cells to do a large screening—one billion cells a day. We discovered another technology on the market, HyperFlask™ by Corning that enabled us to grow multiple layers of cells in the same volume of flask. This is a much more manageable way to keep up with the huge cell culture need,” says Dr. Ren.

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