Gain-of-Signal Assays for Kinases
“With growing interest in evaluating unactive kinases as putative targets, the market is looking for approaches where screening technology is a nonactivity-based approach,” said Sailaja Kuchibhatla, senior vice president of business development at DiscoveRx (www.discoverx.com). “HitHunter™ kinase-binding assays are preferred when both kinase substrate and detection antibody are unknown and kinase activation is not necessary. It is the only high-throughput screening technology currently available for unactive and low-activity kinases.”
DiscoveRx presented two posters, the ADP Hunter Plus and the HitHunter™-b-galactosidase enzyme fragment complementation for kinase binding assays. The ADP Hunter Plus is a nonradioactive, nonantibody approach that measures ADP generation (gain-of-function) in the context of ATP depletion instead of just measuring ATP depletion (loss-of-signal).
“Unlike ATP-depletion assays, ADP-production assays can be performed with whole protein or peptide substrates. ADP output is measured by subsequent ADP-mediated fluorescent signal. The assay conditions have a high tolerance for DTT and are not hampered by background or compound fluorescence,” explained Kuchibhatla.
HitHunter kinase binding assays are based on b-galactosidase-based enzyme fragment complementation. This technology is based on two inactive fragments (large fragment EA and peptide fragment ED) of b-galactosidase enzyme that become active when combined in solution. For kinase binding assays, the ED fragment is coupled to ligands that compete with kinase inhibitors for binding to kinase of interest.
Seth Cohen, Ph.D., director of application sciences at Caliper Life Sciences (www.caliperls.com), presented a tutorial on “Labchip technology from assay development to mechanism of action.” He discussed the use and validation of Caliper’s LabChip® 3000 Drug Discovery System with mobility-shift kinase assays.
“A key feature of the LabChip 3000 system is it allows one to look at enzyme rates in a real-time mode, as one can analyze samples while the kinase reaction is in progress. The LabChip 3000 uses Caliper’s sipper chips for real-time automated sampling.
“Combining real-time kinetics analysis using the LC3000 with robotic automation and design of experimentation software, a wide variety of assay conditions enable rapid kinase assay development. One can, for example, run 370 kinase reaction conditions varying buffer type, pH, detergents, and salts in a single-plate experiment.
“The ability to combine robotics in assay execution along with automated data analysis results in an efficient and noniterative assay-development process,” said Dr. Cohen. Many firms have implemented LabChip technology, notably Pfizer, which is doing compound profiling of kinase inhibitors using the mobility-shift format.