N-Acylpolyamine inhibitors of HDM2 and HDMX binding to p53

Article abstract of DOI:10.1016/j.bmc.2009.10.032

Selective inhibition of protein-protein interactions important for cellular processes could lead to the development of new therapies against disease. In the area of cancer, overexpression of the proteins human double minute 2 (HDM2) and its homolog HDMX has been linked to tumor aggressiveness. Both HDM2 and HDMX bind to p53 and prevent cell cycle arrest or apoptosis in damaged cells. Developing a strategy to simultaneously prevent the binding of both HDM2 and HDMX to p53 is an essential feature of inhibitors to restore p53 activity in a number of different cancers. Inhibition of protein-protein interactions with synthetic molecules is an emerging area of research that requires new inhibitors tailored to mimic the types of interfaces between proteins. Our strategy to create inhibitors of protein-protein interactions is to develop a non-natural scaffold that may be used as a starting point to identify important molecular components necessary for inhibition. In this study, we report an N-acylpolyamine (NAPA) scaffold that supports numerous sidechains in a compact atomic arrangement. NAPAs were constructed by a series of reductive aminations between amino acid derivatives followed by acylation at the resulting secondary amine. An optimized NAPA was able to equally inhibit the association of both HDM2 and HDMX with p53. Our results demonstrate some of the challenges associated with targeting multiple protein-protein interactions involved in overlapping cellular processes.

Full text of DOI:10.1016/j.bmc.2009.10.032

Bioorganic & Medicinal Chemistry 17 (2009) 7884–7893
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Acylpolyamine inhibitors of HDM2 and HDMX binding to p53
Ryo Hayashi a, Deyun Wang b, Toshiaki Hara a, Jaclyn A. Iera b, Stewart R. Durell a, Daniel H. Appella b,
*
a Laboratory of Cell Biology, NCI, NIH, DHHS, Bethesda, MD 20892, United States
b Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective inhibition of protein–protein interactions important for cellular processes could lead to the
development of new therapies against disease. In the area of cancer, overexpression of the proteins
human double minute 2 (HDM2) and its homolog HDMX has been linked to tumor aggressiveness. Both
HDM2 and HDMX bind to p53 and prevent cell cycle arrest or apoptosis in damaged cells. Developing a
strategy to simultaneously prevent the binding of both HDM2 and HDMX to p53 is an essential feature of
inhibitors to restore p53 activity in a number of different cancers. Inhibition of protein–protein interac-
tions with synthetic molecules is an emerging area of research that requires new inhibitors tailored to
mimic the types of interfaces between proteins. Our strategy to create inhibitors of protein–protein inter-
actions is to develop a non-natural scaffold that may be used as a starting point to identify important
molecular components necessary for inhibition. In this study, we report an N-acylpolyamine (NAPA) scaf-
fold that supports numerous sidechains in a compact atomic arrangement. NAPAs were constructed by a
series of reductive aminations between amino acid derivatives followed by acylation at the resulting sec-
ondary amine. An optimized NAPA was able to equally inhibit the association of both HDM2 and HDMX
with p53. Our results demonstrate some of the challenges associated with targeting multiple protein–
protein interactions involved in overlapping cellular processes.
Received 14 August 2009
Revised 14 October 2009
Accepted 15 October 2009
Available online 21 October 2009
Keywords:
HDMX
HDM2
Cancer
Protein–protein interactions
Published by Elsevier Ltd.
1. Introduction
plex formed between HDM2 (residues 17–125 of the N-terminal
domain) and a peptide derived from the transactivation domain
The protein p53 acts as a cellular guardian against proliferation of
damaged cells.1 In healthy cells, p53 is inactivated by several nega-
tive regulators, allowing cells to proliferate.2 Under conditions of
cellular stress or genomic damage, p53 is activated and functions
to promote cell cycle arrest or apoptosis. In the majority of cancers,
p53 activity is defective due either to a genetic mutation in p53 that
prevents DNA binding or to the overexpression of negative regula-
tors of p53 (such as HDM2 and HDMX).3 Overexpression of HDM2
and HDMX has been linked to tumor aggressiveness and drug resis-
tance, and inhibition can restore p53 function and prevent cancer
growth.2,4,5 HDM2 acts as a ubiquitin ligase upon binding to p53, tar-
geting p53 for degradation.2 Recently, several studies have high-
lighted the importance of HDMX as a negative regulator of p53.4,6,7
HDMX binds to p53 at the same site as HDM2, but it lacks ubiquitin
ligase activity and therefore blocks p53 activity without promoting
degradation.8 Although the regulation of HDM2 and HDMX is com-
plex, blocking association of both proteins with p53 seems to be
essential in order to restore p53 activity in a number of cancers.9
The complex formed between HDM2 and p53 has served as a
paradigm for chemists to develop molecules that are tailored to
inhibit protein–protein interactions. A crystal structure of the com-
of p53 (residues 15–29)10 highlighted the following important fea-
tures of this protein–protein interaction: HDM2 has a large hydro-
phobic cleft that binds to p53, p53 adopts an amphipathic
between residues 18–26, and p53 projects three hydrophobic res-
idues from the -helix (F19, W23 and L26) so that they pack tightly
a-helix
a
and deeply in the non-polar cleft of HDM2. The stability of the
p53–HDM2 complex is primarily due to the hydrophobic interac-
tions involving the three cleft-binding residues, as confirmed by
amino acid substitution studies.11 This structure greatly stimulated
the search for HDM2 inhibitors with chemotherapeutic activity,
and much success has been achieved in this area.12,13 The best
inhibitors of HDM2–p53 binding are either small molecules that
are very hydrophobic or oligomeric molecules that arrange hydro-
phobic sidechains into the same three-dimensional arrangement as
the bound p53 helix. One of the best small molecule inhibitors of
HDM2–p53 binding is Nutlin-3.14 This molecule, along with a
few other inhibitors of HDM2, have demonstrated efficacy in mul-
tiple cell-based systems and a few animal models of cancer tu-
mors.15–18 However, Nutlin-3 and the other HDM2 inhibitors
only weakly inhibit HDMX binding to p53, and this deficiency
could ultimately prevent development of Nutlin-3 and other
HDM2 inhibitors into effective chemotherapeutic drugs.6,19
A comparison of the structures of HDM2 and HDMX indicates
the reason that Nutlin-3 shows diminished inhibition of HDMX–
* Corresponding author.
E-mail address: appellad@niddk.nih.gov (D.H. Appella).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.10.032

R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
7885
p53 association.20,21 Within the p53-binding sites of the two pro-
teins, HDMX has a significantly smaller hydrophobic binding cleft
due primarily to the protrusion of the sidechain of tyrosine 96 into
this area. The corresponding sidechain in HDM2 (tyrosine 100) is
rotated away from the binding cleft.20 Nevertheless, the structure
of the complex between the p53 peptide and HDMX is very similar
to the p53–HDM2 complex. In both structures, the same series of
were energy minimized with the CHARMM molecular mechanics
computer program and the CHARMM22 versions of the all-atom
topology and parameter sets.33 The complexes were than further
optimized using the AUTODOCK
4 software package with the
Lamarckian GA algorithm (Scripps Research Institute, La Jolla,
Ca).34 Illustrations of the models were created with the Chimera
software program (University of California, San Francisco, CA).35
Modeling was used to examine the conformational-range of possi-
ble complexes consistent with the experimental results of the dif-
ferent NAPA analogs and to help design better binding analogs. No
attempt was made to predict the ‘best’ complex structure from cal-
culation of the binding energies.
hydrophobic sidechains from the p53
a-helix protrude into the
binding clefts and there are only slight perturbations in the helical
structure to accommodate the differences between the two bind-
ing sites, consistent with the similar affinities of the p53 peptide
for HDM2 and HDMX.22 Based on the structure, it has been pro-
posed that the protrusion of tyrosine 96 into the binding cleft ste-
rically clashes with Nutlin-3 to weaken the binding to HDMX.21
Therefore, the development of inhibitors of both HDM2 and
HDMX binding to p53 is becoming an important new target for
chemists to tackle.23 Recently, the work of Schepartz and co-work-
ers has elegantly demonstrated that helical b-peptides can be
appropriately modified to bind with good affinity to both HDM2
and HDMX.24 Here, we report an alternative scaffold based on
the b-sheet tri-urea template developed by Nowick et al.25 Among
the inhibitors of p53–HDM2, Robinson showed that peptide-based
structures other than a helix can be developed into inhibitors.26 In
this work, cyclic peptides in b-sheet conformations were devel-
oped into very potent inhibitors of HDM2–p53 association.27–29
Based on these precedents, we have developed a scaffold we have
termed N-acylpolyamines (NAPAs, Fig. 1) and we have optimized
the sidechains projecting from the scaffold to inhibit both HDM2
and HDMX binding to a p53 peptide with IC50 values around
2.2. Solid phase NAPA synthesis
See Supplementary data for details on general methods and
preparation of amino aldehydes. TentaGel R resin, Rink type
(25 lmol, substitution 0.17–0.20 mmol/g) was allowed to swell
in NMP in a 10 mL disposable filter polypropylene column (Pierce
Biotechnology Inc., Rockford, IL) for 1 h. b-Alanine was coupled to
the resin by the HBTU–HOBt method. The coupling protocol was
as follows: (i) deprotection of the Fmoc group with 20% piperidine
in NMP for 10 min; (ii) washing with NMP (ꢀ5); (iii) activation of
N-a-Fmoc-protected amino acid (0.25 mmol) with 0.45 M HBTU–
HOBt solution in DMF (0.56 mL, 0.25 mmol), and 2 M DIEA solution
in NMP (0.5 mmol, 500 L) for 10 min; (iv) coupling with the pre-
activated N- -Fmoc-protected amino acid for 40 min; and (v)
l
a
washing with NMP (ꢀ5). The synthetic protocol for urea deriva-
tives was as follows: (i) deprotection of the Fmoc group in the
above condition; (ii) washing with NMP (ꢀ5) and then with dry
2 lM. As illustrated in Figure 1, our general strategy was to project
hydrophobic sidechains from the urea (Ri) to bind into the proteins’
hydrophobic binding clefts while sidechains attached to the carbon
backbone (Ro) would be hydrophilic and exposed to the solvent.
Our results indicate that it is possible to develop non-peptidic syn-
thetic molecules with equal inhibitory activity against both HDM2
and HDMX binding to p53.
DCM (ꢀ5); (iii) addition of N-
a-Fmoc-protected amino aldehyde
(0.25–0.35 mmol) to the resin as a 0.25–0.5 M solution in dry
DCM, followed by acetic acid (7 L, 0.125 mmol); (iv) after shaking
l
for 1 h, all solvent was drained, and the resin was rinsed with dry
DCM (ꢀ5); (v) addition of NaBH(OAc)3 (53 mg, 0.25 mmol) to the
resin as a 0.25 M solution in DCM; (vi) shaking for 2 h, and then
washing with NMP (ꢀ5) and with dry DCM (ꢀ5); (vii) addition of
0.5 M isocyanate solution in dry DCM (0.25–0.50 mmol) and N-
2. Experimental
methylmorphorine (28 lL, 0.25 mmol); and (viii) shaking for 2 h,
and then washing with DCM (ꢀ5) and NMP (ꢀ5). The above steps
2.1. Molecular modeling
were repeated three times. After removing the Fmoc group, 2,4-
dimethoxybenzylisocyanate (21 lL, 0.25 mmol was added to the
Models of the complexes of the different NAPA derivatives and
HDM2 and HDMX were developed to be analogous to a number of
crystal structures of these proteins with peptides and Nutlin. These
included the following entries in the RCSB Protein Data Bank:30
1YCR,10 1RV1,14 2Z5T,21 3DAB and 3DAC,20 3EQS and 3EQY31 and
3G03 and 3FD0.32 The coordinates of the NAPA compounds were
developed with the 2D and 3D editor modules of the Quanta
2006 software package (Accelrys, Inc., San Diego, CA). The models
resin as a 0.5 M solution in dry DCM, followed by N-methylmorph-
orine (28 mL, 0.25 mmol). To add a urea bond or a peptide bond at
the R4 position, isocyanate or carboxylic acid were reacted under
the above conditions. The mixture was shaken for 1–2 h. Final
deprotection and cleavage of the urea derivatives from the resin
were achieved with 94% TFA/5% water/1% TIS for 2 h at room tem-
perature. After removing the resin by filtration, the filtrate was
HDM2 or HDMX
Hydrophobic Sidechains
Ri =
directed into binding cleft
Hydrophilic Sidechains
Ro =
Ri
Ri
Ri
directed away from binding cleft
NH
NH
NH
O
O
O
H
N
N
NH2
N
N
Ro
O
Ro
Ro
O
Ro
Figure 1. Schematic representation of idealized NAPA binding to HDM2 or HDMX with intramolecular hydrogen bonds between the urea sidechains that direct the
hydrophobic sidechains into the binding cleft of the proteins. The hydrophilic sidechains are designed to be solvent exposed.

7886
R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
where ymin, ymax are the observed fluorescence polarization values
when the fluorescein-conjugated p53 peptide was completely
bound or not bound to HDM2 and HDMX, respectively, and x and
z are the competitor concentration and a slope factor, respectively.
The IC50 values for p53(15–29) binding to HDM2 and HDMX were
1–2 lM. For Nutlin-3a the IC50 values were 1 lM and 6–7 lM for
HDM2 and HDMX, respectively. Nutlin compounds were generous
gifts from Dr. L. T. Vassilev and Hoffman-LaRoche, Inc., Nutley, NJ.
concentrated with nitrogen gas, and the crude product was precip-
itated by diethyl ether. Obtained crude products were purified by
preparative HPLC on a Vydac Protein C4 column (22 ꢀ 250 mm
i.d.) using the following solvent systems: (A) water containing
0.05% TFA; (B) acetonitrile containing 0.04% TFA. The purity of
the oligourea was confirmed by analytical HPLC on a Vydac Protein
C4 column (4.6 ꢀ 250 mm i.d.) and an Agilent XDB-C18 column
(4.6 ꢀ 250 mm i.d.) using the same solvent systems as described
above. HPLC-purified urea derivatives were characterized by ESI
or MALDI-TOF mass spectroscopy.
3. Results
2.3. Protein expression
3.1. NAPA synthesis
The HDM2(11–119) and HDMX(1–188) were expressed in Esch-
erichia coli BL21 (DE3) as a GST fusion protein in pGEX4T-1 (GE
Healthcare, Arlington, VA) and purified on glutathione sepharose.
The HDM2(11–119) was cleaved from the GST on the resin using
thrombin. The GST-HDMX(1–188) was eluted with reduced gluta-
thione-containing phosphate buffered saline. The obtained pro-
teins were >90% pure as assessed by SDS–PAGE analysis. The
concentration was determined by A280 and the Bradford protein
assay.
All NAPA molecules were synthesized by solid phase synthesis
on TentaGel resin with a Rink amide linker. The first residue at-
tached to the resin was Fmoc-b-alanine which served as a linker
to facilitate the subsequent steps. After removal of the Fmoc group,
a reductive amination between the primary amine on solid support
and an Fmoc-protected a
-amino aldehyde (Scheme 1)38,39 was car-
ried out using NaBH(OAc)3 as a reducing reagent. Then, an isocya-
nate was reacted with the secondary amine on solid support,
resulting in formation of the urea bond. The sequence of Fmoc
deprotection, reductive amination, and urea formation was re-
peated until the desired oligomer length was achieved. Cleavage
of the NAPA from the resin was accomplished with an aqueous
solution of TFA and TIS as scavengers, and the product was precip-
itated by addition of diethyl ether. The crude material was purified
by reverse phase HPLC using a C4 preparative column.
In the HPLC profiles of each NAPA, multiple peaks with slightly
different retention times were observed and each peak had the
molecular mass of the intended molecule. For several NAPAs, each
peak with the correct molecular weight was purified and then re-
analyzed by HPLC. In each case the purified material eluted at its
original retention time, indicating that each peak represents a con-
stitutional isomer of the same molecule. The most likely explana-
tion for the observation of multiple peaks with the same mass is
that racemization occurred at some point during synthesis. Race-
mization was further confirmed in NAPA 25 where the molecule
2.4. Binding assays
Inhibitory activities of NAPAs were measured by fluorescence
anisotropy competition experiments as previously described.36
After NAPAs were dissolved in water or DMSO as approximately
10ꢁ2 M solution, they were diluted in water to prepare solutions
with several different concentration. Either HDM2(11–119) or
GST-HDMX(1–188) (2.2 lM) was incubated with 30 nM fluores-
cein-labeled human p53(15–29) in 10 mM Tris–HCl (pH 7.6),
150 mM NaCl, 0.50 mM EDTA, and 2 mM b-mercaptoethanol at
room temperature 1 h or 4 °C overnight. Experiments were per-
formed with a 70-fold molar excess of protein over labeled p53-
peptide to ensure that the peptide is completely bound by the pro-
tein. Under these conditions, there is no difference in the minimum
fluorescence polarization values between direct and competition
assays.37 Fluorescence anisotropy was measured at 25 °C on a Bea-
con 2000 (PanVera, Madison, WI). Data points were measured in
triplicate. IC50 values were calculated by nonlinear regression
curve fitting indicated by the following formula using KALEIDA GRAPH
4.0 software (Synergy Software, Reading, PA):
was made from either L- or D-glutamic acid. Isolation of each peak
in the HPLC followed by 1D 1H NMR analysis showed that at least
two HPLC peaks from each synthesis had identical NMR spectra.
Several attempts were made to isolate each HPLC peak of NAPA
25 ( Fig. 7) in sufficient quantity for more detailed analysis by
NMR so that the structure and stereochemistry could be assigned.
The spectra of each of the three peaks from NAPA 25 were complex
and suffered from extensive spectral overlap, which prevents mak-
ðymax ꢁ ymin
Þ
y ¼ ymin
þ
ꢀ
ꢁ
z
x
1 þ
IC50
R1
O
O
1. 20% Piperidine
NMP, 10 min.
N
•
O
NHFmoc
(13-20 eq.)
NHFmoc
N
H
N
H
N
H
R
2.
R
Rink Amide Tentagel Resin
O
N
Me
H
with β-Ala Linker
FmocHN
(10 eq.)
Repeat
O
two times
AcOH (5 eq.), CH2Cl2
3. NaBH(OAc)3 (10 eq.)
R1
R3
O
OH
O
OH
NH
NH
O
O
O
O
Cleave
N
N
NH2
NHFmoc
R
N
H2N
N
H
N
N
H
O
O
HN
O
NH
R2
R1
NH2
Scheme 1. General synthetic strategy for NAPA synthesis.

R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
7887
ing specific assignments to resonances (see Supplementary data).
3.3. Optimization to inhibit HDM2 and HDMX binding to p53
Comparison of the spectra indicate that each HPLC peak represents
a molecule with a similar structure but also indicates that each
peak could itself be a mixture of diastereomers. With three stere-
ogenic centers in each NAPA, the theoretical number of possible
diastereomers for each NAPA is 8. In the absence of more concrete
evidence, it is prudent to assume that each synthesis yields a mix-
ture of all possible diastereomers.
While racemization occurs during synthesis, this does not ap-
pear to be detrimental to inhibition of HDM2 or HDMX association
with p53 (as presented below). Unless otherwise specified, each
NAPA molecule was isolated as a mixture of several HPLC peaks
with identical mass and this mixture of diastereomers was tested
for inhibitory activity. The stereochemistry is purposely left unde-
fined in the molecular structures shown below to highlight this as-
pect of the study. For the most active NAPAs, each peak was
isolated by HPLC and tested individually for inhibition. As de-
scribed below, the stereochemistry of the backbone did not signif-
icantly alter the inhibition properties of the molecule.
To achieve inhibition of HDMX binding to p53, a number of
additional NAPA sidechains were examined in which different
para-substituents at positions X and Y were introduced. Eventually
it was concluded that the central sidechain should be fixed with a
para-bromo phenyl ring (X = Br) as this group routinely afforded
moderate inhibition of HDM2–p53 binding. The next series of stud-
ies focused on variations at position Y as well as small changes in
linker length (Fig. 3), and ultimately it was found that a halogen at
position Y is crucial for inhibition of HDMX–p53. In particular,
NAPA 9, which has a bromine at position Y, inhibited HDM2 and
HDMX binding with p53 two to four times better than a derivative
with a hydrogen at the same position (NAPA 5). Replacing the bro-
mine with a methyl group at the same position (entry 10) did not
significantly affect the inhibitory activity towards HDMX, but
activity against HDM2 decreased by three times. Since the effective
van der Waals radii of methyl and bromo groups are very similar
(1.80 and 1.86 Å, respectively) but their hydrogen bonding propen-
sities are different,40 it is possible that the substituent at this posi-
tion engages in different interactions when binding to HDM2
versus HDMX. When the bromine at position Y was combined with
a slightly longer linker (at position m), modest inhibition of both
protein–protein interactions was achieved (entry 13). For this
and all subsequent studies, inhibition of HDMX–p53 binding and
HDM2–p53 binding was determined for each molecule and IC50
values for both protein–protein interactions are shown.
In the next series of NAPAs, the alkyl hydrophobic sidechain
was replaced with an aromatic group so that different substituents
could be examined at position Z (Fig. 4). Once again, the p-bromo
phenyl group was optimal (entry 17), as other substituents lead
to reduced inhibitory activity towards HDMX. Ultimately it was
determined that a hydrophobic sidechain terminating with a cyclo-
hexyl group was better than an aromatic ring with a substituent at
position Z (entry 18, Fig. 5). These results suggest that the cyclo-
hexyl group may form a hydrophobic interaction on the protein
surface.
With an optimized set of hydrophobic sidechains, the effects of
different modifications at the N-terminus of the NAPAs were ex-
plored. As shown in Figure 5, some hydrophobic sidechains at this
position moderately improved inhibitory activity, while others de-
creased activity. From these results, the NAPA bearing an isopenta-
noyl group (entry 23) had the optimum activity, and this group
was maintained in all subsequent derivatives.
3.2. Initial NAPA design
The initial strategy to develop a NAPA into an inhibitor of
HDM2–p53 binding centered on reproducing the relative position-
ing of the F19, W23 and L26 residues of the p53 peptide that binds
to the protein. Therefore, hydrophobic sidechains were attached to
the three urea groups projecting off the NAPA backbone to orient
them into the hydrophobic binding cleft (as represented by
Fig. 1). The sidechains directly attached to the carbons in the back-
bone were chosen to be hydrophilic to maintain aqueous solubility
and to potentially form stabilizing salt bridges with the protein.
Based on a cursory analysis of the surface of HDM2, it seemed that
the incorporation of two glutamic acid sidechains and a lysine
could result in favorable charge–charge interactions with surface
residues of the protein. For the first round of NAPAs, inhibition of
only p53–HDM2 was examined. Attempts to closely mimic the
hydrophobic groups of p53 (which involved placing an indole side-
chain on the central urea of the NAPA) did not result in any inhibi-
tion of HDM2–p53 association. The synthetic scheme to make this
scaffold allowed numerous other groups to be attached and differ-
ent linker lengths to be explored. As shown in Figure 2, increasing
the distance between the hydrophobic sidechains and the ureas as
well as incorporating different halogens on the aromatic rings
helped to achieve the first weak inhibitor of p53–HDM2 binding
(entry 5). This molecule, however, was not able to inhibit p53–
HDMX binding.
At this point, reoptimization of the sidechains with the aromatic
rings was briefly examined with the goal of reducing the number of
Y
Me
Me
Entry
1.
IC50 (HDM2)a
m
n
o
X
Y
H
H
Cl
H
H
o
O
OH
O
OH
NH
m
1
2
2
2
2
2
2
1
1
1
1
1
2
2
1
1
1
2
2
2
1
> 100
85 10
63 13
Cl
Cl
Cl
Cl
Br
Br
H
NH
2.
NH
O
O
O
3.
N
N
NH2
4.
5.
6.
7.
43
8
H2N
N
H
N
18 2b
O
O
20
38
3
6
H
Br
n
NH2
a All values reported in µM.
b IC50 (HDMX) = 96 4 µM
X
Figure 2. Initial development of NAPAs to inhibit HDM2. The initial NAPA scaffold was modified with halogens and varying lengths of sidechain linkers.

7888
R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
Y
IC50 (HDM2)a IC50 (HDMX)a
Me
Me
OH
Entry
8.
m
2
o
1
2
2
2
2
2
Y
17
9.2 0.4
24
2
41
24
36
3
2
3
Br
Br
Me
NO2
OMe
Br
o
O
9.
2
O
OH
NH
m
NH
10.
2
3
NH
O
O
O
11.
12.
13.
2
2
3
41 10
>100
59
>100
13 1
4
N
N
NH2
H2N
N
H
N
13
1
O
a All values reported in µM.
O
NH2
Br
Figure 3. Extension of NAPAs to inhibit HDMX by introduction of substituents at Y and variation in sidechain lengths.
Z
Br
IC50 (HDM2)a IC50 (HDMX)a
Entry
Z
14.
15.
16.
17.
13
12
10
1
1
1
20
12
8.1 0.4
7.5 0.9
1
1
H
O
OH
O
OH
NH
OMe
NO2
Br
NH
NH
O
O
O
8.2 0.3
a All values reported in µM.
N
N
NH2
H2N
N
H
N
O
O
NH2
Br
Figure 4. Examination of the effects on inhibition of substituents at position Z.
bromine atoms at positions X and Y (Fig. 6). While the bromine at
position X could be replaced with a methyl group without compro-
mising activity for inhibition of HDM2–p53, activity against
HDMX–p53 decreased slightly. Therefore, the bromine at position
X was retained. In contrast, replacement of the bromine atom at
position Y with fluorine resulted in a slight improvement of the
activity for inhibition of HDMX–p53. From this study, the best
NAPA for inhibition of HDM2 and HDMX binding to p53 is the
one represented by entry 25.
3.4. Contributions to activity from stereochemistry, amino acid
sidechains, and ureas
With the NAPA from entry 25 (NAPA 25, Fig. 6) identified as an
optimized inhibitor of HDM2 and HDMX association with p53, ef-
forts were directed toward determining the specific features of this
molecule responsible for the activity. As mentioned previously,
each NAPA oligomer displayed multiple peaks on HPLC purification
that are diastereomers which likely form as a result of racemiza-
IC50 (HDM2)a IC50 (HDMX)a
Br
Entry
18.
19.
R
6.4 1.2
6.5 1.3
7.6 0.4
NH2
PhNH
10.5 0.3
O
OH
O
OH
NH
20.
21.
22.
N
H
17
2
10
8.0 0.5
10
1
NH
NH
O
O
O
6.6 1.2
8.2 0.3
N
H
N
N
NH2
R
N
H
N
H
N
1
O
O
Me
2.8 0.1
2.8 0.2
4.1 0.5
7.5 1.8
23.
24.
Me
NH2
a All values reported in µM.
Br
Figure 5. Optimization of N-terminal group for inhibition of p53 binding to HDM2 and HDMX.

R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
7889
Y
IC50 (HDM2)a IC50 (HDMX)a
Entry
o
2
2
2
3
Y
F
X
O
OH
o
O
OH
NH
25.
26.
27.
28.
2.6 0.2
2.3 0.1
2.9 0.6
2.7 0.1
3.2 0.1
5.1 0.6
Br
NH
NH
O
O
Br
F
F
Me
Me
Me
O
N
N
NH2
Me
N
N
13
1
14
1
H
O
Me
a All values reported in µM.
O
NH2
X
Figure 6. Re-optimization of the sidechains at positions X and Y.
tion. For all the previous inhibition results, multiple HPLC peaks of
each NAPA were collected and tests for inhibition were performed
using a diastereomeric mixture. For NAPA 25, each HPLC peak was
separated and independently tested for inhibition of both HDM2
and HDMX binding to p53. In addition, the same NAPA was made
to L-homo-glutamic acid sidechains (NAPA 34, Fig. 8). HPLC analy-
sis showed this molecule as a single peak, suggesting that racemi-
zation occured at the stereocenters bearing the glutamic acid
sidechains in the previous NAPAs. Unfortunately, the homo-glu-
tamic acid sidechains had a negative impact on inhibitory activity,
especially for HDMX–p53 association. Replacing all the amino acid
sidechains with lysines (NAPA 35) eliminated all inhibitory activ-
ity. Therefore, the arrangement of amino acid sidechains is impor-
tant for the activity of NAPA 25.
In the final series of studies, the importance of the ureas was
investigated by systematically replacing each one with a tertiary
amine (Fig. 9). Eliminating all ureas resulted in a molecule with
no inhibitory activity (entry 36). Replacing one urea with a tertiary
amine resulted in decreased inhibitory activity as well, but replace-
ment of the central urea caused the most significant decrease. For
these studies (entries 37–39), each peak was isolated by HPLC and
each one was tested for activity. In most cases, the activity of each
using
separated and independently tested. As shown in Figure 7A, NAPA
25 made from -Glu showed three peaks by HPLC and the inhibi-
tory activity was similar for each peak (entries 29–31). NAPA 25
made from -Glu showed predominantly two HPLC peaks and each
D-glutamic acid instead of the L-form and HPLC peaks were
L
D
one inhibited similarly (entries 32 and 33). These results indicate
that the stereochemistry of the NAPA backbone does not signifi-
cantly affect inhibitory activity.
The next goal centered on exploring the importance of the
hydrophilic amino acid sidechains to inhibition. Originally, these
sidechains were present to simply promote aqueous solubility. In
the first experiment, the glutamic acid sidechains were changed
F
O
OH
O
OH
NH
A
2
B
NH
NH
O
3
O
a
1
O
N
N
NH2
Me
N
H
N
O
Me
O
NH2
2
1
b
Br
IC50 (HDM2)a IC50 (HDMX)a
Entry
HPLC Peak
29.
30.
31.
32.
33.
a1
a2
a3
b1
b2
1.4 0.3
1.8 0.3
1.8 0.3
1.6 0.4
2.1 1.4
3.3 0.3
4.6 0.6
3.4 0.3
3.3 0.4
4.6 0.6
30
40
Time/min
50
a All values reported in µM.
Figure 7. Separation and inhibitory activity of NAPA 25. (A) HPLC profiles of NAPA 25 synthesized from all
L-amino acids (a) or D-glutamic acid and L-lysine (b). HPLC
separation was performed as described in Experimental section. (B) Structure of NAPA 25 and inhibition data for separated peaks.

7890
R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
F
F
NH2
NH2
O
O
OH
O
OH
NH
NH
NH
NH
O
O
O
O
O
N
N
NH2
N
N
NH2
Me
H2N
N
N
N
N
H
H
O
O
Me
O
NH
O
NH
NH2
NH2
35
IC50 (HDM2)a IC50 (HDMX)a
5.9 0.4 41
No Inhibition No Inhibition
34
Entry
Br
Br
34.
35.
1
a All values reported in µM.
Figure 8. Analog of NAPA 25 with L-homo-glutamic acid and L-lysine.
peak was similar (as previously described for NAPA 25) although
two peaks of entry 38 completely lost the ability to inhibit
HDM2–p53.
groups are removed (Fig. 9). We believe that the hydrogen bonds
help to align all three non-polar residues along the same side of
the inhibitor and therefore maximize solvent exclusion from the
cleft, enhancing binding affinity. In this conformation, the NAPAs
adopt a b-sheet-like conformation that is similar to structures
determined for N,N00-trisubstituted diethylenetriamines.25
3.5. Molecular modeling of the HDM2 and HDMX complex of
NAPA25
Even with the two previous assumptions in place, there are still
many plausible ways to dock NAPA 25 to HDM2 and HDMX due to
the flexibility of the sidechains. The four structures in Figure 11
represent the most likely scenarios for binding based on modeling
To model the complex of NAPA25 with HDM2 and HDMX, a few
general assumptions were made based on the results above and lit-
erature precedent. First, complexes were modeled such that the
hydrophobic sidechains of the NAPAs bind in the clefts of the pro-
teins in a manner similar to the p53 peptide. There is ample prec-
edent in the literature demonstrating that burial of hydrophobic
surface area drives binding of ligands to HDM2, and it is likely that
similar forces direct binding to HDMX.10,11,20,22,31,41 In addition, re-
sults with a number of different NAPAs illustrate that the relative
lengths of hydrophobic sidechains influences binding affinity
(compare NAPAs 8, 9, and 13 in Fig. 3; and 27 and 28 in Fig. 6).
To highlight the importance of sidechain length, Figure 10 provides
a comparison of the hydrophobic groups that project off from the
p53 peptide and NAPA 25 as modeled in an extended conforma-
tion. In this figure, the two horizontal lines represent the bottom
of the hydrophobic cleft of HDM2. In panel A, the relative depths
of the F19, W23 and L26 residues of p53 are presented, with the
tryptophan sidechain reaching the bottom of the hydrophobic cleft
of HDM2. As seen in panel B, these relative distances are approxi-
mately maintained in the extended structure of NAPA 25. From this
perspective, the NAPA molecule’s hydrophobic sidechains fill the
hydrophobic clefts of HDM2 (and HDMX) with the bromine atom
of the central sidechain reaching the bottom of the cleft. Therefore,
all models were constructed with the sidechain of the central urea
group (including the bromine atom) bound in the central pocket of
the hydrophobic cleft. With this sidechain anchored into the bind-
ing pocket, two main conformations of the complex were exam-
ined: one with the long axis of the inhibitor running the length
of the cleft and the other with the molecule rotated 180° around
the static central sidechain.
NAPA 25 with all L-stereochemistry in the backbone (L-NAPA 25).
While other diastereomers were not extensively examined, a cur-
sory examination indicated that backbone stereochemistry has lit-
tle impact on the orientations of the groups at the ends of the urea
sidechains. In this figure, panels A and B represent binding of L-
NAPA 25 to HDM2 in two different orientations that differ by
180° about the central sidechain while panels C and D represent
the same molecule bound in both ways to HDMX. In all cases,
the deepest point of the cleft is filled with the p-bromophenyl side-
chain, which is important for binding to both proteins (Figs. 2 and
6). Although this differs from an indole group, which would be
most similar to W23 of p53, it resembles the crystal structure of
Nutlin 2 bound to HDM2.14 Other studies have also shown that a
halogen at the 6 position of W23 in the p53 peptide nicely fills this
pocket and enhances the binding affinity.41 As the cyclohexane and
p-fluorophenyl sidechains of NAPA 25 (Fig. 6) differ from the F19
and L26 residues of p53,31,32 alternative binding modes that are
not analogous to other known HDM2 ligands likely dictate the
binding of NAPA 25. In panels A and C of Figure 11, the cyclohexane
sidechain is buried in the location occupied by F19 of p53, and the
p-fluorophenyl is in the location of L26. Such a model could explain
the preference for a bromine or fluorine substituent on the phenyl
group of this sidechain (in accordance with the data inFig. 3 for dif-
ferent substituents at position Y). A halogen at this position could
engage in a favorable electrostatic interaction with the hydroxyl
group of the proximal tyrosine residue, Y100 of HDM2 or Y99 of
HDMX, or alternatively, penetrate deeper into smaller sub-cavities
at this location. At the N-terminus of the molecule, the hydropho-
bic sidechain covers extra non-polar surface area at the periphery
of the cleft, which could explain the preference for a non-polar res-
idue at this location (see Fig. 5). This model also demonstrates that
Another assumption for modeling the complexes was that the
three urea groups of the NAPA form two, tandem hydrogen bonds,
as shown in Figures 1 and 10B. Based on the previous work by
Nowick and co-workers,25 this backbone conformation is accessi-
ble. The importance of the ureas is also supported by the results
demonstrating that inhibition is severely weakened when the urea
the negatively-charged glutamic acid residues of
L-NAPA 25 form
salt bridges with H73 and K94 of HDM2 and K50 of HDMX, which

R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
7891
F
F
O
OH
O
OH
O
OH
O
OH
NH
NH
O
O
O
N
N
NH2
N
N
NH2
Me
Me
N
H
N
N
H
N
O
O
Me
Me
O
NH2
NH2
36
37
Br
Br
F
F
O
OH
O
OH
NH
O
OH
O
OH
NH
NH
O
NH
O
O
O
O
N
N
NH2
N
N
NH2
Me
Me
N
N
N
H
N
H
O
O
Me
Me
O
NH2
NH2
39
38
Br
Br
Number of
Number
IC50 (HDMX)a
>100
Entry of Ureas HPLC Peaks IC50 (HDM2)a
36.
37.
0
2
1
2
>100
12
3
31
3
8
3
34
4
38.
2
4
>100
36
22
26
15
6
4
4
4
38
3
23
4
>100
7.3 0.7
6.1 0.4
4.7 0.3
24
19
14
1
3
3
39.
2
3
a Inhibitory values for each HPLC peak are reported in µM.
Figure 9. Effect of replacing urea groups on inhibition of HDM2 and HDMX binding to p53. The arrows designate the location where a urea has been replaced with an amine.
In cases where multiple HPLC peaks were present, each peak was separated and the inhibition tested for each individual peak.
Figure 10. Comparison of the sidechain lengths of (A) HDM2-bound p53 (1YCR.pdb) and (B) extended NAPA 25. The horizontal white lines denote the extents of the F19 and
W23 residues of p53. Only the cleft-binding sidechains of each molecule are shown for clarity. The proposed, intramolecular hydrogen bonds among the three, main urea
groups of NAPA 25 are illustrated by dashed white lines.

7892
R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
Figure 11. Models for binding of
L
-NAPA 25 to HDM2 (1RV1.pdb, chain A) (panels A and B) and HDMX (3EQY.pdb, chain A) (panels C and D). For clarity, the lysine and
glutamine-like sidechains of the NAPA molecule, which are simply exposed to solvent, have been omitted from the illustrations.
could explain why these sidechains are important for inhibition
(consistent with data in Fig. 8).
In the alternative models shown in panels B and D (where the
inhibitor is rotated 180° about the central sidechains), similar salt
the flexibility of the scaffolds can allow the conformation of the
molecules to adapt to slightly different binding pockets. Despite
these advances, most oligomeric inhibitors of protein–protein
interactions are large molecules that would be extremely difficult
to produce in sufficient quantities for drug development. In con-
trast, NAPAs offer the possibility of using a short oligomeric scaf-
fold that is densely functionalized with both hydrophobic and
hydrophilic groups. After several rounds of optimization, a single
trimeric inhibitor was developed that equally inhibits HDM2 and
HDMX binding to p53 with IC50 values in the low micromolar
range. Since the current synthesis yields a diastereomeric mixture
of molecules, clearly an alternative route to these molecules is
needed and new strategies are being examined in this regard. At
the same time, different diastereomers do not have markedly dif-
ferent inhibition properties. This is likely due to the highly flexible
sidechains which can orient the terminal groups in similar confor-
mations regardless of the chirality present in the backbone.
bridges are present between the charged sidechains of L-NAPA 25
and several residues of HDM2 and HDMX. In the complex with
HDM2 shown in panel B, the hydrophobic (leucine-like) sidechain
at the N-terminus of the inhibitor occupies the same location as
L26 of the bound p53 peptide. In this case, the cyclohexane group
covers an additional region of proximally exposed non-polar sur-
face area of the cleft. In the complex with HDMX shown in panel
D, the preferred model has the positions of the cyclohexane and
N-terminal hydrophobic groups switched so that the cyclohexane
occupies the site typically bound by L26 of p53.
4. Discussion
Molecular modeling suggests that the ability of L-NAPA 25 to al-
We have developed a NAPA scaffold that can be used to target
families of closely related protein–protein interactions. The inter-
faces at which proteins recognize one another often consist of a
combination of hydrophobic patches and electrostatic complemen-
tarity.42 Developing inhibitors that mimic such interfaces is notori-
ously challenging. Among the molecules developed to target
HDM2, most approaches use a rigid molecular scaffold to maxi-
mize the hydrophobic contacts with the protein to attain high
affinity and specificity.12,13,17 While these strategies have resulted
in excellent inhibitors of HDM2–p53, the success has been at the
expense of HDMX–p53 inhibition. In the case of Nutlin-3, the dif-
ference in the locations of a single tyrosine between proteins is en-
ough to allow excellent binding to HDM2 and at the same time
significantly curtail binding to HDMX.21 Due to its rigid scaffold,
Nutlin is not able to adapt to the altered binding pocket of HDMX.
Oligomeric molecules, such as the b-peptides developed by Sche-
partz and co-workers,24,43,44 offer the possibility of simultaneously
targeting related families of protein–protein interactions because
ter its conformation between the binding pockets of HDM2 and
HDMX allows for equivalent inhibition of the two proteins. The
models also highlight the ability of this inhibitor to simultaneously
engage in hydrophobic interactions with the protein binding pock-
ets and electrostatic interactions with polar residues that line the
peripheries of the binding clefts. Maximizing both sets of interac-
tions on a scaffold that retains some flexible characteristics may
be an important criterion in future strategies to find inhibitors of
protein–protein interactions.
Acknowledgements
This research was supported in part by the following Intramural
Research Programs of the NIH: NCI Center for Cancer Research,
NIDDK Laboratory of BioorganicChemistry. We gratefully acknowl-
edge Dr. Lisa Jenkins and George Leiman for assistance with the
preparation of this manuscript.

R. Hayashi et al. / Bioorg. Med. Chem. 17 (2009) 7884–7893
7893
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Supplementary data (synthetic procedures and characterization
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