Enzyme Assays

Kinase and phosphatase assay technologies can be divided roughly into those employing whole cells and those employing purified enzymes. Protein kinase enzyme assays require ATP, magnesium (and sometimes manganese) cofactors and a peptide or protein substrate. The requirements for phosphatase enzyme assays are simpler and well described by Montalibet et al. (2005). One must have a method to detect the conversion of substrate by determining either the formation of


HTS Assay Development for Kinases and Phosphatases

Assay Te ch n o I ogy (Commercial and

Alternate Names) Technology Principles Advantages Disadvantages References

Fluorescence polarization (anisotropy) version 1 (InVitroGen PolarScreen)

Fluorescence polarization (anisotropy) version 2 (IMAP)

Scintillation proximity (FlashPlate, SPA)

Fluorescence resonance energy transfer (quenched fluorescence, InVitroGen Z'-LYTE)

Immunosorbent assays (enzyme-linked or fluorescent-linked, cell signaling, PathScan)

Fluorescently labeled substrate peptide binds to anti-phospho antibody after phosphorylation; change in Brownian motion of peptide-antibody complex results in change in anisotropy measured by polarization of incoming light

Fluorophore labeled peptides bind to special detection beads coated with trivalent metal; binding results in change in Brownian motion measured as with FP1 above

Product of reaction is a 33P labeled peptide biotin that can be captured on a detection bead that scintillates from proximity to 33P; dephosphorylation by phosphatases can be detected in signal decrease assay

Peptide labeled with fluorescein and coumarin is quenched until cleavage by protease, modification by phosphorylation, or dephosphorylation by kinase or phosphatase produces resistance to proteolytic cleavage

Antibodies coated onto MTP wells capture kinase or phosphatase substrate; phosphorylation state is detected by anti-phosphopeptide antibody coupled to detector dye; can be read by time-resolved fluorescence (DELFIA) technique

High-throughput, only one labeled substrate required

Versatile without need for antibody

High-throughput, relatively artifact free in imaging-based systems; universal readout for kinases; versatile

Miniaturizeable, ratiometric readout normalizes for pipetting errors; can be applied to kinases and phosphatases

Can be used as sensitive probe for cell lysates in cell-based assays

Susceptible to compound interference; peptide must be relatively small; precludes use of protein substrates

Susceptible to compound interference; peptide must be relatively small; precludes use of protein substrates

Radioactive waste disposal; may be less sensitive than TR-FRET

Coupled assay can be susceptible to protease inhibitors

Lower throughput and wash steps required; must have suitable cell line and antibody pair

Parker (2000); Sills (2002); Newman (2004); Turek-Etienne (2003b)

Turek-Etienne (2003a)

Rodems (2002)

Waddleton (2002); Minor (2003); Zhang (2007)



HTS Assay Development for Kinases and Phosphatases

Assay Technology (Commercial and

Alternate Names) Technology Principles




Kinase-dependent cell growth BaF3

Luciferase-based ATP detection (Promega kinase Glo, Perkin-Elmer Easylite Kinase)

Luminescent oxygen channeling (Perkin Elmer AlphaScreen, Surefire)

Time-resolved Forster resonance energy transfer (version 1: InVitroGen LanthaScreen, Perkin Elmer LANCE, CysBio KinEase)

Cell number or metabolism measured by standard cell detection methods such as ATP detection or MTT dye, Alamar blue; cell growth dependent upon specific tyrosine kinase

ATP-dependent luminescent signal from luciferase conversion of luminal; kinase-dependent depletion of ATP is measured

Anti-phosphotyrosine or -phosphopeptide antibodies bind only to phosphorylated substrate; complex is detected by streptavidin and protein A functionalized beads that when bound together produce channeling of singlet oxygen when stimulated by light; singlet oxygen reacts with acceptor beads to emit photons of lower wavelength than their excitation frequency

Phosphopeptide formation is detected by europium chelate and LI light acceptor dye PKA substrates; dephosphorylation by phosphatases can be detected in signal decrease assay

Very high-throughput cell-based assay

Versatile and non-radioactive

Sensitive, high-throughput; can be applied to cell lysates as substitute for ELISA; proximity distances very large relative to energy transfer; emission frequency lower than excitation frequency, thus eliminating potential artifacts by fluorescent compounds; can be applied to whole cell assays

Very sensitive and miniaturizeable; ratiometric readout normalizes for pipetting errors

Limited for use with tyrosine kinases; cell line must be made

Signal decrease assay; susceptible to luciferase inhibitors

Can be susceptible to interference by compounds that trap singlet oxygen; must work under subdued or specialized lighting

Requires two specialized antibodies; susceptible to interference; low dynamic range for substrate turnover; binding interaction should be within restricted proximity for optimal efficiency

Warmuth (2007)

Koresawa (2004)

Leoprechting (2004); Warner (2004)

Moshinsky (2003); Vogel (2006); Von Ahsen (2006); Schroeter (2008)

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Time-resolved Forster resonance energy transfer (version 2: BellBrook Labs Transcreener, Adapta)

In-Cell Western (Li-COR Cytoblot)

Prompt fluorescence with small molecule substrates (DiFMUP, AnaSpec Sensolyte)

Enzyme fragment complementation (DiscoveRx ED-NSIP HitHunter)

Imaging assays (DiscoveRx PathHunter, Bioimage)

ADP formation by kinase is detected by displacement of red-shifted TR-FRET system between Alexafluor 647-ADP analog and a europium chelated anti-ADP antibody

High-throughput; miniaturizeable; versatile; ratiometric readout

Anti-phospho specific antibody labeled with infrared fluor Only requires one antibody; has potential is used to probe fixed cells on MTP; second color can be used for normalization; binding detected via laser scanning technique

Action of enzyme on substrate converts substrate into fluorescent compound; excellent substrates available for phosphatases

Two fragments of reporter protein fusion are joined via biomolecular interaction, thus reconstituting activity of reporter protein; kinases can be assayed in displacement mode using staurospaurine conjugate to one fragment and kinase fused to second fragment of /3-galactosidase (ED-NSIP); test compound displaces kinase-staurospaurine interaction, and decreases B-gal activity

Can use recombinant green fluorescent or yellow fluorescent fusion proteins to measure kinase-stimulated signalling events such as protein translocation to nucleus or membrane to cytosolic translocations for multiplexing to control for total substrate expression in cells

Versatile; very strong signal

High-throughput; sensitive; amplified enzymatic; chemiluminescent signal less susceptible to interference

Biological assay; can present kinase in native form; examines functional consequences of test compound

Signal decrease assay; binding interaction should be in close proximity (7 to 9 nM)

Multiple wash steps; requires cell fixation; lower throughput

Requires highly purified enzymes, or contaminating phosphatases can be deceptive

Coupled assay; may have interference with /3-galactosidase binding compounds; compound must be competitive with probe

Difficult to develop; lower throughput

Huss (2007)

Chen (2005)

Watanabe (1998); Johnston (2007); Montalibet (2005)

Eglen (2002); Vainshtein (2002)

Nickischer (2006); Traskjr (2006)

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Small molecule substrates for phosphatases


Phosphate +

Small molecule substrates for phosphatases





Antibody dependent ADP detection: fluorescence polarization

[33P]ATP phosphotransfer: SPA, FlashPlate p decay







ATP Luciferin o9

Antibody dependent ADP detection: fluorescence polarization

[33P]ATP phosphotransfer: SPA, FlashPlate p decay


Oxyluciferin PPi Light IC


Antibody-dependent phosphopeptide detection: y^^nM XR-FRET,

AlphaScreen, Fluorescence polarization


FIGURE 1.1 HTS enzyme assay concepts for kinase and phosphatase screening.

phosphopeptide, or phosphoprotein, or the disappearance of adenosine triphosphate (ATP) or the formation of adenosine diphosphate (ADP).

Phosphatase enzyme essays most commonly employ artificial small molecule substrates that become fluorescent by removal of the phosphate moiety. Other methodologies similar to those employed in kinase assays can also be used, in which the removal of phosphate from a peptide or protein substrate can be detected. Figure 1.1 shows the basic principles. Many commercially available kits and published references describe these methodologies (Table 1.1).

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