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| Design of a novel class of peptide inhibitors of CDK/cyclin
activation |
| Cyclin
dependent kinases (CDKs) are key regulators of the cell cycle
progression and therefore constitute excellent targets for
the design of anticancer agents. Most of the inhibitors identified
so far inhibit kinase activity by interfering with the ATP
binding site of CDKs. We recently proposed that the protein/protein
interface and conformational changes required in the molecular
mechanism of CDK2/cyclin A activation were potential targets
for the design of specific inhibitors of cell cycle progression.
To this aim, we have designed and characterized a small peptide,
termed C4, derived from amino-acids 285-306 in a-5 helix of
cyclin A. We demonstrate that this peptide does not interfere
with complex formation, but forms stable complexes with CDK2/cyclin
A. C4 peptide significantly inhibits kinase activity of complexes
harboring CDK2 in a competitive fashion with respect to peptide
substrates, but does not behave as an ATP antagonist. Moreover,
when coupled to the protein transduction domain of TAT, C4
peptide blocks proliferation of tumor cell lines, thereby
constituting a potent lead for the development of specific
CDK/cyclin inhibitors. |
J
Biol Chem. 2005 Jan 13; [Epub ahead of print] |
Design and characterization of peptides derived from cyclin
A
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The sequence of cyclin
A a-5 helix and of the peptide directly derived from
this helix (C1) together with its alanine scan mutants
and other peptides used in this study are listed.
Residues encompassed in a-5 helix of cyclin A are
underlined in grey and mutations are in bold. IC50
values for inhibition of CDK2 by the different peptides
were estimated from doseresponse kinase assays using
Histone H1 as a substrate. Each experiment was performed
independently three times and expressed as mean ±
SE. |
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Inhibition
of tumor cell proliferation by C4-Tat peptide |
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Cells were incubated
with increasing concentrations of C4-Tat for 48 h.
The number of cells was counted with a Counter Coulter.
Each experiment was performed independently three
times and expressed as mean ± SE. |
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Ribbon diagram of CDK2/cyclin
A structure and location of the interface domain.
Ribbon diagram of the structure of CDK2/cyclin A.
CDK2 and cyclin A are in red and blue, respectively.
Cyclin A a5 helix is shown in green. Coordinates of
crystal structures used were extracted from the Brookhaven
Protein Data Bank (code: 1QMZ). The figure was generated
using PyMol software version 0.93 (Delano L W Scientific,
San carlos USA). Insert: Sequence alignments of the
a5 helix of different cyclins (residues 285 - 306).
Sequence homologies are highlighted in yellow. |
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Inhibition
of CDK2/cyclin A kinase activity by C1 peptide.
(A) Increasing concentrations of C1 peptide (0 to
100 µM) were mixed with CDK2/cyclin A prior
to addition of either Histone H1 or GST-Rb substrates.
In vitro kinase reactions were performed as described
in "Experimental Procedures" and phosphorylation
levels of substrates were quantified by autoradiography.
(B) Relative inhibition of substrate phosphorylation
(Histone H1, GST-Rb and the small histone H1-derived
peptide) by C1 peptide. |
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Effect of C4 peptide on formation
of CDK2/cyclin A complex. Increasing concentrations
of C4 and C6 peptides were added to GST-CDK2/cyclin
A (lanes 1 to 5) or GST-cyclin A/CDK2 (lanes 6 &
7). GST-tagged proteins were then precipitated using
glutathione-Sepharose beads. Levels of GST-CDK2 or
CDK2 (panel A) and of GST-cyclin A or cyclin A (panel
B) were determined by western blotting using anti-PSTAIRE
or anti-cyclin A antibodies, respectively. (C) Residual
kinase activities were evaluated as the level of Histone
H1 phosphorylation revealed by autoradiography. |
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Binding of C4 peptide to CDK2/cyclin
A, cyclin A and CDK2 by SPR. Increasing concentrations
of C4 peptide were injected over CDK2 (green) , cyclin
A (blue) or CDK2/cyclin A (red) immobilized on the
chips. The association and dissociation phase data
for a subset of C4 peptide concentrations were fitted
using a simple 1:1 Langmuir model using Biacore Evaluation
software. (insert) Rate constants and Kd derived from
SPR data analysis for C4 peptide binding to the CDK2/cyclin
A complex and to cyclin A. |
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Mechanism of inhibition of CDK2/cyclin
A by C4 peptide. CDK2/cyclin A activity was monitored
as described in the Experimental procedures section
and data were fitted according to a double-reciprocal
plot. (A) Inhibition of CDK2/cyclin A activity by
C4 as a function of ATP. Experiments were performed
with a fixed 15 µM concentration of Histone
H1 in the absence (empty circle) or in the presence
of 0.5 µM (filled circle) and 1 µM (square)
of peptide C4 (B) Inhibition of CDK2/cyclin A activity
by C4 as a function of histone H1. Experiments were
performed with a fixed 0.1 mM concentration of ATP,
in the absence (empty circle) or in the presence of
0.5 µM (filled circle) and 1 µM (square)
of C4. |
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Contacts between CDK2/cyclin
A and the substrate peptide. Ribbon diagram
of the structure of CDK2/cyclin A/peptide, CDK2, cyclin
A and peptide are in red, blue and green respectively.
The 10-residue peptide substrate was docked onto the
structure of CDK2/cyclin A using the structure of
CDK2/cyclinA/substrate peptide reported by Brown et
al (23). Coordinates of crystal structures used were
extracted from the Brookhaven Protein Data Bank (code:
1QMZ). The side chain of key residues in the a3 helix
and the loop connecting a3 and a4 helix are shown
in orange. The figure was generated using PyMol software
version 0.93 (Delano L W Scientific, San carlos USA). |
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Anti-proliferative effect of
C4 peptide on human cancer cells. Exponentially growing
human breast cancer cells (MDA-MB-231) were incubated
in the presence of increasing concentrations of C4
(closed square), C6 (closed triangle), C7 (closed
circle) and C4-Tat (open circle) in the absence of
serum for 45 min, after which the concentration of
serum was adjusted to 10 %. Cells were treated in
the same fashion with Tat (open square) as a control.
After 48 h, the number of cells was counted with a
Counter Coulter. Each experiment was performed independently
three times and expressed as mean ± SE. |
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Keywords: C4, c4, c 4, cyclin a, Cyclin A,
peptide, cdk, C4, c4, c 4, cyclin a, Cyclin A, peptide, cdkC4,
c4, c 4, cyclin a, Cyclin A, peptide, cdk |
Euro