Your target is our aim - Invitrogen

Kinase Profiling
Your target is our aim
SelectScreen Kinase Profiling Service

Kinase Profiling
People and process
make all the difference
SelectScreen Kinase Profiling Service
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Understand compound selectivity and potency across a broad panel
of kinase targets
Evaluate compound activity against disease-relevant kinase mutants
Identify ATP-competitive and allosteric modulators of kinase activity
Invitrogen’s SelectScreen Profiling Services give you the data you need to make smart decisions
sooner. In drug discovery, every delay costs. For fast turnaround of high-quality data, trust the
SelectScreen Kinase Profiling Service. From enzyme and biochemical assay generation to com-
pound screening, we have developed highly controlled processes to ensure data accuracy. Our
commitment to your research means we deliver your data on schedule. Because if you’re sitting
on the next development candidate, wouldn’t you rather know sooner?
Invitrogen’s SelectScreen Kinase Profiling Service brings together the power of our puri-
fied protein kinase targets and our robust Z’-LYTE biochemical assay technology to create the
premier outsourced service on the market.
Built-in quality
All SelectScreen services are performed with reagents manufactured at Invitrogen’s Madison,
Wisconsin Discovery Sciences facility. This ensures the integrity and quality of the reagents. Fur-
thermore, you can purchase all of the reagents used in the service for any internal follow-up work.
Control or reference inhibitor compounds are present for each kinase we assay on every plate
of client compounds. All services are performed in duplicate (n = 2). All services are completely
confidential. Our open business model allows for flexible, volume-based project discounts. With
the addition of our new SelectScreen facility in Scotland, Invitrogen now has a global presence.
This allows for more streamlined compound submission and real-time communication with our
screening teams around the world.

Screening expertise
At Invitrogen, we have more than 10 years of experience in the expression, optimization, and
characterization of protein kinases, and a track record in developing fluorescent, biochemi-
cal, and cell-based assays suitable for HTS, hit-to-lead, and lead optimization. Additionally, our
scientists who are involved in developing and running the SelectScreen service have signifi-
cant screening experience from past employment at major pharmaceutical and biotechnology
companies. We put this experience to work for you, delivering you high-quality results.
High-quality kinases
Invitrogen is the original manufacturer of all kinases used in the SelectScreen Kinase Profil-
ing Service; we ensure the consistency and reliability of each target. All kinases are of human
origin, are quality controlled through mass spectrometry, SDS-PAGE, and radioactivity assays,
and are the same proteins sold in the Invitrogen Discovery Sciences catalog. The kinases on the
SelectScreen panel have been selected based on the following criteria:
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Therapeutic relevance
Pathway biology
Phylogenetic diversity
The selection of kinases on the panel is continually increasing and many of the kinases we offer
are unique to Invitrogen Discovery Sciences. Please visit www.invitrogen.com/kinaseprofiling
for the most up-to-date list.
www.invitrogen.com
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Kinase Profiling
Technology behind the SelectScreen service
The SelectScreen service is built around the Z´-LYTE assay platform, a technology ideal
for kinase screening applications. This biochemical fluorescence resonance energy trans-
fer (FRET) assay employs a coupled enzymatic reaction which is based on the differential
sensitivity of phosphorylated and nonphosphorylated peptides to proteolytic cleavage by
a site-specific protease (Figure 1). This technology is also available for purchase from the
Invitrogen catalog, making the same robust reagents used in the SelectScreen Kinase
Profiling Service available for your in-house, follow-up assays.
A stringent validation process ensures the highest-quality data possible. Every 384-
well plate we screen contains multiple controls that determine whether the plate passes
or fails. These include:
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ADP
ATP
Duplicate value difference determinations, n = 2
Control wells to assess assay interference—a protease control and a fluorescence
control—used to alert clients about potential interference
Phosphorylation controls
Control inhibitor IC 50 determination for each kinase
Kinase reaction
C
C
Substrate
P
FRET
Kinase
10–40% of substrate
is phosphorylated
F
F
C
C
P
FRET
P
FRET
Development reaction
F
Developing
Reagent
F
Development reagent, which recognizes and
cleaves nonphosphorylated peptide, is added
C
C
FRET
F
OH
F
F
C
Emission ratio (445 /520 )
C
P
Detection
F
C OH F
PhosphorylatedNonphosphorylated
peptide peptide
Figure 1—Schematic of the Z´-LYTE assay. In a 10 µl kinase reaction, the kinase transfers the γ-phosphate of
ATP to a single serine, threonine, or tyrosine residue in the synthetic peptide substrate (2 µM). The peptide is
labeled with two fluorophores (coumarin and fluorescein)—one at each end, to make up a FRET pair. In the
development reaction, 5 µl of a site-specific protease recognizes and cleaves nonphosphorylated peptides.
Phosphorylation of peptides suppresses cleavage by the protease. Cleavage disrupts FRET between the
coumarin and the fluorescein on the peptide. Uncleaved, phosphorylated peptides maintain the FRET signal.
During detection, a ratiometric readout of donor emission over acceptor emission quantitates reaction
progress. The ratio is low if the peptide is phosphorylated, and high if the peptide is nonphosphorylated.
Compounds that inhibit kinase activity will therefore produce a high ratio, and are easily distinguished from
potential protease inhibitors that produce a low ratio.

Flexible approach to screening
Clients can choose from multiple parameters to design the opti-
mal project to meet their screening goals. Options include:
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Screening mode—profiling can be performed in single-
concentration inhibition determinations, or dose response
titrations (3-fold serial dilutions at the starting concentration
of the customer’s choosing)
ATP concentration—clients may choose from 3 ATP
concentrations: 100 µM, 10 µM, or the K m[app]
Compound supply—the service offers clients the ability
to screen small collections of lead compounds, or larger
chemical arrays and libraries
Turnaround time—results of the service are returned to the
client within two weeks
Enzyme selection—clients have the freedom to select
any number of kinases from Invitrogen’s SelectScreen
panel. This panel is constantly expanding; visit
www.invitrogen.com/kinaseprofiling for updates.
Proven performance
To demonstrate the value of this service, the SelectScreen kinase
profiling service was used in several experiments. The assays and
results are discussed below.
Understand compound selectivity across a broad
panel of kinase targets
The sample data illustrate the inhibitory profiles of Gleevec® and
Sutent® compounds against the Invitrogen SelectScreen kinase
panel of 224 kinases grouped by subfamily (Figure 2).
Compound
Gleevec ®
Compound
Sutent ®
100
80
60
40
20
0
–20
100
80
60
40
20
0
–20
Concentration
1 µM
TK
TKL
STE
CK1
AGC
CAMK
CMGC
Concentration
1 µM
TK
TKL
STE
CK1
AGC
CAMK
CMGC
Figure —Inhibitory profiles of Gleevec® and Sutent® against the Invitrogen
SelectScreen kinase panel. Gleevec® and Sutent® were screened at a single
concentration (1 µM) in the SelectScreen service against 224 kinases at
K m[app] for ATP. Kinases are grouped by family around the graph. The percent
inhibition of each kinase is represented by the distance from the center (0%).
These graphs show the difference in selectivity between these two clinically
approved kinase inhibitors.
www.invitrogen.com
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Kinase Profiling
Evaluate compound activity against disease-relevant kinase mutants
IC 50 curves were generated for the kinase inhibitor Gleevec® against the related mutant
forms of the kinase ABL1 (Figure 3). As was predicted from patient populations bearing
these mutations, Gleevec® potently inhibits the wild-type form of the enzyme, but shows
significantly reduced potency against some mutants, with no effect on the “gatekeeper
mutant” T315I.
Identify ATP-competitive and allosteric modulators of kinase activity
The SelectScreen Kinase Profiling Service can be used to identify and annotate classical
ATP-competitive inhibitors (Type I) as well as next-generation allosteric modulators (Type II
and III inhibitors). We examined the effect of a well-known Type I inhibitor, SB203580, and
a Type II inhibitor, BIRB796, on the activity of MAPK14 (p38 alpha). Figure 4 demon-
strates that the potency of the Type II inhibitor BIRB796 is increased ~4-fold following a
20 minute preincubation prior to assay. In contrast, there is no effect with preincubation
on the potency of SB203580, demonstrating the different binding kinetics of these two
inhibitor types. (Preincubation can be incorporated into SelectScreen projects on a
custom basis.)
% inhibition
110
100
90
80
70
60
50
40
30
20
10
0
IC 50 of ABL1, ABL1(E255K), ABL1(G250E),
ABL1(T315I), and ABL1(Y253F) with
Gleevec ® at 100 µM ATP
ABL1 IC = 300 nM
50
ABL1(E255K) IC = >3,000 nM
50
ABL1(G250E) IC = >3,000 nM
50
ABL1(T315I) IC = >10,000 nM
50
ABL1(Y253F) IC = >3,000 nM
50
–10
10 –1 10 0 10 1 10 2 10 3 10 4 10 5
[Gleevec ® ] (nM)
Figure 3—Effect of Gleevec® on the activity of various mutant forms of ABL1. The data show that small amino acid
changes in the catalytic site of the kinase may have dramatic effects on inhibitor sensitivity.

A B
Type I
Compound [ATP] Preincubation IC 50 Z´
SB203580 100 µM None 22.4 0.72
SB203580 100 µM 20 min 22.6 0.62
% inhibition
% inhibition
120
100
80
60
40
20
0
MAPK14 (p38 alpha) : ATP 100 µM
SB203580
–20
1 10 100 1,000 10,000
120
100
80
60
40
20
0
Concentration (nM)
MAPK14 (p38 alpha) : ATP 100 µM
SB203580
–20
1 10 100 1,000 10,000
Concentration (nM)
Concentration (nM)
Figure 4—Preincubation of compounds prior to assay initiation can dramatically increase the apparent potency of “slow on-rate” compounds. The potency of SB203580 (A)
and BIRB796 (B) against MAPK14(p38 alpha) was tested +/– pre-incubation for 20 min. This data shows the expected increase in potency of the “slow on-rate” Type II
allosteric inhibitor, BIRB796.
Type II
Compound [ATP] Preincubation IC 50 Z´
BIRB796 100 µM None 471 0.72
BIRB796 100 µM 20 min 118 0.62
% inhibition
% inhibition
120
100
80
60
40
20
0
MAPK14 (p38 alpha) : ATP 100 µM
BIRB796
–20
1 10 100 1,000 10,000
120
100
80
60
40
20
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MAPK14 (p38 alpha) : ATP 100 µM
BIRB796
–20
1 10 100 1,000 10,000
Concentration (nM)
www.invitrogen.com
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How to order:
The SelectScreen Kinase Profiling Service is straightforward and
efficient to use. No subscription or enrollment is necessary. Proj-
ects proceed through the following steps:
1.
2.
3.
4.
Fill out the SelectScreen submission form available online
at www.invitrogen.com/kinaseprofiling.
Receive confirmation from Invitrogen with scope of the proj-
ect and pricing.
Supply your purchase order number and prepare com-
pounds for submission to Invitrogen.
Receive your data analysis results as an electronic file.
The protocol and assay conditions used in the SelectScreen
Profiling Services, as well as the compound submission guide-
lines, are available to view on the corresponding website for each
profiling service.
Additional SelectScreen
compound screening services
available from Invitrogen
Invitrogen offers a suite of screening services to support
your discovery research:
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SelectScreen Cell-Based Pathway Profiling
Service—more than 14 pathway assays avail-
able for cellular selectivity profiling and potency
determinations
SelectScreen Cell-Based Nuclear Receptor
Profiling Service—cell-based steroidal and non-
steroidal family nuclear receptor assays available for
triaging compounds
SelectScreen Library Screening Service—screen
large library collections against any of Invitrogen’s
kinase, nuclear receptor, GPCR, or pathway assays
SelectScreen Custom P450 Profiling Service—
determine your lead compound’s inhibitory profile
against five key CYP450 isozymes
SelectScreen Custom Screening Service—let us
build the assay for you and then screen your com-
pounds
Learn more at
www.invitrogen.com/profilingpartner.
©2007 Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept
the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. Gleevec® is a trademark of
Novartis. Sutent® is a trademark of Pfizer Inc. B-069350-r1 0407
www.invitrogen.com