A kinase is an enzyme that aids in the transfer of a phosphate from ATP to another specific molecule. There are more than 550 members of the human kinase enzyme protein family. They regulate many activities in the human body. Drug development relies heavily on kinase screening assay techniques to identify new drug targets. The kinase assay is used to test potential drugs or test compounds to see how they affect kinase activity, and thus enable drug discovery progress. Compounds that inhibit kinase activity in vitro can be further studied to see if they have pharmacological characteristics that make them suitable as a drug. These features include potent inhibition of a specific kinase, lack of toxicity, and the ability to remain stable in the body long enough to be effective.
Figure 1 Kinase Enzymes
Kinase activity assay techniques involve a variety of approaches. Quantifying kinase activity requires detecting either the formation of the phosphorylated product or measuring the amount of ATP used or ADP formed. Regardless of which kinase assay principle is chosen, there are several different kinase assay in vitro formats and readout options available.
This kinase assay protocol tracks the transfer of the radioisotope 32P from ATP, [γ-32P] to the substrate. The amount of phosphorylated substrate that is formed is quantified by scintillation counting.
Limitations: Disposal of radioactive reagents is costly, requires special environmental, health and safety considerations, and must be carefully documented to meet regulations.
This approach measures the binding of small molecules to the kinase of interest, usually by monitoring signal from a fluorescent tracer. When the tracer is bound to the kinase, fluorescence resonance energy transfer (FRET) occurs and when the tracer is displaced, FRET is reduced.
A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation.
This kinase assay principal allows investigation of a broad variety of kinases, regardless of the acceptor substrate. kinase assay kits also lower the cost of assay development because one set of assay reagents can be used for all targets. Because ADP is detected directly there is a lower risk of compound interference from coupling agents.
Screen for small molecule inhibitors
Determine enzymatic activity
Profile inhibitor potency/determine IC50 values
Measure residence time/off-rate/dissociation of lead molecules with target
Inhibitor selectivity profiling
Mechanism of action studies
Given the therapeutic and commercial success of small-molecule enzyme inhibitors, as exemplified by kinase inhibitors in oncology, a major focus of current drug discovery and development efforts is on enzyme targets. Understanding the process of an enzyme-catalyzed reaction can help to conceptualize different types of inhibitors and to inform the design of screens to identify desired mechanisms. Exploiting this information allows the thorough evaluation of diverse compounds, providing the knowledge required to efficiently optimize leads towards differentiated candidate drugs. CD ComputaBio provides kinase assays for HTS drug screening efforts. Reagents and instrumentation are readily available for your requirements. Please contact us for more service details.
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