Drug Discovery
Drug Discovery
Review of biochemical assays for protein kinase drug discovery
Kinase
Peptide substrate + ATP
Hu Li
Mg2+
Phospho-peptide + ADP
Fig. 1 Schematic depiction of kinase reaction
Group I assays: phosphopeptide detection
About the Authors: Dr. Hu Li is a
Manager of Biological Reagents and Assay
Development in Molecular Discovery
Research, GlaxoSmithKline. He has 12
years experience in the field of drug
discovery focusing on small molecular
candidates spanning from target
identification to assay development and
Group II assays: Detection of ATP depletion
variety of assay technologies and handson experience in many assay platforms
Kinase-Glo®
Easylite-Kinase™
PKLight™
Group III assays: Detection of ADP production
High Throughput Screening (HTS) and
lead optimization. He has knowledge of a
Radioactive-based
Filter binding
SPA, FlashPlate®
Non-radioactive-based
IMAP®
LanthaScreen™
KinEASE™ (LANCE®)
AlphaScreen® PhosphoSensor
Z’-LYTE™ Kinase Assay
Caliper technology
Omnia® Kinase Assay
HitHunter™ EFC Kinase Assays
Antibody-based
Transcreener®
Adapta®
Non-antibody-based
ADP Quest™
ADP-Glo™
across many target classes including
kinases, GPCRs, Nuclear Receptors and
novel enzymes. Dr. Li is a member of NIH
INTRODUCTION
study section of Assay Development for
High Throughput Molecular Screening
(R21) and HTS assay review (R03). Dr. Li
obtained his PhD degree in biochemistry
from Bryn Mawr College in 1996, an
MS degree in bioinorganic chemistry
from Academia Sinica in 1989, and a
BS degree in inorganic chemistry from
Nanjing University in 1986. Dr. Li is a lifetime member of SAPA.
24
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
Protein kinases are enzymes that transfer a γ-phosphate group
from ATP to their substrate (e.g. protein, peptide), generating
phosphorylated protein/peptide and ADP as products (Fig. 1).
They play a pivotal role in all aspects of cellular physiology such
as growth, differentiation, and metabolism. Their involvement
in pathological conditions such as cancer, inflammatory diseases,
neuronal disorders, and metabolic disorders make kinases important targets for drug discovery1,2.
Identification of protein kinase modulators through high
throughput screening (HTS) has become a common strategy for
kinase drug discovery in both academia and the pharmaceutical
industry. To feed the increasing demand, vendors have commercialized a plethora of kinase assays that are readily available
to users. These assays measure kinase activity by detecting either
increase of the products (e.g. phosphopeptide, ADP) or decrease
of reactants (e.g. ATP) of a kinase reaction.
Several reviews published on kinase assay technologies
in the past few years 3,4,5 do not cover the most recent
advances in this field and do not emphasize the utility of
the following aspects: HTS vs profiling vs mode of action
(MOA) studies. This review will briefly summarize assays
that are being commonly employed in protein kinase
drug discovery, introduce a few novel assays emerging on
the market, and discuss the pros and cons of each assay
in relation to its application.
Kinase assays can be roughly categorized into three
groups based on their detection modality: Group I: Detection of phosphopeptide; Group II: Measurement of
ATP consumption; Group III: Quantification of ADP
production. In each group, there exist multiple readouts
based on which technology is being used, including
radioactivity, fluorescence, luminescence, fluorescence
polarization (FP), time-resolved fluorescence resonance
energy transfer (TR-FRET).
GROUP I ASSAYS: PHOSPOPEPTIDE DETECTION
accommodate screening of large collections of synthetic
compounds led to the development of the Scintillation
Proximity Assay (SPA)6. This method utilizes scintillation beads coated with a variety of materials to capture
phosphorylated products which incorporate 33P-ATP.
Typically, a peptide is biotinylated so that it can be
captured by streptavidin coated beads. Before the debut
of imaging readers such as LeadSeeker (Amersham
Pharmacia) or ViewLux (Perkin Elmer), blue-shifted
polyvinyl toluene (PVT) and yttrium silicate (YSi) beads
were used with Topcount. These beads were replaced
with red-shifted polystyrene (PS) and yttrium oxide (YO)
beads which can be read on detectors equipped with
a CCD camera such as LeadSeeker and ViewLux. The
CCD camera-equipped imagers provide much faster
reading speed and the red-shifted beads are subject to
less interference by color compounds than blue-shifted
beads in a screening collection. Alternatively, as shown in
Fig. 2, kinase substrate can be captured on the FlashPlate
surface coated with scintillant. Upon kinase reaction, the
incorporated 33P on the substrate is brought into close
proximity with the scintillant coating on FlashPlate. The
resulting luminescent signal is detected on a microplate
scintillation counter or luminescent reader. These two
methods are both high-throughput and can be used for
HTS. However, the bead-based SPA is preferred due to
its “mix-and-read” nature, which is easy to automate on
HTS platforms.
Radioactivity-based
Filter binding assay (FBA)
Quantification of phosphorylated peptide in
a kinase reaction provides a direct measurement of kinase activity. Filter binding assays
are considered the “gold standard” compared with non-radiometric methods. In this
format, 33P-ATP is spiked in a kinase reaction and the resulting 33P-phosphopeptide is
captured on a nitrocellulose filter. The filter
is dried and radioactivity is counted on a
scintillation counter (Topcount, MicroBeta).
However, due to low throughput and radioFig. 2. Illustration of FlashPlate Technology (source: Perkin Elmer Life Sciactivity usage, this method is rarely used for
ences)
HTS. Many reagent companies and Contract
Research Organizations (CROs) utilize these
assays for compound profiling services. These methods
Non-radioactive-based
are also often used in MOA studies in the lead optimization phase of drug discovery.
Although FlashPlate and bead-based SPA both provide
adequate throughput for HTS, their high cost, and raScintillation Proximity Assay (SPA and FlashPlate®)
dioactivity have prompted development of assays using
The call for a higher throughput radiometric assay to
fluorescence-based technologies.
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
25
Drug Discovery
IMAP®
IMAP (Immobilized Metal Affinity Phosphorylation)
7,8,9
was commercialized by Molecular Devices, now
part of MDS Analytical Technologies. This assay uses
fluorescently-labeled peptide (available either in fluorescein or TAMRA) as a kinase substrate. After a kinase
phosphorylation step, the phosphopeptide is captured by
a metal chelated bead as shown in Fig. 3A. The binding
to large molecular weight beads results in a change in
anisotropy of the fluoropeptide, i.e. higher fluorescence
polarization (FP) signal compared with lower FP signal
generated by unbound fluorescently labeled peptide.
®
As a further innovation on IMAP®-FP, IMAP beads
were spiked with terbium to provide TR-FRET measurement (Fig. 3B). Upon excitation at 337nm, Tb (donor)
transfers energy to the fluorescent label on the peptide
and generates FRET signal. The readout of this assay is
calculated from the emission ratio of fluorescent label to
that of Tb, which is 520nm/490nm for fluorescein and
570nm/550nm for TAMRA, respectively.
Drug Discovery
IMAP® assays are applicable to both serine/threonine
and tyrosine kinases and there are many labeled peptides available from MDS Analytical Technologies. In
addition, both of these formats are homogeneous, high
throughput, and can be easily miniaturized into 384 or
1536 format. The Progressive Binding Buffer system
allows the use of peptides containing multiple acidic
residues and has high ATP tolerance. However, higher
percentage conversion of substrate is required to generate robust signal in IMAP®-FP. The TR-FRET readout
offers the following advantages over IMAP®-FP: (1)
Robustness at low % phosphorylation; (2) Flexibility in
substrate size (can use full size protein); (3) flexibility in
substrate concentration; (4) Direct determination of apparent substrate Km; (5) Less fluorescence interference.
LanthaScreen™
LanthaScreen™ is a relatively new method commercialized by Invitrogen. The principle of LanthaScreen™ is
described in Fig. 4.
HO
FL
ce
S/T/Y
eq
es
tid
p
Pe
n
ue
FL
PO4
Kinase, ATP
S/T/Y
pt
Pe
u
eq
es
id
Mg2+
ce
en
+
Tb
Em: 520nm
Energy transfer
Ex. 337nm
FL
PO4
Tb
Fig.3A. Illustration of IMAP®-FP assay (source: Molecular
ce
S/T/Y
eq
es
tid
p
Pe
n
ue
Devices)
Fig. 4. LanthaScreen™ assay principle. Tb: Terbium; SA:
Streptavidin; S/T/Y: Serine/Threonine/Tyrosine; FL: Fluorescein
Fig. 3B. Illustration of IMAP®-TR-FRET assay (source:
Molecular Devices)
26
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
Like IMAP®, this assay utilizes a fluorescein-labeled substrate. Once the kinase reaction is quenched with EDTA,
terbium (Tb)-labeled anti-phospho antibody is added
to the mixture to capture the phosphorylated product.
Upon exciting at 337nm, Tb emits at 490nm, which
transfers energy to the fluorescein molecule tagged to
the substrate resulting in an increased FRET signal. The
long decay time after excitation of Tb allows for time-resolved measurement. Signal from this assay is calculated
as the ratio of the acceptor (fluorescein) emission to the
donor (terbium) emission, 520nm/490nm. The amount
of antibody bound to the phospho-peptide is directly
proportional to the amount of phosphorylated substrate
in the reaction. This is a simple ratiometric-based “mixand-read” method, amenable for HTS. However, the
short emission wavelength of fluorescein at 520nm has
a potential for interference from colored compounds.
In addition, specific antibody is needed to capture the
phosphorylated peptide, which limits the scope of its
application.
KinEASE™ /LANCE®
Using similar technology, Perkin Elmer and CisBio
market KinEASE™ and LANCE®, respectively. The
LANCE® assay uses europium (Eu) chelate as the
donor to couple with acceptors such as the fluorescent
protein allophycocyanin (APC or XL-665). The donor
Eu-chelate is usually conjugated with phospho-specific
antibody which binds phosphorylated product. The
acceptor molecule is linked to streptavidin (SA), which
binds to the fluoro-tagged biotinylated peptide. The
formation of this large complex brings donor in close
proximity to acceptor. Excitation at 337nm will lead to a
FRET transfer from donor to acceptor, which emits at
665nm. For KinEASE™ assay, the only difference is the
chelate. A europium ion is caged within a tris-bipyridine,
called cryptate. This complex improves reagent stability in acidic environment and against EDTA, which is
often used to stop the reaction. Both assays are homogeneous, very sensitive, and easy to miniaturize. The
longer emission wavelength of the acceptor at 665nm
avoids potential interference from colored compounds.
However, the complicated setup involving Eu-phosphospecific antibody, SA-acceptor, biotin-peptide requires
multiple optimization steps, which generally increases
assay development time and costs.
AlphaScreen® PhosphoSensor
Fig. 5. Illustration of AlphaScreen® PhosphoSensor assay
(source: Perkin Elmer Life Sciences)
cascade of activation steps among different fluorophores
within the acceptor bead, a fluorescence signal at 520620nm is emitted and detected. The advantages of this
assay are that it is homogenous, it does not require the
use of antibodies and the substrate can be a full-length
protein. AlphaScreen® technology offers advantages over
TR-FRET in that the coupling distance for donor and
acceptor is much longer (~200nm vs ~10nm). However,
due to the light sensitivity of the beads, it may be tricky
to control assay variability in day to day operation. In
addition, the Envision reader requires an additional AlphaScreen® capability.
Z’-LYTE™ Kinase Assay
In the Z’-LYTE™ assay format, the peptide substrate is
tagged with a donor fluorophore (coumarin) and acceptor fluorophore (fluorescein) at each end which makes
up a FRET pair 12. This peptide also contains a built-in
protease cleavage site. Once the kinase reaction is complete, a cocktail with protease is added for detection.
The AlphaScreen® PhosphoSensor is an antibody-free
kinase assay. The assay relies on the use of a biotinylated
kinase substrate with a pair of donor and acceptor beads
10,11
as depicted in Fig. 5.
Typically, the donor bead is coated with streptavidin,
which binds to a biotinylated substrate, and the acceptor
bead is conjugated with the antiphospho Lewis metal
chelate (LMC3+). Once substrate is phosphorylated by
a kinase, it brings the donor and acceptor beads into
close proximity (<200nm). Upon excitation at 680nm,
a photosensitizer in the donor bead converts ambient oxygen into the excited singlet state, which diffuses
across the acceptor bead and reacts with thioxene and
thus generates chemiluminescence at 370nm. Through a
Fig. 6. Illustration of Z’-LYTE™ Kinase Assay (source:
Invitrogen)
The assay principle is depicted in Fig. 6. In the absence
of phosphorylation, the peptide is cleaved by a site-specific protease. However, phosphorylation of the peptide
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
27
Drug Discovery
Caliper technology
Caliper technology (Caliper Life Sciences, Hopkinton,
MA) is based on the micro-fluidic mobility shift using
fluorescently labeled peptide substrate. Upon kinase
phosphorylation, the product has a different net charge
compared to the unphosphorylated peptide. As a result,
the product and substrate have different mobility in an
electric field and they generate different signals when
they reach the detector. One unique feature of Caliper’s
Technology is that both unphosphorylated and phosphorylated products are accurately detected simultaneously and the reaction process can be monitored. Advantages of this technology include straight-forward data
interpretation, elimination of interference compounds,
and real-time kinetic study. This assay is suitable for
profiling and MOA studies. Like other assay methods,
it has limitations, too, including low throughput (not
applicable for HTS), high substrate turnover to generate an adequate signal, well-thought peptide design to
allow substantial charge-to-mass ratio different between
substrate and product, and purchase of single-purpose
equipment to perform the detection.
Omnia® Kinase Assay
The majority of reported kinase assays have an endpoint readout which makes them suitable for HTS and
profiling where throughput is a key factor. Recently, a
novel method has been reported by Shults and Imperiali
13,14
and commercialized by Invitrogen, which monitors
kinase reactions in kinetic mode. The principal of this
assay is shown in Fig. 7.
This is essentially a chelation-enhanced fluorescence reporter system generated by Sox amino acid 15. The engineered reporter contains three elements: a kinase recognition motif, a Sox-tagged β-turn sequence (two amino
acids, typically a proline and glycine) and a Serine/
Threonine/Tyrosine residue which bridges the former.
Upon kinase phosphorylation, the phosphorylated Serine/Threonine/Tyrosine along with Sox forms a binding
pocket for Mg2+, which produces a strong fluorescent
28
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
Antibody-based
360nm
β-tum sequence
N
O
O
O
N
Mg2+
O
O
Ki
na
se
re
co
gn
iti
on
m
ot
if
O
SO
X
/Y
S/
T
/Y
S/
T
se
re
co
gn
iti
on
m
ot
if
OH
Mg
2+
Transcreener®
β-tum sequence
Kinase, ATP
SO
X
Ki
na
by a kinase renders it immune to the protease cleavage
and therefore leaves the peptide intact. Since the assay
signal is measured by a ratio of donor emission vs acceptor emission, the unphosporylated peptide generates
higher signal than phosphorylated peptide. Hence, the
inhibition of a kinase reaction results in signal increase.
This assay offers homogeneity, is non-radioactive, and
amenable to HTS. However, false positives against the
protease need to be triaged.
Drug Discovery
485nm
Fig. 7. Illustration of Omnia® assay principle
signal at 485nm. The difference in affinities to Mg2+
before and after phosphorylation of the engineered
peptide produces a significant difference in fluorescence
intensity, which is the key to the assay. Since the peptide
motif can be tailored to target a desired kinase, this assay
can be versatile. The big advantage of this assay is that
kinase activity is monitored kinetically. This is ideal for
detailed mechanistic studies. In order to overcome short
wavelength, a concern for autofluorescent compound
interference, the assay is ideally performed kinetically
for a few minutes to measure the slope from each well,
therefore the throughput becomes an issue, i.e. it hinders
this assay’s applicability for HTS. Invitrogen is working
on far-red probe, which would make it suitable for endpoint detection and more applicable to HTS.
HitHunter™EFC
HitHunter™EFC is a chemiluminescence-based assay
that utilizes Enzyme Fragment Complementation (EFC)
technology16. In the absence of kinase reaction products,
ED (enzyme donor)-conjugated phosphopeptide label
binds to a high affinity antibody, which prevents it from
binding to inactive EFC enzyme to form an active EFC
enzyme. However, upon kinase reaction, the phospopeptide generated in a kinase reaction will displace EDconjugated phosphopeptide bound to the antibody in a
quantitative manner. As a result, the ED fragment will
bind to inactive EFC enzyme fragment to form an active
enzyme that will hydrolyze its substrate (Fig. 8). This is
a homogenous signal increase assay that can be easily
scalable into 384- or 1536-format. Since the assay signal
is amplified by the enzyme, it is very sensitive and the
chemiluminescence nature eliminates optical interference
of fluorescent compounds.
However, the assay requires an antibody with high affinity towards phosphopeptide.
Fig. 8. Illustration of HitHunter™EFC kinase assay (source:
DiscoveRx)
Bellbrooks is the first vendor to commercialize an ADP
detection kit, called Transcreener®, which uses FP-based
detection. The assay works as follows (Fig. 9): Labeled
ADP binds to a high affinity ADP antibody resulting in
high FP signal. ADP produced in a kinase reaction will
compete with the labeled ADP for antibody-binding and
decrease the FP signal.
GROUP II ASSAYS: DETECTION OF ATP DEPLETION
Kinases convert ATP to ADP by transferring a
γ-phosphate group of ATP to an acceptor molecule and
therefore quantification of unused ATP in a kinase reaction can be used as a indirect measurement of activity
of purified kinase enzymes17. With this assay technology,
luciferase utilizes the high energy bonds of ATP to convert luciferin to oxyluciferin and gives off light during
the process. Although there are a number of commercial
kits available such as Kinase-Glo® (Promega), easyliteKinaseTM (Perkin Elmer) and PKLightTM (Cambrex), the
assay principle is the same (see below).
Fig. 9. Schematic principle of Transcreener® ADP FP assay
Adapta®
Using a similar approach, Invitrogen is now marketing
the Adapta assay.
luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP + light
This is a very energy efficient process and reduction of
ATP results in a reduction in the production of photons.
The biggest advantage of this type of assay is that it is
universal (applicable to all kinases) and does not require
antibodies. The luminescence readout also avoids fluorescent compound interference. These assays are costeffective and scalable into 384 and 1536 formats.
GROUP III ASSAYS: DETECTION OF ADP PRODUCTION
More recent advances use coupled-enzyme systems to
detect kinase activity via quantification of ADP production from a kinase reaction18. Compared with ATP
detection, ADP quantification assays provide better
sensitivity due to lower background. There are two types
of platform technologies for quantifying ADP production: antibody-based and antibody-free. The former are
competitive assays, where ADP generated in a kinase
reaction directly competes off ADP-tracer, whereas the
latter are signal increase assays which provide a high S/B
ratio (signal to background) window.
Fig. 10. Schematic principle of the Adapta® Universal Kinase
Assay (source: Invitrogen)
The difference between the Adapta® and Transcreener®
assays is that the Adapta’s ADP antibody is conjugated
with Europium instead of Tb. Instead of detecting FP
signal change, this assay measures TR-FRET signal from
Europium to Alexa Fluor tagged ADP. Both formats
are homogenous and easy to automate. Since the assays
measure ADP instead of phosphopeptide, it applies to
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
29
Drug Discovery
both serine/threonine and tyrosine kinases. The
drawback are that it is a signal decrease assay,
requires specific ADP antibody, and requires
greater than 10% conversion of ATP to ADP in
order to be robust.
Drug Discovery
ATPase reaction
(ATP)
(+Pi)
(+substrate-P)
Table 1. Summary of common kinase assa
Reagent ll (step2)
ADP
Enzyme ll
Enzyme I inhibitor
ATP
Luciferase
Luciferin
Light
ATP
Non-antibody-based
ADP Quest™
Kinase reaction
(Substrate+ATP)
or
Kinase autophosphorylation
(ATP)
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
Assay name
Phosphorylated
Filter binding
peptide
assay
Phosphorylated
ATP
Antibody-free ADP detection uses a coupled
enzyme system. ADP Quest™ (DiscoverX,
Fremont, CA) is a fluorescent intensity-based
Fig. 11. Schematic representation of ADP-Glo assay
method to detect the accumulation of ADP by a
coupling enzyme system including pyruvate kinase
Component II also contains an inhibitor to the enzyme
(PK), pyruvate oxidase (POX), and horseradish
in Component I preventing it from degrading newly
peroxidase (HRP). In the first coupling step after kinase
converted ATP from ADP.
reaction, ADP and phosphoenolpyruvate are converted
by pyruvate kinase to ATP and pyruvate. Subsequently,
Due to the extremely low background and signal inpyruvate is converted by pyruvate oxidase to hydrogen
crease nature of this assay compared with Kinase-GloTM,
peroxide (H2O2), which is used to oxidize the fluorescent
ADP-GloTM has demonstrated the following advantages:
substrate Amplex Red (10-acetyl-3,7-dihydroxyphenoxextreme sensitivity (0.1 pmoles of ADP), broad ATP
azine) in the presence of HRP, resulting in the producconcentration (1uM to 5mM), and applicability to all
tion of highly fluorescent resorufin which emits fluokinases. As a result, this assay can be potentially used for
rescence signal at 590nm. The advantages of this assay
HTS, profiling, as well as MOA studies.
are that it can be monitored kinetically, accommodate
moderate ATP concentration (1-300uM), and is applicable to all kinases. However, the coupling reagents may
SUMMARY
need to be treated with catalase beads to reduce high
background.
The abundance of off-the-shelf kinase assays on the
market presents a great challenge for scientists who
ADP-Glo™
start a new lead discovery program. Since each assay has
Although Kinase-Glo® and other ATP-depletion kits
its pros and cons, one has to consider many factors in
with the same principles are commonly used for HTS
choosing an assay that is fit-for-purpose. These include,
due to their universal applicability for kinases, its shortbut are not limited to, (1) Throughput (2) Sensitivity
comings of high background and large turnover of
(3) Tolerance to high ATP concentration (4) Cost (5)
substrate have limited its use for kinases with slow
Adaptability in automation (6) Instrumentation. One
turnover and MOA assays. To circumvent this problem,
may have to evaluate more than one platform assay
Said Goueli and colleagues at Promega developed a
before finally choosing one. Table 1 is a summary of
novel method that retains the advantages but eliminate
some of the common assays discussed in this review
the drawback of Kinase-Glo® assay. The principle of the
with my personal opinion with regards to their pros
assay is depicted in Fig. 11.
and cons. It is apparent that a single assay that can be
used for all purposes during a lifetime of a kinase drug
The simple “mix-and-read” cocktails contain two major
discovery program is hard to find, because the criteria
components and they are added to a complete kinase
of a fit-for-purpose assay change at stages of the proreaction in two steps. In the first step, Component I is
gram. For instance, the focus of an HTS assay is its high
added. It contains an enzyme to remove unreacted ATP
throughput, robustness, and affordability, but a modein a kinase reaction. In the second step, Component II is
of-action (MOA) assay emphasizes its sensitivity, more
added. This component contains a mixture of enzymes
directness (less artifacts), and kinetic readout. The good
that convert ADP (product of the kinase reaction) back
news is that it should be possible to find assays from the
to ATP and luciferease and its substrate to convert
available repertoire that are appropriate to each stage of
ATP to AMP in a two-step reaction and gives off light.
discovery.
30
Analyte
peptide
Phosphorylated
peptide
FlashPlate
SPA
Assay
technology
Assay principle
Pros
Luminescence
33P-labeled phospho-peptide is captured on nitrocellulose paper
phosphorylated peptide,
direct measurement of
sensitive
Luminescence
Luminescence
33P-lableled phospho-peptide is captured on a FlashPlate
coated with scintillant
medium to high throughput,
relatively low compound
interference
Reaction product is a 33P-lableled phospho-peptide, which
high throughput,
can be captured on a detection bead, which scintillates fromo
homogeous, relatively low
proximity to 33P.
compound interference
Fluorophore-labeled peptides (Fluorescein or TAMRA) binds to
Phosphorylated
peptide
IMAP
peptide
LANCE/
peptide
kinEASE
Phosphorylated
peptide
AlphaScreen
radioative waste, limited sensitivity
compared with TR-FRET
radioative waste disposal, limited
sensitivity compared with TR-FRET
peptide must be relatively small,
FRET
TR-FRET, IMAP beads are spiked with Tb, which produce TR-
requirement
cannot use protein substrate for
TR-FRET
which recognizes phosphorylated fluorescein-conjugated
Europium-chelated (cryptate) donor linked with anti-phospho
TR-FRET
antibody. Biotinylated substrate Streptavidin-allophycocyanin
as acceptor
AlphaScreen
Typical
application
MOA, profiling
MOA, profiling
HTS, profiling
Susceptible to compound interference,
Versatile, no antibody
substrate as acceptor
Phosphorylated
mutl-washing steps
trivalent metal coated beads. Binding creates change in FP. For
Terbium-chelated donor beads coated with anti-phospho antibody
LanthaScreen
low throughput, radioactive waste,
FP or TR-
FRET readout.
Phosphorylated
Cons
HTS, profiling
FP mode
Sensitive, ratiometric
readout, high throughput
Requires specific antibody
HTS
Sensitive, ratiometric
readout, high throughput,
less interference from
Requires specific antibody
HTS, profiling,
MOA
compounds
Donor and acceptor beads are brought to proximity by
highly sensitive, high
conjugates coated on beads and analylyte. Upon excitation,
throughput, long excitation
Requires specific Antibody pairs,
donor bead excites oxygen. The singlet oxygen reacts with the
and shorter emission reduce
asssay variability, cost
acceptor beads to give off signal.
compound interference
HTS, profiling
FRET-peptide tagged with donor (coumarin) and acceptor
Phosphorylated
peptide
Z’ Lyte
FRET
(fluorescein) at each end as the kinase substrate. Site-specific
ratiometric, high throughput,
Coupled assay can be susceptible to
protease cleavage of the peptide results signal increase.
hemogeneous
protease inhibitor compounds
On-chip- or off-chip-based assay, Chip-based separation of
accurate detection, less
Needs special substrate; expensive,
product and substrate based on charge/mass ratio
interference
special instrument
HTS, profiling
Phosphorylation of the peptide is immune to protease cleavage.
Phosphorylated
peptide
Phosphorylated
peptide
Phosphorylated
peptide
ATP detection
ATP detection
Caliper
Caliper
Omnia/SOX
Fluorescence
HitHunter EFC
EFC
Kinase-Glo
Easylite kinase
Luminescence
Luminescence
ATP detection
Pklight
Luminescence
ADP detection
Transcreener
FP
Chelation-enhanced fluorescence reporter system
kinetic readout, antibody
Fluorescent compound interference,
free
requires engineered peptide substrate
kinase generated phosphopeptide competes with ED- labeled
Mix and read approach,
phosphopeptide, and therefore frees ED to form active EFC
red-shift tracer to reduce
enzyme
compound interference
ATP-dependent luminescent signal from luciferase conversion of
luciferin. Kinase depedent depletion of ATP is quantified
ATP-dependent luminescent signal from luciferase conversion of
luciferin. Kinase depedent depletion of ATP is quantified
ATP-dependent luminescent signal from luciferase conversion of
luciferin. Kinase depedent depletion of ATP is quantified
ADP from the kinase reaction competes with fluorophore-labeled
anti-ADP antibody
Adapta
TR-FRET
from TR-FRET. ADP from the kinase reaction completes with
ADP Quest
Fluorescence
ADP detection
ADP-Glo
Luminescence
ADP detection using a coupling enzyme system including
pyrovate kinase, pyruvate oxidase, HRP and Amplex Red
Quantification of kinase dependent ADP production by converting
it to ATP and ATP is measured by luciferease
HTS, profiling
inhibition of luciferase, high substrate
HTS, profiling
conversion
Signal decrease, false positive due to
Versatile, non-radioactive
inhibition of luciferase, high substrate
HTS, profiling
conversion
Versatile, non-radioactive
Signal decrease, false positive due to
inhibition of luciferase
HTS, profiling
Signal decrease, Antibody dependent,
Versitile, non-radioative
limited ATP concentration, high
HTS
product turnover required
Versitile, non-radioative
fluorophore-labeled ADP
ADP detection
MOA
Signal decrease, false positive due to
Versatile, non-radioactive
Europium chelated anti-ADP antibody binds to tracer-ADP to
ADP detection
Require special antibody pair, not
suitable for high ATP concentration
profiling, MOA
Versatile, high throughput
Highly sensitive, low
background, versatile, high
throughput
Signal decrease assay, Antibody
dependent
HTS
Limited ATP concentration, false
HTS, profiling,
positive due to coupling enzymes
MOA
False positive due to coupling
HTS, profiling,
enzymes
MOA
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
31
Drug Discovery
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Drug Discovery
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Cell-based Assay Strategies for Kinase Inhibitor Discovery
Jun Xian
Antibody-based technology
About the Authors: Dr. Jun Xian is
Associate Director of Partners Center
for Drug Discovery at Brigham and
Women’s Hospital and Assistant
Director of Cancer Drug Discovery at
Regular cell line
“Sandwich” ELISA
Electrochemiluminescence (ECL)
DELFIA® TRF Assays
AlphaLISA®
Luminex®
Fluorescence-activated cell sorting (FACS)
High content screening (HCS)
In-cell western
FACE™ method
Over expression/fusion substrate
LanthaScreen™ Cellular Assays
Inducible vectors
Dana Farber/ Harvard Medical School
cancer center. He is also an Instructor
at Harvard Medical School. His group is
guiding academic researchers working
on early stages of drug discovery for
their novel targets. From 2000 to 2003,
Dr. Xian was the screening manager at
Genome Therapeutics Co., where he
was in charge the internal compound
library and screening process. He was
a Project Leader in High-throughput
Antibody free technology
Engineering cell lines
Cignal™ Reporter Assay
CellSensor® Reporter Assay
PathHunter™
Label free detections
xCELLigence System
CellKey™ System
Epic® System
Process Group at Cereon Genomics, LLC
(Monsanto) from 1998 to 2000. Prior to
join Cereon, he was a research scientist
at Hybridon, Inc. from 1996 to 1998.
Dr. Xian received his BS and MS in
Chemistry from South China University
of Technology in 1983 and 1986. He got
his PhD in Chemistry from Wichita State
University in 1994, followed by postdoctoral training in Biology Division at
California Institute of Technology from
1994 to 1996.
32
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
Introduction
Cell-based assays monitor changes, such as morphological, gene
expression and protein modification, in living cells in response to a
wide variety of biological, chemical or physical stimuli including receptor activation, drug action, external nutrition change and stress. The
market for these assays and their applications is growing rapidly, with
new advances in technology platforms and high throughput screening
systems.
As the important cellular regulatory proteins in cell signal transduction
pathway, kinases mediate a number of physiological and pathological changes in cell function by the phosphorylation modification of
Serine/Threonine or Tyrosine residues in proteins. Inappropriate or
deregulated kinase function is often associated with disease states, such
Tr e n d s i n B i o / P h a r m a c e u t i c a l I n d u s t r y
33