M. M. Madden et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1472–1475
1473
MeO
residues are solvent exposed and do not make critical contacts
with either Mdm2 or Mdmx (Fig. 1a).11 After mapping these resi-
dues to the PDI helix wheel model (Fig. 1b), three possible stapling
schemes emerged: i,i+4 stapling between Glu-4 and Asp-8 and be-
tween His-5 and Gln-9, and i,i+7 stapling between Glu-4 and Thr-
OMe
N
N
N
N
N
N
O
O
O
O
11.12 To expedite the synthesis of stapled peptides, alkene-(
a) and
NH
NH
NH
NH
302 nm
tetrazole-modified lysine (b) were prepared from Fmoc-Lys-OH
(Scheme S1 in Supplementary data) and used directly in solid-
phase peptide synthesis. After cleavage from the resin and purifica-
tion by reverse-phase HPLC, linear peptides were exposed to a
handheld UV lamp (UVM-57, 302 nm, 115 V, 0.16 A) in a mixed
acetonitrile/water (1:1) solvent for 2 h (Scheme 1). Three stapled
peptides, 1–3, embodying three stapling patterns were obtained
(Table 1). The conversions from linear precursors to stapled prod-
ucts were 82% for 1, 75% for 2, to 89% for 3, based on HPLC analysis
(Figs. S1–S3 in Supplementary data).
ACN/H2O
-N2
Ac-L-T-F-α-H-Y-W-β-Q-L-T-S-NH2
Ac-L-T-F-α-H-Y-W-β-Q-L-T-S-NH2
PDI-1
1
Scheme 1. Representative synthesis of stapled peptide 1 using a photoinduced 1,3-
dipolar cycloaddition reaction.
lysine, respectively.
a and b denote the alkene and tetrazole-modified
Previously, PDI was determined to inhibit the full-length p53
binding to Mdm2 and Mdmx with IC50 values of 44 nM and
550 nM, respectively, in an ELISA assay.11b To gauge the effect of
stapling on inhibitory activity, stapled PDI analogs 1–3 were eval-
uated and their inhibitory activity data were collected in Table 1.
Compared to PDI, 1 with the cross-linker connecting residues 4–8
showed similar activities against both Mdm2 and Mdmx. The other
i,i+4 stapled PDI analog 2 with the cross-linker connecting residues
5–9 showed roughly fourfold drop in Mdm2 activity and threefold
drop in Mdmx activity. Meanwhile, the i,i+7 stapled PDI analog 3
showed sevenfold increase in Mdm2 activity together with a slight
improvement in Mdmx activity. To discern the effect of stapling
from that of residue substitution, we determined the activity of
200-fold increase in the Mdm2 activity and 20-fold increase in
the Mdmx activity.
Since higher positive charges generally lead to improved cellu-
lar uptake,5d,13 we prepared a series of positively charged PDI ana-
logs by substituting the remaining non-essential residues (His-5,
Ala-8, Gln-9 and Thr-11) in 1 and 3 with either one (1a, 3a and
3b) or two arginines (1b, 3c and 3d) and determined their inhibi-
tory activities (Table 1). Compared to their parent peptides, these
positively charged analogs showed 2–6-fold drop in activity
against Mdm2 and small changes against Mdmx (Table 1), indicat-
ing that arginine substitutions were largely tolerated. Furthermore,
we probed the effect of increased hydrophobicity at the binding
surface of PDI by replacing the canonical Trp with 6-chlorotrypto-
phan to derive 3e.14 Interestingly, 3e showed roughly twofold in-
crease in Mdm2 activity but twofold decrease in Mdmx activity
relative to 3d (Table 1), which can be attributed to the interaction
between 6-chlorine and Phe-86 of Mdm2, but not Mdmx, as de-
tected previously.14b
its linear precursor, PDI-4a11b, with IC50 values of 1200 nM and
6300 nM against Mdm2 and Mdmx, respectivley. By comparing
the activity of 3 with that of PDI-4
a
11b, stapling led to roughly
To examine whether stapled PDI analogs penetrate into cancer
cells, we took advantage of the intrinsic fluorescence of the pyraz-
oline cross-linker6 and monitored their cellular uptake by fluores-
cence microscopy. Our initial study revealed that the charge
neutral stapled peptide 3 was not cell-permeable (Fig. S6 in
Supplementary data). To our delight, treatment of U2OS cells with
+1 charged stapled peptides 3a and 3b gave rise to a punctuated
fluorescence pattern (Fig. 2). This distribution pattern suggests that
stapled peptides are taken up by cancer cells via a pinocytotic
pathway where the majority of peptides are likely trapped in the
endosomes.15 On the other hand, treatment of U2OS cells with
+2 charged stapled peptides 3c, 3d and 3e produced more diffusive
fluorescence patterns, suggesting that increased positive charge
may facilitate the peptides to escape the acidic endosomes. Nota-
bly, stapled peptide 3c in which two arginines form a continuous
positive patch in an i,i+4 fashion showed a strong and uniform
fluorescence distribution in the cytosol, indicating that the charge
locations are also important in addition to the total charge. This
charge location dependency also suggests that the observed intra-
cellular fluorescence was not an artifact derived from the cell fixa-
tion. It is noteworthy that U2OS cells treated with a linear
fluorescent peptide carrying +2 charge, 4 (see Table 1 for se-
quence), under identical conditions did not produce any intracellu-
lar fluorescence, confirming that stapling is essential for cellular
uptake (Fig. 2). In our preliminary circular dichroism study, stapled
peptide 3e showed 21% helicity compared to 13% for peptide 4 of
the same sequence (Fig. S7), indicating that stapling indeed in-
creases helicity, which is partly responsible for the enhanced cellu-
lar uptake.
a
F3
F3
W7
L10
W7
L10
PDI:Mdmx
PDI:Mdm2
b
2
6
T
Y
9
Q
10
L
L
O
H
S
5
Mdm2/
Mdmx
3
F
N
N
Me
O
O
12
HN
NH
W
7
L
α
β
L
1
A
T
E
4
11
8
L
L
Figure 1. (a) Crystal structures of PDI bound to Mdm2 (PDB code: 3G03) and Mdmx
(PDB code: 3FDO). The proteins were rendered in surface model and PDI was shown
in ribbon and tube model. The side chains of three canonical residues, F3, W7 and
L10, were shown in blue. (b) Helical wheel diagram of PDI viewed from the N- to C-
terminus. The five possible stapling sites were colored in red. The pyrazoline cross-
linker structure formed after the cycloaddition reaction is shown on the right.
To assess whether enhanced cellular uptake imparts in vivo
activity, U2OS cells stably expressing a p53-dependent luciferase
Madden, Michael M.
