Screening for Activity and Selectivity
With over 500 kinases in the human genome, it is important to know the specificity profile of development candidates, but this can be time consuming and labor intensive. Ambit's approach is to build ATP-site dependent competition binding assays in an innovative way.
Each kinase or kinase domain is cloned and inserted into a proprietary T7 phage genome. The T7 phage are then allowed to infect E. coli cells. When the phage replicate, the kinase protein "sticks out" from the T7 phage.
"It's like a GFP fusion but instead of GFP, there's a T7 phage," explains Lockhart. In addition to the T7 phage-kinase fusion, the competitive binding assays also employ immobilized bait ligands and the kinase inhibitors to be tested.
Ambit allows the three components to interact in 96-well plates, with separate assays for more than 180 human kinases. After a washing step, the bound T7 phage are eluted and quantified using PCR, which is both sensitive (detection down to 10 molecules of protein per well) and quantitative (proportional to the number of bound molecules).
Ambit has used this technology to profile the selectivity of many kinase inhibitors currently on the market and in development.For example, the company recently discovered that Vertex Pharmaceuticals' (Cambridge, MA) VX-680, currently in Phase I clinical trials as an Aurora inhibitor, also hits Abl and multiple Gleevec-resistant forms of Abl. Information like this provides "clear directions on new uses and new treatment options," says Dr. Lockhart.
Ambit is also using its screening method to identify new kinase inhibitors. "We built a focused collection of small molecules with druglike properties that we think have a high probability of hitting the ATP site of some kinases or a kinase," says Dr. Lockhart.
By screening this library against the panel of human kinases, they came up with a series of potent FLT3 inhibitors with attractive specificity profiles. They are currently selecting a lead to go into formal IND enabling studies.
The question of specificity remains a key issue for small molecule drug discovery in general.
"Some of the activity of small molecules may be based on the fact that they are not selective," says Steve Arkinstall, Ph.D., vp, research, Serono Research Institute (Rockland, MA).
"Companies always strive for selectivity, but if you have really selective molecules they may be less efficacious in some assays modeling certain aspects of disease. The biology is starting to tell us that it's targeting multiple receptors, which that may be a more effective way of controlling angiogenesis. The same is true for tumor cell survival. You may need to target more than one pathway to get results."
According to Brian Healey, Ph.D., head of lead discovery at Serono Research Institute, these considerations have led to a "definite change in how people are focusing their chemistry effort."
As one way of addressing biological complexity, Serono has chosen to pursue high content screening and analysis methods, those where researchers are "measuring multiple endpoints simultaneously and/or where image content or microscopy is used," says Dr. Healey.
His team has developed a new automated high throughput screen for angiogenesis inhibitors. Serono's assay is based on the BD Biosciences (San Jose, CA) endothelial cell tube formation assay system, in which human umbilical vascular endothelial cells or human myometrial vascular endothelial cells are seeded into 96-well plates that contain BD Bioscience's matrigel.
In the presence of appropriate growth factors, the endothelial cells organize into tubular vasculature. Dr. Healey developed an image-processing algorithm using the Molecular Devices' (Sunnyvale, CA) MetaMorph image-analysis software, "compressing 3-D multi-plane images of the microvasculature into a single enhanced image to yield a robust quantitation of tube length."
Combined with a robotic screening system that employs an integrated Discovery-1 automated microscope, the imaging algorithm allows high throughput screening of angiogenesis inhibitors. "Moving to cell-based assays is a way to get closer to the pharmacology," explains Dr. Healey.