Probing the structural requirements of peptoids that inhibit HDM2-p53 interactions

Article abstract of DOI:10.1021/ja056344c

Many cellular processes are controlled by protein-protein interactions, and selective inhibition of these interactions could lead to the development of new therapies for several diseases. In the area of cancer, overexpression of the protein, human double minute 2 (HDM2), which binds to and inactivates the protein p53, has been linked to tumor aggressiveness and drug resistance. In general, inhibition of protein-protein interactions with synthetic molecules is challenging and currently remains a largely uncharted area for drug development. One strategy to create inhibitors of protein-protein interactions is to recreate the three-dimensional arrangement of side chains that are involved in the binding of one protein to another, using a nonnatural scaffold as the attachment point for the side chains. In this study, we used oligomeric peptoids as the scaffold to begin to develop a general strategy in which we could rationally design synthetic molecules that can be optimized for inhibition of protein-protein interactions. Structural information on the HDM2-p53 complex was used to design our first class of peptoid inhibitors, and we provide here, in detail, the strategy to modify peptoids with the appropriate side chains that are effective inhibitors of HDM2-p53 binding. While we initially tried to develop rigid, helical peptoids as HDM2 binders, the best inhibitors were surprisingly peptoids that lacked any helix-promoting groups. These results indicate that starting with rigid peptoid scaffolds may not always be optimal to develop new inhibitors.

Full text of DOI:10.1021/ja056344c

Published on Web 01/19/2006
Probing the Structural Requirements of Peptoids That Inhibit
HDM2-p53 Interactions
†
†
‡
,§
Toshiaki Hara, Stewart R. Durell, Michael C. Myers, and Daniel H. Appella*
Contribution from the Laboratory of Cell Biology, NCI, National Institutes of Health, DHHS,
Bethesda, Maryland 20892, Department of Chemistry, Northwestern UniVersity,
EVanston, Illinois 60208, and Laboratory of Bioorganic Chemistry, NIDDK,
National Institutes of Health, DHHS, Bethesda, Maryland 20892
Received September 14, 2005; E-mail: appellad@niddk.nih.gov
Abstract: Many cellular processes are controlled by protein-protein interactions, and selective inhibition
of these interactions could lead to the development of new therapies for several diseases. In the area of
cancer, overexpression of the protein, human double minute 2 (HDM2), which binds to and inactivates the
protein p53, has been linked to tumor aggressiveness and drug resistance. In general, inhibition of protein-
protein interactions with synthetic molecules is challenging and currently remains a largely uncharted area
for drug development. One strategy to create inhibitors of protein-protein interactions is to recreate the
three-dimensional arrangement of side chains that are involved in the binding of one protein to another,
using a nonnatural scaffold as the attachment point for the side chains. In this study, we used oligomeric
peptoids as the scaffold to begin to develop a general strategy in which we could rationally design synthetic
molecules that can be optimized for inhibition of protein-protein interactions. Structural information on the
HDM2-p53 complex was used to design our first class of peptoid inhibitors, and we provide here, in detail,
the strategy to modify peptoids with the appropriate side chains that are effective inhibitors of HDM2-p53
binding. While we initially tried to develop rigid, helical peptoids as HDM2 binders, the best inhibitors were
surprisingly peptoids that lacked any helix-promoting groups. These results indicate that starting with rigid
peptoid scaffolds may not always be optimal to develop new inhibitors.
3
Introduction
(residues 15-29), which greatly stimulated the search for
HDM2 inhibitors with chemotherapeutic activity.4 In the
-6
Over the last 20 years, many connections between cancer and
the protein p53 have been established. In normal cells, p53 can
be inactivated due to association with the protein human double
minute 2 (HDM2). When p53 is inactive, cells are able to grow
and proliferate. Under conditions of cellular stress or genomic
damage, p53 activity is turned on. Once activated, p53 functions
as a transcription factor to promote production of other proteins
that result in cell cycle arrest or apoptosis. In the majority of
cancers, the mechanism of action of p53 is defective. In one-
half of all cancers, p53 acquires a genetic mutation that prevents
DNA binding and therefore inhibits p53 from acting as a
transcription factor. In a large number of other cancers, wild-
type p53 protein is present, but the cancer cells overexpress
negative regulators of p53, such as HDM2. Overexpression of
HDM2 has been linked to tumor aggressiveness and drug
resistance, and inhibition of HDM2 can restore p53 function
and prevent cancer growth.1,2
crystal, the N-terminal domain of HDM2 (residues 17-125)
binds a conserved fragment of the N-terminal, transactivation
domain of p53. The HDM2 fragment has two structurally similar
subdomains arranged to form a large, nonpolar cleft that binds
the p53 fragment. The latter forms an amphipathic R-helix
between residues 18-26, while both ends are in extended
conformations. As seen in Figure 1, the tandem F19, W23, and
L26 residues of p53 pack tightly and deeply in the nonpolar
crevice of HDM2. Within this complex, there are no salt-bridges,
and only two intermolecular hydrogen bonds. The stability of
the complex is primarily due to the hydrophobic interactions
involving the three cleft-binding residues, as confirmed by amino
7
acid substitution studies.
Considerable effort has been expended to exploit HDM2 as
a molecular target to kill cancer cells, leading to greater
understanding of the variety of pathways and mechanisms in
which it is involved (both dependent and independent of p53).6
The structural detail of HDM2 was first revealed in 1996 by
a crystal structure of the complex formed between HDM2 and
a peptide derived from the transactivation domain of p53
Inhibition of HDM2 binding to p53 has recently emerged as a
(
3) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine,
A. J.; Pavletich, N. P. Science 1996, 274, 948-953.
(
(
(
4) Shair, M. D. Chem. Biol. 1997, 4, 791-794.
5) Ch e` ne, P. Mol. Cancer Res. 2004, 2, 20-28.
†
Laboratory of Cell Biology, NIH.
Northwestern University.
Laboratory of Bioorganic Chemistry, NIH.
6) Buolamwini, J. K.; Addo, J.; Kamath, S.; Patil, S.; Mason, D.; Ores, M.
Curr. Cancer Drug Targets 2005, 5, 57-68.
‡
§
(7) B o¨ ttger, A.; B o¨ ttger, V.; Garcia-Echeverria, C.; Chene, P.; Hochkeppel,
H. K.; Sampson, W.; Ang, K.; Howard, S. F.; Picksley, S. M.; Lane, D. P.
J. Mol. Biol. 1997, 269, 744-756.
(
(
1) Momand, J.; Wu, H. H.; Dasgupta, G. Gene 2000, 242, 15-29.
2) Ch e` ne, P. Nat. ReV. Cancer 2003, 3, 102-109.
10.1021/ja056344c CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 1995-2004
9
1995

A R T I C L E S
Hara et al.
systems, including “high-affinity” tri-peptoid ligands for two
7-transmembrane G-protein-coupled receptor proteins, peptide-
peptoid hybrid ligands for Src homology 3 (SH3) protein
domains, and tri-peptoid blockers of the Vanilloid receptor
subunit 1 (VR1) and N-methyl-D-aspartate (NMDA) receptor
channels associated with pain and neurodegeneration.12
Another important feature of peptoids is that the backbone
can be sterically biased to form a helical conformation by
incorporating chiral side chains into the oligomer.13 A rigid
helical scaffold can predetermine the docked conformation to
a protein, thus providing more favorable entropy for binding.
This has been exploited to form amphipathic peptoid helices to
mimic the antibacterial Magainin peptide and the lung surfactant
Protein C for extracellular medicinal applications.14 Recently,
X-ray crystal structure studies have confirmed that the helical
structure is preserved for a wide variety of side chain sizes,
Figure 1. Cross-sectional slice of the crystal structure of the p53 peptide
(blue) bound to HDM2 (green). Arrows highlight the three hydrophobic
15
including both aromatic and aliphatic classes. The chiral-
residues on the p53 peptide that are critical for binding.
peptoid helix is similar to a type-I polyproline helix, in which
the amide bonds are cis, and the carbonyl groups point with
the oxygens toward the N-terminus, causing the electrostatic
dipole moment to be the reverse of standard R-helices. Because
the helical conformation is dictated by steric constraints, and
not hydrogen bonds, as with a regular protein, it is able to persist
in both aqueous and nonpolar solvents and under a broad range
4
common strategy to restore wild-type p53 activity. In this
context, a variety of effective HDM2 inhibitors have been
independently identified either from natural products or using
5,8-10
structure-based drug design and/or combinatorial screening.
The best inhibitors of HDM2-p53 binding are either small
molecules that are very hydrophobic or oligomeric molecules
that arrange hydrophobic side chains into the same three-
dimensional arrangement as the bound p53 helix. While a
number of these have been demonstrated to normalize levels
of p53 and kill cancer cells in culture, relatively few have
demonstrated biological activity in animal studies, and none has
been employed in clinical trials. Therefore, the development of
additional HDM2 inhibitors is still necessary.
In this paper, we describe our development of a competitive
binding inhibitor based on oligomeric peptoids. The peptoid
monomer (or N-substituted glycine) is similar to a regular amino
acid, except that the side chain is attached to the backbone
nitrogen atom instead of the R-carbon. Peptoids are a
particularly attractive choice for making peptide-like compounds
because a wide variety of side chains can be attached to the
peptoid backbone and peptoids are resistant to proteolytic
digestion. For these reasons, peptoids have been studied as
potential agonists and antagonists in a number of biological
11,16
of pH and temperature conditions.
Helical polypeptoids have
well-characterized circular dichroism (CD) spectra, similar to
a regular protein R-helix, which provides a rapid probe of the
conformational state. In addition, at least some peptoids are
pharmacologically biocompatible and are currently being ex-
1
7
amined for a variety of therapeutic purposes.
In this study, we reveal several important molecular features
of HMD2-binding peptoids that are required to achieve binding
to HDM2. Most of the initial peptoid designs were guided by
molecular modeling of a helical peptoid bound to HDM2;
however, substantial experimentation with new peptoid side
chains was essential to obtain HDM2 binding. Direct experi-
mental comparisons of the binding affinity of our peptoid
inhibitors with that of the p53 peptide, as well as the recently
11
1
8
developed small molecule antagonist Nutlin-3 and another
9
HDM2-binding peptoid isolated from a combinatorial screen,
are also presented. While we approached the design of peptoid
inhibitors starting from a stable helix that contained chiral side
chains at every position, to increase aqueous solubility we had
to incorporate achiral side chains with polar functional groups.
Unfortunately, chiral versions of these side chains are not easily
synthesized. Therefore, as more achiral side chains were
incorporated in the peptoids to promote aqueous solubility,
helical stability was diminished. In this study, our results
(
(
8) Klein, C.; Vassilev, L. T. Br. J. Cancer 2004, 91, 1415-1419.
9) Alluri, P. G.; Reddy, M. M.; Bachhawat-Sikder, K.; Olivos, H. J.; Kodadek,
T. J. Am. Chem. Soc. 2003, 125, 13995-14004.
10) (a) Duncan, S. J.; Gr u¨ schow, S.; Williams, D. H.; McNicholas, C.; Purewal,
R.; Hajek, M.; Gerlitz, M.; Martin, S.; Wrigley, S. K.; Moore, M. J. Am.
Chem. Soc. 2001, 123, 554-560. (b) Kumar, S. K.; Hager, E.; Pettit, C.;
Gurulingappa, H.; Davidson, N. E.; Khan, S. R. J. Med. Chem. 2003, 46,
(
2
4
2
813-2815. (c) Galatin, P. S.; Abraham, D. J. J. Med. Chem. 2004, 47,
163-4165. (d) Sakurai, K.; Chung, H. S.; Kahne, D. J. Am. Chem. Soc.
004, 126, 16288-16289. (e) Kritzer, J. A.; Lear, J. D.; Hodsdon, M. E.;
Schepartz, A. J. Am. Chem. Soc. 2004, 126, 9468-9469. (f) Fasan, R.;
Dias, R. L. A.; Moehle, K.; Zerbe, O.; Vrijbloed, J. W.; Obrecht, D.;
Robinson, J. A. Angew. Chem., Int. Ed. 2004, 43, 2109-2112. (g) Zhong,
H.; Carlson, H. A. Proteins 2004, 58, 222-234. (h) Grasberger, B. L.; et
al. J. Med. Chem. 2005, 48, 909-912. (i) Yin, H.; Lee, G. I.; Park, H. S.;
Payne, G. A.; Rodriguez, J. M.; Sebti, S. M.; Hamilton, A. D. Angew.
Chem., Int. Ed. 2005, 44, 2704-2707. (j) Ding, K.; et al. J. Am. Chem.
Soc. 2005, 127, 10130-10131. (k) Murray, J. K.; Farooqi, B.; Sadowsky,
J. D.; Scalf, M.; Freund, W. A.; Smith, L. A.; Chen, J.; Gellman, S. H. J.
Am. Chem. Soc. 2005, 127, 13271-13280. (l) Kritzer, J. A.; Leudtke, N.
W.; Harker, E. A.; Schepartz, A. J. Am. Chem. Soc. 2005, 127, 14584-
(12) (a) Zuckermann, R. N.; et al. J. Med. Chem. 1994, 37, 2678-2685. (b)
Nguyen, J. T.; Porter, M.; Amoui, M.; Miller, W. T.; Zuckermann, R. N.;
Lim, W. A. Chem. Biol. 2000, 7, 463-473. (c) Planells-Cases, R.; et al. J.
Pharmacol. Exp. Ther. 2002, 302, 163-173. (d) Garcia-Martinez, C.; et
al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2374-2379.
(13) Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L., Jr.; Dill, K.
A.; Zuckermann, R. N.; Cohen, F. E. Fold Des. 1997, 2, 369-375.
(14) (a) Patch, J. A.; Barron, A. E. J. Am. Chem. Soc. 2003, 125, 12092-12093.
(b) Wu, C. W.; Seurynck, S. L.; Lee, K. Y.; Barron, A. E. Chem. Biol.
2003, 10, 1057-1063.
1
4585. (m) Tsukamoto, S.; Yoshida, T.; Hosono, H.; Ohta, T.; Yokosawa,
(15) Wu, C. W.; Kirshenbaum, K.; Sanborn, T. J.; Patch, J. A.; Huang, K.;
Dill, K. A.; Zuckermann, R. N.; Barron, A. E. J. Am. Chem. Soc. 2003,
125, 13525-13530.
H. Bioorg. Med. Chem. Lett. 2005, 16, 69-71.
(
11) (a) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.; Bradley,
E. K.; Truong, K. T.; Dill, K. A.; Cohen, F. E.; Zuckermann, R. N. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 4303-4308. (b) Kirshenbaum, K.;
Zuckermann, R. N.; Dill, K. A. Curr. Opin. Struct. Biol. 1999, 9, 530-
(16) Sanborn, T. J.; Wu, C. W.; Zuckermann, R. N.; Barron, A. E. Biopolymers
2002, 63, 12-20.
(17) Gibbons, J. A.; et al. J. Pharmacol. Exp. Ther. 1996, 277, 885-899.
(18) Vassilev, L. T.; et al. Science 2004, 303, 844-848.
5
35.
1996 J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006

Peptoids That Inhibit HDM2−p53 Interactions
A R T I C L E S
surprisingly demonstrate that fewer chiral side chains in the
peptoid result in better HDM2 binding.
Experimental Section
1
. Molecular Modeling. Atomic coordinates for all of the models
Figure 2. Synthetically prepared amines that were incorporated into
were generated and energy minimized with the CHARMM molecular
mechanics computer program, combined with the CHARMM22
peptoids to promote interactions with HDM2 or promote aqueous solubility.
1
9a
9
(
*Ethanolamine has been previously incorporated into peptoids, but in this
1
9b
versions of the all-atom topology and parameter sets to describe the
residues. Whereas the HDM2 model could be formed by the common
amino acid residues already present in the package, the topologies for
the different peptoids were “manually” created. This was accomplished
by taking side chain topology fragments from the default set of common
residues and grafting them onto the amide nitrogen of glycine.
Additional customizations were made to create the stereogenic centers
and/or alter other aspects of the side chains. It should be emphasized
that no attempt was made to use the potential energy functions to make
de novo predictions of the conformation of the peptoid beyond the
obvious covalent geometry, or the binding affinity of the real peptoid
to HDM2 in solution.
work we introduced a t-Bu ether protecting group.)
9 (which will be abbreviated as Flu-p53(15-29)) was used.22 The
2
degrees of displacement of the labeled peptide from HDM2 at a series
of concentrations of added peptoid were measured, and then the IC50
values for inhibition were calculated by regression analysis. Isothermal
titration calorimetry was performed according to previously published
procedures,23 and experimental details are provided in the Supporting
Information. Nutlin compounds were generous gifts from Dr. L. T.
Vassilev and Hoffman-LaRoche, Inc., Nutley, NJ.
Results
1. Initial Peptoid Design. To reproduce the relative position-
The primary design goal was to create a helical polypeptoid with
phenylalanine, tryptophan, and leucine side chains along one side to
reproduce as closely as possible the relative orientations of the F19,
W23, and L26 residues of the bound p53 peptide, which are the most
important for binding. To start, the backbone of the polypeptoid was
manipulated into the ideal helical conformation described by Armand
and co-workers using molecular graphics software. We always chose
to use right-handed helices, which are formed from the S enantiomers
of chiral peptoid monomers. The model was then docked to HDM2 by
overlaying the cleft-binding residues of p53 in the crystal structure.
Energy minimization was then used to remove atomic overlaps and
optimize any available hydrogen bonds and/or salt-bridges.
ing of the F19, W23, and L26 residues of the bound p53
fragment, it was necessary to account for the structural differ-
ences between the ideal type-1 polyproline helix of the peptoid
and the standard R-helix of regular peptides. One difference is
that the peptoid helix is more tightly wound than that of the
peptide, with only 3.0 residues per turn as compared to 3.6.
Thus, simply transposing the peptide sequence to the peptoid
results in a helix in which the alignment of these three residues
is lost. Fortunately, this could be corrected by deleting one
position between the phenylalanine and tryptophan residues in
the peptoid sequence, thus going from (i,i+4) to (i,i+3). Because
the tryptophan and leucine are already one position closer in
the p53 sequence, the misalignment in the peptoid helix
conformation is less. In addition, this part of the bound p53
helix is already in a distorted, less tightly wound conformation.
Thus, it was not necessary to alter the spacing between these
two residues in the peptoid sequence. The other relevant
structural difference between the two helices is that the side
chains project out at different angles. This has a critical effect
on how they orient and fit in the contours of the hydrophobic
cleft of HDM2. Fortunately, this problem could be overcome
by adding an extra methylene group to both the phenylalanine
and the tryptophan peptoid side chains, which allows for greater
orientational variation of the phenyl and indole rings.
7,20
13
As described in the Results, the procedure was iteratively repeated
with additional modifications to the peptoid model to enhance the
overlap of the cleft-binding residues and increase the solubility and
binding affinity. It should be emphasized that no attempt was made to
use the calculated binding energies of the models as a predictor of the
binding affinity of the real polypeptoid to HDM2 in solution.
Unfortunately, the current state of potential energy functions and
achievable time-scales of dynamic simulations are insufficient to
accurately sample the conformational space and calculate the relevant
thermodynamic factors (especially of the hydrophobic effect). Rather,
the three-dimensional computer models served to examine the steric
goodness-of-fit of the peptoid to HDM2 and to devise chemical
improvements for the subsequent rounds of synthesis and testing.
2
. Peptoid Synthesis. All peptoids were synthesized using the
2
1
submonomer approach, using commercially available amines or
synthetically prepared ones. In some cases, new amines were made
that had not previously been incorporated into peptoids. These were
specifically made to adjust the properties of the peptoids to promote
interactions with HDM2 or to enhance aqueous solubility. The new
amines incorporated into peptoids are shown in Figure 2, and the details
of the synthetic work are described in the Supporting Information.
As shown in Figure 3, these modifications allow for a
relatively close structural reproduction of the main p53 binding
residues. Modeling of the complex demonstrates that only the
phenyl and indole rings need to fit into tightly contoured wells.
In contrast, the region of the HDM2 cleft to which the leucine
binds is relatively shallow and extended, and thus able to accom-
modate a repositioning of this third hydrophobic residue. This
is consistent with the peptide mutagenesis results of B o¨ ttger and
3
. Binding Assays. The protein HDM2 was expressed following
3
published procedures, which are summarized in the Supporting
Information. To determine the ability of each peptoid to inhibit p53-
HDM2 association, a reported fluorescence polarization competition
assay with a fluorescein-labeled p53 peptide composed of residues 15-
7
co-workers, in which the phenylalanine and tryptophan were
essential for activity, but the leucine could be changed to iso-
leucine, methionine, or valine and still maintain partial activity.
2
. Initial Peptoids - Synthesis, Secondary Structure,
Aqueous Solubility, and Binding. The first step was to build
(
19) (a) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.;
Swaminathan, S.; Karplus. M. J. Comput. Chem. 1983, 4, 187-217. (b)
MacKerell, A. D.; et al. J. Phys. Chem. B 1998, 102, 3586-3616.
(22) (a) Knight, S. M. G.; Umezawa, N.; Lee, H.-S.; Gellman, S. H.; Kay, B.
K. Anal. Biochem. 2002, 300, 230-236. (b) Zhang, R.; Mayhood, T.; Lipari,
P.; Wang, Y.; Durkin, J.; Syto, R.; Gesell, J.; McNemar, C.; Windsor, W.
Anal. Biochem. 2004, 331, 138-146.
(20) Garc ´ı a-Echeverr ´ı a, C.; Ch e` ne, P.; Blommers, M. J.; Furet, P. J. Med. Chem.
2
000, 43, 3205-3208.
(21) Figliozzi, G. M.; Goldsmith, R.; Ng, S. C.; Banville, S. C.; Zuckermann,
R. N. Methods Enzymol. 1996, 267, 437-447.
(23) Houtman, J. C. D.; et al. Biochemistry 2004, 43, 4170-4178.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006 1997

A R T I C L E S
Hara et al.
Figure 3. Structural comparison of the three hydrophobic cleft-binding residues of the bound p53 peptide (green structure) and the designed peptoid helix
based on peptoid PS2 in the Supporting Information). The peptoid helix shown is the result of energy minimization while docked in the binding cleft of
(
HDM2. Peptoid side chains are designated with a P followed by the amino acid side chain that they mimic. (Additional side chains of the peptoid and
peptide are omitted for clarity.)
Figure 4. (A) Model of peptoid 2 docked to HDM2. The surface of HDM2 is shown as an electrostatic potential energy map. HDM2 amino acids with
which the peptoid side chains could interact are labeled. (B) Side view showing hydrophobic peptoid side chains (green) bound in the HDM2 crevice. HDM2
is shown as a transparent surface.
a plain, helical peptoid from 10 chiral (S)-N-(1-phenylethyl)-
glycine subunits (peptoid PS1, Supporting Information) to serve
as a reference point for the CD spectrum of a helical peptoid.
In acetonitrile, peptoid PS1 has a far-UV CD spectrum typical
of a peptoid in a right-handed helical conformation, with two
minima at 219 and 205 nm, and a maximum at 193 nm (Figure
S3, Supporting Information). Our first attempt to make an
HDM2-binding peptoid centered on introducing achiral pheny-
lalanine, tryptophan, and leucine peptoid analogue subunits at
positions 3, 6, and 9 of peptoid PS1, as is called for in the orig-
inal design (see peptoid PS2, Supporting Information). In aceto-
nitrile, the CD spectrum of peptoid PS2 retains the characteristic
structure of a helix, but with the magnitude of molar ellipticity
reduced approximately 30% as compared to peptoid PS1 (Figure
S3, Supporting Information). This likely reflects the reduction
in the number of chiral subunits in the peptoid decamer and
indicates a less stable helical conformation in solution. Data
on the binding of peptoid PS2 to HDM2 could not be obtained
due to the poor aqueous solubility of this peptoid. To make a
more water-soluble analogue, three achiral peptoid glutamic-
acid residues were substituted into the sequence to create peptoid
1 (Figure 5). Still assuming a helical conformation for the bound
inhibitor, these new side chains were designed to form salt-
bridges with the K94, H96, and K51 residues of HDM2 at the
edges of the binding cleft (modeling not shown). In an effort
to keep at least one chiral subunit at the C-terminus, the
phenylalanine, tryptophan, and leucine analogues were shifted
to positions 2, 5, and 8, and the glutamic acid analogues were
put at positions 4, 7, and 9. Peptoid 1 was found to be partially
soluble in water (pH 7.2, 10 mM Tris HCl, 10 mM NaCl, 0.5
mM EDTA), but was inactive in the fluorescence polarization
binding assay (results not shown). With the number of chiral
subunits reduced to only 4 out of 10, the CD spectrum of peptoid
1 indicated a further destabilization of the helical conformation
in aqueous solution, especially by the large diminution of the
molar ellipticity of the positive peak at 193 nm (Figure 6).
1998 J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006

Peptoids That Inhibit HDM2−p53 Interactions
A R T I C L E S
Figure 5. Initial peptoids synthesized to test for HDM2 binding, helicity, and water solubility.
Synthesis of the phosphonate-containing peptoid monomer relied
on the development of two new building block amines (see
Supporting Information for synthesis). As predicted, peptoid 2
was more soluble in water using the same buffer conditions as
for peptoid 1, but a slightly more basic pH of 7.6 was optimal
for solubility. However, the new side chains resulted in a further
destabilization of the helical conformation in solution, as
indicated by further reduction in the magnitude of the two
negative peaks and complete elimination of the positive peak
in the CD spectrum as compared to peptoid PS1 (Figure 6).
Fortunately, peptoid 2 was the first to demonstrate at least weak
(IC50 ) 188 µM) competitive binding to HDM2 (Figure 11A
and Table 1). This binding may be due to the greater electrostatic
attraction of the phosphonates to the positively charged lysine
residues around the cleft of HDM2, as indicated by the modeling
of peptoid 2 bound to HDM2 presented in Figure 4. In this
model, the phosphonate side chains of the peptoid interact
favorably with two lysines and one histidine on the surface of
the protein, while the indole side chain of the peptoid forms a
hydrogen bond to the carbonyl oxygen of leucine 54.
Figure 6. CD spectra of peptoids 1-4 and 10. The spectra were measured
in Tris buffer (10 mM Tris, 10 mM NaCl, 0.5 mM EDTA, pH 7.6) at room
temperature. Peptoid concentration was 60 µM for all samples.
Interestingly, the two negative peaks at 219 and 205 nm were
approximately the same as for peptide PS2.
To further increase the aqueous solubility, peptoid 2 was
synthesized with three phosphonates replacing the carboxylates.
In an effort to increase the hydrophilicity of the phenylethyl
side chains, peptoid 3 was synthesized with para-nitro groups
J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006 1999

A R T I C L E S
Hara et al.
Figure 7. Peptoids synthesized to probe the importance of hydrophobic interactions (numbering continues from Figure 5).
on the phenyl rings, that is, using (S)-N-(p-nitrophenylethyl)-
glycine peptoid subunits. This analogue has been previously
examined and found to promote the same helical conformation
as the nonsubstituted peptoids.11 Interestingly, the CD spectrum
of peptoid 3 in solution was found to significantly differ from
that of peptoid 2, with total abolition of the negative peak at
that peptoid 3 is not in a helical conformation in aqueous
solution. Fortunately, however, peptoid 3 showed a significant
increase in the binding affinity to HDM2, with an IC50 of 15
µM (Figure 11A and Table 1), only 5 times larger than that of
the p53 peptide. While the exact reasons for the improvements
in binding are unclear, it is possible that the nitro groups promote
alternative conformations of the peptoid that are more comple-
mentary to the HDM2 binding site and/or that favorable
hydrogen bonds between the nitro groups and proximal residues
on HDM2 are formed. With the improvements in binding seen
with peptoid 3, we pursued this structure as a lead compound
and made additional modifications to reveal the essential features
and enhance the binding affinity.
2
19 nm (Figure 6). Rather, the spectrum resembles that of an
acetylated monomer of (S)-N-(p-nitrophenylethyl)glycine in
acetonitrile shown in Figure 4 of the paper published by
Kirshenbaum and co-workers,1 which also has a single
negative peak at approximately 205 nm and a molar ellipticity
1a
-
3
2
of approximately 17 × 10 deg cm /dmol. Because the
monomer is not physically able to form a helix, this suggests
2000 J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006

Peptoids That Inhibit HDM2−p53 Interactions
A R T I C L E S
Figure 8. Effects of peptoid chirality on HDM2 binding (numbering continues from Figure 7).
Figure 9. Effect of replacing a p-nitrophenyl group on HDM2 binding.
Figure 10. Structures of Nutlin-3 and the Alluri peptoid used to compare binding to HDM2.
3
. Charged Peptoid Residues. To attain biological activity,
sulfonamide substitution at position 9, retained good water
solubility, and the binding affinity to HDM2 was largely
unaffected as compared to peptoid 3 (Figure 11A and Table 1).
In addition, the CD spectrum was very similar (Figure 6).
However, additional sulfonamide substitutions at positions 4 and
7 resulted in peptoids that were insoluble in aqueous solutions.
Therefore, the sequence of two phosphonates and one sulfona-
mide, as in peptoid 4, was incorporated in the development of
subsequent peptoids.
it will ultimately be necessary for the peptoid to cross cell
membranes. To facilitate this, we sought to reduce the number
of phosphonate groups so that there was a minimum of anionic
charge. Therefore, peptoids were made in which the phosphonate
side chains were selectively replaced with sulfonamides (see
peptoid 4 in Figure 5 and peptoids PS3 and PS4 in Figure S1
of the Supporting Information). Although sulfonamides are
neutral in overall charge, the two oxygens on the sulfur have
partial negative charges that may still interact with the proximal
positively charged residues of HDM2. Peptoid 4, with one
4. Peptoid Length. The next series of peptoids were designed
to probe the effect of oligomer length on HDM2 binding.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006 2001

A R T I C L E S
Hara et al.
Figure 11. Binding and inhibitory activity of peptoids, p53 peptide, and Nutlin-3. (A) Fluorescence polarization assay: Displacement of HDM2 bound
Flu-p53(15-29) by peptoids and reported competitors. Compounds were titrated to preincubated HDM2 (2.2 µM)-Flu-p53(15-29) (30 nM). (B) ITC
assay: Binding isotherm for the interaction of HDM2 with peptoid 13. The titration involved the injection of 7.5 µL of the peptoid solution (125 µM) into
HDM2 (8.5 µM). All experiments were performed at 25 °C in Tris buffer (10 mM Tris HCl, 150 mM NaCl, 1 mM â-mercaptoethanol, 0.5 mM EDTA, pH
7
.6), and the data were analyzed as described in the Supporting Information.
Table 1. IC50 Values Obtained from Fluorescence Polarization
with a methyl group decreased the binding affinity to HDM2,
supporting the structural model. As compared to peptoid 4 (IC50
of 17.8 µM), the most significant loss in binding affinity was
seen when the mimic of the phenylalanine side chain was
replaced with a methyl group (peptoid 5, IC50 of 59.8 µM). This
effect was less pronounced when side chains mimicking the
tryptophan and leucine were replaced with methyl groups
entry
IC50
(µ
M)a
entry
IC50 (µ
M)a
peptoid
peptoid
10
11
12
13
1
1
2
3
4
5
6
7
8
9
NBb
99 ( 0.8
19.5 ( 1.7
18.2 ( 1.7
6.6 ( 0.7
12.7 ( 1.2
NB
188 ( 30
15.3 ( 1.0
17.8 ( 1.0
59.8 ( 3.7
28.3 ( 2.1
25.3 ( 2.9
26.8 ( 2.9
29.7 ( 4.6
14
b
Alluri
Nutlin
3a
(peptoids 6 and 7, IC50 values of 28.3 and 25.3 µM, respec-
tively). This is in contrast to the work of B o¨ ttger and co-
1.2 ( 0.3
17.7 ( 3.3
7
3b
workers, who found the tryptophan side chain to have the most
stabilizing effect. In the second set of peptoids, scrambling the
positions of the hydrophobic side chains in the peptoid sequence
(peptoids 8 and 9) lowered the binding affinity to a similar
degree, as seen in peptoids 6 and 7. Finally, peptoid 10 was
synthesized by analogy to the work of Garc ´ı a-Echeverr ´ı a and
co-workers, who obtained significant improvement in binding
affinity by substituting chlorine at the 6 position of the
a
Concentration necessary to displace 50% of HDM2-bound Flu-
p53(15-29). For comparison, the p53(15-29) peptide was bound with an
IC50 ) 3.0 ( 0.3 µM. NB ) no detectable binding.
b
Adding an extra (S)-N-(p-nitrophenylethyl)glycine residue to the
N- and C-termini of peptoid 4 does not have a significant impact
on the binding affinity to HDM2 (peptoids PS5, PS6, and Table
S1 in the Supporting Information). However, the binding affinity
was significantly reduced with a shorter peptoid that lacked the
N- and C-terminal (S)-N-(p-nitrophenylethyl)glycines of peptoid
5,20
tryptophan indole moiety. They hypothesize that the chlorine
increases the intermolecular van der Waals contact by filling a
proximal cavity below the indole ring at the bottom of the
HDM2 binding cleft. Similarly, peptoid 10 showed a significant
decrease in the IC50 value from 17.8 to 9.9 µM (Figure 11A
and Table 1). It should be noted, however, that the effect of the
chloro-substitution was much larger for the peptides, with
approximately 2 orders of magnitude reduction in the IC50
4
(peptoid PS7). In this case, the IC50 increased to 222 µM,
similar to that of peptoid 2. Therefore, the peptoid length was
maintained at 10 residues for subsequent development, with the
chiral (S)-N-(p-nitrophenylethyl)glycine residues maintained at
the N- and C-termini.
5
. Cleft-Binding Residues. To test the assumption that the
2
0
value. Nevertheless, these results are consistent with the
arrangement (i.e., p53 wild-type arrangement) of hydrophobic
residues predicted to be energetically preferred for binding.
hydrophobic phenylalanine, tryptophan, and leucine side chain
mimics of peptoid 4 each bind in the nonpolar cleft of HDM2,
peptoids 5-10 were designed and synthesized (Figure 7). In
this series of molecules, peptoids 5-7 each have a single
hydrophobic, cleft-binding side chain replaced with a methyl
group, while peptoids 8 and 9 have the order of these side chains
scrambled, and peptoid 10 has a chlorine-substituted indole side
chain. For the first set, replacing the hydrophobic side chain
6. Residue Chirality. Although the CD spectrum of peptoid
4 suggests the absence of a helix in solution, the remaining four
chiral residues in the sequence may still provide a conforma-
tional bias to the final, docked peptoid structure. To investigate
the potential effects, peptoid 11 was synthesized with the
2002 J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006

Peptoids That Inhibit HDM2−p53 Interactions
A R T I C L E S
Table 2. Kd from Isothermal Titration Calorimetry
Discussion
d
K (µ
M)a
entry
These results demonstrate that we have successfully devel-
oped an all-peptoid decamer that competes with the wild-type,
N-terminal p53 peptide for the binding site of the N-terminal
domain fragment of HDM2. Although not currently better than
the p53 peptide, the IC50 and dissociation constant for peptoid
peptoid
10
3
1.75 ( 0.22
1.23 ( 0.31
0.62 ( 0.12
1
p53(15-29) peptide
a
Titrations were performed in 10 mM Tris HCl, 150 mM NaCl, 1 mM
â-mercaptoethanol, 0.5 mM EDTA, pH 7.6. All data were fit to a single
site binding curve with 1:1 stoichiometry, from which Kd was calculated.
1
3 are only approximately 2-fold larger (Figure 11, Tables 1
and 2). Our structure-based design approach, which involved
iterative cycles of computer model building, led us to the initial
peptoids that showed only weak binding to HDM2. Three
synthetic modifications to the side chains of the initial peptoids
allowed significant improvement in the binding affinity. The
first major improvement came by introducing three phosphonate-
containing side chains at positions 4, 7, and 9 to increase the
aqueous solubility and, according to modeling, potentially form
salt-bridges with positively charged residues around the HDM2
binding cleft (Figure 4). Subsequent substitutions with neutral
sulfonamides showed that the third phosphonate group does not
significantly affect binding (peptoid 4). The next major im-
provement came with the addition of para-nitro substitutions
on the phenyl rings of the four (S)-N-(1-phenylethyl)glycine side
chains at positions 1, 3, 6, and 10 (peptoid 3). As described in
the Results, the enhanced affinity may be due to a number of
factors, including favorable interactions with proximal residues
of HDM2. Support for this comes from the finding that deletion
of both the N- and C-terminal (S)-N-(p-nitrophenylethyl)glycine
residues drastically reduces the binding affinity, and in the
modeled complex these nitro groups could interact with the
positively charged K94 and R97 residues of HDM2. Further-
more, replacement of the p-nitrophenyl group at position 3 with
a hydroxyethyl group (peptoid 14) also decreased the HDM2
binding affinity, indicating that this central p-nitrophenyl group
interacts favorably with HDM2. The final improvement came
with the substitution of chlorine at the 6 position of the indole
ring of the tryptophan peptoid analogue (peptoids 10 and 13).
A similar result, although of greater magnitude, was found in
the development of peptide-based inhibitors of the same HDM2
stereochemistry reversed for the first and third chiral p-
nitrophenylethyl side chains (i.e., at positions 1 and 6), and
peptoid 12 was synthesized with all achiral analogues (Figure
8
). In fact, none of these modifications had a significant effect
on the binding affinity to HDM2 (Figure 11A and Table 1). In
addition, the aqueous solubility of peptoid 12 in buffer was the
same (1.8 mM) as its chiral predecessor (peptoid 4). To
investigate further, peptoid 13 was synthesized as an achiral
version of peptoid 10. In this case, with the 6-chlorotryptophan
substitution present, removal of the chirality resulted in a
relatively small increase in the binding affinity, that is, a
reduction in IC50 from 9.9 to 6.6 µM (Figure 11A and Table
1
). Thus, the best peptoid inhibitor we obtained to this point
lacked any of the originally designed stereocenters and contained
the 6-chloro substitution on the tryptophan peptoid side chain.
To eliminate any concerns that peptoid 13 could be aggregating
into dimers, trimers, or tetramers, analytical unltracentrifugation
of this peptoid strongly indicated that it is present as a monomer
under the aqueous conditions used to test the binding (see the
Appendix in the Supporting Information for details).
7
. Role of One Central p-Nitro Group. To assess whether
the central p-nitrophenylethyl groups play a role in binding to
HDM2, one of these groups was changed to a hydroxy ethyl
side chain, as seen in peptoid 14 (Figure 9). The binding of
this peptoid to HDM2 was 2-fold weaker than that of peptoid
1
3, indicating that this side chain does contribute to binding.
8
. Comparison with Other Molecules and Isothermal
2
0
target.
Titration Calorimetry (ITC). Additional experiments were
performed to directly compare the affinities of peptoids 10 and
The reduced binding affinities of peptoids 5-9 as compared
to peptoid 4 (Table 1) indicate that the peptoid side chains that
mimic the phenylalanine, tryptophan, and leucine of the p53
peptide each play roles in stabilizing the complex, similar to
the p53 peptide. Furthermore, the improvement in binding
affinity seen with the introduction of the 6-chloroindole side
chain (peptoid 10) has some correlation with the improvement
in binding seen with HDM2-binding peptides containing 6-chlo-
rotryptophan. The structural explanation provided by Garc ´ı a-
Echeverr ´ı a and co-workers is that the chlorine fills a cavity in
1
3 to those of three other HDM2-binding molecules in the
1
8
literature, that is, the a and b enantiomers of Nutlin-3 and the
best HDM2-binder found from screening a large library of
peptoids (Figure 10). We will refer here to the latter as the
9
“Alluri peptoid”. Under the same conditions, Nutlin-3a and -3b
bound HDM2 with IC50 values of 1.2 and 17.7 µM, respectively,
as compared to 3.0 µM for the control p53 peptide (Table 1).
Interestingly, while the IC50 value we obtained for the b
enantiomer of Nutlin-3 was close to that previously reported
by Vassilev and co-workers (i.e., 13.6 µM), they obtained a
value approximately 1 order of magnitude smaller for the a
enantiomer (i.e., 0.09 µM).18 In contrast, we did not detect any
competitive binding for the Alluri peptoid using the fluorescent
assay. Thus, the Alluri peptoid likely binds to the N-terminus
of the HDM2 fragment at an alternate location than the p53
binding cleft. To further confirm the binding association of
peptoids 10 and 13 to HDM2, isothermal titration calorimetry
was performed to obtain dissociation constants (Kd) for the
peptoids and the p53 peptide. As shown in Table 2 (and Figure
20
the binding cleft below the indole moiety. While it is tempting
to assume that the three hydrophobic peptoid residues all bind
in the nonpolar cleft of HDM2, consistent with early peptide
7
3
studies, the crystal structure, and our computer generated
model, this may not be the case. For all of these control peptoids
(peptoids 5-10), the changes in binding affinity are significantly
less dramatic than the corresponding changes observed in
HDM2-binding peptides. Furthermore, the requirement for nitro
groups to be present on several of the peptoid side chains
indicates that there are important peptoid-HDM2 interactions
that do not involve the hydrophobic side chains.
1
1B), the relative differences in Kd values are similar to the
IC50 data obtained from fluorescence polarization.
Another major question concerns the conformation of the
J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006 2003

A R T I C L E S
Hara et al.
peptoid backbone when bound to HDM2. Although initially
designed with chiral (S)-N-(1-phenylethyl)glycines to have a
right-handed type-I polyproline helical conformation, our op-
timizations led to peptoid 13, which is devoid of chiral side
chains. Because of the synthetic difficulty, it is not feasible at
the present time to make a derivative of peptoid 13 in which
each side chain contains a helix-promoting stereogenic carbon
at every position. Because the steric bias provided by each (S)-
N-(1-phenylethyl)glycine has been calculated to be as small as
Our study has identified many important details about using
a peptoid oligomer to mimic a specific bioactive peptide. In
contrast to our initial predictions, the achiral peptoids were the
best HDM2 binders rather than the peptoids with helical
structures. Such results indicate that peptoids can adopt con-
formations that are nonhelical, which are important for binding
to proteins. While the results we present in this paper do not
compete with many recently published studies of HDM2
5,10,20
inhibition with regard to drug development,
they do reveal
0
.5 kcal/mol between right- and left-handed helical conforma-
a number of important elements to consider when using peptoids
to inhibit protein-protein interactions. Our future structural
studies will likely guide our development of additional peptoid
side chains that can further enhance binding to HDM2 as well
as identify new ways to design peptoids to inhibit other protein-
protein interactions.
1
3
tions in solution, it is understandable that mixing chiral and
achiral groups results in peptoids that are not fully helical in
solution, as indicated by the CD data. While it is possible that
a helical conformation in the peptoid may be induced by binding
to HDM2, alternate peptoid conformations could also be induced
by the energetics of binding. Indeed, through modeling we were
able to identify other, nonhelical conformations of the peptoid
backbone that could be relevant to binding HDM2 (results not
shown). Therefore, the helical peptoid conformation shown in
Figure 4 may not be very accurate, but it does demonstrate that
stabilizing, intermolecular interactions can form between the
peptoid’s two phosphonate-containing residues, the N- and
C-terminal nitrophenyl groups, and, by analogy, the sulfonamide
group. This model also places the hydrophobic side chains into
the binding cleft of HDM2. We are currently attempting to
determine the crystal structure of this complex to answer these
questions and provide a more confident basis to design further
optimizations. In addition, it will show if the peptoid binding
induces a conformation in the HDM2 fragment different from
that in the original crystal structure with the wild-type p53
peptide.24
Acknowledgment. This research was supported in part by
the following Intramural Research Programs of the NIH: NCI
Center for Cancer Research and NIDDK Laboratory of Bioor-
ganic Chemistry. M.C.M. was supported by a graduate student
fellowship from Abbott Laboratories while at Northwestern
University. We are grateful to Dr. Vassilev at Hoffman LaRoche
for providing a sample of the Nutlins. We would also like to
thank Dr. Marc S. Lewis for performing the analytical ultra-
centrifugation experiments.
Supporting Information Available: Synthetic procedures and
characterization for all new peptoid monomers and peptoid
oligomers. Experimental procedures for circular dichroism,
fluorescence polarization, and isothermal titration calorimetry.
Complete author lists for refs 10h, 10j, 12a, 12c, 12d, 17, 18,
1
9b, and 23. This material is available free of charge via the
Internet at http://pubs.acs.org.
(24) Schon, O.; Friedler, A.; Freund, S.; Fersht, A. R. J. Mol. Biol. 2004, 336,
1
97-202.
JA056344C
2004 J. AM. CHEM. SOC.
9
VOL. 128, NO. 6, 2006