Assay Tec hnology

Kinase Assay Development
A Strategy for Comprehensive Kinase Profilingfor Drug Discovery

Masaki Gouda, PhD,
Carna Biosciences

Kinase Preparations forAssay Development

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18 human protein kinase genes have been identified in the human kinome [1]. It is well known that protein kinases are key elements in intracellular signaling pathways that control many physiological processes.
Furthermore, it has been demonstrated that the activity of protein kinases
are altered in several human diseases such as cancer and autoimmune disorders. In order to develop good therapies based on small molecule inhibitors
while avoiding unanticipated side effects, it is crucial that the targeted kinaseis specifically and selectively modulated. Profiling techniques, in which thesame molecule is assessed across a broad panel of kinases, is an accepted
method to evaluate the specificity and selectivity of drug candidates. For
assaying kinase activity, there are multiple strategies available. In this article, I
introduce the platform technology of the mobility shift assay (MSA), for profiling multiple kinases that Carna Biosciences has adopted to use in our kinase profiling service.

In order to develop a well behaving kinase assay, the purified protein kinase
needs to be produced and isolated in away that retains its activity. We have so
far produced over 300 human protein kinases and corresponding assays. We andother research groups have noticed thatrecombinant kinase proteins expressed
in insect cells with a histidine-tag can potentially contain host cell-derived kinase
activity [2]. Excluding the possibility ofthis contaminated activity is critical in kinase assay development to establish accurate kinase profiling. One way to eliminate this issue is to select the specificsubstrate for each kinase carefully. The
other way is to express the kinases witha GST-tag instead. Isolating kinases with aconsistent phosphorylation state is essential to avoid batch to batch differences. In addition, for kinetic analysis it isespecially important that highly active kinases should be used that give a linear
response. Thereat, the kinases ought tobe highly activated by including their upstream kinases [3] or dephosphorylatingwith a phosphatase [4] if necessary.

Assay Development in MSA Platform
The gold standard method for kinase
assays is to monitor the incorporationof 33P labelled adenosine triphosphate(ATP) as a measure of substrate phosScreening 2/2008 15

Assay Tec hnology
Fig.1: Assay optimisation for
the Mobility Shift Assay.1-1)
Peak represents the substrate
and product. The peak heightis measured and the % conversion is determined by thefollowing equation; [productpeak/(product peak + substrate peak)] x 100. 1-2) Real
time monitoring for enzyme
linearity 1-3) Km determination with Michaelis-Menten
plot 1-4) IC50 value wasdetermined by a known kinaseinhibitor. The ATP concentration used was equivalent tothe Km.

Fig. 2: Substrate optimisation. The chargeof the peptide is modified to obtain asharp peak for the MSA format.

phorylation. There are an increasing
number of assay formats that have more
recently been developed for measuring
kinase activity, radioactive and non-radioactive, antibody-based, and non-antibody based. Many factors go into selecting an appropriate format. The trend
in recent years has been shifting tomore homogeneous assay formats [5].
A very promising non-radioactive andnon-antibody-based technology that relies on fluorescence intensity is Caliper’s mobility shift assay (MSA). This is atechnology based on microfluidics on a
microchip involving the electrophoretic
separation of a fluorescently labelled
substrate and the phosphorylated product. Among several assay platforms,
MSA is one of the most accurate methods. In this system, the kinase reaction
takes place in a 384-well plate, and acapillary sipper takes the sample into
the microchip as the separation device

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for the substrate and the phosphorylated product detected via laser-induced fluorescence. Based on the separation principle of the assay, MSA is
unlikely to be affected by interference
from a compound’s autofluorescence.
The steps taken in developing an assay
using MSA are illustrated in figure 1. Anexample of the peaks generated isshown in figure 1-1. The amount ofproduct formed is determined by calculating the ratio of the product peak tothe substrate peak. The peak separation
condition should be carefully optimised
to obtain the ideal peaks (fig. 2). This assay method, however, is strongly affected by the nature of the peptide, especially the charge. Therefore, thesubstrate design is critical to the success of the assay. One of the remarkable features of the MSA platform is theability to measure real-time kinetics bysipping samples at various time inter-vals from a single well as shown in figures 1-2. Compared to other assay
methods, MSA allows simplified realtime kinetic analysis and accelerated
assay development and optimisation.
The calculation of Km values from initial velocities and the determination of
enzyme linearity are shown in figures
1-3. To validate the assay, we measure
the inhibition by a known kinase inhibitor (fig.1-4). To date, we have beenable to develop 209 assays with thisMSA platform.

Profiling Data of Kinase
Inhibitors in the Clinic
Since a protein substrate cannot be usedwith the MSA format, we use an ELISA
format to assay kinases using a protein
substrate. In order to help validate our
profiling methods, we have profiled twodrugs, Gleevec and Tarceva against 247

Assay Tec hnology

Fig. 3: Kinase profiling results of Gleevec and Tarceva. Each compound was tested at I mM with the ATP concentration at the Km.

kinases as shown in figure 3. At 1 mM
concentration, Gleevec inhibited by morethan 80 % the following kinases: ABL,PDGFRα PDGFRβ, KIT, KIT[V650G],
EGFR[L858R], DDR1, DDR2 and LCK.
Whereas,Tarceva at 1 mM concentration
inhibited by more than 80 % the following kinases: EGFR, EGFR[L858R],
FGFR3[K650M], FLT3, HER4, LOK,
MAP2K5, PDGFRα [T674I] and SLK.
These results obtained at Carna Biosciences are consistent to what has already been reported [6, 7].

Conclusions
The MSA platform is a powerful tool forthe evaluation of kinase activity. Any
drug discovery kinase program requires
the determination of a compound’s selectivity across a broad panel of kinases.
With this information, the prediction ofunwanted side effects is possible. Furthermore, any small molecule regardlessof its target, kinase or otherwise, should
be profiled against a variety of targets
including kinases to learn its potential liabilities.

Acknowledgement
I thank members of our R & D group,
product and manufacturing group, andDr. Yusuke Kawase, and Carolyn Foley. I
also thank Dr. Marcie Glicksman for

helpful discussions regarding this manuscript.

References[1] Manning G., et al.: Science 298, 1912-34(2002)[2] Woods YL, et al.: Biochem J 355, 597-607(2001)[3] Fleming Y, et al.: Biochem J 15, 145-54(2000)[4] Bhat RV, et al.: Proc Natl Acad Sci USA
97, 11074-11079 (2000)[5] Bonetta L, et al.: Nature methods 2, 225228 (2005)[6] Zhang, P, et al.: Molecular Cancer 1-9(2003)[7] Fedorov, O, et al.: Proc. Natl. Acad. Sci.
USA 104, 20523-8 (2007)

E www.eMagazineSCREENING.com

Contact:Masaki Gouda, PhD
Research and Development
Carna Biosciences IncKobe, Japan
Tel.: +81 78 302 7091
Fax: +81 78 302 7086
masaki.gouda@carnabio.com
www.carnabio.com

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