Fluorogenic peptide substrates for serine and threonine phosphatases

Article abstract of DOI:10.1021/ol1003065

Figure presented A new fluorescent assay for Ser/Thr protein phosphatases has been developed. Hydrolysis of a phosphoSer residue liberates the Ser hydroxyl group, which induces a cyclization reaction on the N-terminal carbamate and releases a fluorescent reporter. Sequence selectivity is observed using several peptide substrates against alkaline phosphatase (ALP), bacteriophage protein phosphatase (-PPase), and vaccinia H1 related phosphatase (VHR). These studies suggest that the assay could be a useful tool for profiling the substrate specificities of medicinally important phosphatases.

Full text of DOI:10.1021/ol1003065

ORGANIC
LETTERS
2010
Vol. 12, No. 9
1936-1939
Fluorogenic Peptide Substrates for
Serine and Threonine Phosphatases
Fengtian Xue† and Christopher T. Seto*
Department of Chemistry, Brown UniVersity, ProVidence, Rhode Island 02912
Christopher_Seto@brown.edu
Received February 4, 2010
ABSTRACT
A new fluorescent assay for Ser/Thr protein phosphatases has been developed. Hydrolysis of a phosphoSer residue liberates the Ser hydroxyl
group, which induces a cyclization reaction on the N-terminal carbamate and releases a fluorescent reporter. Sequence selectivity is observed
using several peptide substrates against alkaline phosphatase (ALP), bacteriophage λ protein phosphatase (λ-PPase), and vaccinia H1 related
phosphatase (VHR). These studies suggest that the assay could be a useful tool for profiling the substrate specificities of medicinally important
phosphatases.
Serine and threonine protein phosphatases (Ser/Thr PPases)
are key regulators of signal transduction and play essential
roles in cell proliferation, division, and apoptosis in eukary-
otes.1 The specificities of PPases are controlled by a number
of regulatory proteins that form heterodimeric complexes
with the catalytic domains. These regulatory proteins influ-
ence both catalytic activity and subcellular localization.2 The
abnormal function of Ser/Thr PPases are implicated in a
variety of human diseases including asthma, myocardial
infarction, and immunosuppression.3 Consequently, there is
a great deal of interest in imaging their activity in ViVo,
understanding their specificities, and discovering selective
inhibitors as therapeutic agents. New assays that are highly
sensitive, amenable to high throughput screening applica-
tions, and can be used to profile the substrate specificities
of PPases would be valuable tools to accomplish these goals.
To date, the substrate specificities of Ser/Thr PPases have
not been comprehensively examined; published work em-
ploys a limited number of variants of known peptide
sequences.4 However, it is clear from inhibition studies with
natural products and their analogs that a high degree of
selectivity can be engineered into inhibitors.3 Peptide sub-
strates show significant differences in activities for particular
sequences. Thus, important information may be gained about
the biological activity of Ser/Thr PPases by studying their
(3) (a) McCluskey, A.; Sakoff, J. A. MinireV. Med. Chem. 2001, 1, 43.
(b) Sakoff, J. A.; McCluskey, A. Curr. Pharm. Des. 2004, 10, 1139. (c)
Honkanen, R. E.; Golden, T. Curr. Med. Chem. 2002, 9, 2055. (d) Baba,
Y.; Hirukawa, N.; Sodeoka, M. Bioorg. Med. Chem. 2005, 13, 5164. (e)
Aggen, J. B.; Humphrey, J. M.; Gauss, C.-M.; Huang, H.-B.; Nairn, A. C.;
Chamberlin, A. R. Bioorg. Med. Chem. 1999, 7, 543. (f) Wipf, P.;
Cunningham, A.; Rice, R. L.; Lazo, J. S. Bioorg. Med. Chem. 1997, 5,
165. (g) Hart, M. E.; Chamberlin, A. R.; Walkom, C.; Sakoff, J. A.;
McCluskey, A. Bioorg. Med. Chem. Lett. 2004, 14, 1969. (h) McConnell,
J. L.; Wadzinski, B. E. Mol. Pharmacol. 2009, 75, 1249. (i) Bajsa, J.; Duke,
S. O.; Tekwani, B. L. Curr. Drug Targets 2008, 9, 997.
† Current Address: Department of Chemistry, University of Louisiana
at Lafayette, Lafayette, LA 70504.
(4) (a) Pinna, L. A.; Donella-Deana, A. Biochim. Biophys. Acta 1994,
1222, 415. (b) Agostinis, P.; Goris, J.; Waelkens, E.; Pinna, L. A.; Marchiori,
F.; Merlevede, W. J. Biol. Chem. 1987, 262, 1060. (c) Donella-Deana, A.;
Krinks, M. H.; Ruzzene, M.; Klee, C.; Pinna, L. A. Eur. J. Biochem. 1994,
219, 109. (d) Ansai, T.; Dupuy, L. C.; Barik, S. J. Biol. Chem. 1996, 271,
24401. (e) Agostinis, P.; Goris, J.; Pinna, L. A.; Marchiori, F.; Perich, J. W.;
Meyer, H. E.; Merlevede, W. Eur. J. Biochem. 1990, 189, 235.
(1) (a) Gee, C. E.; Mansuy, I. M. Cell. Mol. Life. Sci. 2005, 62, 1120.
(b) Rusnak, F.; Mertz, P. Physiol. ReV. 2000, 80, 1483. (c) Tonks, N. K.;
Neel, B. G. Cell 1996, 87, 356. (d) Hunter, T. Cell 1995, 80, 225. (e)
Zolnierowicz, S.; Bollen, M. EMBO J. 2000, 19, 483.
(2) Terrak, M.; Kerff, F.; Langsetmo, K.; Tao, T.; Dominguez, R. Nature
2004, 429, 780.
10.1021/ol1003065  2010 American Chemical Society
Published on Web 04/01/2010

sequence selectivities. These data will be useful for the design
of synthetic PPase inhibitors. Traditional PPase assays
employ malachite green to detect inorganic phosphate, or
use 32P-labeled substrates. More recent assays have been
developed that rely on changes in fluorescence, fluorescence
polarization, or luminescence or changes in a substrate’s
susceptibility to cleavage by proteases.5 Herein, we report a
new PPase assay that incorporates a number of attractive
features including high sensitivity, ability to measure kinetics
in real time, and substrates that contain a natural phospho-
serine residue rather than an activated phosphate ester such
as p-nitrophenyl phosphate. This new assay is also amenable
to substrate profiling and high throughput screening applica-
tions.
Figure 2. Structures of the substrates.
undergoes fast, spontaneous cyclization (rate )115 nM s-1)
to liberate 7-hydroxycoumarin.
We examined the kinetics of substrate hydrolysis using
three phosphatases: alkaline phosphatase (ALP), bacterioph-
age λ protein phosphatase (λ-PPase), and Vaccinia H1 related
phosphatase (VHR). ALP is a nonspecific enzyme that
hydrolyzes phosphate esters from a wide variety of substrates.
λ-PPase is homologous with the N-terminal half of protein
phosphatase 1 (PP1) and is commonly used as a model
PPase. Finally, VHR is a duel specificity phosphatase that
hydrolyzes both phosphoTyr and phosphoSer/phosphoThr-
containing substrates. Figure 3 shows the fluorescence spectra
Figure 1. Outline of the Ser/Thr PPase assay.
Figure 1 shows the general outline of the assay. The
substrates incorporate a coumaryl group connected to the
N-terminus of a phosphoSer-containing peptide through a
carbamate linkage. Hydrolysis of the phosphate ester by a
phosphatase generates the free Ser hydroxyl group, which
undergoes a spontaneous intramolecular 5-membered ring
closure onto the carbonyl group of the carbamate. This
cyclization liberates 7-hydroxycoumarin, which generates a
strong fluorescent signal.
During the development of this assay, we required
substrates that are stable under the aqueous conditions of
the enzymatic reactions but give a fast ring closure upon
hydrolysis of the phosphate ester. We first examined the
stability of model substrates 1 and 2. The carbamate linkage
in compound 1a hydrolyzed in pH 8 buffer with a half-life
of 30 min, while compound 2a was fully hydrolyzed after
only several minutes. Since carbamate hydrolysis occurs by
deprotonation of the N-H group and elimination of 7-hy-
droxycoumarin to give the corresponding isocyanate, replac-
ing the N-H with an N-Me group should decrease the rate
of background hydrolysis. We found that the N-Me deriva-
tives 3 and 5 were stable (hydrolysis rate <0.5 nM s-1). By
contrast, 4a, which contains a free Ser hydroxyl group,
Figure 3. Fluorescence spectra of compound 5a (50 mM in Tris
buffer, pH 8.0) before (brown points and insert) and after (blue
points) treatment with ALP (λex ) 340 nm, λem ) 452 nm).
of the simplest substrate 5a. Fluorescence intensity increases
by 650-fold upon hydrolysis of the substrate by ALP. These
results demonstrate that the assay has a large dynamic range,
and will provide good sensitivity. We also examined
substrates such as 5b that liberate p-nitrophenol. Hydrolysis
of these compounds can be monitored by UV spectroscopy.
Kinetic parameters for 5a and 5b with ALP are shown in
Table 1.
(5) (a) Noble, J. E.; Ganju, P.; Cass, A. E. G. Anal. Chem. 2003, 75,
2042. (b) Zhu, Q.; Huang, X.; Chen, G. Y. J.; Yao, S. Q. Tetrahedron Lett.
2004, 45, 707. (c) Pastula, C.; Johnson, I.; Beechem, J. M.; Patton, W. F.
Comb. Chem. High Throughput Screening 2003, 6, 341. (d) Tremblay, M. S.;
Zhu, Q.; Marti, A. A.; Dyer, J.; Halim, M.; Jockusch, S.; Turro, N. J.; Sames,
D. Org. Lett. 2006, 8, 2723. (e) Kupcho, K.; Hsiao, K.; Bulleit, B.; Goueli,
S. A. J. Biomol. Screen. 2004, 9, 223. (f) Sahoo, H.; Hennig, A.; Florea,
M.; Roth, D.; Enderle, T.; Nau, W. M. J. Am. Chem. Soc. 2007, 129, 15927.
(g) Balakrishnan, S.; Zondlo, N. J. J. Am. Chem. Soc. 2006, 128, 5590.
To increase noncovalent interactions of the substrates with
phosphatases, we prepared tripeptides 6 and 7. These
Org. Lett., Vol. 12, No. 9, 2010
1937

Table 1. Kinetic Data for Substrates 5-7
kcat/Km
(M-1s-1
)
enzyme substrate
kcat (s-1
)
Km (µM)
ALP
5a
5b
6a
6b
7a
6a
6b
7a
6a
6b
7a
0.17 ( 0.02
0.62 ( 0.7
380 ( 70
820 ( 400
41 ( 13
430
760
5.5 ( 0.6
134 000
19 600
58 500
135
7.9 ( 0.9
4.1 ( 0.3
400 ( 120
70 ( 20
λ-PPase
0.015 ( 0.001
0.018 ( 0.02
0.0031 ( 0.0004
0.041 ( 0.004
0.083 ( 0.007
NAa
130 ( 40
770 ( 320
170 ( 40
220 ( 80
790 ( 210
NAa
23
18
VHR
186
105
NAa
a No detectable activity with 2 mM 7a over 30 min.
substrates were synthesized using Fmoc solid phase peptide
synthesis procedures. The N-terminus of each peptide was
capped with p-nitrophenyl chloroformate or coumaryl chlo-
roformate, prepared from 7-hydroxycoumarin and triphos-
gene. The sequence of substrate 6 (pSVA) is derived from
the regulatory subunit of protein kinase A, which is a known
substrate for VHR.6 In compound 7a, this sequence has been
changed to pSEE to alter the electrostatic interactions with
the enzymes. Compounds 6a, 6b, and 7a are all excellent
substrates for ALP (Table 1). The tripeptides 6a and 6b show
300-fold and 25-fold improvement in kcat/Km over their single
amino acid analogs 5a and 5b. Two general observations
can be made comparing the coumaryl and p-nitrophenyl
substrates. First, the kcat/Km values for the coumaryl-
containing substrate 6a are higher than for the p-nitrophenyl
analog 6b. This difference typically reflects the higher affinity
of 6a for the phosphatases when compared to 6b. Second,
the accuracy of the kinetic measurements is higher with the
fluorescent substrate 6a.
Figure 4. (Top) Model showing the interactions between substrate
6a and the electrostatic surface potential of VHR (C124S). (Bottom)
Detailed view showing substrate 6a docked into the active site.
with the acidic patch, and in particular with Asp92. This
electrostatic repulsion causes 7a to be a nonsubstrate for
VHR.
As a final validation of this assay we measured the activity
of two know inhibitiors that have been reported in the
literature. RK-682 (Figure 5) has a reported IC50 value of
The tripeptides are also substrates for λ-PPase. This
enzyme shows a 6-fold preference for the pSVA sequence
in 6a. By contrast, VHR is highly selective. Compound 6a,
which contains the natural sequence from the regulatory
subunit of protein kinase A, is a substrate with a kcat/Km value
of 186 M-1 s-1. On the other hand, compound 7a with the
sequence pSEE does not react with VHR, even at high
concentrations and over extended reaction times. We per-
formed computer modeling studies (Hyperchem) to rational-
ize these observations. As shown in Figure 4, substrate 6a
was docked into the active site of VHR (C124S). We used
the crystal structure of a bound phosphopeptide (PDB 1J4X)
as a guide to correctly position the substrate in the active
site.6b This model suggests that the coumaryl group of the
substrate binds in a pocket defined by Arg158 and Asn41,
while the phosphate ester of the phosphoSer side chain
contacts Arg130 and Ser124. The side chains of Val and
Ala interact with the acidic patch on the phosphatase. If
substrate 6a is replaced by 7a, the side chains of the two
Glu residues on 7a form repulsive electrostatic interactions
Figure 5. Structure of RK-682.
2.0 µM against VHR at pH 6.5 using p-nitrophenyl phosphate
as the substrate.7 Using 6a as the substrate, we measured
the IC50 value to be 3.6 µM at pH 8.0. Sodium vanadate
inhibits alkaline phosphatase with a Ki of 2.1 µM.8 Using
compound 5b as the substrate, we measured the IC50 value
to be 1.6 µM. In both cases, the similarity between reported
(6) (a) Yuvaniyama, J.; Denu, J. M.; Dixon, J. E.; Saper, M. A. Science
1996, 272, 1328. (b) Schumacher, M. A.; Todd, J. L.; Rice, A. E.; Tanner,
K. G.; Denu, J. M. Biochemistry 2002, 41, 3009.
(7) Hamaguchi, T.; Sudo, T.; Osada, H. FEBS Lett. 1995, 372, 54.
(8) Whisnant, A. R.; Gilman, S. D. Anal. Biochem. 2002, 307, 226.
1938
Org. Lett., Vol. 12, No. 9, 2010

and measured inhibition demonstrates that this new fluores-
cent assay for PPases provides accurate inhibition data.
We have developed a new continuous assay for Ser/Thr
PPases that relies on a cyclization reaction to liberate a
fluorescent reporter. The assay shows good sensitivity, and
the substrates are straightforward to prepare by solid phase
peptide synthesis. We have observed sequence selectivity
using several peptide substrates against ALP, λ-PPase, and
VHR. In its current form, the assay is useful for probing
interactions between a phosphatase and the amino acids that
are on the C-terminal side of the phosphoSer residue of the
substrate. We are currently working to modify the substrates
so that they provide information about interactions with
N-terminal residues as well. This assay should be a useful
tool for profiling the substrate specificities of a variety of
medicinally important phosphatases.
Acknowledgment. We thank the National Institutes of
Health NIGMS (GM57327) for financial support of this work
and Michael Zompa for synthesis of compound 1a.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL1003065
Org. Lett., Vol. 12, No. 9, 2010
1939