Achieving cell penetration with distance-matching cysteine cross-linkers: A facile route to cell-permeable peptide dual inhibitors of Mdm2/Mdmx

Article abstract of DOI:10.1039/c1cc13320a

We report the design of bisarylmethylene bromides as a new class of rigid, distance-matching cysteine cross-linkers. By cross-linking a peptide dual inhibitor of Mdm2/Mdmx containing cysteines at i,i+7 positions, dramatic enhancement in cell permeability was achieved, along with increased helicity and biological activity.

Full text of DOI:10.1039/c1cc13320a

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Achieving cell penetration with distance-matching cysteine cross-linkers:
a facile route to cell-permeable peptide dual inhibitors of Mdm2/Mdmxw
a
a
b
b
a
Avinash Muppidi, Zhiyong Wang, Xiaolong Li, Jiandong Chen and Qing Lin*
Received 3rd June 2011, Accepted 1st July 2011
DOI: 10.1039/c1cc13320a
We report the design of bisarylmethylene bromides as a new
class of rigid, distance-matching cysteine cross-linkers. By
cross-linking a peptide dual inhibitor of Mdm2/Mdmx containing
cysteines at i,i+7 positions, dramatic enhancement in cell
permeability was achieved, along with increased helicity and
biological activity.
cysteine cross-linkers, and their effect on cell permeability of a
bioactive peptide (Fig. 1).
In order to evaluate the effect of cysteine cross-linking on
cell permeability, we decided to use a peptide dual inhibitor of
p53-Mdm2/Mdmx interactions (PDI) as our model peptide
because (i) PDI has shown to be an excellent dual inhibitor of
1
3
the p53-Mdm2 and the p53-Mdmx interactions, two validated
cancer targets; and (ii) the crystal structures of PDI and its
analogs in complexes with Mdm2/Mdmx have been solved
A growing number of chemical strategies have been developed
recently to convert peptides into cell-permeable ligands targeting
1
intracellular protein-protein interactions. These include
14
13
recently. However, PDI was found to be cell impermeable,
2
the use of alternative backbones such as b-peptides, the
diminishing its therapeutic potential. Since PDI (sequence =
LTFEHYWAQLTS) does not encode cysteine, we reasoned
that we could replace two residues at the solvent exposed face
of the helix with cysteines followed by the cysteine-specific
cross-linking. Inspection of the PDI-Mdm2/Mdmx complex
structures revealed that Glu-4 and Thr-11 are solvent exposed
and could be substituted without substantial loss of inhibitory
3
incorporation of a,a-dialkylamino acids such as Aib, the side
chain-to-side chain cross-linking via the lactam, hydro-
4
5
carbon, and heterocyclic bridges, the backbone-to-side chain
6
7
cross-linking, and the low molecular weight peptidomimetics
8
such as terphenyl-based helix mimics. Though these strategies
have generated peptide analogs with improved cell permeability,
nonnatural amino acids were typically required. For example,
hydrocarbon-based cross-linking requires the introduction of
two chiral a,a-dialkylamino acids carrying olefin side chains
and subsequent ruthenium-catalyzed ring closing metathesis
1
4
activity. Besides, a heterocyclic cross-linker has been successfully
6
c
applied to these two sites. Thus, we prepared L,L-dicysteine
substituted PDI analog 1 and found that the substitutions
were largely tolerated based on the ELISA data (compare 1 to
PDI in Table 1). We also prepared a D,L-dicysteine analog 3
5a,5b,9
produces a mixture of Z- and E-olefin linkages.
One effective strategy to impart cell permeability is to fuse
by placing D-Cys at position 4 with an aim to examine
5b,15
configurational effect.
1
0
the peptide with cell penetrating peptide sequences; however,
significant mass is added to the peptide. In search of an atom-
economical strategy, we were intrigued by an earlier report by
ELISA data showed that D,L-dicysteine
substitution was very well tolerated; indeed, more than 3-fold
increase in Mdm2 activity was observed (Table 1).
1
1
Woolley and co-workers in which alkylation of dicysteines
located at i,i+11 positions of a peptide with distance-matching
dichloroacetamides resulted in a substantial increase in peptide
helicity; however, the effect of cysteine cross-linking on
cell permeability was not demonstrated. Since the increased
To identify suitable rigid cross-linkers that selectively alkylate
dicysteines at the i,i+7 positions of PDI with a distance
˚
of 11.9 A between two b-carbons for the L,L-configuration
˚
(Fig. 1) and 10.9 A for the D,L-configuration, we explored
1
2
helicity generally leads to improved cell penetration,
we envisioned that with a proper choice of rigid, hydrophobic
cross-linkers we could obtain the cross-linked peptides
with improved cell permeability. Herein, we report the
design of a pair of bisarylmethylene bromides as rigid i,i+7
a
Department of Chemistry, State University of New York at Buffalo,
Buffalo, New York 14260, USA. E-mail: qinglin@buffalo.edu;
Fax: +01 (716) 645 6963; Tel: +01 (716) 645 4254
Department of Molecular Oncology, H. Lee Moffitt Cancer Center,
b
Tampa, Florida 33612, USA
w Electronic supplementary information (ESI) available: Full experi-
mental details and compound characterization data. See DOI:
Fig. 1 Structures of the rigid cysteine cross-linkers Bph and Bpy
(
left), and a model of Bpy cross-linked peptide dual inhibitor (right)
˚
10.1039/c1cc13320a
with two cysteines separated by a distance of 11.9 A.
9
396 Chem. Commun., 2011, 47, 9396–9398
This journal is c The Royal Society of Chemistry 2011

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a
Table 1 Sequence and inhibitory activity of the cross-linked peptides
solubility and higher bioactivities than Bph cross-linked ones,
we prepared the N-terminally fluorescein-conjugated 1b, 2b,
3
Mdm2 IC50 Mdmx IC50
b, and 4b along with PDI, 1, and 2 as controls. As shown in
Fig. 2, linear fluorescein-labeled PDI and 1 exhibited minimal
17
cell penetration relative to the DMSO control. As expected,
Name Sequence
Charge (nM)
(nM)
b
PDI
1
LTFEHYWAQLTS
LTFCHYWAQLCS
ꢀ1
0
0
0
0
+2
44
500
57 ꢁ 4.6
39 ꢁ 3.2
35 ꢁ 4.5
ND
1800 ꢁ 180
4800 ꢁ 1100
890 ꢁ 120
ND
0
0
c
substituting two arginines into PDI led to a 2.5-fold increase in
mean fluorescence (compare 2 to 1). On the other hand,
cysteine cross-linking by Bpy caused greater increase in mean
fluorescence (B6-fold for 1b over 1, B5-fold for 2b over 2),
indicating that the effect of cross-linking on enhancing the cell
permeability is greater than that of positive charge. Moreover,
these two effects appear additive as the positively charged, Bpy
cross-linked 2b showed additional 2-fold increase compared to
1
1
2
2
2
3
3
3
4
4
a
b
LTFC HYWAQLC S
d
LTFC HYWAQLC S
0
0
0 0
LTFCRYWARLCS
0
0
a
b
LTFC RYWARLC S
0
240 ꢁ 13
240 ꢁ 16
13 ꢁ 1.3
8.3 ꢁ 0.5
5.4 ꢁ 0.4
210 ꢁ 19
100 ꢁ 8.2
5500 ꢁ 980
1500 ꢁ 150
340 ꢁ 27
22 ꢁ 4.0
14 ꢁ 2.0
4500 ꢁ 890
1200 ꢁ 87
0
0 0
LTFC RYWARLC S +2
e
LTFcHYWAQLCS
0 0
LTFc HYWAQLC S
0
0
0
+2
+2
a
b
a
b
0
0
0 0
LTFc HYWAQLc S
LTFc RYWARLC S
0
0
0
0
0 0
LTFc RYWARLC S
1
b. Similar enhancement was observed for peptides 3b and 4b
a
ELISA was repeated three times to derive average IC50 values along
b
with standard deviations. IC50 values were taken from ref. 13 where
with the D,L-configuration (Fig. 2b). To determine the extent
by which structural change upon Bph/Bpy cross-linking
contributes to cell permeability, we performed the CD measure-
ment of the cross-linked peptides. Compared to the linear
c
the same assay was performed. C denotes Bph-linked cysteine.
0
d
0 0
C
e
denotes Bpy-linked cysteine. c denotes D-cysteine. ND, no data.
1
2-mer peptides PDI, 1, and 3 which showed 11–13% helicity,
0
various aryl methylene bromides and found 4,4 -bis-
0
cysteine cross-linking caused modest increase in percent helicity
(up to 20%, see Fig. S5). Overall, a positive correlation
between helicity and permeability appears to exist as peptides
with higher helicity generally showed higher cell permeability.
To corroborate the FACS result, a confocal microscopy
study was carried out and the data were collected in Fig. 3a. In
the FITC channel, the positively charged, Bpy cross-linked
peptides 2b and 4b showed the strongest cellular fluorescence,
in excellent agreement with the FACS data; however, cellular
debris was detected for 2b and 4b treated cells in the DIC
channel, indicating apparent cytotoxicity. This cytotoxicity
was further confirmed by the ATP assay (Fig. 3b), which
was attributed to the positive charge. To gain an insight into
the possible mechanism of cell permeation, we examined the
subcellular localization of 3b, the most potent, cell-permeable,
bromomethyl-biphenyl (Bph) and 6,6 -bis-bromomethyl-
0
[3,3 ]bipyridine (Bpy) provide nearly perfect matches. In a
Langevin dynamic simulation study, the sulfur-to-sulfur dis-
˚
tances range from 10.5 to 12.2 A for Bph and Bpy scaffolds,
˚
respectively, with the median distances center around 11.6 A
(
Fig. S1 in ESI). Bph is commercially available while Bpy
can be conveniently prepared through a two-step procedure
Scheme S1). To evaluate the efficiency of the cross-linking
(
reaction, we incubated the fully deprotected peptide 1 or 3
at 1 mM concentrations with 1.5 equiv of Bph or Bpy in a
mixed solution of acetonitrile/30 mM ammonium bicarbonate,
pH 8.5. HPLC traces of the crude products showed that the
reactions proceeded to completion within 2 h with greater than
9
5% conversion (Fig. S2).
While the initial cross-linked peptides (1a, 1b, 3a, 3b) were
charge-neutral, we also prepared the positively charged analogs
2a, 2b, 4a, 4b) by placing two arginines at the solvent exposed
(
side. Then, we performed ELISA with these compounds to
gauge the cross-linking effect on their inhibitory activity
(
Table 1 and Fig. S3). All charge-neutral cross-linked peptides
showed modest increase in Mdm2 activity, but variable
effect on Mdmx activity relative to their linear counterparts
(
compare 1a/1b to 1; 3a/3b to 3). Interestingly, cysteine cross-
linked peptides 3a and 3b with D,L-configuration exhibited a
5- and 24-fold increase in Mdmx activity, respectively
Table 1). In a molecular modeling study, the D,L-dicysteine-
1
(
linked Bpy in 3b appears to bind snugly onto a nearby
hydrophobic pocket while the L,L-dicysteine-linked Bpy does
not (Fig. S4). This type of cross-linker–protein surface inter-
action was observed recently in the crystal structure of Mcl-1
1
6
bound to a hydrocarbon cross-linked peptide. Meanwhile,
all positively charged peptides showed significantly reduced
activities compared to their neutral counterparts (compare 2a
to 1a, 2b to 1b, 4a to 3a, and 4b to 3b in Table 1), indicating
that arginine substitutions at positions 5 and 9 were not well
tolerated.
Next, we examined the effect of cysteine cross-linking on cell
permeability using fluorescence activated cell sorting (FACS).
Because Bpy cross-linked peptides showed superior water
Fig. 2 Flow cytometry analysis of HeLa cells after treatment with
10 mM fluorescein-conjugated peptides. (a) Representative histograms;
(b) Bar graph showing normalized mean fluorescence.
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Chem. Commun., 2011, 47, 9396–9398 9397

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cells stably expressing a p53-dependent luciferase reporter
(
Fig. S6).
In summary, we have designed bisarylmethylene bromides
as a new class of rigid cysteine cross-linkers. By cross-linking
PDI analogs containing two cysteines at i,i+7 positions,
significant enhancement in cell permeability was achieved
without apparent cytotoxicity, along with modest increases
in helicity and bioactivity. Since dicysteines can be readily
introduced into proteins, this cysteine cross-linking chemistry
could also be potentially useful for improved delivery of cross-
linked proteins into cells.
We gratefully acknowledge Pardee Foundation (to Q.L.)
and the National Institutes of Health (CA 118210 to J.C.) for
financial support.
Notes and references
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8
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398 Chem. Commun., 2011, 47, 9396–9398
This journal is c The Royal Society of Chemistry 2011