| May
1, 2005 |
| By:
Richard
M. Eglen |
| Pharmaceutical
Discovery |
|
The DiscoveRx HitHunter ™ technology provides a novel approach for
kinase high-throughput screening. This homogeneous assay platform
produces a positive chemiluminescent signal that is highly scalable to
several microtiter plate formats and is markedly free from issues of
optical interference. Collectively, the approach is readily configured
for HTS assays at Ser, Thr and Tyr kinases.
Table I. The kinase group names
are: AGC containing PKA, PKG, PKC families; CAMK containing
calcium/calmodulin-dependent protein kinases; CK1 containing
casein kinase 1; CMGC containing CDK, MAPK, GSK3, CLK families;
TK containing tyrosine kinases. Those residues underlined and in
boldface represent the sites of phosphorylation.
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Introduction Tyr, Ser and Thr
kinases are major targets in drug discovery (1). A novel kinase
screening assay format in which a positive chemiluminescent signal is
generated in proportion to the amount of substrate phosphorylated
recently has been developed at DiscoveRx Corp. (Fremont, CA, USA),
providing an approach that is easily automated, scalable to high-density
microtiter plate formats and markedly resistant to compound
interference. The HitHunter™ kinase assays are based on the principle
of β-Galactosidase (β-Gal) enzyme fragment complementation (EFC),
in a fashion analogous to that underlying the widely-used HitHunter™
cAMP assays (2, 3, 4).
Table II. The assay protocol
essentially comprises a kinase reaction step and an EFC (enzyme
fragment complementation) detection step, since several reagents
are pooled in the assay for simultaneous additions. The EFC
detection step, for example, comprises the addition of EA and
b-Gal substrates. The total time for the assay is approximately
3 h and can be configured for 96-, 384- or 1536-well plate
formats.
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In a HitHunter™ assay, substrate phosphorylation is measured in a
competitive immunoassay format using proprietary antibodies that are
highly selective for the phosphorylated (over the non-phosphorylated
substrate), thereby allowing screening assays to be configured to cover
a large proportion of the human kinome (Table I). EFC occurs when a
peptide containing amino acids from the α region of β (enzyme
donor [ED]) combines to an otherwise inactive β peptide fragment
(enzyme acceptor [EA]), forming an active enzyme that hydrolyzes a
substrate to produce a chemiluminescent signal (2, 5). A key feature of
EFC assays is that signal generated by β turnover is directly
proportional to the concentration of labeled phosphopeptide to be
detected.
Figure 1. A standard curve for a
HitHunter™ Ser/Thr EFC kinase assay using the phospho-AMARA
substrate. Values are mean ± s.e. mean, n = 3; experiments were
performed in 384-well microtiter plates.
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Experimental Conditions
A HitHunter™ kinase assay typically comprises a kinase assay step and
a phosphopeptide detection step (Table II). Firstly, the kinase is
allowed to phosphorylate the peptide substrate, in either the presence
or absence of inhibitor. Since kinase reaction conditions are somewhat
specific, the ATP and substrate concentrations can be titrated for
optimal activity. Practically, it has been found that temperatures of
ambient to 37° C for 1 h generally are appropriate for most kinase EFC
assays. Secondly, the phosphopeptide produced by the kinase is measured
by displacement of the ED-phosphopeptide conjugate from the
phosphopeptide antibody. Once displaced, the ED-phosphopeptide
complements with EA and generates a chemiluminescent signal.
Figure 2. An enzyme curve for a
HitHunter™ Ser/Thr EFC kinase assay using the AMPK kinase.
Values are mean ± s.e. mean, n = 3; experiments were performed
in 384-well microtiter plates.
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Results The
HitHunter™ assay produces a positive chemiluminescent signal that
increases as the kinase product increases. Routinely, ratios are
produced that are 7- to 10-fold above background, resulting in assays of
both high precision (Z' factors of greater than 0.7) and, due to β
turnover, at low kinase concentrations. A typical calibration curve for
the Ser/Thr kinase assay (Figure 1) shows a concentration-dependent
increase in chemiluminescent signal over a wide phosphopeptide
concentration range (2-200 nM).
Figure 3. A staurosporine
inhibition curve for a HitHunter ™ Ser/Thr EFC kinase assay
using the AMPK kinase. Values are mean® s.e. mean, n = 3;
experiments were performed in 384-well microtiter plates.
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A concentration-response curve for
AMPK kinase is shown in Figure 2, and a staurosporine inhibitor curve is
shown in Figure 3.
Table III. Characteristics of
DiscoverRx HitHunter™ kinase assays
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Conclusions HitHunter™
kinase assays provide a unique approach to kinase HTS, with several
advantages over existing homogeneous kinase assays formats (Table 3).
β-gal EFC generates an amplified signal, allowing the kinase assay
to be conducted in an automated setting. As a chemiluminescent signal is
generated in the assay, it also provides a robust assay platform without
marked issues of optical interference. The homogeneous format is readily
scalable and easily configured to enable HTS assays at a variety of Ser,
Thr and Tyr kinases.
References
1. P. Cohen, Nat. Rev. Drug Disc. 1, 309-315 (2002).
2. R.M. Eglen and R. Singh,
Combin. Chem. & HTS 6, 313-387 (2003).
3. R. Golla and R. Seethala, J.
Biomol. Screen. 7, 515-525 (2002).
4. M. Weber, M. Ferrer, W. Zheng et
al., Assay and Drug Dev. 2, 39-50 (2004).
5. R.M. Eglen, Assay and Drug
Dev. 1, 97-104 (2002).
DiscoveRx Corp.
42501 Albrae St.
Fremont, CA 94538 USA
www.discoverx.com
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