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Protein kinase profiling assays: a technology review
Drug Discovery Today: Technologies
Vol. 18, No. nullC 2015
Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Boston, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Profiling used in lead optimization and drug discovery
Protein kinase profiling assays: a technology review Yuren Wang*, Haiching Ma Reaction Biology Corp., One Great Valley Parkway, Suite 2, Malvern, PA 19355, USA
Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.
Introduction Protein kinases are one of the largest families of evolutionarily related proteins and comprise one of the most abundant gene families in humans. The human kinome comprises 518 protein kinases and approximately 20 lipid kinases [1]. Protein kinases act on proteins, phosphorylating them on their serine, threonine, tyrosine, or histidine residues. Phosphorylation can modify a protein functional activity in cells in many ways [2–4]. During the last couple of decades, much clinical evidence has demonstrated that the protein kinases represent some of the most effective therapeutic targets in various types of cancer [5]. Over 20 kinase inhibitors for the treatment of cancers and inflammatory diseases have been approved by the FDA, beginning in 2001 with the approval of *Corresponding author.: Y. Wang ([email protected]) 1740-6749/$ ß 2015 Elsevier Ltd. All rights reserved.
Section editor: Haiching Ma – Reaction Biology Corporation, Malvern, PA, USA. imatinib [6]. The market for kinase inhibitors is expected to continue to grow, with worldwide sales forecasted to reach approximately $20 billion in 2015 [7]. In addition, knowledge of the off-target effects of a kinase inhibitor can be important to understand their biological mechanism particularly for non-selective compounds, to anticipate their potential toxicities, as well as to discover unexpected activities which could lead to different chemical design and possibly novel therapeutic opportunities [8]. Due to the large size of the protein kinase family, many screening technologies which are compatible for highthroughput screening (HTS) against comprehensive panels of kinases and their mutants have been extensively pursued and developed [9–11]. However, the plethora of assay formats also poses a challenge to decide which assay platform or technology will be the best fit. Various factors such as the cost, efficiency, and how convenient the assay is to use, as well as low rates of false positive/negative results should be considered as well. In the sections below, we will discuss the most commonly used kinase assay methods along with their pros and cons in general drug screening applications.
Kinase functional assays Kinase functional assays, also known as activity-based assays or enzymatic assays, directly or indirectly quantify the catalytic product production (i.e., the phosphorylated substrate, ADP or g-phosphate) [10,11]. In a survey of drug discovery labs and companies worldwide in 2013, functional assays were the most preferred format when compared to
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binding-based and cellular assays [12]. Functional assay platforms include radiometric, fluorescence-based, luminescence-based, and mobility shift assays.
Radiometric filtration binding assay The radioisotope filtration binding assay is the most traditional method and is considered the gold standard for kinase profiling assays [10]. It is the only format that directly detects the true product without modified substrates or coupling enzymes. In this assay, test compounds are incubated with kinase, substrate, required cofactors, and radioisotope-labeled ATP such as 32P-g-ATP or 33P-g-ATP. Following incubation of the reaction mixture, radioisotope-labeled catalytic product is spotted onto p-81 phosphocellulose filter papers, which are subsequently washed to remove unreacted radioactive ATP. The incorporation of this radiolabeled phosphate into the kinase substrate is assayed to detect the kinase phosphoryl transfer activity, which is directly proportional to the amount of phosphorylated substrate. The major advantage of this method is that it is a universal kinase assay method which can be used for all protein kinases without limitations. This method does not require any special substrate labeling or modification, and detection is free from interference from fluorescent compounds and unreacted radioisotope. However, the use of radioisotopes and the filter washing and separation steps present a major limitation to applying this technology for large-scale HTS. Both Reaction Biology Corporation (RBC)’s HotSpotSM [13] and Eurofins’s KinaseProfilerTM [14] use this technology for profiling large kinase panels. The HotSpotSM technology developed by RBC is a miniaturized kinase assay platform which reduces the consumption of radioisotope materials, kinase targets, substrates, and compounds, which reduces costs and allows for rapid screening and profiling of compounds. As such, this technology is easily adaptable for use in HTS. Currently RBC offers the largest protein kinase panel in the market with 578 kinases including 366 wild type kinases, 175 mutant kinases, 20 atypical kinases, and 17 lipid kinases for profiling service [13]. Several large scale kinase profiling efforts for known kinase inhibitors using these assay formats have been successfully conducted, and the results provide very useful information on the selectivity of kinase drugs and inhibitors against the human kinome [8,15].
Scintillation proximity assay (SPA) SPA is a radiometric, activity-based assay utilizing radioisotope-labeled ATP (either 32P-g-ATP or 33P-g-ATP) [16,17]. The radiolabeled phosphate is transferred by kinase to the protein or peptide substrate which is captured on the surface of scintillation beads by affinity methods. The most common method to capture the substrate on SPA beads is to biotinylate the substrate and use streptavidin-coated beads. It is also possible to use glutathione-coated beads with a GST fusion e2
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tagged substrate, or Copper His-tag beads for substrates containing the six histidine motif. When the radioisotope is brought into close proximity with the scintillant in the beads, through binding to the substrate, scintillation is triggered. One advantage of this assay format is that there are no wash steps required, making the format HTS friendly. Disadvantages of this format include the requirement for substrate modification and the potential for signal interference when high ATP concentrations are used. Because [33P] is used in SPA, the beads need to be packed by a quick centrifugation after the reaction to reduce background caused by non-proximity effect of [33P]. PerkinElmer also offers two types of scintillant-coated microtiter plates, Scintiplates1 and Flashplates1, for direct assays that eliminate the need to add the SPA beads [18]. The interior of each well in the plates is permanently coated with a thin layer of polystyrene-based scintillant. ProQinase (Freiburg, Germany) uses the FlashPlate1 technology for its PanQinase1 profiling services [19].
Fluorescence intensity assay (FI) To facilitate the HTS for kinase inhibitors, many convenient and automation friendly ‘mix and read’ assays have been developed that use fluorescence emission as a detection method. FI assays are one of the most common fluorescence-based methods for detecting enzyme activity, and are widely used among kinase assays that use protease-based detection reactions. For example, Promega’s ProFluor1 assay utilizes a rhodamine-110 fluorophore conjugated peptide substrate [20]. The non-phosphorylated peptide can be digested by a proprietary endopeptidase to release free fluorescent dye rhodamine-110, but the phosphorylated peptide is resistant to such digestion. However, only a few FI based kinase assay kits are available for kinases including Src-family and PKA, simply because it is difficult to develop peptide substrates that can be used by both the kinases and the special peptidase in the reaction. Several other FI based kinase assays detect the kinase activity by measuring one of the reaction products such as ADP. BellBrook provides a homogenous Transcreener ADP2 FI Assay which detects the ADP production upon the kinase activity by IRDye-linked antibody against ADP [21]. Several other assays of ADP production use linked reactions involving pyruvate kinase, pyruvate oxidase, and horseradish peroxidase. This approach is commercially available from DiscoveRx as ADP HunterTM HS and ADP QuestTM HS which are universal and sensitive assays [22]. However, the multistep reactions involve many enzymes, which can complicate the hit conformation process. The Omnia1 Kinase Assay is a new technology which monitors kinase reactions in kinetic mode by utilizing a chelation-enhanced fluorescence reporter system. It contains a kinase-specific peptide substrate which includes a chelation-enhanced fluorophore (CEF) called Sox, an unnatural amino acid. Upon the phosphorylation by a kinase, the phosphorylated peptide forms a structure to chelate
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Mg2+, and produces a strong fluorescent signal at 485 nm. The assay can be used to select for both ATP-competitive and ATP non-competitive (allosteric) inhibitors. It is also ideal for detailed mechanistic studies for kinase inhibitors since it delivers real-time data. This assay is currently commercialized by Life Technology [23].
Fluorescence polarization assay (FP) FP assays utilize a technology that monitors a fluorescent molecular movement and rotation. When excited with polarized light, a molecule with high molecular weight will have a slower rotational movement compared with a molecule with low molecular weight. Therefore, when a molecule is linked to a fluorescence tracer, the polarized fluorescent signal will be correlated to the size of the molecule. FP assays are widely employed in kinase inhibitor screening. Several companies have developed assay kits using this method, including BellBrook’s TranscreenerTM ADP FP assay [24], Life Technology’s Far-Red PolarScreenTM FP Assay [25], and Molecular Devices’s IMAPTM FP assay [26]. The TranscreenerTM ADP FP assay uses an ADP-specific antibody to detect the conversion of ATP to ADP by the target kinase. By contrast, the PolarScreenTM system is designed to detect the production of phosphorylated substrates using substrate-specific antibodies. The IMAPTM assay uses the high affinity of trivalent metal-containing nanoparticles (beads) to phosphor groups of a fluorescently labeled substrate. The binding of the substrate to the beads is correlated to enzyme activity, and can be detected using FP as a readout. Carna’s QSS AssistTM FP Assay Kit also uses the IMAPTM technology [27]. The advantages of the FP assay include a mix and read approach that is automation friendly and the availability of many assay kits on the market. However, the cost of developing new assays is relatively high, especially for the methods that use specific antibodies and tracers. Therefore, the approaches adapted by IMAPTM and TranscreenerTM are more generally applicable to a limited number of kinases. Similar to other types of fluorescence-based detection methods, FP detection has relatively high rates of false positives and false negatives because of the fluorescence interferences from fluorescent tracers, labeled substrates, and colored and fluorescence compounds [28].
Fluorescence resonance energy transfer (FRET) and Alpha technology FRET involves a non-radiative energy transfer from a donor fluorophore to a close-proximity acceptor fluorophore. The advantages of this method are its homogenous format and simple application to HTS. Life Technology has developed a full panel of synthetic FRET-peptide substrates labeled with a donor (i.e., coumarin) and acceptor (i.e., fluorescein) for its ZLyteTM system [29]. In a non-phosphorylated state, these peptides can be digested by a reporter protease, which will disrupt the FRET pair. A benefit of the FRET assay is its
ratiometric method to quantify kinase reactions by calculating the ratio of donor emissions to acceptor emissions after excitation of the donor fluorophore. This method reduces the well-to-well variations in FRET-peptide concentration and signal intensities. However, this format requires that compounds be screened for inhibitory action against the protease coupling enzyme. Users must also be cautious of interference due to auto-fluorescence from compounds or substrates, though this concern may be addressed by performing ratiometric measurements. Another specific challenge for the FRET system is to design the specific substrate sequences for specific kinases, so Invitrogen uses only a limited number of synthetic substrates in this format. Perkin Elmer developed new Alpha technologies including AlphaScreenTM and AlphaLISA1, which use a different approach of biotinylated kinase substrates together with the proprietary latex donor bead coated with streptavidin and acceptor beads linked with antiphosphate antibody [30]. When both donor and acceptor beads are in close proximity via interaction between the phosphor-substrate and antiphosphate antibody, a photosensitizer in the donor upon laser excitation at 680 nm converts ambient oxygen to the excited singlet state, which diffuses across to react with thioxene in the acceptor generating chemiluminescence at 370 nm. This in turn further activates fluorophores on the same acceptor bead to emit fluorescence signal at 520–620 nm. AlphaScreen1 and AlphaLISA1 technologies can be used to perform tyrosine, serine/threonine, and lipid kinase assays. Substrate capture by the beads is achieved using protein tags (GST, 6His), chemical labels (biotin, digoxygenin, FITC), or by using specific antibodies to detect unmodified endogenous proteins. Both AlphaScreen and AlphaLISA technologies can be used for biochemical assays, though AlphaLISA beads can also be used when working with more-complex matrices such as serum samples or cell lysates. In general, the production of specific antibodies against phosphorylated substrates for the AlphaScreenTM method, especially for antiphosphorylated serine/threonine peptides is a big challenge.
Time-resolved fluorescence assay (TRF) Time-resolved fluorescence assays use fluorophores with long fluorescence decay time. The fluorescence is monitored as a function of time after excitation by a flash of light. Lanthanide ions, such as europium, samarium, and terbium, are commonly used in this technology owing to their longer emission lifetimes (hundreds of microseconds versus several nanoseconds for conventional organic fluorophores). TRF detection, therefore, will reduce background fluorescence from chemical compounds and increase detection sensitivity. Perkin Elmer’s DELFIA1 kits are such TRF-based assays [31]; however, this specific assay involves fluorophore-conjugated substrate binding, separation, and development steps for detection. In DELFIA1 assays, the kinase reactions are carried www.drugdiscoverytoday.com
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out with a biotinylated substrate, and the reaction mixture is then transferred into the capture plate coated with streptavidin. The plate is washed multiple times before europiumlabeled antibody is added to detect phosphorylated substrates. An enhancement solution is then added to dissociate the europium ions into solution, which forms a highly fluorescent chelate with components from the enhancement solution. The assay is sensitive and free of interference from compound fluorescence or fluorophore labeling. However, it may not be a time and cost efficient method owing to the multistep process of transferring, washing, developing, and detecting kinase activity. In addition, antibody selection and development is another challenge for serine/threonine kinase assays.
Time-resolved fluorescence resonance energy transfer (TR-FRET) and homogeneous time resolved fluorescence (HTRF) technology TR-FRET technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. A popular TR-FRET kinase assay employs a peptide substrate, labeled with an acceptor fluorophore, and an anti-phosphopeptide detection antibody, labeled with a donor fluorophore. In this manner, only phosphorylated substrate will exhibit TR-FRET. Several TR-FRET assay kits are available such as PerkinElmer’s Lance1 TR-FRET kinase assay [32] and Life Technology’s LanthaScreenTM TR-FRET kinase assay [33]. The Lance1 assay uses europium chelate as the donor to link with antiphosphorylated biotinylated-substrate antibodies and uses straptavidin–allophycocyanin as the acceptor. Life Technology has developed the LanthaScreenTM reagent system, in which the terbium chelate is used as the donor and the small molecule fluorescein is used as the acceptor. The peptide substrate is labeled with fluorescein directly. On completion of the kinase reaction, terbium-labeled antiphosphopeptide antibody is added for detection, with no need to add additional acceptor molecules. In this manner, the cost of reagents is reduced. Other advantages of TR-FRET assays include the low buffer and media interference due to the dual-wavelength detection, high sensitivity, and robustness that can be miniaturized into the 384 and 1536-well plate formats. However, due to the challenge of developing specific detection antibodies, only peptide substrates are compatible with this format. HTRF is a generic assay technology to TR-FRET, exampled by the HTRF1 KinEASETM assay which was developed by Cisbio [34]. The donor europium is in the cryptate form instead of chelate form, which contains a europium ion caged within a tris-bipyridine structure to improve reagent stability in acidic media and to avoid potential competing chelating activities from other bioreagents such as Mn2+. The donor e4
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and acceptor can be formed by using antibodies against either phosphorylated substrate. The HTRF assay has benefits such as low enzyme consumption and compatibility with a wide range of ATP concentrations (up to mM). Using the same HTRF technology, Cisbio further developed a HTRF1 Transcreener1 ADP assay which is a universal method for identifying and characterizing the activity of all kinases. The ADP formation in the kinase reaction is detected by a specific monoclonal antibody labeled with Eu3+ cryptate, and directly correlates with the amount of phosphorylated substrate in kinase assays. By comparison, the Transcreener1 ADP2 TRFRET Red Assay uses Terbium chelate and produces a far-red, TR-FRET signal [35]. The use of a far-red tracer can significantly minimize interference from fluorescent compounds and light scattering. The ADP methods are enzyme-free detections and compatible with any substrate.
Fluorescence lifetime technology (FLT) Fluorescence lifetime (FLT) technologies are being increasingly viewed as an alternative to ‘traditional’ fluorescencebased readouts. It is a robotic, antibody-free, homogeneous assay platform which enables the user to avoid interference from fluorescent compounds within a screening library. Recent efforts have successfully addressed target class applicability, assay validation, and assay reagent availability issues using FLT. Almac applied this technology in a FLEXYTETM protein kinase assay platform which provides a robust and economical platform for screening and profiling of kinase activity [36]. In this method, a specially designed peptide substrate labeled with fluorescent dye is applied and phosphorylated by the kinase. Then a proprietary Lifetime Modulator (LiM) or a chelate (SMC) reagent is added to complex the phosphate group which causes a reduction in fluorescence lifetime of up to 5 ns. This assay is only currently available for some Ser/Thr kinases and Tyr kinases since limited generic peptide substrates are available. The FLT reader situation has also improved with the launch of a new dedicated FLT plate reader from Edinburgh Instruments able to generate a large signal window with excellent precision using Almac’s FLEXYTE1 assay kits.
Luminescence-based assay Luminescence-based assays measure the depletion of ATP using luciferase, which, in the presence of ATP, converts luciferin to oxiluceiferin, resulting in emission of light. Luminescence has been widely adapted in a variety of kinase assays mainly under the extensive development by companies such as Promega and PerkinElmer. Most luminescencebased kinase assays utilize the firefly protein luciferase which converts the substrate luciferin into oxiluciferin to release a yellow-green photon of light with a spectral maximum of 560 nm in the presence of ATP. Promega’s Kinase-GloTM luminescent kinase assay detects kinase activity by measuring
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the depletion of ATP, making it similar to technologies that measure the production of ADP [37]. This method is a universal biochemical assay that can be used for any combination of kinase and substrate, regardless of the nature of the substrate such as peptide, protein, sugar, and lipids. PerkinElmer’s ATPLiteTM Luminescence method uses the same approach [38]. Though the luminescence-based assay format eliminates the concern of signal interference by fluorescent compounds, compounds need to be screened for inhibitory activity against luciferase. Additionally, the format demonstrates low sensitivity when low ATP concentrations are used.
after which the reaction mixtures are sipped into the microfluidic chip for separation and detection (off-chip assay). In general, the on-chip format requires a high conversion rate (20%) within a short reaction time, which may make the off-chip approach a better choice for the observation of enzyme kinetics within the linear range. One advantage of the off-chip assay is that the reaction mixture can be sipped and separated at different time points to give a real-time kinetic measurement.
Kinase binding assays
ELISA-based detection was widely used before fluorescencebased methods gained wide popularity. In this format, the substrate is captured to a matrix and detected by a specific antiphosphorylated substrate antibody. The chemical compounds are washed away before detection; therefore, the final signal is not affected by the compound’s fluorescence. The detection can also be very sensitive when using antibodies labeled with highly fluorescent dyes. However, the requirement of separation with multiple washing steps limits ELISA’s use in HTS applications. In addition, the development of a specific antibody that recognizes some serine/threonine kinase substrates presents another challenge for its application in kinase profiling. Many companies such as Life Technology, Cell Signaling, Abcam, Cell Biolabs, RayBiotech, etc., offer many ELISA based kinase assay kits for some specific kinases.
Binding assays quantitatively measure the binding of small molecules to the kinase protein, rather than measuring catalytic product. The binding assays are valuable in determining the target engagement and binding affinity. Unlike functional assays, the kinase binding assay can be performed with either active or non-activated kinase preparations. On the other hand, because binding assays are usually performed without ATP or substrate, they are generally unable to detect substrate-specific inhibitors which are of special interest in drug discovery due to their potential for high selectivity. Binding assays are also unlikely to detect inhibitors that interact with domains other than the kinase domain, including the pleckstrin homology (PH) domain, which has emerged as a highly selective anti-cancer target. Therefore, depending on the binding site of compounds on the kinase, the binding activity of compounds does not always translate into the activity on the kinase function.
Mobility shift assay
KinomeScanTM technology
The mobility shift assay takes advantage of the fact that the phosphorylated peptide substrate is more negatively charged than the same substrate in an unphosphorylated state. Consequently, when a mixture of these peptides is introduced to electrophoresis, they differ in mobility. PerkinElmer Company (former Caliper Life Sciences) developed a microfluidic chip-based assay called LabChip EZ Reader MSA assay for kinases [39]. In this approach, labeled substrates are set up in polypropylene microplates. As the kinase reaction is being run in the microplate, small volumes of each assay are ‘sipped’ up into the LabChip microfluidic chip. The LabChip multi-sipper chips comprise small channels connected to upstream and downstream electrodes. As the reaction moves through these channels, the reaction components are separated into substrate and product (or starting material and end material) via differences in their mobility. These differences in mobility are related to the charge/mass ratio of each component (substrate, product, etc.). As each component reaches the detection window on the chip, a fluorescent peak is recorded. PerkinElmer Company provides the ProfilerPro Kinase selectivity assay kit on this technology (on-chip assay). Caliper has also introduced a second method that allows the reactions to be performed in conventional microtiter plates
The KinomeScanTM technology is originally developed by Ambit (San Diego, CA) and later licensed by DiscoveRx [40]. In this platform, the standard kinase inhibitors have been biotinylated and immobilized with Streptavidin-coated magnetic beads. The assay is then carried out by combining the DNA-tagged kinases which are either displayed on the surface of modified T7 phage or in cell culture and test compounds in binding buffers. A test compound with a high binding affinity for the target kinase will prevent the kinase binding to the bead-bound biotinylated ligands, which will then be eluted and quantified by real-time quantitative PCR. The KinomeScanTM technology has been successfully applied to screen a panel of 72 known kinase inhibitors [41].
Enzyme-linked immunosorbent assay (ELISA)
LanthaScreen1 Eu Kinase Binding Assay Invitrogen (ThermoFisher Scientific) develops a LanthaScreen1 Eu Kinase Binding Assay which detects a specific interaction of a Eu-anti-epitope tag antibody bound to the kinase of interest [42]. Binding of an Alexa Fluor1 conjugate or ‘tracer’ to a kinase is detected by addition of a Eu-labeled anti-tag antibody. Binding of the tracer and antibody to a kinase results in a FRET readout, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. Invitrogen’s kinase www.drugdiscoverytoday.com
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Table 1. Overview of common kinase profiling assay platforms Technology
Examples SM
Mechanisms 32
33
Pros
Cons Radioisotope safety management Washing step required
Radioisotope Filter Binding assay
HotSpot KinaseProfilerTM
P or P Direct transfer of radiolabeled phosphate to substrate
Scintillation Proximity Assay
FlashPlate1 SPA1
32P or 33P Direct transfer of radiolabeled phosphate to substrate
Detects true catalytic product Homogenous No washing step required
Fluorescence Intensity Assay
ProFluor1 Transcreener1 ADP HunterTM Omnia1
Homogenous HTS compatible Omnia1 delivers real-time data Ideal for mechanistic study
Need to develop peptide substrates Requires counter screening against coupling enzyme Higher fluorescence interference
Time Resolved Fluorescence Assay
DELFIA1
Long fluorescence Lanthanide ions
Low background fluorescence Low compound interference
Washing and developing steps Modified substrates Antibody development
Fluorescence Polarization Assay
TranscreenerTM PolarScreenTM IMAPTM HitHunterTM PolarScreenTM KinEASETM QSS AssistTM
Fluorescence Monitors a fluorescent molecular movement and rotation
Homogenous HTS compatible Versatile
Costly to develop reagents (antibody, labeled substrate) Relatively high rates of false positives and false negatives
Fluorescence Resonance Energy Transfer (FRET)
Z-LyteTM
Fluorescence A non-radiative energy transfer from a donor fluorophore to a close-proximity acceptor fluorophore
HTS friendly Homogenous High sensitivity Lower assay variation
Synthetic peptide substrate Requires counter screening against protease
Time-resolved Fo¨rster-Resonance Energy Transfer Assay (TR-FRET)
Lance1 TR-FRET LanthaScreenTM KinEASETM TranscreenerTM
Fluorescence FRET with fluorophores of long decay times Antibody-based detection
HTS friendly Homogenous High sensitivity Low assay variability
Antibody development Peptide substrates Substrate modification for fluorophore labeling
Fluorescence lifetime Assay (FLT)
FLEXYTETM
Fluorescence Reduction in fluorescence lifetime of a peptide substrate labeled with fluorescent dye
Homogenous HTS compatible Antibody free Low compound interference
Need specific designed peptide substrate Only apply to some kinases
Luminescence Assay
Kinase-GloTM ATPLiteTM
Coupling assay with luciferase to detect kinase activity (indirect assay format)
Avoids fluorescence interference from compounds HTS friendly Homogenous
Requires counter screening against coupling enzyme Low sensitivity with low ATP dose
Mobility Shift
Caliper technology
Chip-based separation of phosphorylated product from unphosphorylated substrate based on charge/mass ratio
HTS friendly Less interference Measurement of real-time kinetics
Special peptide substrate only Special instrument and analysis High cost
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Fluorescence Varied mechanisms Coupling enzymes Omnia1 uses a chelation-enhanced fluorescence reporter system
Detects true catalytic product No substrate modification Apply to all protein kinases No fluorescence interference HotSpotSM, a low-volume biologically relevant format, is HTS friendly
Radioisotope safety management Substrate modification High ATP limitation Limit to some protein kinases
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Table 1 (Continued ) Technology Competition Binding Assay
Examples TM
KinomeScan LanthaScreen1
Mechanisms
Pros
Cons
Compounds compete with probe/tracer molecule for kinase binding
Amenable to partially purified kinases Measures binding to inactive kinases HTS friendly Kd determination
tracers are based on ATP-competitive kinase inhibitors, making them suitable for detection of any compounds that bind to the ATP site or to an allosteric site altering the conformation of the ATP site. This assay is a simple mix-and-read assay, with no development steps. It is also a useful assay to evaluate kinetics of compound binding particularly for the slow on-rate compounds which are preferentially binding to a conformation of the kinase that is in equilibrium with other forms.
Conclusion Kinase profiling seeks to determine the specificity of a compound against a large panel of diverse kinases. As such, a profiling assay format should be applicable to all kinases within the panel, and immune from interferences both from detections and compounds. By comparison of the assay formats discussed above, the radioisotope-based filtration binding assay stands out as one of the most favorable choices for kinase profiling activity assays because it can universally apply to all protein kinases to measure their functional activities. As such, the radioisotope-based filtration binding assay is often referred to as the ‘gold standard’ method, compared to other assay formats such as fluorescence-based and luminescent platforms. It has been reported that the radioisotope based kinase assay has a relatively lower rate of false positives compared to the fluorescence based assays such as the TR-FRET method [43]. The fluorescence based methods pose significant challenges due to fluorescence interference caused by fluorescent compounds and fluorescently-labeled substrates, antibodies, and tracers. More critically, owing to the modified reaction components and the variety of detection methods, fluorescence based assays with different formats for screening the same library and target could create strikingly different sets of inhibitors [43,44]. Therefore, confirmation assays to validate the initial hits are necessary to reduce the false positive and false negative rates. Binding based assays quantitatively measure the binding of small molecules to the kinase, mostly on the ATP-binding site. In contrast to the functional assays, the binding-based assays do not require ATP in the reaction and therefore the results may not be affected by ATP concentrations. However, it should be noted that the binding affinity data such as the Kd value generated from binding assays may be distinct from Ki values generated from functional based assays because of the different nature of the assay platforms [45]. Therefore, the
Does not measure the catalytic product Need probes or tracer molecules No ATP or substrate No detection of inhibitors that are substrate-specific or bind to non-kinase domains
Kd values and Ki values using these approaches should not be compared directly without understanding the reaction conditions in each assay. In conclusion, the major forms of kinase assay methods and technologies along with their advantages and challenges are discussed and further summarized in Table 1. Although there is no single technology that is sufficient to satisfy all kinase drug discovery needs, the radioisotope filter binding assay remains the gold standard assay for kinase profiling as it is an activity based assay that directly detects the true catalytic product, does not use modified substrates or detection antibodies, does not require counter screening with low compound interference, and most importantly can apply to all protein kinases in a HTS format.
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[16] Ahsen OV, Bc¸mer U. High-throughput screening for kinase inhibitors. ChemBioChem 2005;6:481–90. [17] Available from: http://www.perkinelmer.com/Catalog/Category/ID/ SPA%20Protein%20Kinase%20Assays [18] Geist C, et al. A homogeneous protein kinase assay performed on 384-well streptavidin-coated flashplate HTS PLUS, Available from http:// www.perkinelmer.com/CMSResources/Images/44-73831FSP_File13 HomogeneousProteinKinase.pdf [19] Available from: http://proqinase.com/content/view/127 [20] Available from: http://www.promega.com/products/cell-signaling/ protein-phosphatase-assays/profluor-ser_thr-ppase-assay/ [21] Available from: http://www.bellbrooklabs.com/products-services/ transcreener-hts-assays/adp2-assays/adp2-assays-fi [22] Available from: http://www.discoverx.com/product-data-sheets-3-tab/ 90-0088 [23] Available from: http://www.lifetechnologies.com/us/en/home/ life-science/drug-discovery/target-and-lead-identification-andvalidation/kinasebiology/kinase-activity-assays/omnia-kinase-assays. html [24] Available from http://www.bellbrooklabs.com/products-services/ transcreener-hts-assays/adp2-assays/adp2-assays-fp [25] Kevin L, et al. Redder is better: far-red polarscreenTM FP Kinase Assays; Available from: http://tools.lifetechnologies.com/downloads/ Redder_formated_poster.pdf [26] Available from: http://www.moleculardevices.com/reagents-supplies/ assay-kits/enzymes/imap-assays [27] Available from: http://www.carnabio.com/english/product/assay-fp.html [28] Kashem MA, Nelson RM, Yingling JD, Pullen SS, Prokopowicz III AS, Jones JW, et al. Three mechanistically distinct kinase assay compared: measurement of intrinsic ATPase activity identified the most comprehensives set of ITK inhibitors. J Biomol Screen 2007;12: 70–83. [29] Available from: http://www.lifetechnologies.com/us/en/home/ life-science/drug-discovery/target-and-lead-identificationand-validation/kinasebiology/kinase-activity-assays/z-lyte. html?cid=fl-zlyte [30] Available from http://www.perkinelmer.com/Resources/ TechnicalResources/ApplicationSupportKnowledgebase/ AlphaLISA-AlphaScreen-no-washassays/alpha_kinase_assays.xhtml
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[31] Available from: http://www.perkinelmer.com/catalog/category/id/ delfia%20phosphotyrosine%20and%20phosphoserine%20threonine% 20reagents [32] Available from: http://www.perkinelmer.com/catalog/category/id/ lance%20ultra [33] Available from: http://www.lifetechnologies.com/us/en/home/ life-science/drug-discovery/target-and-lead-identification-and-validation/ kinasebiology/kinase-activity-assays/lanthascreen-tr-fret-toolbox.html [34] Available from: http://www.cisbio.com/drug-discovery/ kinases-cellular-phospho-total-protein-assays [35] Available from: http://bellbrooklabs.com/products-services/transcreenerhts-assays/adp2-assays/adp2-assays-tr-fret-red [36] Available from: http://www.almacgroup.com/ api-services-chemical-development/flexyte-assays/kinases/ [37] Available from: http://www.promega.com/products/cell-signaling/ kinase-assays/kinase_glo-luminescent-kinase-assays/ [38] Available from; http://www.perkinelmer.com/catalog/product/id/ 6016941 [39] Available from http://www.perkinelmer.com/Resources/ TechnicalResources/ApplicationSupportKnowledgebase/LabChip/ labchip_ezreader.xhtml [40] Available from: http://www.discoverx.com/technologies-platforms/ competitive-binding-technology/kinomescan-technology-platform [41] Davis MI, Hunt JP, Herrgard S, Ciceri P, Wodicka LM, Pallares G, et al. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 2011;29:1046–5142. [42] http://www.thermofisher.com/us/en/home/life-science/drug-discovery/ target-and-lead-identification-and-validation/kinasebiology/ kinase-activity-assays/lanthascreentm-eu-kinase-binding-assay. html?ICID=ta-lm-lanthascreen kinase binding assays-LanthaScreen1 Eu Kinase Binding Assay [43] Ahsen OV, Schmidt A, Klotz M, Parczyk K. Assay concordance between SPA and TR-FRET in highthroughput screening. J Biomol Screen 2006;11:606–16. [44] Wu X, Sills MA, Zhang JH. Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability. J Biomol Screen 2005;9:581–9. [45] Buolamwini JK, Kamath S. Molecular-kinase interaction map, Opinion and comment section. Nat Biotechnol 2005;11:1346–8.
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Editors-in-Chief Kelvin Lam – Simplex Pharma Advisors, Inc., Boston, MA, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Profiling used in lead optimization and drug discovery
Protein kinase profiling assays: a technology review Yuren Wang*, Haiching Ma Reaction Biology Corp., One Great Valley Parkway, Suite 2, Malvern, PA 19355, USA
Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.
Introduction Protein kinases are one of the largest families of evolutionarily related proteins and comprise one of the most abundant gene families in humans. The human kinome comprises 518 protein kinases and approximately 20 lipid kinases [1]. Protein kinases act on proteins, phosphorylating them on their serine, threonine, tyrosine, or histidine residues. Phosphorylation can modify a protein functional activity in cells in many ways [2–4]. During the last couple of decades, much clinical evidence has demonstrated that the protein kinases represent some of the most effective therapeutic targets in various types of cancer [5]. Over 20 kinase inhibitors for the treatment of cancers and inflammatory diseases have been approved by the FDA, beginning in 2001 with the approval of *Corresponding author.: Y. Wang ([email protected]) 1740-6749/$ ß 2015 Elsevier Ltd. All rights reserved.
Section editor: Haiching Ma – Reaction Biology Corporation, Malvern, PA, USA. imatinib [6]. The market for kinase inhibitors is expected to continue to grow, with worldwide sales forecasted to reach approximately $20 billion in 2015 [7]. In addition, knowledge of the off-target effects of a kinase inhibitor can be important to understand their biological mechanism particularly for non-selective compounds, to anticipate their potential toxicities, as well as to discover unexpected activities which could lead to different chemical design and possibly novel therapeutic opportunities [8]. Due to the large size of the protein kinase family, many screening technologies which are compatible for highthroughput screening (HTS) against comprehensive panels of kinases and their mutants have been extensively pursued and developed [9–11]. However, the plethora of assay formats also poses a challenge to decide which assay platform or technology will be the best fit. Various factors such as the cost, efficiency, and how convenient the assay is to use, as well as low rates of false positive/negative results should be considered as well. In the sections below, we will discuss the most commonly used kinase assay methods along with their pros and cons in general drug screening applications.
Kinase functional assays Kinase functional assays, also known as activity-based assays or enzymatic assays, directly or indirectly quantify the catalytic product production (i.e., the phosphorylated substrate, ADP or g-phosphate) [10,11]. In a survey of drug discovery labs and companies worldwide in 2013, functional assays were the most preferred format when compared to
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binding-based and cellular assays [12]. Functional assay platforms include radiometric, fluorescence-based, luminescence-based, and mobility shift assays.
Radiometric filtration binding assay The radioisotope filtration binding assay is the most traditional method and is considered the gold standard for kinase profiling assays [10]. It is the only format that directly detects the true product without modified substrates or coupling enzymes. In this assay, test compounds are incubated with kinase, substrate, required cofactors, and radioisotope-labeled ATP such as 32P-g-ATP or 33P-g-ATP. Following incubation of the reaction mixture, radioisotope-labeled catalytic product is spotted onto p-81 phosphocellulose filter papers, which are subsequently washed to remove unreacted radioactive ATP. The incorporation of this radiolabeled phosphate into the kinase substrate is assayed to detect the kinase phosphoryl transfer activity, which is directly proportional to the amount of phosphorylated substrate. The major advantage of this method is that it is a universal kinase assay method which can be used for all protein kinases without limitations. This method does not require any special substrate labeling or modification, and detection is free from interference from fluorescent compounds and unreacted radioisotope. However, the use of radioisotopes and the filter washing and separation steps present a major limitation to applying this technology for large-scale HTS. Both Reaction Biology Corporation (RBC)’s HotSpotSM [13] and Eurofins’s KinaseProfilerTM [14] use this technology for profiling large kinase panels. The HotSpotSM technology developed by RBC is a miniaturized kinase assay platform which reduces the consumption of radioisotope materials, kinase targets, substrates, and compounds, which reduces costs and allows for rapid screening and profiling of compounds. As such, this technology is easily adaptable for use in HTS. Currently RBC offers the largest protein kinase panel in the market with 578 kinases including 366 wild type kinases, 175 mutant kinases, 20 atypical kinases, and 17 lipid kinases for profiling service [13]. Several large scale kinase profiling efforts for known kinase inhibitors using these assay formats have been successfully conducted, and the results provide very useful information on the selectivity of kinase drugs and inhibitors against the human kinome [8,15].
Scintillation proximity assay (SPA) SPA is a radiometric, activity-based assay utilizing radioisotope-labeled ATP (either 32P-g-ATP or 33P-g-ATP) [16,17]. The radiolabeled phosphate is transferred by kinase to the protein or peptide substrate which is captured on the surface of scintillation beads by affinity methods. The most common method to capture the substrate on SPA beads is to biotinylate the substrate and use streptavidin-coated beads. It is also possible to use glutathione-coated beads with a GST fusion e2
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tagged substrate, or Copper His-tag beads for substrates containing the six histidine motif. When the radioisotope is brought into close proximity with the scintillant in the beads, through binding to the substrate, scintillation is triggered. One advantage of this assay format is that there are no wash steps required, making the format HTS friendly. Disadvantages of this format include the requirement for substrate modification and the potential for signal interference when high ATP concentrations are used. Because [33P] is used in SPA, the beads need to be packed by a quick centrifugation after the reaction to reduce background caused by non-proximity effect of [33P]. PerkinElmer also offers two types of scintillant-coated microtiter plates, Scintiplates1 and Flashplates1, for direct assays that eliminate the need to add the SPA beads [18]. The interior of each well in the plates is permanently coated with a thin layer of polystyrene-based scintillant. ProQinase (Freiburg, Germany) uses the FlashPlate1 technology for its PanQinase1 profiling services [19].
Fluorescence intensity assay (FI) To facilitate the HTS for kinase inhibitors, many convenient and automation friendly ‘mix and read’ assays have been developed that use fluorescence emission as a detection method. FI assays are one of the most common fluorescence-based methods for detecting enzyme activity, and are widely used among kinase assays that use protease-based detection reactions. For example, Promega’s ProFluor1 assay utilizes a rhodamine-110 fluorophore conjugated peptide substrate [20]. The non-phosphorylated peptide can be digested by a proprietary endopeptidase to release free fluorescent dye rhodamine-110, but the phosphorylated peptide is resistant to such digestion. However, only a few FI based kinase assay kits are available for kinases including Src-family and PKA, simply because it is difficult to develop peptide substrates that can be used by both the kinases and the special peptidase in the reaction. Several other FI based kinase assays detect the kinase activity by measuring one of the reaction products such as ADP. BellBrook provides a homogenous Transcreener ADP2 FI Assay which detects the ADP production upon the kinase activity by IRDye-linked antibody against ADP [21]. Several other assays of ADP production use linked reactions involving pyruvate kinase, pyruvate oxidase, and horseradish peroxidase. This approach is commercially available from DiscoveRx as ADP HunterTM HS and ADP QuestTM HS which are universal and sensitive assays [22]. However, the multistep reactions involve many enzymes, which can complicate the hit conformation process. The Omnia1 Kinase Assay is a new technology which monitors kinase reactions in kinetic mode by utilizing a chelation-enhanced fluorescence reporter system. It contains a kinase-specific peptide substrate which includes a chelation-enhanced fluorophore (CEF) called Sox, an unnatural amino acid. Upon the phosphorylation by a kinase, the phosphorylated peptide forms a structure to chelate
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Mg2+, and produces a strong fluorescent signal at 485 nm. The assay can be used to select for both ATP-competitive and ATP non-competitive (allosteric) inhibitors. It is also ideal for detailed mechanistic studies for kinase inhibitors since it delivers real-time data. This assay is currently commercialized by Life Technology [23].
Fluorescence polarization assay (FP) FP assays utilize a technology that monitors a fluorescent molecular movement and rotation. When excited with polarized light, a molecule with high molecular weight will have a slower rotational movement compared with a molecule with low molecular weight. Therefore, when a molecule is linked to a fluorescence tracer, the polarized fluorescent signal will be correlated to the size of the molecule. FP assays are widely employed in kinase inhibitor screening. Several companies have developed assay kits using this method, including BellBrook’s TranscreenerTM ADP FP assay [24], Life Technology’s Far-Red PolarScreenTM FP Assay [25], and Molecular Devices’s IMAPTM FP assay [26]. The TranscreenerTM ADP FP assay uses an ADP-specific antibody to detect the conversion of ATP to ADP by the target kinase. By contrast, the PolarScreenTM system is designed to detect the production of phosphorylated substrates using substrate-specific antibodies. The IMAPTM assay uses the high affinity of trivalent metal-containing nanoparticles (beads) to phosphor groups of a fluorescently labeled substrate. The binding of the substrate to the beads is correlated to enzyme activity, and can be detected using FP as a readout. Carna’s QSS AssistTM FP Assay Kit also uses the IMAPTM technology [27]. The advantages of the FP assay include a mix and read approach that is automation friendly and the availability of many assay kits on the market. However, the cost of developing new assays is relatively high, especially for the methods that use specific antibodies and tracers. Therefore, the approaches adapted by IMAPTM and TranscreenerTM are more generally applicable to a limited number of kinases. Similar to other types of fluorescence-based detection methods, FP detection has relatively high rates of false positives and false negatives because of the fluorescence interferences from fluorescent tracers, labeled substrates, and colored and fluorescence compounds [28].
Fluorescence resonance energy transfer (FRET) and Alpha technology FRET involves a non-radiative energy transfer from a donor fluorophore to a close-proximity acceptor fluorophore. The advantages of this method are its homogenous format and simple application to HTS. Life Technology has developed a full panel of synthetic FRET-peptide substrates labeled with a donor (i.e., coumarin) and acceptor (i.e., fluorescein) for its ZLyteTM system [29]. In a non-phosphorylated state, these peptides can be digested by a reporter protease, which will disrupt the FRET pair. A benefit of the FRET assay is its
ratiometric method to quantify kinase reactions by calculating the ratio of donor emissions to acceptor emissions after excitation of the donor fluorophore. This method reduces the well-to-well variations in FRET-peptide concentration and signal intensities. However, this format requires that compounds be screened for inhibitory action against the protease coupling enzyme. Users must also be cautious of interference due to auto-fluorescence from compounds or substrates, though this concern may be addressed by performing ratiometric measurements. Another specific challenge for the FRET system is to design the specific substrate sequences for specific kinases, so Invitrogen uses only a limited number of synthetic substrates in this format. Perkin Elmer developed new Alpha technologies including AlphaScreenTM and AlphaLISA1, which use a different approach of biotinylated kinase substrates together with the proprietary latex donor bead coated with streptavidin and acceptor beads linked with antiphosphate antibody [30]. When both donor and acceptor beads are in close proximity via interaction between the phosphor-substrate and antiphosphate antibody, a photosensitizer in the donor upon laser excitation at 680 nm converts ambient oxygen to the excited singlet state, which diffuses across to react with thioxene in the acceptor generating chemiluminescence at 370 nm. This in turn further activates fluorophores on the same acceptor bead to emit fluorescence signal at 520–620 nm. AlphaScreen1 and AlphaLISA1 technologies can be used to perform tyrosine, serine/threonine, and lipid kinase assays. Substrate capture by the beads is achieved using protein tags (GST, 6His), chemical labels (biotin, digoxygenin, FITC), or by using specific antibodies to detect unmodified endogenous proteins. Both AlphaScreen and AlphaLISA technologies can be used for biochemical assays, though AlphaLISA beads can also be used when working with more-complex matrices such as serum samples or cell lysates. In general, the production of specific antibodies against phosphorylated substrates for the AlphaScreenTM method, especially for antiphosphorylated serine/threonine peptides is a big challenge.
Time-resolved fluorescence assay (TRF) Time-resolved fluorescence assays use fluorophores with long fluorescence decay time. The fluorescence is monitored as a function of time after excitation by a flash of light. Lanthanide ions, such as europium, samarium, and terbium, are commonly used in this technology owing to their longer emission lifetimes (hundreds of microseconds versus several nanoseconds for conventional organic fluorophores). TRF detection, therefore, will reduce background fluorescence from chemical compounds and increase detection sensitivity. Perkin Elmer’s DELFIA1 kits are such TRF-based assays [31]; however, this specific assay involves fluorophore-conjugated substrate binding, separation, and development steps for detection. In DELFIA1 assays, the kinase reactions are carried www.drugdiscoverytoday.com
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out with a biotinylated substrate, and the reaction mixture is then transferred into the capture plate coated with streptavidin. The plate is washed multiple times before europiumlabeled antibody is added to detect phosphorylated substrates. An enhancement solution is then added to dissociate the europium ions into solution, which forms a highly fluorescent chelate with components from the enhancement solution. The assay is sensitive and free of interference from compound fluorescence or fluorophore labeling. However, it may not be a time and cost efficient method owing to the multistep process of transferring, washing, developing, and detecting kinase activity. In addition, antibody selection and development is another challenge for serine/threonine kinase assays.
Time-resolved fluorescence resonance energy transfer (TR-FRET) and homogeneous time resolved fluorescence (HTRF) technology TR-FRET technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. A popular TR-FRET kinase assay employs a peptide substrate, labeled with an acceptor fluorophore, and an anti-phosphopeptide detection antibody, labeled with a donor fluorophore. In this manner, only phosphorylated substrate will exhibit TR-FRET. Several TR-FRET assay kits are available such as PerkinElmer’s Lance1 TR-FRET kinase assay [32] and Life Technology’s LanthaScreenTM TR-FRET kinase assay [33]. The Lance1 assay uses europium chelate as the donor to link with antiphosphorylated biotinylated-substrate antibodies and uses straptavidin–allophycocyanin as the acceptor. Life Technology has developed the LanthaScreenTM reagent system, in which the terbium chelate is used as the donor and the small molecule fluorescein is used as the acceptor. The peptide substrate is labeled with fluorescein directly. On completion of the kinase reaction, terbium-labeled antiphosphopeptide antibody is added for detection, with no need to add additional acceptor molecules. In this manner, the cost of reagents is reduced. Other advantages of TR-FRET assays include the low buffer and media interference due to the dual-wavelength detection, high sensitivity, and robustness that can be miniaturized into the 384 and 1536-well plate formats. However, due to the challenge of developing specific detection antibodies, only peptide substrates are compatible with this format. HTRF is a generic assay technology to TR-FRET, exampled by the HTRF1 KinEASETM assay which was developed by Cisbio [34]. The donor europium is in the cryptate form instead of chelate form, which contains a europium ion caged within a tris-bipyridine structure to improve reagent stability in acidic media and to avoid potential competing chelating activities from other bioreagents such as Mn2+. The donor e4
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and acceptor can be formed by using antibodies against either phosphorylated substrate. The HTRF assay has benefits such as low enzyme consumption and compatibility with a wide range of ATP concentrations (up to mM). Using the same HTRF technology, Cisbio further developed a HTRF1 Transcreener1 ADP assay which is a universal method for identifying and characterizing the activity of all kinases. The ADP formation in the kinase reaction is detected by a specific monoclonal antibody labeled with Eu3+ cryptate, and directly correlates with the amount of phosphorylated substrate in kinase assays. By comparison, the Transcreener1 ADP2 TRFRET Red Assay uses Terbium chelate and produces a far-red, TR-FRET signal [35]. The use of a far-red tracer can significantly minimize interference from fluorescent compounds and light scattering. The ADP methods are enzyme-free detections and compatible with any substrate.
Fluorescence lifetime technology (FLT) Fluorescence lifetime (FLT) technologies are being increasingly viewed as an alternative to ‘traditional’ fluorescencebased readouts. It is a robotic, antibody-free, homogeneous assay platform which enables the user to avoid interference from fluorescent compounds within a screening library. Recent efforts have successfully addressed target class applicability, assay validation, and assay reagent availability issues using FLT. Almac applied this technology in a FLEXYTETM protein kinase assay platform which provides a robust and economical platform for screening and profiling of kinase activity [36]. In this method, a specially designed peptide substrate labeled with fluorescent dye is applied and phosphorylated by the kinase. Then a proprietary Lifetime Modulator (LiM) or a chelate (SMC) reagent is added to complex the phosphate group which causes a reduction in fluorescence lifetime of up to 5 ns. This assay is only currently available for some Ser/Thr kinases and Tyr kinases since limited generic peptide substrates are available. The FLT reader situation has also improved with the launch of a new dedicated FLT plate reader from Edinburgh Instruments able to generate a large signal window with excellent precision using Almac’s FLEXYTE1 assay kits.
Luminescence-based assay Luminescence-based assays measure the depletion of ATP using luciferase, which, in the presence of ATP, converts luciferin to oxiluceiferin, resulting in emission of light. Luminescence has been widely adapted in a variety of kinase assays mainly under the extensive development by companies such as Promega and PerkinElmer. Most luminescencebased kinase assays utilize the firefly protein luciferase which converts the substrate luciferin into oxiluciferin to release a yellow-green photon of light with a spectral maximum of 560 nm in the presence of ATP. Promega’s Kinase-GloTM luminescent kinase assay detects kinase activity by measuring
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the depletion of ATP, making it similar to technologies that measure the production of ADP [37]. This method is a universal biochemical assay that can be used for any combination of kinase and substrate, regardless of the nature of the substrate such as peptide, protein, sugar, and lipids. PerkinElmer’s ATPLiteTM Luminescence method uses the same approach [38]. Though the luminescence-based assay format eliminates the concern of signal interference by fluorescent compounds, compounds need to be screened for inhibitory activity against luciferase. Additionally, the format demonstrates low sensitivity when low ATP concentrations are used.
after which the reaction mixtures are sipped into the microfluidic chip for separation and detection (off-chip assay). In general, the on-chip format requires a high conversion rate (20%) within a short reaction time, which may make the off-chip approach a better choice for the observation of enzyme kinetics within the linear range. One advantage of the off-chip assay is that the reaction mixture can be sipped and separated at different time points to give a real-time kinetic measurement.
Kinase binding assays
ELISA-based detection was widely used before fluorescencebased methods gained wide popularity. In this format, the substrate is captured to a matrix and detected by a specific antiphosphorylated substrate antibody. The chemical compounds are washed away before detection; therefore, the final signal is not affected by the compound’s fluorescence. The detection can also be very sensitive when using antibodies labeled with highly fluorescent dyes. However, the requirement of separation with multiple washing steps limits ELISA’s use in HTS applications. In addition, the development of a specific antibody that recognizes some serine/threonine kinase substrates presents another challenge for its application in kinase profiling. Many companies such as Life Technology, Cell Signaling, Abcam, Cell Biolabs, RayBiotech, etc., offer many ELISA based kinase assay kits for some specific kinases.
Binding assays quantitatively measure the binding of small molecules to the kinase protein, rather than measuring catalytic product. The binding assays are valuable in determining the target engagement and binding affinity. Unlike functional assays, the kinase binding assay can be performed with either active or non-activated kinase preparations. On the other hand, because binding assays are usually performed without ATP or substrate, they are generally unable to detect substrate-specific inhibitors which are of special interest in drug discovery due to their potential for high selectivity. Binding assays are also unlikely to detect inhibitors that interact with domains other than the kinase domain, including the pleckstrin homology (PH) domain, which has emerged as a highly selective anti-cancer target. Therefore, depending on the binding site of compounds on the kinase, the binding activity of compounds does not always translate into the activity on the kinase function.
Mobility shift assay
KinomeScanTM technology
The mobility shift assay takes advantage of the fact that the phosphorylated peptide substrate is more negatively charged than the same substrate in an unphosphorylated state. Consequently, when a mixture of these peptides is introduced to electrophoresis, they differ in mobility. PerkinElmer Company (former Caliper Life Sciences) developed a microfluidic chip-based assay called LabChip EZ Reader MSA assay for kinases [39]. In this approach, labeled substrates are set up in polypropylene microplates. As the kinase reaction is being run in the microplate, small volumes of each assay are ‘sipped’ up into the LabChip microfluidic chip. The LabChip multi-sipper chips comprise small channels connected to upstream and downstream electrodes. As the reaction moves through these channels, the reaction components are separated into substrate and product (or starting material and end material) via differences in their mobility. These differences in mobility are related to the charge/mass ratio of each component (substrate, product, etc.). As each component reaches the detection window on the chip, a fluorescent peak is recorded. PerkinElmer Company provides the ProfilerPro Kinase selectivity assay kit on this technology (on-chip assay). Caliper has also introduced a second method that allows the reactions to be performed in conventional microtiter plates
The KinomeScanTM technology is originally developed by Ambit (San Diego, CA) and later licensed by DiscoveRx [40]. In this platform, the standard kinase inhibitors have been biotinylated and immobilized with Streptavidin-coated magnetic beads. The assay is then carried out by combining the DNA-tagged kinases which are either displayed on the surface of modified T7 phage or in cell culture and test compounds in binding buffers. A test compound with a high binding affinity for the target kinase will prevent the kinase binding to the bead-bound biotinylated ligands, which will then be eluted and quantified by real-time quantitative PCR. The KinomeScanTM technology has been successfully applied to screen a panel of 72 known kinase inhibitors [41].
Enzyme-linked immunosorbent assay (ELISA)
LanthaScreen1 Eu Kinase Binding Assay Invitrogen (ThermoFisher Scientific) develops a LanthaScreen1 Eu Kinase Binding Assay which detects a specific interaction of a Eu-anti-epitope tag antibody bound to the kinase of interest [42]. Binding of an Alexa Fluor1 conjugate or ‘tracer’ to a kinase is detected by addition of a Eu-labeled anti-tag antibody. Binding of the tracer and antibody to a kinase results in a FRET readout, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. Invitrogen’s kinase www.drugdiscoverytoday.com
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Table 1. Overview of common kinase profiling assay platforms Technology
Examples SM
Mechanisms 32
33
Pros
Cons Radioisotope safety management Washing step required
Radioisotope Filter Binding assay
HotSpot KinaseProfilerTM
P or P Direct transfer of radiolabeled phosphate to substrate
Scintillation Proximity Assay
FlashPlate1 SPA1
32P or 33P Direct transfer of radiolabeled phosphate to substrate
Detects true catalytic product Homogenous No washing step required
Fluorescence Intensity Assay
ProFluor1 Transcreener1 ADP HunterTM Omnia1
Homogenous HTS compatible Omnia1 delivers real-time data Ideal for mechanistic study
Need to develop peptide substrates Requires counter screening against coupling enzyme Higher fluorescence interference
Time Resolved Fluorescence Assay
DELFIA1
Long fluorescence Lanthanide ions
Low background fluorescence Low compound interference
Washing and developing steps Modified substrates Antibody development
Fluorescence Polarization Assay
TranscreenerTM PolarScreenTM IMAPTM HitHunterTM PolarScreenTM KinEASETM QSS AssistTM
Fluorescence Monitors a fluorescent molecular movement and rotation
Homogenous HTS compatible Versatile
Costly to develop reagents (antibody, labeled substrate) Relatively high rates of false positives and false negatives
Fluorescence Resonance Energy Transfer (FRET)
Z-LyteTM
Fluorescence A non-radiative energy transfer from a donor fluorophore to a close-proximity acceptor fluorophore
HTS friendly Homogenous High sensitivity Lower assay variation
Synthetic peptide substrate Requires counter screening against protease
Time-resolved Fo¨rster-Resonance Energy Transfer Assay (TR-FRET)
Lance1 TR-FRET LanthaScreenTM KinEASETM TranscreenerTM
Fluorescence FRET with fluorophores of long decay times Antibody-based detection
HTS friendly Homogenous High sensitivity Low assay variability
Antibody development Peptide substrates Substrate modification for fluorophore labeling
Fluorescence lifetime Assay (FLT)
FLEXYTETM
Fluorescence Reduction in fluorescence lifetime of a peptide substrate labeled with fluorescent dye
Homogenous HTS compatible Antibody free Low compound interference
Need specific designed peptide substrate Only apply to some kinases
Luminescence Assay
Kinase-GloTM ATPLiteTM
Coupling assay with luciferase to detect kinase activity (indirect assay format)
Avoids fluorescence interference from compounds HTS friendly Homogenous
Requires counter screening against coupling enzyme Low sensitivity with low ATP dose
Mobility Shift
Caliper technology
Chip-based separation of phosphorylated product from unphosphorylated substrate based on charge/mass ratio
HTS friendly Less interference Measurement of real-time kinetics
Special peptide substrate only Special instrument and analysis High cost
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Fluorescence Varied mechanisms Coupling enzymes Omnia1 uses a chelation-enhanced fluorescence reporter system
Detects true catalytic product No substrate modification Apply to all protein kinases No fluorescence interference HotSpotSM, a low-volume biologically relevant format, is HTS friendly
Radioisotope safety management Substrate modification High ATP limitation Limit to some protein kinases
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Table 1 (Continued ) Technology Competition Binding Assay
Examples TM
KinomeScan LanthaScreen1
Mechanisms
Pros
Cons
Compounds compete with probe/tracer molecule for kinase binding
Amenable to partially purified kinases Measures binding to inactive kinases HTS friendly Kd determination
tracers are based on ATP-competitive kinase inhibitors, making them suitable for detection of any compounds that bind to the ATP site or to an allosteric site altering the conformation of the ATP site. This assay is a simple mix-and-read assay, with no development steps. It is also a useful assay to evaluate kinetics of compound binding particularly for the slow on-rate compounds which are preferentially binding to a conformation of the kinase that is in equilibrium with other forms.
Conclusion Kinase profiling seeks to determine the specificity of a compound against a large panel of diverse kinases. As such, a profiling assay format should be applicable to all kinases within the panel, and immune from interferences both from detections and compounds. By comparison of the assay formats discussed above, the radioisotope-based filtration binding assay stands out as one of the most favorable choices for kinase profiling activity assays because it can universally apply to all protein kinases to measure their functional activities. As such, the radioisotope-based filtration binding assay is often referred to as the ‘gold standard’ method, compared to other assay formats such as fluorescence-based and luminescent platforms. It has been reported that the radioisotope based kinase assay has a relatively lower rate of false positives compared to the fluorescence based assays such as the TR-FRET method [43]. The fluorescence based methods pose significant challenges due to fluorescence interference caused by fluorescent compounds and fluorescently-labeled substrates, antibodies, and tracers. More critically, owing to the modified reaction components and the variety of detection methods, fluorescence based assays with different formats for screening the same library and target could create strikingly different sets of inhibitors [43,44]. Therefore, confirmation assays to validate the initial hits are necessary to reduce the false positive and false negative rates. Binding based assays quantitatively measure the binding of small molecules to the kinase, mostly on the ATP-binding site. In contrast to the functional assays, the binding-based assays do not require ATP in the reaction and therefore the results may not be affected by ATP concentrations. However, it should be noted that the binding affinity data such as the Kd value generated from binding assays may be distinct from Ki values generated from functional based assays because of the different nature of the assay platforms [45]. Therefore, the
Does not measure the catalytic product Need probes or tracer molecules No ATP or substrate No detection of inhibitors that are substrate-specific or bind to non-kinase domains
Kd values and Ki values using these approaches should not be compared directly without understanding the reaction conditions in each assay. In conclusion, the major forms of kinase assay methods and technologies along with their advantages and challenges are discussed and further summarized in Table 1. Although there is no single technology that is sufficient to satisfy all kinase drug discovery needs, the radioisotope filter binding assay remains the gold standard assay for kinase profiling as it is an activity based assay that directly detects the true catalytic product, does not use modified substrates or detection antibodies, does not require counter screening with low compound interference, and most importantly can apply to all protein kinases in a HTS format.
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