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.