Rational design of cyclic peptide modulators of the transcriptional coactivator CBP: A new class of p53 inhibitors

Article abstract of DOI:10.1021/ja107761h

The CREB binding protein (CBP) is a human transcriptional coactivator consisting of several conserved functional modules, which interacts with distinct transcription factors including nuclear receptors, CREB, and STAT proteins. Despite the importance of CBP in transcriptional regulation, many questions regarding the role of its particular domains in CBP functions remain unanswered. Therefore, developing small molecules capable of selectively modulating a single domain of CBP is of invaluable aid at unraveling its prominent activities. Here we report the design, synthesis, and biological evaluation of conformationally restricted peptides as novel modulators for the acetyl-lysine binding bromodomain (BRD) of CBP. Utilizing a target structure-guided and computer-aided rational design approach, we developed a series of cyclic peptides with affinity for CBP BRD significantly greater than those of its biological ligands, including lysine-acetylated histones and tumor suppressor p53. The best cyclopeptide of the series exhibited a K_d of 8.0 μM, representing a 24-fold improvement in affinity over that of the linear lysine 382-acetylated p53 peptide. This lead peptide is highly selective for CBP BRD over BRDs from other transcriptional proteins. Cell-based functional assays carried out in colorectal carcinoma HCT116 cells further demonstrated the efficacy of this compound to modulate p53 stability and function in response to DNA damage. Our results strongly argue that these CBP modulators can effectively inhibit p53 transcriptional activity by blocking p53K382ac binding to CBP BRD and promoting p53 instability by changes of its post-translational modification states, a different mechanism than that of the p53 inhibitors reported to date.

Full text of DOI:10.1021/ja107761h

COMMUNICATION
pubs.acs.org/JACS
Rational Design of Cyclic Peptide Modulators of the Transcriptional
Coactivator CBP: A New Class of p53 Inhibitors
Guillermo Gerona-Navarro,† Yoel-Rodríguez,†,‡ Shiraz Mujtaba,† Antonio Frasca,† Jigneshkumar Patel,†
Lei Zeng,† Alexander N. Plotnikov,† Roman Osman,† and Ming-Ming Zhou*,†
†Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1677, New York,
New York 10029, United States
‡Department of Natural Sciences, Hostos Community College of CUNY, Bronx, New York 10451, United States
S Supporting Information
b
ABSTRACT: The CREB binding protein (CBP) is a
human transcriptional coactivator consisting of several
conserved functional modules, which interacts with distinct
transcription factors including nuclear receptors, CREB, and
STAT proteins. Despite the importance of CBP in tran-
scriptional regulation, many questions regarding the role of
its particular domains in CBP functions remain unanswered.
Therefore, developing small molecules capable of selectively
Figure 1. (A) Structure of cyclopeptides developed in this study, and a
linear p53-K382ac peptide. (B) Stereoview of the representative structure of
modulating a single domain of CBP is of invaluable aid at
unraveling its prominent activities. Here we report the
design, synthesis, and biological evaluation of conforma-
tionally restricted peptides as novel modulators for the
acetyl-lysine binding bromodomain (BRD) of CBP. Utiliz-
ing a target structure-guided and computer-aided rational
design approach, we developed a series of cyclic peptides
with affinity for CBP BRD significantly greater than those of
its biological ligands, including lysine-acetylated histones
and tumor suppressor p53. The best cyclopeptide of the
series exhibited a Kd of 8.0 μM, representing a 24-fold
improvement in affinity over that of the linear lysine 382-
acetylated p53 peptide. This lead peptide is highly selective
for CBP BRD over BRDs from other transcriptional pro-
teins. Cell-based functional assays carried out in colorectal
carcinoma HCT116 cells further demonstrated the efficacy
of this compound to modulate p53 stability and function in
response to DNA damage. Our results strongly argue that
these CBP modulators can effectively inhibit p53 trans-
criptional activity by blocking p53K382ac binding to CBP
BRD and promoting p53 instability by changes of its post-
translational modification states, a different mechanism than
that of the p53 inhibitors reported to date.
the CBP BRD/p53-K382ac complex for the 50 ns MD simulations.
Recent studies show that upon DNA damage CBP is recruited
by the tumor suppressor p53 to modify chromatin and aid
transcription activation of p53 target genes. This coactivator
recruitment process is facilitated by the bromodomain (BRD) of
CBP binding to p53 at the C-terminal acetylated lysine 382
(K382ac).4,5 The molecular basis of this CBP BRD/p53 recogni-
tion was defined by the three-dimensional solution structure
of CBP BRD bound to a lysine 382-acetylated p53 peptide
(p53-K382ac).5 Using this complex structure and following
a target structure-guided design, we have identified two cyclic
peptides that selectively inhibit CBP’s acetylated p53 binding
activity in cells under stress conditions. These cyclopeptide
ligands represent the most potent CBP BRD chemical ligands
reported to date (Figure 1A).
Our rational ligand design began with performing molecular
dynamics (MD) simulations on the NMR structure of the CBP
BRD bound to p53-K382ac (PDB id: 1JSP). In this complex
the p53-K382ac peptide lies across a pocket formed between the
ZA and BC loops in the CBP BRD and adopts a β-turn like
conformation with the K382ac being at the beginning of the
turn5 (Figure 1B). This β-turn like conformation of the p53
peptide is a distinctive feature of the CBP BRD/p53-K382ac
recognition as compared to other BRD structures and likely plays
a pivotal role in ligand specificity and affinity.4,5
The MD simulations showed that the two ends of the
octapeptide were considerably more flexible than K382ac and
its flanking residues, which are anchored in the binding pocket
of the CBP BRD (Supporting Information, Figure 1A). We
computed the distance distribution curves between the Cβ atoms
of residues R379 and H380 on the N-terminal side of K382ac
and the residues on the C-terminal side of the β-turn (L383,
uman transcriptional coactivator CREB binding protein
H
(CBP) functions to physically bridge many DNA-binding
transcription factors to the basal transcription machinery.1
Despite its importance as a master nuclear integrator of tran-
scriptional responses, many questions about CBP functions and
regulation remain unanswered.2,3 Thus, chemical probes able to
modulate its specific domains are of great interest, as such
molecules would constitute powerful tools to systematically
elucidate the biological functions of CBP with respect to
endogenous proteins in cells.
Received: August 28, 2010
Published: January 27, 2011
r
2011 American Chemical Society
2040
dx.doi.org/10.1021/ja107761h J. Am. Chem. Soc. 2011, 133, 2040–2043
|

Journal of the American Chemical Society
COMMUNICATION
Figure 2. Structure and binding affinity to CBP BRD of the synthesized peptide ligands.
M384, and F385, Supporting Information, Figure 1B). The
results suggested that the β-turn like conformation could be sta-
bilized by cyclizing the linear peptide through residues M384 and
either R379 or H380, and by means of a linker two or three atoms
long. Since none of these residues showed important contribu-
tions to the binding energy, we reasoned that they could be
replaced by cysteines that could then anchor cyclization of the
peptide.
The synthesis of these two cyclic peptides was carried out on
solid phase (Supporting Information, Scheme 1). To explore the
molecular determinants of CBP BRD/p53-K382ac binding as
well as to validate our binding model, we prepared another four
cyclopeptides, which resulted from the combination of linking
through either R379 or H380 and F385 or L383. The affinities of
the six cyclic peptides (a-f) for CBP BRD were next evaluated
in vitro using a competition fluorescent polarization (FP) assay.
The cyclization of p53-K382ac between positions H380 and
F385, M384, or L383 (d-f), resulted in a major loss of the p53
binding, indicating that H380 is essential for the CBP BRD/p53
interaction (Supporting Information, Table 1). This result is in
agreement with our previous study using mutant p53 peptides.5
Conversely, incorporation of conformational constrains through
R379 and F385, M384, or L383 led to a 5-, 9-, or 2-fold increase
(b, a, c) in binding affinity, respectively, over that of the linear
p53-K382ac peptide (Figure 2). The best peptide (a or 1)
exhibited an IC50 of 22.6 μM. This result is fully aligned with
the predictions made in our molecular modeling, thereby validat-
ing our model.
by further MD simulations studies of the CBP BRD/4 complex
(Supporting Information).
We then synthesized the cyclic H380W peptide 4 and
determined its binding affinity using the same FP competition
assay described above. As predicted by our molecular modeling
studies, this peptide showed improved affinity over the histidine-
substituted one (1), with an IC50 of 8.0 ( 0.3 μM, thus proving
to be the most active peptide of the series (Figure 2). This result
represents a 24-fold improvement in affinity as compared to that
of the linear p53-K382ac peptide. We next studied the contribu-
tion of the conformational effect on the affinity of 4. To this end,
we measured the binding to CBP BRD of its linear analogue (5),
using the FP competition assay. Indeed, the weaker IC50
obtained for this linear peptide confirmed that the cyclization
does make an important contribution to the binding affinity with
a free energy change of -1.1 kcal/mol (ΔΔG = ΔGcyclic
-
ΔGlinear) (Figure 2). Therefore, the overall improvement ob-
served in the affinity of this cyclopeptide is the result of two
combined effects: the modification made to the peptide sequence
and the stabilization of the bioactive conformation by means of
the cyclization.
To understand the molecular basis of CBP BRD/4 recogni-
tion, we performed NMR titration of the BRD binding to both
4 and the linear p53-K382ac peptide. Comparison of two-
dimensional (2D) 1H-15N HSQC (heteronuclear single quan-
tum coherence) spectra of CPB BRD with and without a peptide
(molar ratio of 1:3) revealed that binding of the cyclized peptide
4 led to more extended chemical shift changes of protein NMR
resonances than that of the linear p53 peptide,5 thus confirm-
ing the stronger nature of the former interaction (Supporting
Information, Figure 2). Overall, a similar set of protein resi-
dues exhibited the major perturbations upon peptide binding,
confirming the similarity of the binding mode of the two
peptides.
The molecular determinants of the CBP BRD/1 interac-
tion were further studied by MD simulation (Supporting In-
formation). In distinction from the CBP BRD/p53-K382ac
binding,6 in this cyclic octapeptide, H380 was found to be sitting
in a different site of the acetyl-lysine-binding pocket interacting
with key residues such as Tyr1167, Asn1168, and Arg1169 in the
BC loop of CBP BRD (Supporting Information, Figure 1D).
As such, we envisioned that replacing H380 by a tryptophan
could enhance peptide binding. This hypothesis was supported
Next, we studied the specificity of the CBP-BRD/4 association
(most potent cyclopeptide) over other BRDs by FP, using a
fluorescein-labeled analogue of it (Fl-4), which was synthesized
2041
dx.doi.org/10.1021/ja107761h |J. Am. Chem. Soc. 2011, 133, 2040–2043

Journal of the American Chemical Society
COMMUNICATION
as described in the Supporting Information. The binding affinity
of this fluorescent cyclopeptide for CBP-BRD, as determined in a
direct in vitro FP assay (Supporting Information, Figure 3A), was
almost identical to the IC50 value previously calculated for 4. This
result validates our in vitro competition FP data. The selectivity of
this peptide was assessed against BRDs from coactivator PCAF
(p300-CBP associated factor) and a BET family protein BRD4
(BRD-containing protein 4). Neither PCAF BRD nor the BRD4
BRDs showed significant interactions with the cyclopeptide Fl-4,
thus demonstrating the specificity of the CBP BRD/4 association
(Supporting Information, Figure 3A).
cells. Since our goal was to test this cyclic peptide in a functional
assay, we next focused our efforts in preparing more stable cyclic
analogs of 4. Specifically, we reasoned that cyclization through
a thioether-like linker would provide improved stability while
retaining the binding properties of the parent cyclic peptide.
Thus, we synthesized bis-thioether linked analogs of 4 by using
a previously reported chemoselective strategy6 with a modifica-
tion. This procedure involved treating linear unprotected dicys-
teine-containing peptides with polybromobenzyl derivatives in
CH3CN/NH4HCO3 solution at pH 7.8. In this study, we used
instead, dibromoalkyl analogs as alkylating agents. At first, the
previously reported methodology did not produce the expected
cyclic peptides, and very little reaction was observed after 24 h
stirring at room temperature. However, microwave irradiation
at 100 °C for 10 min afforded the desired cyclic bis-thioether
peptides in almost quantitative yields (Scheme 1).
While cyclic peptides are considerably more stable than their
linear counterparts, disulfide bridges could be reduced inside
Scheme 1. Cyclization to bis-thioether linked peptides.
[NaHCO3 (20 mM, pH = 7.8)/CH3CN (7:1)]
The affinity of the bis-thioether cyclic peptides 6-8 for CBP
BRD was measured in the FP competition assay, of which 7 and 8
showed weak affinities (IC50 > 90 μM), whereas 6 was almost as
potent as the parent peptide 4 (Figure 2). These results further
highlight the importance of the conformational effect on the
binding of these cyclopeptides to CBP BRD.
Finally, we characterized the lead peptide’s ability to modulate
p53 functions in cells, including p53-induced p21 activation, p53
stability, and p53 association with CBP upon stimulation. For
p53-based p21-luciferase assay, colorectal carcinoma HCT116
cells were cotransfected with the p21 promoter in tandem with
luciferase and renilla luciferase plasmids. Doxorubicin treatment
of the HCT116 cells caused activation of p21 reflected by
Figure 3. (A) Inhibition of p53-induced p21 luciferase expression by the peptide ligands. (B) Effects of the p53 peptides on endogenous p53 and p21
in HCT116 cells after doxorubicin treatment. (C) Inhibition of overexpressed HA-CBP and Flag-p53 interactions in HEK293T cells by the peptides,
4 and 6.
2042
dx.doi.org/10.1021/ja107761h |J. Am. Chem. Soc. 2011, 133, 2040–2043

Journal of the American Chemical Society
COMMUNICATION
luciferase activity (Figure 3A). This p53-induced p21 activation
showed a dose-dependent inhibition by the cyclic peptides 4 and
6, but not by the linear peptides. We next investigated effects of
these peptides on the level of endogenous p53 and p21 proteins
in HCT116 cells. The HCT116 cells were incubated with the
peptide ligands for 12 h prior to the treatment with doxorubicin
(300 ng/mL) for the next 24 h. The Western blots showed that,
whereas the linear peptides were almost inactive, the cyclic peptides
4 and 6 effectively reduced the protein level of endogenous p53 and
p21 (Figure 3B). These results indicated that inhibition of lysine-
acetylated p53 association with CBP results in a decreased stability
of p53, likely due to deacetylation and ubiquitination-mediated
protein degradation, leading to a reduced activation of p21. We
then assessed how these peptides inhibit p53/CBP interaction by
overexpressingHA-CBP and Flag-p53 in human embryonic kidney
(HEK) 293T cells. The HA-CBP pull-down and Western blot
analyses revealed that, consistent with its enhanced stability of
cyclization, 6 showed much better ability in inhibiting doxorubicin-
induced p53/CBP binding than did 4 (Figure 3C). The apparently
less profound inhibitory effects of 6 or 4 in this experiment than
those seen in the p53 luciferase study could be due to differences in
the peptide cell permeability with different cell lines, and also
readout sensitivity of the two assays.
Diversity in Health-Related Research Program (G.G.-N.) and
grants from the National Institutes of Health (M.-M.Z. and S.M.).
’ REFERENCES
(1) Kwok, R. P.; Lundblad, J. R.; Chrivia, J. C.; Richards, J. P.;
Bachinger, H. P.; Brennan, R. G.; Roberts, S. G.; Green, M. R.;
Goodman, R. H. Nature 1994, 370 (6486), 177–178.
(2) Goodman, R. H.; Smolik, S. Genes Dev. 2000, 14, 1553–1577.
(3) Chan, H. M.; Thangue, N. B. J. Cell Sci. 2001, 114, 2363–2373.
(4) Zeng, L.; Zhang, Q.; Gerona-Navarro, G.; Moshkina, N.; Zhou,
M. M. Structure 2008, 16, 643–652.
(5) Mujtaba, S.; He, Y.; Zeng, L.; Yan, S.; Plotnikova, O.;
Sachchidanand; Sanchez, R.; Zeleznik-Le, N. J.; Ronai, Z.; Zhou,
M. M. Mol. Cell 2004, 13, 251–263.
(6) Timmerman, P.; Beld, J.; Puijk, W. C.; Meloen, R. H. Chem-
BioChem 2005, 6, 821–824.
(7) Sachchidanand; Resnick-Silverman, L.; Yan, S.; Mutjaba, S.;
Liu, W. J.; Zeng, L.; Manfredi, J. J.; Zhou, M. M. Chem. Biol. 2006, 13,
81–90.
(8) Strom, E.; Sathe, S.; Komarov, P. G.; Chernova, O. B.; Pavlovska,
I.; Shyshynova, I.; Bosykh, D. A.; Burdelya, L. G.; Macklis, R. M.;
Skaliter, R.; Komarova, E. A.; Gudkov, A. V. Nat. Chem. Biol. 2006, 2 (9),
474–479.
(9) Nayak, S. K.; Panesar, P. S.; Kumar, H. Curr. Med. Chem. 2009,
16 (21), 2627–2640.
The mechanism of action of our peptidic ligands in p53
inhibition differs from that of the p53 inhibitors known to date
that may act by blocking mitochondrial trans-localization of
p53 in transcription-dependent or -independent pathways.8,9
Because excess p53 activity has been reportedly linked to diseases
such as neurodegenerative disorders (Alzheimer’s disease,
Parkinson’s disease and multiple sclerosis)9,10 and infectious9
and autoimmune diseases,9 chemical ligands capable of temporal
and selective inhibition of p53 are of great interest. Further, recent
studies have catapulted p53 to the center of stem cell biology,
showing that disruption of the p53/p21 pathway enhances efficiency
of reprogramming somatic cells into induced pluripotent stem (iPS)
cells by overexpression of transcription factors Oct4, Sox2, Klf4, and
c-Myc.11-17 In these studies, p53 inhibition was achieved by using an
siRNA gene knockdown approach. The p53 inhibitors would be an
attractive technology for facilitating iPS production through inhibit-
ing endogenous p53 in a temporal and revisible manner. Therefore
our study opens doors for future investigations that examine the
utility of this new class of p53 inhibitors in disease models in which a
temporal down-regulation of p53 may be beneficial, as well as for
studies of the role of p53 acetylation in transcriptional activity in
different biological systems.
(10) Li, J.; Ghiani, C.; Kim, J. Y.; Liu, A.; Sandoval, J.; DeVellis, J.;
Casaccia-Bonnefil, P. J. Neurosci. 2008, 28, 6118–6127.
(11) Banito, A.; Rashid, S. T.; Acosta, J. C.; Li, S.; Pereira, C. F.; Geti,
I.; Pinho, S.; Silva, J. C.; Azuara, V.; Walsh, M.; Vallier, L.; Gil, J. Genes
Dev. 2009, 23, 2134–2139.
(12) Hanna, J.; Saha, K.; Pando, B.; van Zon, J.; Lengner, C. J.;
Creyghton, M. P.; van Oudenaarden, A.; Jaenisch, R. Nature 2009, 462,
595–601.
(13) Hong, H.; Takahashi, K.; Ichisaka, T.; Aoi, T.; Kanagawa, O.;
Nakagawa, M.; Okita, K.; Yamanaka, S. Nature 2009, 460, 1132–1135.
(14) Kawamura, T.; Suzuki, J.; Wang, Y. V.; Menendez, S.; Morera,
L. B.; Raya, A.; Wahl, G. M.; Belmonte, J. C. Nature 2009, 460, 1140–
1144.
(15) Li, H.; Collado, M.; Villasante, A.; Strati, K.; Ortega, S.;
Canamero, M.; Blasco, M. A.; Serrano, M. Nature 2009, 460, 1136–
1139.
(16) Marion, R. M.; Strati, K.; Li, H.; Murga, M.; Blanco, R.; Ortega,
S.; Fernandez-Capetillo, O.; Serrano, M.; Blasco, M. A. Nature 2009,
460, 1149–1153.
(17) Utikal, J.; Polo, J. M.; Stadtfeld, M.; Maherali, N.; Kulalert, W.;
Walsh, R. M.; Khalil, A.; Rheinwald, J. G.; Hochedlinger, K. Nature 2009,
460, 1145–1148.
’ ASSOCIATED CONTENT
S
Supporting Information. Schemes S1-S3, Figures
b
S1-S4, Table S1, experimental procedures for the peptide
synthesis, computational protocols, binding and cell-based as-
says. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
ming-ming.zhou@mssm.edu
’ ACKNOWLEDGMENT
This work was supported by the PSC-CUNY Research Award
6357-0041 (Y.R.), the NCI’s Research Supplements to Promote
2043
dx.doi.org/10.1021/ja107761h |J. Am. Chem. Soc. 2011, 133, 2040–2043