Cell-permeable bicyclic peptide inhibitors against intracellular proteins

Article abstract of DOI:10.1021/ja503710n

Cyclic peptides have great potential as therapeutic agents and research tools but are generally impermeable to the cell membrane. Fusion of cyclic peptides with a cyclic cell-penetrating peptide produces bicyclic peptides that are cell-permeable and retain the ability to recognize specific intracellular targets. Application of this strategy to protein tyrosine phosphatase 1B and a peptidyl-prolyl cis-trans isomerase (Pin1) isomerase resulted in potent, selective, proteolytically stable, and biologically active inhibitors against the enzymes.

Full text of DOI:10.1021/ja503710n

Subscriber access provided by UZH Hauptbibliothek / Zentralbibliothek Zuerich
Communication
Cell-Permeable Bicyclic Peptide Inhibitors against Intracellular Proteins
Wenlong Lian, Bisheng Jiang, Ziqing Qian, and Dehua Pei
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja503710n • Publication Date (Web): 27 Jun 2014
Downloaded from http://pubs.acs.org on July 1, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Cell-Permeable Bicyclic Peptide Inhibitors against Intracellu-
lar Proteins
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Wenlong Lian, Bisheng Jiang, Ziqing Qian, and Dehua Pei*
Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210,
USA
Supporting Information Placeholder
In this work, we report a potentially general approach to
ABSTRACT: Cyclic peptides have great potential as thera-
designing cell-permeable cyclic peptidyl inhibitors against
intracellular proteins such as PTP1B.
peutic agents and research tools but are generally imperme-
able to the cell membrane. Fusion of the cyclic peptides with
a cyclic cell-penetrating peptide produces bicyclic peptides
that are cell permeable and retain the ability to recognize
specific intracellular targets. Application of this strategy to
protein tyrosine phosphatase 1B and peptidyl prolyl cis-trans
isomerase Pin1 resulted in potent, selective, proteolytically
stable, and biologically active inhibitors against the enzymes.
We recently discovered cyclo(FΦRRRRQ) (cFΦR4, where Φ
is L-naphthylalanine) as a novel class of cell-penetrating pep-
tide (CPP).11 Unlike previous CPPs, which are typically linear
peptides and entrapped in the endosome, cFΦR4 efficiently
escapes from the endosome into the cytoplasm. Short pep-
tide cargos (1-7 aa) can be delivered into mammalian cells by
incorporating them into the cFΦR4 ring. Encouraged by this
finding, we explored the possibility of developing bifunction-
al cyclic peptides containing both cell-penetrating and tar-
get-binding sequences as cell-permeable inhibitors against
intracellular proteins. To generate specific inhibitors against
PTP1B, we synthesized a one-bead-two-compound library on
spatially segregated ChemMatrix resin,12 in which each bead
displayed a bifunctional cyclic peptide on its surface and
contained the corresponding linear peptide in its interior as
an encoding tag [Scheme 1 and Figure S1 in Supporting In-
formation (SI)]. The bifunctional cyclic peptides all featured
the CPP motif FΦR4 (or its inverse sequence RRRRΦF) on
one side and a random pentapeptide sequence (X1X2X3X4X5)
on the other side, where X2 represents a 9:1 (mol/mol) mix-
ture of Tyr and F2Pmp while X1 and X3-X5 are any of the 24
amino acids that included 10 proteinogenic L-amino acids
(Ala, Asp, Gln, Gly, His, Ile, Pro, Ser, Tyr, Trp), 5 unnatural
α-L-amino acids [F2Pmp, L-4-fluorophenylalanine (Fpa), L-
norleucine (Nle), L-phenylglycine (Phg), L-pipecolic acid
(Pip)], and 9 α-D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu,
D-β-naphthylalanine (D-Nal), D-Phe, D-Pro, D-Thr, and D-
Val]. The library has a theoretical diversity 6.6 x 105. The use
of 9:1 Tyr/F2Pmp ratio at the X2 position, together with a 5-
fold reduction of the surface peptide loading, reduced the
amount of F2Pmp-containing peptides at the bead surface by
50-fold, increasing the stringency of library screening.13
Screening 100 mg of the library (~300,000 beads/compounds)
against Texas red-labeled PTP1B resulted in 65 positive
beads, which were individually sequenced by partial Edman
degradation-mass spectrometry (PED-MS)14 to give 42 com-
plete sequences (Table S1).
Cyclic peptides (and depsipeptides) exhibit a wide range of
biological activities.1 Several innovative methodologies have
recently been developed to synthesize cyclic peptides, either
individually2 or combinatorially,3 and screen them for biolog-
ical activity. A particularly exciting application of cyclic pep-
tides is the inhibition of protein-protein interactions
(PPIs),4,5 which remain challenging targets for conventional
small molecules. However, a major limitation of cyclic pep-
tides is that they are generally impermeable to the cell mem-
brane, precluding any application against intracellular tar-
gets, which include most of the therapeutically relevant PPIs.
Although formation of intramolecular hydrogen bonds6 or
Nα-methylation of the peptide backbone7 can improve the
membrane permeability of certain cyclic peptides, alternative
strategies to increase the cell permeability of cyclic peptides
are clearly needed.
Protein-tyrosine phosphatase 1B (PTP1B) is a prototypical
member of the PTP superfamily and plays numerous roles
during eukaryotic cell signaling. Because of its role in nega-
tively regulating insulin and leptin receptor signaling, PTP1B
is a valid target for treatment of type II diabetes and obesity.8
A large number of PTP1B inhibitors have been reported,9
however, none of them have succeeded in the clinic. Design-
ing PTP inhibitors is challenging because most of the phos-
photyrosine (pY) isosteres such as difluorophosphonomethyl
phenylalanine (F2Pmp)10 are impermeable to the cell mem-
brane. Additionally, because all PTPs share a similar active
site, achieving selectivity for a single PTP has been difficult.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Scheme 1. Evolution of a Cell-Permeable PTP1B Inhibitor
Page 2 of 5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Three representative hit sequences [D-Thr-D-Asn-D-Val-
F2Pmp-D-Ala-Arg-Arg-Arg-Arg-Nal-Phe-Gln (inhibitor 1),
Ser-D-Val-Pro-F2Pmp-His-Arg-Arg-Arg-Arg-Nal-Phe-Gln
(inhibitor 2), and Ile-Pro-Phg-F2Pmp-Nle-Arg-Arg-Arg-Arg-
Nal-Phe-Gln (inhibitor 3)] were resynthesized and purified
ring to a minimal size which, according to the previously
observed trend,11 should result in more efficient cellular up-
take. The bicyclic system should be able to accommodate
cargos of any size, because incorporation of the latter does
not change the size of CPP ring and, therefore, should not
Scheme 2. Solid-Phase Synthesis of Inhibitor 4
Reagents: a) standard Fmoc chemistry; b) trimesic acid, HBTU;
c) Pd(PPh3)4, Nꢀmethylaniline; d) PyBOP; e) TFA.
by HPLC. All three peptides are competitive PTP1B inhibi-
tors, with peptide 2 being most potent (IC50 = 31 ± 3 nM)
(Table S2 and Figure S2). Unfortunately, inhibitor 2 showed
no significant activity in cellular assays. Confocal microscop-
ic analysis of human cells treated with fluorescein isothiocy-
anate (FITC)-labeled inhibitor 2 indicated poor cellular up-
take of the peptide (Figure 1a). Although disappointing, this
result was not entirely unexpected. Our previous study
showed that as the size of the cargo inserted into the cFΦR4
ring increased, the cellular uptake efficiency of the cyclic
peptides decreased dramatically.11 We reasoned that larger
rings are more conformationally flexible and may bind less
tightly to the cell surface receptors (e.g., membrane phos-
pholipids) during endocytosis. The negatively charged
F2Pmp may also interact intramolecularly with the FΦR4
motif and interfere with its CPP function.
Figure 1. (a) Liveꢀcell confocal microscopic images (same Zꢀ
section) of A549 lung cancer cells after treatment for 2 h with 5
ꢀM FITCꢀlabeled inhibitor 2 (top panel) or 4 (bottom panel) and
endocytosis marker dextranRho (1.0 mg/mL). (b) Lineweaverꢀ
Burk plot showing competitive inhibition of PTP1B by 0 (○), 28
(●), 56 (□), and 112 nM inhibitor 4 (▲). (c) Sensitivity of variꢀ
ous PTPs to inhibitor 4 (the y axis values are pNPP hydrolysis
rates by each PTP relative to that in the absence of inhibitor).
Data shown in (b) and (c) are representative data sets.
affect the delivery efficiency of the cyclic CPP. The use of a
rigid scaffold (e.g., trimesic acid) may also help keep the CPP
and cargo motifs away from each other and minimize any
mutual interference. The smaller rings of a bicyclic peptide,
To improve the cell permeability of inhibitor 2, we ex-
plored a bicyclic system in which the CPP motif is placed in
one ring whereas the target-binding sequence constitutes the
other ring (Scheme 1). The bicyclic system keeps the CPP
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
compared to its monocyclic counterpart, should result in
cence throughout the cytoplasm and nucleus as well as fluo-
1
2
3
4
5
6
7
8
greater structural rigidity and improved metabolic stability.
rescence puncta, indicating that a fraction of the inhibitors
reached the cytoplasm and nucleus while the rest was likely
entrapped in the endosomes. Incubation of inhibitor 4 in
human serum for 24 h at 37 °C resulted in ~10% degradation,
whereas 91% of inhibitor 2 was degraded under the same
condition (Figure S3). Overall, inhibitor 4 compares favora-
bly with the small-molecule PTP1B inhibitors reported to
date9 with respect to potency, selectivity over the highly sim-
ilar TCPTP (17-fold), and cell permeability (Table S4).
To convert the monocyclic PTP1B inhibitor 2 into a bicyclic
peptide, we replaced the Gln residue (used for attachment to
the solid support and peptide cyclization) with (S)-2,3-
diaminopropionic acid (Dap) and inserted a second Dap res-
idue at the junction of CPP and PTP1B-binding sequences (C-
terminal to His) (Scheme 1). Synthesis of the bicycle was ac-
complished by the formation of three amide bonds between
a trimesic acid and the N-terminal amine and the side chains
9
Inhibitor 4 was next tested for its ability to perturb PTP1B
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Conversion of Impermeable Pin1 Inhibitor into
a Cell-Permeable Bicyclic Inhibitor
Figure 2. Inhibition of PTP1B in vivo. (a) AntiꢀpY immunoblot
of global pY protein levels in A549 cells after treatment with 0ꢀ5
ꢀM inhibitor 4 for 2 h. (b) SDSꢀPAGE analysis (Coomassie blue
staining) of the same samples from (a) shows uniform sample
loading in all lanes. (c) Effect of inhibitor 4 on insulin receptor
phosphorylation at Tyr1162 and Tyr1163 sites. HepG2 cells were
treated with indicated concentrations of inhibitor 4 for 2 h and
then stimulated with insulin (100 nM) for 5 min, followed by
SDSꢀPAGE and immunoblotting with antiꢀIRpY1162/pY1163 antiꢀ
body. (d) Quantitation of IR pY levels from (c) (data shown are
the mean ± SD from five independent experiments).
function during cell signaling. Treatment of A549 cells with
inhibitor
4 (0-5 ꢀM) resulted in dramatic and dose-
dependent increase in the pY levels of a large number of pro-
teins, consistent with the broad substrate specificity of
PTP1B15 (Figure 2a). Analysis of the same samples by Coo-
massie blue staining showed similar amounts of proteins in
all samples (Figure 2b), indicating that the increased pY lev-
els reflected increased phosphorylation (or decreased PTP
reaction) instead of changes in the total protein levels. Re-
markably, the increase in tyrosine phosphorylation was al-
ready apparent at 8 nM inhibitor 4. Interestingly, further
increase in inhibitor concentration beyond 1 ꢀM reversed the
effect on tyrosine phosphorylation, an observation that was
also made previously by Zhang and co-workers with a differ-
ent PTP1B inhibitor.16 To obtain further evidence that the
intracellular PTP1B was inhibited by peptide 4, we monitored
the pY level of insulin receptor (IR), a well-established PTP1B
substrate in vivo,8 by immunoblotting with specific antibod-
ies against the pY1162pY1163 site. Again, treatment with inhibi-
tor 4 caused dose-dependent increase in insulin receptor
phosphorylation up to 1 ꢀM inhibitor and the effect leveled
off at higher concentrations (Figure 2c,d). Taken together,
our data indicate that bicyclic inhibitor 4 efficiently entered
mammalian cells and inhibited PTP1B in vivo. The decreased
phosphorylation at higher inhibitor concentrations may be
caused by nonspecific inhibition of other PTPs (which may in
turn down regulate protein tyrosine kinases). It may also
reflect the pleiotropic roles played by PTP1B, which can both
negatively and positively regulate the activities of different
protein kinases.17
of the two Dap residues (Scheme 2).5 Briefly, the linear pep-
tide was synthesized on Rink amide resin using the standard
Fmoc chemistry and Nβ-alloxycarbonyl (Alloc)-protected
Dap. After removal of the N-terminal Fmoc group, the ex-
posed amine was acylated with trimesic acid. Removal of the
Alloc groups with Pd(PPh3)4 followed by treatment with Py-
BOP afforded the desired bicyclic structure. To facilitate la-
beling with fluorescent probes, a lysine was added to the C-
terminus. The bicyclic peptide (peptide 4) was deprotected
by TFA and purified to homogeneity by HPLC.
Bicyclic peptide 4 acts as a competitive inhibitor of PTP1B,
with a KI value of 37 ± 4 nM (Figure 1b). It is highly selective
for PTP1B. When assayed against p-nitrophenyl phosphate
(pNPP) as substrate (500 ꢀM), inhibitor 4 had IC50 values of
30 ± 4 and 500 ± 250 nM for PTP1B and TCPTP, respectively
(Figure 1c and Table S3). It exhibited minimal inhibition of
any of the other PTPs tested (≤10% inhibition of HePTP,
SHP-1, PTPRC, PTPH1, or PTPRO at 1 ꢀM inhibitor concen-
tration). Gratifyingly, inhibitor 4 has greatly improved cell
permeability over peptide 2, as detected by live-cell confocal
microscopy of A549 cells treated with FITC-labeled inhibitor
4 (Figure 1a). The treated cells showed both diffuse fluores-
To test the generality of the bicyclic approach, we applied
it to design cell permeable inhibitors against peptidyl prolyl
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
2000, 122, 5891-5892. (c) Sun, Y.; Lu, G.; Tam, J. P. Org. Lett.
cis-trans isomerase Pin1, a potential target for treatment of a
variety of human diseases including cancer,18 for which po-
tent, selective, and biologically active inhibitors are still lack-
ing.19 Thus, we fused a previously reported monocyclic pep-
tide (5), which is a potent inhibitor against Pin1 in vitro (KD
258 ± 66 nM) but membrane impermeable,20 with cFΦR4
(Scheme 3). In addition, we replaced the L-Tyr at the pThr+3
position with an Arg to improve the aqueous solubility. The
resulting bicyclic peptide 6 bound Pin1 with a KD value of 130
± 44 nM (Table S5 and Figure S4). Insertion of a D-Ala at the
pThr+5 position to increase the separation between the Pin1-
binding and cell-penetrating motifs improved the inhibitor
potency by ~2-fold (KD = 72 ± 21 nM for inhibitor 7). Inhibitor
7 competed with FITC-labeled inhibitor 5 for binding to Pin1
(Figure S5), indicating that they both bind to the Pin1 active
site. Substitution of D-Thr for D-pThr of inhibitor 7 reduced
its potency by ~10-fold (KD = 620 ± 120 nM for inhibitor 8,
Table S5), whereas further replacement of the pipecolyl resi-
due with D-Ala abolished Pin1 inhibitory activity (peptide 9).
As expected, bicyclic inhibitors 7-9 are cell permeable (Figure
S6). Treatment of HeLa cells with inhibitor 7 resulted in
dose-dependent inhibition of cell growth (45% inhibition
after 3-day treatment at 20 ꢀM inhibitor 7), whereas the im-
permeable inhibitor 5 and inactive peptide 9 had no effect
(Figure S7). Peptide 8 also inhibited cell growth, but to a
lesser extent than inhibitor 7. Finally, treatment of HeLa cells
with inhibitor 7 dramatically increased the cellular levels of
promyelocytic leukemia protein (PML), an established Pin1
substrate destabilized by Pin1 activity (Figure S8).21
2001, 3, 1681-1684. (d) Kohli, R. M.; Walsh, C. T.; Burkart, M. D.
Nature 2002, 418, 658-661. (e) Qin, C.; Bu, X.; Zhong, X.; Ng, N.
L. J.; Guo, Z. J. Comb. Chem. 2004, 6, 398-406. (f) Turner, R. A.;
Oliver, A. G.; Lokey, R. S. Org. Lett. 2007, 9, 5011-5014. (g) Hili,
R.; Rai, V.; Yudin, A. K. J. Am. Chem. Soc. 2010, 132, 2889-2891.
(h) Lee, J.; McIntosh, J.; Hathaway, B. J.; Schmidt, E. W. J. Am.
Chem. Soc. 2009, 131, 2122-2124. (i) Frost, J. R.; Vitali, F.; Jacob,
N. T.; Brown, M. D.; Fasan, R. ChemBioChem 2013, 14, 147-160.
(3) For examples, see: (a) Eichler, J.; Lucka, A. W.; Pinilla, C.;
Houghten, R. A. Mol. Divers. 1996, 1, 233-240. (b) Giebel, L. B.;
Cass, R. T.; Milligan, D. L.; Young, D. C.; Arze, R.; Johnson, C. R.
Biochemistry 1995, 34, 15430-15435. (c) Scott, C. P.; Abel-Santos,
E.; Wall, M.; Wahnon, D. C.; Benkovic, S. J. Proc. Natl. Acad.
Sci. USA 1999, 96, 13638-13643. (d) Millward, S. W.; Takahashi,
T. T.; Roberts, R. W. J. Am. Chem. Soc. 2005, 127, 14142 -14143.
(e) Sako, Y.; Morimoto, J.; Murakami, H.; Suga, H. J. Am. Chem.
Soc. 2008, 130, 7232-7234. (f) Li, S.; Marthandan, N.; Bowerman,
D.; Garner, H. R.; Kodadek, T. Chem. Commun. 2005, 581-583.
(g) Joo, S. H.; Xiao, Q.; Ling, Y.; Gopishetty, B.; Pei, D. J. Am.
Chem. Soc. 2006, 128, 13000-13009. (h) Heinis, C.; Rutherford,
T.; Freund, S.; Winter, G. Nat. Chem. Biol. 2009, 5, 502-507. (i)
Tse, B. N.; Snyder, T. M.; Shen, Y.; Liu, D. R. J. Am. Chem. Soc.
2008, 130, 15611-15626.
(4) For examples, see: (a) Leduc, A.-M.; Trent, J. O.; Wittliff, J. L.;
Bramlett, K. S.; Briggs, S. L.; Chirgadze, N. Y.; Wang, Y.; Burris,
T. P.; Spatola, A. F. Proc. Natl. Acad. Sic. USA 2003, 100, 11273-
11278. (b) Millward, S. W.; Fiacco, S.; Austin, R. J.; Roberts, R.
W. ACS Chem Biol 2007, 2, 625-634. (c) Tavassoli, A.; Lu, Q.;
Gam, J.; Pan, H.; Benkovic, S. J.; Cohen, S. N. ACS Chem. Biol.
2008, 3, 757-764. (d) Wu, X.; Upadhyaya, P.; Villalona-Calero,
M. A.; Briesewitz, R.; Pei, D. Med. Chem. Commun. 2013, 4, 378-
382. (e) Birts, C. N.; Nijjar, S. K.; Mardle, C. A.; Hoakwie, F.;
Duriez, P. J.; Blaydes, J. P.; Tavassoli, A. Chem. Sci. 2013, 4,
3046-3057. (f) Kawakami, T.; Ishizawa, T.; Fujino, T.; Reid, P. C.;
Suga, H.; Murakami, H. ACS Chem. Biol. 2013, 8, 1205-1214.
(5) Lian, W.; Upadhyaya, P.; Rhodes, C. A.; Liu, Y.; Pei, D. J. Am.
Chem. Soc.2013, 135, 11990-11995.
(6) Rezai, T.; Bock, J. E.; Zhou, M. V.; Kalyanaraman, C.; Lokey, R.
S.; Jacobson, M. P. J. Am. Chem. Soc.2006, 128, 14073-14080.
(7) (a) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem.
Res. 2008, 41, 1331-1342. (b) White, T. R.; Renzelman, C. M.;
Rand, A. C.; Rezai, T.; McEwen, C. M.; Gelev, V. M.; Turner, R.
A.; Linington, R. G.; Leung, S. S. F.; Kalgutkar, A. S.; Bauman, J.
N.; Zhang, Y. Z.; Liras, S.; Price, D. A.; Mathiowetz, A. M.; Ja-
cobson, M. P.; Lokey, R. S. Nat. Chem. Biol. 2011, 7, 810-817.
(8) (a) Elchelby, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.;
Collins, S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-
Hagen, J.; Chan, C. C.; Ramanchandran, C.; Gresser, M. J.;
Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544–1548.
(b) Zabolotny, J. M.; Bence-Hanulec, K. K.; Stricker-Krongrad,
A.; Haj, F.; Wang, Y.; Minokoshi, Y.; Kim, Y. B.; Elmquist, J. K.;
Tartaglia, L. A.; Kahn, B. B.; Neel, B. G. Dev. Cell 2002, 2, 489-
495.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In conclusion, we have developed a potentially general ap-
proach to designing cell-permeable bicyclic peptides against
intracellular targets. Our preliminary studies show that re-
placement of the PTP1B-binding motif with other peptide
sequences of different physicochemical properties also re-
sulted in their efficient delivery into cultured mammalian
cells. The availability of a general intracellular delivery
method should greatly expand the utility of cyclic peptides in
drug discovery and biomedical research.
ASSOCIATED CONTENT
Supporting Information
Experimental details and additional data. This material is
available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
(9) He, R.; Zeng, L.-F.; He, Y.; Zhang, Z.-Y. in New Therapeutic
Strategies for Type 2 Diabetes: Small Molecule Approaches. Ed.
R. M. Jones, RSC Publishing 2012, pp142.
(10) Burke Jr., T. R.; Kole, H. K.; Roller, P. P. Biochem. Biophys. Res.
Commun. 1994, 204, 129-134.
pei.3@osu.edu.
Notes
The authors declare no competing financial interests.
(11) Qian, Z.; Liu, T.; Liu, Y.-Y.; Briesewitz, R.; Barrios, A. M.;
Jhiang, S. M.; Pei, D. ACS Chem. Biol. 2013, 8, 423-431.
(12) Liu, R.; Marik, J.; Lam, K. S. J. Am. Chem. Soc. 2002, 124, 7678-
7680.
(13) Chen, X.; Tan, P. H.; Zhang, Y.; Pei, D. J. Comb. Chem. 2009, 11,
604−611.
(14) Thakkar, A.; Wavreille, A.-S.; Pei, D. Anal. Chem. 2006, 78,
5935−5939.
ACKNOWLEDGMENT
This work was supported by NIH (GM062820 and CA132855).
REFERENCES
(1) Pomilio, A. B.; Battista, M. E.; Vitale, A. A. Curr. Org. Chem.
2006, 10, 2075-2121.
(2) For examples, see: (a) Meutermans, W. D. F.; Golding, S. W.;
Bourne, G. T.; Miranda, L. P.; Dooley, M. J.; Alewood, P. F.;
Smythe, M. L. J. Am. Chem. Soc. 1999, 121, 9790-9796. (b)
Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc.
(15) Ren, L.; Chen, X.; Luechapanichkul, R.; Selner, N. G.; Meyer, T.
M.; Wavreille, A.-S.; Chan, R.; Iorio, C.; Zhou, X.; Neel, B. G.;
Pei, D. Biochemistry 2011, 50, 2339.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
(16) Xie, L.; Lee, S. Y.; Andersen, J. N.; Waters, S.; Shen, K.; Guo, X.
(19) More, J. D.; Potter, A. Bioorg. Med. Chem. Lett. 2013, 23, 4283-91.
(20) Liu, T.; Liu, Y.; Kao, H.-Y.; Pei, D. J. Med. Chem. 2010, 53, 2494-
2501.
L.; Moller, N. P.; Olefsky, J. M.; Lawrence, D. S.; Zhang, Z. Y. Bi-
ochemistry 2003, 42, 12792-12804.
1
2
3
4
(21) Reineke, E. L.; Lam, M.; Liu, O.; Liu, Y.; Stanya, K. J.; Chang, K.
S.; Means, A. R.; Kao, H. Y. Mol. Cell. Biol. 2008, 28, 997ꢀ1006.
(17) Lessard, L.; Stuible, M.; Tremblay, M. L. Biochim. Biophys. Acta
2010, 1804, 613.
(18) Lu, K. P.; Zhou, X. Z. Nat. Rev. Mol. Cell Biol. 2007, 8, 904-916.
5
6
7
Insert Table of Contents artwork here
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment