On-bead fluorescence assay for serine/threonine kinases

Article abstract of DOI:10.1021/ol052125k

(Chemical Equation Presented) A novel fluorescence-based assay for serine/threonine kinases is described. Base-mediated β-elimination of the phosphate moiety and the Michael addition of a thiol-containing fluorescent molecule allows convenient and efficie

Full text of DOI:10.1021/ol052125k

ORGANIC
LETTERS
2005
Vol. 7, No. 25
5565-5568
On-Bead Fluorescence Assay for
Serine/Threonine Kinases
Shoji Akita, Naoki Umezawa, and Tsunehiko Higuchi*
Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity, 3-1
Tanabe-dori, Mizuho-ku, Nagoya, Japan
higuchi@phar.nagoya-cu.ac.jp
Received September 3, 2005
ABSTRACT
A novel fluorescence-based assay for serine/threonine kinases is described. Base-mediated
â
-elimination of the phosphate moiety and the
Michael addition of a thiol-containing fluorescent molecule allows convenient and efficient detection of the enzyme activity. This approach
may be broadly applicable to various serine/threonine kinases.
Protein kinases catalyze phosphoryl group transfer from
adenosine triphosphate (ATP) to serine, threonine, and/or
tyrosine residues of target proteins. Protein phosphorylation
is one of the most common posttranslational modifications
and plays an important role in intracellular signal transduc-
tion. Protein kinases ultimately regulate many aspects of
cellular function, including proliferation, differentiation, the
cell cycle, metabolism, transcription, and apoptosis.1 Protein
kinase genes form one of the largest gene families in
eukaryotes; in humans they are estimated to account for 1.5-
2.5% of all genes.2 A key feature of protein kinases is
substrate specificity, which is mainly determined by the
primary sequence around the phosphorylation site of the
target proteins. As a result, great efforts have been made to
identify the potential kinase substrates, including combina-
torial synthesis of peptide libraries with radioisotope-based
or antibody-based detection.3 Protein kinases are also becom-
ing attractive targets for drug discovery, since many of them
are associated with a wide variety of diseases, such as
cancers, cardiovascular disease, and inflammation.4 There
is a great impetus to develop small-molecular inhibitors
specific to a particular kinase, for both therapeutic reasons5
and enzyme functional analysis.6
Conventional assay for protein kinase activity is based on
the transfer of 32P from γ-32P-ATP to the target peptides or
proteins.7 This method is widely used, but radiolabel
represents a potential risk to human health and to the
environment. In addition, the long exposure time needed for
sensitive detection of 32P does not lend itself to high-
throughput applications. The use of antibodies directed
against phosphorylated residues is also popular,8 but the
specificity of the antibodies is problematic in some cases.
Several fluorescent probes have been developed recently,9-11
such as Zn2+-based artificial phosphate receptors9 and peptide
(4) (a) Bridges, A. J. Chem. ReV. 2001, 101, 2541. (b) Noble, M. E. M.;
Endicott, J. A.; Johnson, L. N. Science 2004, 303, 1800.
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Circulation 2004, 109, 1196. (b) Daub, H.; Specht, K.; Ullrich, A. Nat.
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Wood, J. L.; Morgan, D. O.; Shokat, K. M. Nature 2000, 407, 395.
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MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760.
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Bioorg. Med. Chem. Lett. 2002, 12, 2085.
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S. Science 2002, 298, 1912. (b) Manning, G.; Plowman, G. D.; Hunter, T.;
Sudarsanam, S. Trends Biochem. Sci. 2002, 27, 514.
(3) (a) Pearson, R. B.; Kemp, B. E. Methods Enzymol. 1991, 200, 62.
(b) Songyang, Z.; Blechner, S.; Hoagland, N.; Hoekstra, M. F.; Piwnica-
Worms, H.; Cantley, L. C. Curr. Biol. 1994, 4, 973. (c) Wu, J.; Ma, Q. N.;
Lam, K. S. Biochemistry 1994, 33, 14825.
10.1021/ol052125k CCC: $30.25
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Published on Web 11/10/2005

substrate bearing a phosphate sensor.10 These probes are
elegantly designed, but their affinity and sensitivity could
be affected by the amino acid sequence of the target
phosphopeptide.9b Therefore, a convenient assay method
would become a powerful tool for kinase research. Here we
report the novel fluorescence-based assay for serine/threonine
kinases, which is applicable to high-throughput screening.
Our approach is summarized in Figure 1. The key reactions
screening. We adopted solid-supported peptide substrate and
fluorescence detection to achieve a rapid, safe, and sensitive
assay.
The thiol-containing fluorescent molecules were designed
and synthesized according to Scheme 1. We selected
Scheme 1 Synthesis of Compounds 4 (a) and 9 (b)
Figure 1. Summary of the assay.
coumarins as the fluorophore because of their high fluores-
cence quantum yield and low hydrophobicity. Their relatively
hydrophilic nature is expected to reduce nonspecific interac-
tion with the solid support; such an interaction would
potentially lower the reliability. We designed the hydroxy-
coumarin derivative 4 and the aminocoumarin derivative 9.
To examine their reactivity, the reaction was performed with
methyl vinyl ketone as a model Michael acceptor under basic
conditions.
Both 4 and 9 gave the desired compounds 10 and 11,
respectively. As shown in Figure 2, the fluorescence intensity
of 10 was much stronger than that of 4, suggesting that the
fluorescence from nonreacted dye is weaker than that from
the reacted dye. Compound 4 was found to be fairly stable
in the buffer at pH 9.4; almost no disulfide product was
detected by HPLC (see Supporting Information). Similar
fluorescence spectra were obtained with aminocoumarin
derivatives 9 and 11 (see Supporting Information). This
characteristic will reduce the background fluorescence from
the dye nonspecifically bound to the solid support. The
fluorescence properties of coumarin derivatives are known
to be influenced by the degree of intramolecular charge
transfer (ICT)13 and/or photoinduced electron transfer (PET)14a
from the substituents to the coumarin ring. Although the
are base-mediated â-elimination of the phosphate moiety and
the Michael addition of a thiol-containing fluorescent
molecule, which enable the selective transformation of
phosphoserine and phosphothreonine residues into fluores-
cent derivatives. Although several researchers have used
similar chemistry to enrich and quantify phosphopeptides,
identification of the sites of phosphorylation typically relied
on tandem mass spectrometry to sequence individual pep-
tides.12 MS spectrometry is not necessarily suitable for
quantification and is difficult to apply to high-throughput
(9) (a) Ojida, A.; Mito-oka, Y.; Inoue, M.; Hamachi, I. J. Am. Chem.
Soc. 2002, 124, 6256. (b) Ojida, A.; Mito-oka, Y.; Sada, K.; Hamachi, I. J.
Am. Chem. Soc. 2004, 126, 2454.
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Lawrence, D. S. J. Biol. Chem. 2002, 277, 11527. (c) Shults, M. D.;
Imperiali, B. J. Am. Chem. Soc. 2003, 125, 14248.
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Sci. U.S.A. 2001, 98, 14997. (b) Ting, A. Y.; Kain, K. H.; Klemke, R. L.;
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(b) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379. (c)
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Org. Lett., Vol. 7, No. 25, 2005

To examine the conditions for on-bead assay, Kemptide
and phosphorylated Kemptide were synthesized on TentaGel
S NH2 resin; the peptides were deprotected but remained
bound on the resin under the normal deprotection/cleavage
conditions. It was found that the conditions optimized for
the solution-phase reaction were applicable with slight
modification. Dye 4 usually gave results slightly better than
those with 9 (see Supporting Information).
Next we tried to detect PKA (catalytic subunit) activity.
As a solid support, we chose TentaGel and PEGA800 resin,
since they swell well in aqueous solution and several on-
bead enzyme assays using them have already been reported.3c,17
The substrate peptide synthesized on resin was treated with
PKA, base, and dye 4, followed by observation under a
fluorescence microscope. Observation of the beads was
performed in glycine/NaOH buffer (pH 9.4), since the
fluorescence of 10 was strong and stable in the pH range of
9-10 (see Supporting Information). Our initial experiment
with these resins, however, did not work well. Kemptide resin
both treated with and without PKA showed little fluorescence
(see Supporting Information). Only PEGA1900 resin was
found to be applicable for PKA enzyme reaction among the
resins we tried, probably because PKA was too big to
permeate into regular resin, including TentaGel or PEGA800
.
Figure 2. (a) Reaction of 4 and 9 with methyl vinyl ketone. (b)
Emission spectra (ex 360 nm) of 4 and 10 (5 µM) in 100 mM
glycine/NaOH buffer (pH 9.4) containing 0.1% DMSO.
The molecular weight cutoff for PEGA1900 is around 70 kDa,
whereas the cutoff for PEGA800 is around 35 kDa (the
molecular weight of PKA is 40 kDa). The fluorescence from
the beads treated with PKA increased as the incubation time
with PKA was increased (Figure 3a); this result indicated
reason for the weak fluorescnece of thiol-containing cou-
marins is not entirely clear, the PET mechanism is supposed
to be appropriate. The formation of 10 from 4 led to little
change in the absorbance maxima but increased the fluo-
rescence intensity, which is a characteristic feature of PET-
type fluorescent probes.14
We first optimized the conditions of â-elimination and
Michael addition in solution. As a peptide substrate, we chose
Kemptide (Ac-LRRASLG-NH2), which is the well-known
target sequence of cAMP-dependent protein kinase (PKA).15
Both Kemptide and phosphorylated Kemptide (Ac-LRRA-
pSLG-NH2) were synthesized with standard Fmoc solid-
phase methods. The reactions were monitored by HPLC (see
Supporting Information), and conditions were found under
which both â-elimination and Michael addition proceeded.
Although Ba(OH)2 was reported as a good reagent for
â-elimination,16 NaOH worked better in our case. It was
confirmed that 4 and 9 were selectively introduced into the
phosphorylated Kemptide but not into Kemptide itself. The
products were characterized by MALDI-MS (see Supporting
Information).
Figure 3. (a) Detection of PKA activity. Beads are incubated with
PKA (30 units mL-1) for 2, 4, or 12 h at room temperature and
then treated with base and 4. (b) Effect of IP. Beads were incubated
with IP (0-500 µM) and PKA (30 units mL-1) for 10 h at room
temperature and then treated with base and 4. All fluorescence
images (ex 360 nm, em >425 nm) were taken in 100 mM glycine/
NaOH buffer (pH 9.4).
(13) Setsukinai, K.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. J.
Chem. Soc., Perkin Trans. 2 2000, 2453.
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Kawai, T.; Irie, M.; Imato, T. Bioorg. Med. Chem. 2005, 13, 1131. (b)
Tanaka, K.; Miura, T.; Umezawa, N.; Urano, Y.; Kikuchi, K.; Higuchi, T.;
Nagano, T. J. Am. Chem. Soc. 2001, 123, 2530. (c) Kenmoku, S.; Urano,
Y.; Kanda, K.; Kojima, H.; Kikuchi, K.; Nagano, T. Tetrahedron 2004,
60, 11067. (d) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T.
J. Am. Chem. Soc. 2004, 126, 3357.
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that the PKA activity could be successfully monitored as
fluorescence intensity. Longer incubation time was needed
Org. Lett., Vol. 7, No. 25, 2005
5567

compared with the solution-phase reaction; the phosphory-
lation rate of Kemptide on solid support was supposed to be
much slower. Although the enzyme reaction rate is decreased,
solid-support-based techniques could provide the potential
for the simultaneous screening of thousands of enzyme
substrates/inhibitors in a single experiment.3c,17,18 We ad-
ditionally examined the effect of kinase inhibitor. The PKA-
specific inhibitory peptide (IP; Ac-GRTGRRNAI-NH2)19 was
added at various concentrations (0-500 µM) and incubated
with PKA. After the usual procedure, the fluorescence
intensity of the beads was observed; it became weaker after
addition of the inhibitor, and the fluorescence intensity was
inversely correlated with the inhibitor concentration (Figure
3b). This result confirmed that our method can be used for
inhibitor screening. Also, these data in Figure 3 can be easily
quantified (see Supporting Information).
Figure 4. Detection of CK I activity. Beads were incubated (a)
without kinase, (b) with PKA (100 units mL-1), or (c) with CK I
(100 kunits mL-1) at 30 °C for 20 h. Subsequent treatment was
the same as described in the legend to Figure 3.
In summary, we have developed a versatile fluorescence-
based assay for serine/threonine kinase. PKA and CK I
activities were readily and selectively detected by means of
this method. Also, the assay was proven to be suitable for
kinase inhibitor screening. Our method is expected to be
useful in the high-throughput format, e.g., with microarrays
and microplates, and should be a valuable tool for kinase
research.
To test the generality of our method, we next applied it to
detect casein kinase I (CK I) activity. CK I substrate peptide
(Ac-IGDDDDAYSDTETTEA-resin),20 which is highly an-
ionic (Kemptide is cationic), was synthesized on PEGA1900
beads, and the beads were incubated with CK I or PKA. As
a negative control, beads incubated without kinase were also
prepared. The subsequent procedure was the same as for the
PKA assay, except for the â-elimination conditions; a slightly
higher concentration of NaOH was required to complete the
reaction. Greater fluorescence intensity was observed on the
beads incubated with CK I compared with the negative
control and the beads incubated with PKA (Figure 4). Thus,
two different kinase activities were selectively detected by
our method, suggesting that the method is applicable to
various protein kinases.
Acknowledgment. This research was supported in part
by a Grant-in-Aid for Young Scientists (B) (15790072 and
17790093) to N.U., a Grant-in-Aid for University and Society
Collaboration (12793009) to T.H. from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT),
Japan, and a Grant-in-Aid for Research in Nagoya City
University to N.U. The authors thank Cypress International
Co., Ltd. for the donation of PEGA1900 resin. We also thank
Prof. Naohide Hirashima (Nagoya City University) and Prof.
Yasuteru Urano (the University of Tokyo) for helpful
comments.
(17) Meldal, M.; Svendsen, I.; Breddam, K.; Auzanneau, F.-I. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3314.
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M. Acc. Chem. Res. 2003, 36, 370. (b) Panicker, R. C.; Huang, X.; Yao, S.
Q. Comb. Chem. High Throughput Screening 2004, 7, 547.
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D. A. J. Biol. Chem. 1989, 264, 8802.
Supporting Information Available: Experimental pro-
cedures, spectral data for synthesized compounds, and other
detailed results. This material is available free of charge via
the Internet at http://pubs.acs.org.
(20) Marin, O.; Meggio, F.; Sarno, S.; Andretta, M.; Pinna, L. A. Eur.
J. Biochem. 1994, 223, 647.
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