Cyclin A probes by means of intermolecular sensitization of terbium-chelating peptides

Article abstract of DOI:10.1021/ja803520q

Intermolecular sensitization of lanthanide ions was effectively implemented in the development of fluorescent sensors targeting cyclin A. A chelating unit has been conjugated to peptides containing a known cyclin A binding motif (CBM). Upon interaction of the modified terbium-chelating peptides with the cyclin A substrate recruitment groove, the Tb³⁺ ion is placed in the vicinity of the Trp²¹⁷ side chain, which results in efficient intermolecular terbium sensitization and specific long wavelength fluorescent emission from the metal center. Copyright

Full text of DOI:10.1021/ja803520q

Published on Web 07/01/2008
Cyclin A Probes by Means of Intermolecular Sensitization of
Terbium-Chelating Peptides
†
§
‡
†
,†
Elena Pazos, Daniel Torrecilla, Miguel V a´ zquez L o´ pez, Luis Castedo, Jos e´ L. Mascare n˜ as,*
§
,†
Anxo Vidal, and M. Eugenio V a´ zquez*
Departamento de Qu ´ı mica Org a´ nica, Departamento de Qu ´ı mica Inorg a´ nica, and Departamento de Fisiolog ´ı a,
UniVersidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Received May 12, 2008; E-mail: eugeniov@usc.es
Cell cycle progression is tightly regulated by the activity of
several kinase complexes (kCDKs) formed by the association of
1
cyclin-dependent kinases (CDK) and a regulatory cyclin unit.
Genetic evidence supports a strong correlation between alterations
2
in the regulation of kCDKs and abnormal cell proliferation,
therefore these complexes represent attractive targets for the
3
development of tumor suppression agents. One of the most
promising strategies for obtaining new kCDK inhibitors consists
of targeting the cyclin A substrate recruitment element called the
4
,5
cyclin binding groove (CBG). This task might greatly benefit
from the existence of fluorescent probes that provide a signal upon
specific binding to the groove. Moreover, such sensors might also
be of unique utility to signal the presence of the protein in complex
cellular environments. Herein we describe the design and synthesis
of an efficient and selective cyclin A fluorescent sensor.
Figure 1. Proposed interaction of the terbium-chelating octapeptide 1, with
the binding groove of cyclin A based on the X-ray structure of cyclin A
1
3
bound to a peptide inhibitor. This interaction places the metal center near
217
the Trp side chain (∼14 Å). The Lys residue modified in control peptides
2
and 3 is also labeled.
Lanthanide-based probes have received a great deal of attention
a
6
Scheme 1. Designed Terbium-Chelating Peptides
in recent years owing to their unique spectroscopic properties. In
these applications, lanthanide ions are usually excited through
intramolecular energy transfer from aromatic residues in their
7
,8
vicinity. In contrast with the widespread application of such
intramolecular sensitization, the use of intermolecular lanthanide
sensitization processes is very scarce and essentially limited to the
9,10
development of chemical sensors for aromatic molecules or ions.
The CBG of cyclin A is partly defined by a Trp residue (217),
which is involved in hydrophobic contacts with the binding partners
through its indole side chain. We realized that such a Trp could be
used as an antenna to sensitize a lanthanide ion incorporated at an
appropriate position of a cyclin-binding peptide, no further than
a
Key CBM residues are in bold. Dap ) 2,3-diaminopropanoic acid
residue.
1
5-20 Å from the Trp. The design of the peptides was based on
oriented away from the surface of the protein, and peptide 3,
featuring the DOTA attached to the shorter side chain of a 2,3-
diamino propanoic acid (Dap) residue introduced in the same
position. We also synthesized the control peptide 4, which lacks
the DOTA macrocycle (Scheme 1).
the well-known consensus sequence for specific cyclin A binding
(
CBM, cyclin binding motif), Arg/Lys-Xaa-Leu, where Xaa is any
11,4
amino acid.
Fine-tuning of this core model sequence on the basis
1
2
of reported peptide library screenings led us to design peptide 1,
3+
which incorporates the Tb chelating macrocycle DOTA (1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid) at the N-terminus
of the optimized CBM.
Detailed synthetic procedures are given in the Supporting
Information. In short, peptides were assembled following standard
15
Fmoc solid phase peptide synthesis protocols, and the DOTA unit
Preliminary molecular modeling studies based on the X-ray
t
1
3
was then introduced into the peptide scaffold as the tri Bu-protected
structures of cyclin A bound to small peptides suggested that the
octapeptide 1 would place the metal center in relative proximity to
acid while the peptides were still attached to the solid support. For
peptides 2 and 3 an orthogonal (alloc) protection scheme was used
to allow selective modification of the Lys and Dap side chains,
respectively. HPLC-purified peptides were subsequently complexed
with TbCl in HEPES buffer to give the desired chelates, which
3
were purified again by HPLC.
Control fluorescence experiments confirmed that 1 was essentially
nonfluorescent upon irradiation at 280 nm. Cyclin A displayed a
strong Trp fluorescence emission band under these conditions, but
2
17
14
the sensitizing aromatic Trp antenna, while keeping intact the
structural requirements for high-affinity binding and avoiding
unwanted steric clashes with the protein surface (Figure 1). To study
the influence of the metal ion location relative to the Trp antenna,
we also synthesized peptide 2, in which the chelating DOTA unit
is attached to a nonessential Lys side chain in the peptide that is
†
Departamento de Qu ´ı mica Org a´ nica.
§
the emission remained unchanged upon addition of up to 10 equiv
Departamento de Fisiolog ´ı a.
‡
3+
Departamento de Qu ´ı mica Inorg a´ nica.
3
of TbCl or chelated terbium in the form of DOTA[Tb ] complex.
9
652 9 J. AM. CHEM. SOC. 2008, 130, 9652–9653
10.1021/ja803520q CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
suitable for biological studies. The methodology should be of
general utility to obtain sensors for other biomolecular systems.
Current work is focused on refining the design to increase the
affinity constant for cyclin A and hence improve the energy transfer
efficiency in order to enable the detection of endogenous levels of
cyclin in cell lysates. We are also studying the use of this approach
17
to discover specific cyclin inhibitors by using competition assays.
Acknowledgment. This work was supported by the Spanish
MEC (CTQ2006-01339, SAF2006-06868 and SAF2007-61015),
Consolider Ingenio (CSD2007-00006), the Human Frontier Science
Program (CDA0032/2005-C), and by the Xunta de Galicia
(
PGIDIT06PXIB209018PR and GRC2006/132). E.P. thanks the
Spanish MEC for her PhD fellowship. M.E.V., A.V., and M.V.L.
thank the Spanish MEC for their Ramon y Cajal contracts. A.V. is
also a recipient of a Consolidation Grant from the Xunta de Galicia.
We also thank Dr. Enrique P e´ rez-Pay a´ (Centro de InVestigaci o´ n
Pr ´ı ncipe Felipe, Valencia) for his generous gift of the cyclin A
protein construct and Robert P. Fisher (Memorial Sloan-Kettering
Cancer Center, New York) for the cyclin A sample used in
preliminary experiments.
Figure 2. Fluorescence spectra of 1 (1 µM, 10 mM HEPES buffer, pH
.5, 100 mM NaCl, λexc ) 280 nm) with increasing amounts of cyclin A.
The insert shows the plot of the fluorescence emission intensity at 545 nm
with the best-fitting binding curve (see Supporting Information for curve
fitting model and details).
7
In contrast, the addition of successive aliquots of a cyclin A solution
(
37 µM stock) to a 1 µM solution of peptide 1 resulted in the
3
+
appearance of the three characteristic Tb emission bands, with
the most intense emission at 545 nm. This effect could only be
observed when both cyclin A and 1 were simultaneously present
in the solution. The increased emission at 545 nm followed a typical
saturation profile that allowed the calculation of a binding constant
of 895 ( 190 nM (Figure 2, insert). Peptide 2, which contains
the DOTA ligand in the Lys side chain, also exhibited the typical
lanthanide emission upon cyclin A addition, albeit with weaker
intensity (Supporting Information). This suggests that in this
Supporting Information Available: Synthetic and spectroscopic
details and protein expression procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
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JA803520Q
J. AM. CHEM. SOC. 9 VOL. 130, NO. 30, 2008 9653