Amber Dance is an award-winning freelance science writer based in Southern California. She is the ALS (Lou Gehrig’s disease) reporter for the Alzheimer Research Forum. She contributes to The Scientist and Nature journals, and has written about topics ranging from record-breaking rocks to bizarre new ant species.
Kinases are major players in biology, sprinkling phosphate groups throughout the cell to regulate such baseline activities as growth, division and death. Drug makers are eager to develop kinases inhibitors to treat a variety of conditions, especially cancer, and scientists can use myriad assays to test those compounds, including both biochemical and cell-based platforms.
The types of assays are well established, says Carolyn Pettersson, senior manager of product development at Life Technologies. Now, companies continue to add kinases to their platforms, from ABL1 to ZIPK, working up to humankind’s “kinome” of more than 500 enzymes.
Biochemical kinase assays frequently work for high-throughput screens, costing as little as pennies per well. Many researchers prefer to outsource kinase work, especially for large panels of drug candidates or kinases. For example, Reaction Biology runs kinase assays for customers at any stage in the drug-discovery process. Similarly, Life Technologies conducts kinase experiments for clients in its SelectScreen program. After researchers have identified their favorite candidate drugs, they can test the compounds in cell-based assays, a more physiological format.
Kinase classic
Classic kinase assays that measure the transfer of radioactive phosphate from ATP to substrate, like those protocols used by Reaction Biology for its clients, remain the “gold standard,” says Haiching Ma, chief science officer at the company. There is no need to modify the substrate or incorporate additional detection mechanisms. That means a lower rate of false negatives and false positives, Ma says.
Others believe nonradioactive alternatives work perfectly well and without the dangers and inconvenience of radioactivity. “Fluorescent kinase assays have become widely accepted over the last decade,” notes Pettersson.
Activity assays
Fluorescent assays include Life Technologies’ LanthaScreen Kinase Activity Assay, which is based on fluorescence resonance energy transfer (FRET). The protocol mixes the kinase with a fluorescein-labeled substrate and ATP. After the reaction, users add an antibody specific for the phosphorylated substrate and tagged with a terbium fluorophore. If the antibody finds its substrate, FRET occurs, creating a readable signal.
Life Technologies also offers the Z’-LYTE assay, which uses peptide substrates with a fluorophore on each end. While the peptide remains intact, FRET occurs. Phosphorylation by the substrate’s kinase protects it from cleavage by a protease. If the substrate remains unphosphorylated, it is cleaved, eliminating the FRET signal. Life Technologies recommends LanthaScreen for a first pass and Z’-LYTE for secondary screens.
Binding assays
Products like Z’-LYTE measure kinase activity, but others simply ask whether a compound of interest binds the kinase at all. Generally, a molecule that binds to a kinase’s active or allosteric site is likely to inhibit it, says Abhishek Saharia, senior product manager at DiscoveRx. For example, the leukemia drug Gleevec works by binding inactive Abl kinase, preventing it from adopting its active conformation.
DiscoveRx’s KINOMEscan service, for drug-development stages from high-throughput screens to selectivity and potency testing, measures kinase-inhibitor binding only. A bait molecule that binds the kinase’s active site is bound to a bead. If the drug binds the kinase as well, it competes with the bait or alters the active site, reducing the amount of kinase attached to the beads. To quantitate how much kinase stays stuck to the baited beads, DiscoveRx has tagged each kinase with DNA. Quantitative PCR tallies the amount of kinase present.
Life Technologies’ LanthaScreen Eu Kinase Binding Assay, for target screening, works on a similar principle. It starts with a fluorescently labeled tracer that binds the kinase. If an inhibitor that also binds the kinase is present, it outcompetes the tracer, kicking it off the kinase and blocking FRET between the tracer and a fluorescently labeled antibody.
Universal assays
Suppose your favorite kinase has not yet made the list in kinase-assay catalogs. Universal assays typically measure ADP production as kinases pull phosphate groups off ATP, and they work for any kinase-substrate pair. These are particularly useful for lipid kinases, because lipids are more difficult than peptides to modify for standard assays like Z’-LYTE, says Steve Riddle, senior staff scientist at Life Technologies.
Life Technologies’ Adapta platform, for primary and secondary screens, is one such universal assay. Again, it relies on a FRET signal, this time between an ADP-like tracer and an anti-ADP antibody. If real ADP is produced, it displaces the tracer, disrupting the FRET signal. Another option is DiscoveRx’s ADP Hunter assay, appropriate for high-throughput screening. A pair of enzymes converts ADP to a fluorescent molecule so it can be measured.
Alternatively, many companies can create custom assays for kinases not already in their catalogs.
Cell-based assays
For most scientists, biochemical assays are the “frontline” experiments, says Ma. They are typically less expensive, and more high-throughput-compatible, than cell-based assays. In-cell assays make good secondary tools to confirm that the reactions in the test tube match those in a living cell.
DiscoveRx’s InCell Hunter platform, launched in 2012, is one cell-based assay. It is compatible with high-throughput screens but is best suited for optimizing a drug’s specificity and potency in cells, Saharia says. InCell Hunter is based on the principle that when an inhibitor binds a kinase, it alters the stability of the enzyme—sometimes for the better, sometimes for the worse, says Saharia. By detecting the amount of kinase present, users can infer whether a candidate compound bound the kinase.
To measure the kinase, InCell Hunter culture lines include a kinase linked to a piece of the beta-galactosidase enzyme. At the end of the experiment, users add the other half of the beta-galactosidase and its substrate. Active beta-galactosidase hydrolyzes the substrate, producing a chemiluminescent signal proportional to the amount of kinase in the cell.
These are just a few of the many kinase assays available as kits or services. Make sure you understand how the assay works and what you are measuring, so you can select the right assay for your needs, Ma recommends.