May 15, 2015 (Vol. 35, No. 10)

Kinase Assays that are Compatible with High-Throughput Screening Techniques Are Essential

Kinases play critical roles in cellular function and disease pathology. They can have stimulatory or inhibitory effects on multiple targets. As a result, researchers are investigating the entire family of kinases (kinome) through large-scale genomic, proteomic, and chemical biological analyses. The goals are to get a better handle on kinase function, identify new kinase inhibitors for cancer therapy, and screen candidates for side-effects on other pathways.

To help researchers get the most out of these assays, GEN has put together this roundup, which presents advice collected from several kinase assay experts.

GEN: What are favored kinase assays for large-scale screening of numerous samples?

Mr. Bosse: Favored high-throughput kinase assays include biochemical assays relying on purified enzymes and substrates, or more biologically relevant cellular assays. Biochemical assays can be performed using fluorescent technologies (such as lanthanide chelate excite, or LANCE, for time-resolved fluorescence resonance energy transfer, or TR-FRET) or chemiluminescent technologies (such as AlphaScreen).

SureFire is a popular technology to measure kinase activity from cellular extracts. Live cell kinase assays can be executed using bioluminescence energy transfer (BRET), high-content screening (HCS), or phenotypic label-free techniques (such as Epic).

Dr. Goueli: The ADP-Glo™ assay is widely used for high-throughput screening of kinase inhibitors. The assay is universal, which means it is applicable for all classes of kinases with a variety of substrates ranging from peptide, proteins, and lipids to sugars and alcohols.

This also makes it ideal for profiling lead compounds after screening. The add-and-read format makes it amenable to a broad range of plate sizes. The assay is robust with a Z' of over 0.8, which indicates high reproducibility.

ADP-Glo is very sensitive, requires small amount of enzymes, and does not require antibodies or the use of radioactive nucleotides. These features make it ideal for screening kinases for novel modulators with high efficiency.

Dr. Noah: One of the most favored kinase assays for large-scale screening is a phospho-specific antibody-based assay that utilizes a precoated substrate readily phosphorylated by a specific protein kinase. It is a nonradioactive approach used for the detection of protein kinase activity in partially purified, purified, or crude enzyme preparations from any species.

It is a flexible colorimetric assay that can be used as an endpoint or a kinetic assay readout in a 96-well plate format with easy sample-handling protocols. This assay format can be easily automated to allow screening of chemical libraries for the identification of new kinase inhibitors.

Dr. Vogel: For highest throughput applications, simplicity, cost, and robustness are primary considerations. Many scientists have found TR-FRET-based kinase binding assays to be ideal for this application, since these assays directly measure the ability of a compound to bind to a kinase, and are independent of kinase activity. As a result, these assays do not require any optimization of reaction conditions, such as substrate and ATP concentrations or reaction time. In addition to cost reductions associated with the lack of requirement for substrate, the format is readily miniaturized to a low-volume 384- or 1,536-well format.

GEN: What are the best assays for oncological targets?

Mr. Bosse: Oncological kinase targets can be divided in two categories: extracellular targets (receptor tyrosine kinases or RTKs) and cytosolic targets. RTKs are normally characterized by measuring tyrosine phosphorylation with technologies such as scintillation proximity assay (SPA), LANCE, or AlphaScreen. Cytosolic targets encompass tyrosine and serine/threonine kinases also assayed with the same assay technologies.

Dr. Goueli: Oncological targets, which include growth factor receptor tyrosine kinases, soluble tyrosine kinases, and lipid kinases, require an assay that is universal and tolerates a broad range of ATP and substrate concentrations. ADP-Glo has these features since it is universal and homogenous. It is very fast, convenient to carry out, and less costly. It can be used in any research lab without the need for radioactivity training and licensing or any concerns about the quality of the antibodies. It is also very sensitive, which makes it ideal for these targets, which tend to have low activity in vitro.

Dr. Noah: Kinase assays are used in cancer research to study oncogenic signaling. In particular, the Akt/PKB pathway is a popular target since it sits at the crossroads of both oncogenic and tumor suppressor signaling networks. Nonradioactive kinase activity assays targeted at Akt, PKA, and PKC provide a safe, rapid, and reliable method for the screening of inhibitors or activators. Due to the high degree of homology in the ATP-binding pocket between Akt, PKA, and PKC, orthogonal assays should be used to verify the specificity of therapeutic compounds against the target of interest.

Dr. Vogel: When screening for compounds to specifically target oncology-linked kinase mutations, it is important to screen against both the mutant kinase and the wild-type kinase to minimize eventual toxicities. After narrowing down compounds based on biochemical assays, most researchers move on to cell-based assays in which the full-length target kinase is present in its native environment, and phosphorylation of a cellular substrate is measured. Although this has historically been accomplished using western blots or ELISAs, the use of GFP-fusions substrates allows detection of kinase activity in solution by using lanthanide-labeled phosphospecific antibodies that form a FRET pair with the phosphorylated substrate.

This image depicts the dichloroacetic acid (DCA) molecule. Salts of DCA are being investigated as potential drugs because they inhibit pyruvate dehydrogenase kinase, which has been investigated as a new therapeutic target in oncology. []

GEN: What are the disadvantages of fluorescence-based kinase assays, and how can they be addressed?

Mr. Bosse: Disadvantages include artifacts due to prompt fluorescence and color quenching. Prompt fluorescence can be avoided using time-resolved fluorescence (TRF, TR-FRET) or by opting for chemiluminescent assays. Color quenching can addressed using radiometric technologies such as 33P detection via standard scintillation or scintillation proximity.

Dr. Goueli: Fluorescent-based assays suffer rom the liability of generating large number of false hits and the concern about the reliability of the hits generated from a screen. This becomes a big issue when screening large and diverse libraries of compounds. Bioluminescent-based assays such as ADP-Glo circumvent these problems and have been shown to generate very low numbers of false hits.

The fluorescent-based assays also rely on selective and sensitive probes, the design of which requires modification of the substrates and incur higher cost. One of the main drawbacks of current fluorescent assays is the intolerance for high concentrations of substrates since they are used as probes and cannot be used at high concentrations in the assays.

Dr. Noah: Fluorescence-based kinase assays are cost effective, do not require radioactivity, and allow for miniaturization. As a result, they increase the overall throughput of screening compounds in kinase assays.

However, the main disadvantages of this format are signal quenching of fluorescent compounds and interference due to precipitation. Red-shifted substrates have been developed to help with these issues. Such assays contain a substrate that reads in the red end of the spectrum as opposed to the green end. This approach allows one to avoid interference at the lower wavelengths caused by some compounds. Often such substrates are designed to have better solubility allowing for a lower Km to be used, which is very advantageous for kinase inhibitor screening.

Dr. Vogel: As with any optical technique, fluorescence-based kinase assays can be impacted by optical interference caused by colored compounds absorbing excitation or emission light, insoluble compounds scattering light, or autofluorescent compounds causing abnormally high readings in assay wells.

To an extent, time-resolved fluorescence-based assays overcome many of these impacts. The ratiometric readout can compensate for the presence of compounds that absorb light, and the time-resolved format means that the assay signal can be measured after interference from scattered light or autofluorescent compounds decays.

However, depending on the degree of interference (since most compounds are screened at micromolar concentrations, and assay components are present at nanomolar concentrations), severely interfering compounds can be flagged by looking at the raw assay data before it is processed to a ratiometric readout. 

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