Non-Peptidic Cell-Penetrating Motifs for Mitochondrion-Specific Cargo Delivery

Article abstract of DOI:10.1002/anie.201811940

Mitochondrial dysfunction is linked to a variety of human illnesses, but selective delivery of therapeutics into the mitochondrion is challenging. Now a family of amphipathic cell-penetrating motifs (CPMs) is presented, consisting of four guanidinium groups and one or two aromatic hydrophobic groups (naphthalene) assembled through a central scaffold (a benzene ring). The CPMs and CPM-cargo conjugates efficiently enter the interior of cultured mammalian cells and are specifically localized into the mitochondrial matrix, as revealed by high-resolution confocal microscopy. With a membrane-impermeable peptide as cargo, the CPMs exhibited ≥170-fold higher delivery efficiency than previous mitochondrial delivery vehicles. Conjugation of a small-molecule inhibitor of heat shock protein 90 to a CPM resulted in accumulation of the inhibitor inside the mitochondrial matrix with greatly enhanced anticancer activity. The CPMs showed minimal effect on the viability or the mitochondrial membrane potential of mammalian cells.

Full text of DOI:10.1002/anie.201811940

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
International Edition Chemie
www.angewandte.org
Accepted Article
Title: Non-Peptidic Cell-Penetrating Motifs for Mitochondrion-Specific
Cargo Delivery
Authors: Dehua Pei, George Appiah Kubi, Ziqing Qian, Souad Amiar,
Ashweta Sahni, and Robert V. Stahelin
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201811940
Angew. Chem. 10.1002/ange.201811940
Link to VoR: http://dx.doi.org/10.1002/anie.201811940
http://dx.doi.org/10.1002/ange.201811940

Angewandte Chemie International Edition
10.1002/anie.201811940
COMMUNICATION
Non-Peptidic Cell-Penetrating Motifs for Mitochondrion-Specific Cargo
Delivery
George Appiah Kubi, Ziqing Qian, Souad Amiar, Ashweta Sahni, Robert V. Stahelin, and Dehua Pei*
Abstract: Mitochondrial dysfunction is linked to a variety of human
illnesses, but selective delivery of therapeutics into the mitochondrion
has been challenging. We report herein a family of amphipathic cell-
penetrating motifs (CPMs) consisting of four guanidinium groups and
one or two aromatic hydrophobic groups (e.g., naphthalene) assembled
through a central scaffold (e.g., a benzene ring). The CPMs and CPM-
cargo conjugates efficiently enter the interior of cultured mammalian
cells and are specifically localized into the mitochondrial matrix, as
revealed by high-resolution confocal microscopy. With a membrane-
impermeable peptide as cargo, the CPMs exhibited ≥170-fold higher
delivery efficiency than previously reported mitochondrial delivery
vehicles. Conjugation of a small-molecule inhibitor of heat shock
protein 90 to a CPM resulted in accumulation of the inhibitor inside the
mitochondrial matrix and greatly enhanced its anticancer activity. The
CPMs showed minimal effect on the viability or the mitochondrial
membrane potential of mammalian cells.
Mitochondria carry out a wide range of vital biochemical functions in
eukaryotic cells including ATP production, calcium homeostasis, cell
death, growth, differentiation, and catabolism and anabolism of
secondary metabolites.[1] Mitochondrial dysfunction is linked to many
human diseases such as cardiovascular diseases (e.g., atherosclerosis,
ischemia/reperfusion injury, heart failure, stroke), aging and
neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s
disease, Huntington’s disease, amyotrophic lateral sclerosis, and
Friedreich’s ataxia), and metabolic diseases (e.g., diabetes and
obesity).[2] Therapeutic intervention of mitochondrial diseases is
challenging, because a drug molecule must traverse the plasma as well
as the two mitochondrial membranes before reaching the mitochondrial
matrix. Current approaches to delivering bioactive cargo molecules into
Figure 1. Design and synthesis of CPMs. a) Comparison of the
strctures of a cyclic cell-penetrating peptide (CPP9) and non-
peptidic CPMs. b) Solid-phase synthesis of CPM1-miniPEG-Lys.
SPPS, solid-phase peptide synthesis.
and cationic liposome-based[11] transporter systems. In this work, we
report a new class of non-peptidic cell-penetrating motifs (CPMs)
capable of delivering small molecules and peptides into the
mitochondrial matrix with unprecedented specificity and efficiency.
We previously discovered a family of small amphipathic cyclic
peptides as highly efficient cell-penetrating peptides (CPPs), which are
capable of delivering a wide variety of cargo molecules (e.g., small
the mitochondria involve lipophilic cations [e.g., triphenylphosphonium
[4]
(
TPP) ion],[3] cationic peptidomimetics and oligosaccharides, or
mitochondrial-penetrating peptides (MPPs).[5-8] To the best of our
knowledge, these mitochondrial transporters have only been used to
deliver membrane-permeable small-molecule cargoes (e.g., anticancer
agents) into the mitochondrial matrix. To deliver larger, impermeable
cargoes into the mitochondria, other investigators have fused the cargo
[12,
molecules, peptides and proteins) into the cytosol of mammalian cells.
13]
The cyclic CPPs typically consisted of two aromatic hydrophobic
amino acids and 3 or 4 arginine residues of both L- and D-configurations
e.g., CPP9 in Figure 1a). To gain further insight into the structure-
(
activity relationship and potentially discover still more active cell-
penetrating molecules, we designed a non-peptidic CPM consisting of 4
guanidinium groups anchored to a rigid scaffold (e.g., a benzene ring),
to mimic the 4 arginine residues of cyclic CPPs (Figure 1a, CPM1). An
aromatic hydrophobic amino acid [e.g., naphthylalanine (Nal)] was also
(
e.g., peptide nucleic acids) to naturally occurring mitochondrial
targeting sequences (MTSs),[9] which are recognized by the
mitochondrial import machinery, or employed nanoparticle-based[10]
appended to the benzene ring to render the CPM amphipathic, a feature
[*]
G. Appiah Kubi, Dr. Z. Qian, A, Sahni, Prof. Dr. D. Pei
Department of Chemistry and Biochemistry
The Ohio State University
[13]
critical for efficient cytosolic entry.
To facilitate fluorescent
labeling/cargo attachment and minimize any potential mutual
interference between the CPM and the dye/cargo, a flexible linker,
miniPEG-Lys, was added to the carboxyl group of Nal (Figure 1b).
CPM1-miniPEG-Lys was readily synthesized on solid phase, starting
with the miniPEG-Lys linker. After the sequential addition of Fmoc-Nal
and 3,5-bis(bromomethyl)benzoic acid (Bmb) by standard peptide
chemistry, the four guanidinium groups were installed by treating the
resin-bound Bmb moiety with excess 1,1’-(azanediylbis(hexane-6,1-
diyl))diguanidine under mildly basic condition (pH 9). CPM1-miniPEG-
484 West 12th Avenue, Columbus, OH 43210 (USA)
E-mail: pei.3@osu.edu
Dr. S. Amiar, Prof. Dr. R. V. Stahelin
Department of Medicinal Chemistry and Molecular Pharmacology
Purdue University
575 W Stadium Ave, West Lafayette, IN 47907 (USA)
Supporting information for this article is given via a link at the end of
the document
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201811940
COMMUNICATION
Lys was cleaved from the resin and deprotected (at the lysine side chain)
by treatment with trifluoroacetic acid (TFA).
To test whether CPM1 can enter the cytosol of mammalian cells, we
labeled CPM1-miniPEG-Lys at its lysine side chain with a pH-sensitive
dye, naphthofluorescein (NF; pKa = 7.8), which is minimally fluorescent
when entrapped inside the acidic endosomal/lysosomal compartments
a)
700
6
5
4
3
2
00
00
00
00
00
(
pH 4.5-6.5) but becomes highly fluorescent upon escaping from the
100
0
[14]
endosome/lysosome. Flow cytometry analysis of HeLa cells treated
for 2 h with 5 M CPM1 (see Figure S1 for detailed structure) gave a
relative mean fluorescence intensity (MFI) of 404% [relative to CPP9
(
NF
NF
100%), which is one of the most active CPPs known to date[13]] (Figure
CPMTMR
MitoGreen
Merge
2
a), suggesting that CPM1 efficiently enters the cytosol. To confirm
b)
c)
d)
R = 0.73
cytosolic entry, we labeled CPM1-miniPEG-Lys with a pH-insensitive
dye, tetramethylrhodamine (TMR), and examined HeLa cells treated
with 2 µM CPM1
TMR
by live-cell confocal microscopy. Surprisingly,
CPM1TMR exhibited exclusively punctate fluorescence in the
cytoplasmic region (Figure 2b). These seemingly conflicting results led
us to hypothesize that after cytosolic entry, CPM1 might become
associated with subcellular structures such as the mitochondria, since
certain lipophilic cations had previously been shown to accumulate
inside the mitochondrial matrix.[3,4,6-8,15] Indeed, incubation of HeLa
cells with CPM1TMR and a mitochondrion-specific dye (MitoTracker
Green) showed co-localization of the two, with a Pearson’s correlation
coefficient of 0.73 (Figure 2b). In contrast, CPM1TMR overlapped poorly
with an endosomal marker, Alexa Fluor 488-labeled dextran, showing a
correlation coefficient of 0.35 (Figure S2a). The fluorophore did not
affect the mitochondrial localization of CPM1, since fluorescein-labeled
CPM1 also co-localized with MitoTracker Red (Pearson’s correlation
coefficient = 0.70; Figure S2b and vide infra).
R = 0.73
R = 0.89
In an attempt to improve the cytosolic entry efficiency and/or
mitochondrial-targeting specificity of CPM1, we added a second
hydrophobic amino acid, D-phenylalanine (D-Phe) or D-Nal, to the
CPM1 structure to give CPM2 and CPM3, respectively (Figure 1a).
Previous studies had shown that an alternating stereochemical
configuration of the two hydrophobic residues improved the cytosolic
entry efficiency of cyclic CPPs.[13] Incorporation of a D-amino acid
should also improve the proteolytic stability of the CPMs. As a control,
we also generated CPM4, in which the Nal residue in CPM1 was
replaced with an alanine (Figure S1). Flow cytometry analysis of HeLa
cells treated for 2 h with 5 M NF-labeled CPM1-4 gave relative MFI
Figure 2. Cellular entry and mitochondrial localization of CPMs. (a) Cytosolic
entry efficiency of CPM1-4 in to HeLa cells as analyzed by flow cytometry. MFI
values of cells treated with 5 M NF-labeled CPM1-4 for 2 h are relative to that
of CPP9 (100%). Data reported are the mean ± SD of three independent
experiments. Control, untreated cells. (b) Live-cell confocal microscopic
TMR
images of HeLa cells after 2 h treatment with 2 M CPM1
(red) and 15 min
incubation with MitoTracker Green. A merged image is shown on the right with
the R value representing Pearson’s correlation coefficient for co-localization.
(c) Same as (d) but with CPM2. (d) Same as (d) but with CPM3.
negative charges at physiological pH) and its cellular entry was again
monitored by confocal microscopy. CPM3Alexa488 efficiently entered
HeLa cells and co-localized with MitoTracker Red (Pearson’s
correlation coefficient = 0.90-0.97), demonstrating the ability of CPMs
to deliver negatively charged cargoes into the mitochondria (Figure S3).
Confocal imaging of HeLa cells treated with CPM3Alexa488 for shorter
periods of time (e.g., 15 min) produced a punctate fluorescence pattern
of minimal overlap with that of MitoTracker Red (Pearson’s correlation
coefficient = 0.12; Figure S4). This observation suggests that the CPMs
enter cells by endocytic mechanisms and are largely entrapped inside
endosomes/lysosomes after 15 min of treatment. On the other hand, once
escaping from the endosome, translocation of CPMs from the cytosol
into the mitochondria appears to be a rapid event.
To test whether the CPMs can deliver membrane-impermeable
cargoes into the mitochondria, we designed an all D-peptide, ala-asn-
ala-asn-ala-asn-miniPEG-Lys (ananan), as a mock cargo molecule. The
choice of a D-peptide was to minimize any proteolytic degradation
during experimentation and simplify data interpretation. The D-peptide
was conjugated with CPM3 at its N-terminus and labeled with TMR at
values of 404%, 549%, 506%, and 21%, respectively (relative to
CPP9NF), confirming the critical role of hydrophobic groups for efficient
cellular entry (Figure 2a). Live-cell confocal microscopic imaging
showed that CPM2TMR and CPM3TMR co-localized with MitoTracker
Green in HeLa cells, with Pearson’s correlation coefficients of 0.73 and
0
.89, respectively (Figure 2c,d). Interestingly, while each MitoTracker
TMR
Green signal was matched by a corresponding CPM
reverse was not true. A small fraction of CPM2TMR (and CPM3TMR
signal, the
)
signals was not matched by MitoTracker Green signals. These signals
were likely derived from CPMs still entrapped inside the endosomal
and/or lysosomal compartments. CPM3 appears to be more efficient in
endosomal escape than CPM1 and CPM2, resulting in better co-
localization with the MitoTracker (Figure 2b-d). As expected, CPM4
showed much weaker intracellular fluorescence (Figure 2a) and poorer
co-localization with MitoTracker Green than CPM1-3 (R = 0.52; Figure
S2c). CPM3 was selected for further studies, because of its high cellular
uptake efficiency and mitochondrial-targeting specificity.
To ascertain that mitochondrial localization is not caused by the
TMR label, CPM3 was labeled with Alexa Fluor 488 (which carries two
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201811940
COMMUNICATION
the C-terminal lysine (Figure S1). HeLa cells were treated with the
peptides and MitoTracker Green, and imaged by live-cell confocal
microscopy. CPM3-anananTMR produced punctate fluorescence, which
overlapped well with that of MitoTracker Green, having a Pearson’s
correlation coefficient of 0.80 (Figure 3a). A small amount of the peptide
appeared to remain entrapped inside the endosomal/lysosomal
compartments, causing the correlation coefficient to be <1. As expected,
cells treated with the unconjugated D-peptide (Ac-anananTMR) showed
no detectable intracellular fluorescence (Figure 3b). For comparison, we
also conjugated the D-peptide with two of the most widely used (and
a) CPM3-anananTMR
MitoGreen
Merge
R = 0.80
most efficient) mitochondrion targeting agents, TPP and (F
is cyclohexylalanine and r is D-arginine; Figure S1).[6, 8] Under the
same condition, (F r)
-anananTMR showed partial co-localization with
x 3
r) (where
F
x
x
3
Ac-anananTMR
MitoGreen
Merge
b)
MitoTracker Green, as indicated by a Pearson’s correlation coefficient
of 0.47 (Figure 3c), which is similar to the previously reported values.[6]
x 3
A substantial fraction of (F r)
-anananTMR was localized in subcellular
structures other than the mitochondria, most likely the endosomes and/or
lysosomes. TPP-anananTMR showed excellent co-localization with
MitoTracker Green (Pearson’s correlation coefficient = 0.74) but
produced very weak intracellular fluorescence (Figure 3d). Next, the
three peptide conjugates were labeled with NF and their
cytosolic/mitochondrial entry efficiencies into HeLa cells were
quantitated by flow cytometry. Consistent with the results obtained with
CPM3NF (Figure 2a), CPM3-anananNF entered the cytosol/mitochondria
with remarkable efficiency, which was 170- and 1300-fold higher than
(
F
x
r)
3
-anananTMR
MitoGreen
Merge
c)
R = 0.47
that of (F
x
r)
3
-anananNF and TPP-anananNF, respectively (Figure 3e).
TPP-anananTMR
MitoGreen
Merge
d)
e)
The sub-mitochondrial localization of the peptide was evaluated by
high-resolution 3D-SIM imaging of HeLa cells treated with CPM3-
TMR
ananan
and transfected with the mitochondrial marker Grx2-mito-
[16]
roGFP. The latter produces a green fluorescent protein (GFP) inside
the mitochondrial matrix. CPM3-anananTMR appeared to be highly
distributed throughout the mitochondrial network (Figure 4a). The
20
m
R = 0.74
7
000
zoomed insets (Figure 4b-d) showed highly similar fluorescence patterns
for CPM3-anananTMR and mitochondrial GFP. Inner lamellar membrane
6000
5000
4
000
accumulation is observed with dark regions and internal thin fluorescent
gaps, corresponding to the structure of mitochondrial cristae (Figure 4b-
d bottom panels). Treatment of HEK293 cells with CPM3-anananTMR
and Grx2-mito-roGFP exhibited similar mitochondrial fluorescence
patterns (data not shown). Similar mitochondrial structures were also
previously observed where different mitochondrion probes and markers
3000
2
1
000
000
0
Control
CPM3
(F
x
r)
3
TPP
Figure 3. Mitochondrial delivery of peptidyl cargo by CPM3, (F
TPP. (a) Upper panel, live-cell confocal microscopic images of HeLa cells
after treatment with 2 M CPM3-ananan
for 15 min. Lower panel, zoomed-in images of the boxed areas in the
upper panel. (b) Same as (a) but cells were treated with 2 M Ac-
x 3
ananan . (c) Same as (a) but cells were treated with 2 M (F r) -
ananan . (d) Same as (a) but cells were treated with 2 M TPP-
ananan . Different imaging conditions were used in a-d in order to
x 3
r) , and
TMR
for 2 h and MitoTracker Green
were analyzed by SD-SIM imaging.[17] These data demonstrate that
CPM3-anananTMR is efficiently delivered into the mitochondrial matrix.
TMR
We next tested whether CPMs can deliver a biologically active cargo
into the mitochondrial matrix. Geldanamycin (GA) is a potent small-
molecule inhibitor of heat shock protein 90 (Hsp90) and has anti-
proliferative activity against cancer cells.[18,19] Interestingly, treatment of
cancer cells with GA causes only partial loss of cell viability, even at
saturating concentrations. Delivery of GA into the mitochondrial matrix
with a targeting moiety and subsequent inhibition of mitochondrial
Hsp90, however, were previously shown to induce rapid apoptosis and
cause complete killing of cancer cells.[19] We covalently attached GA to
CPM3 (Figure 5a) and assessed the anti-proliferative activity of CPM3-
GA, GA alone, or CPM3 alone against HeLa cells. In agreement with
previous reports,[19] GA caused a maximum of ~40% inhibition in HeLa
cell viability at saturating concentrations, although the maximum was
reached at a low inhibitor concentration (0.3 M; Figure 5b). In contrast,
CPM3-GA reduced the viability of HeLa cells in a dose-dependent
fashion (IC50 ~4 M) and completely killed the cancer cells at 10 M
concentration. Annexin V and propidium iodine staining of the treated
TMR
TMR
x 3
detect the TMR signals. (e) Cytosolic entry efficiency of CPM3-, (F r) -,
NF
and TPP-ananan into HeLa cells as analyzed by flow cytometry. Data
shown are the mean ± SD of three independent experiments. Control,
untreated cells. Scale bars, 20 m (10 m for the lower panel in a and b).
cells indicated apoptotic cell death (Figure S5). CPM3 alone showed no
significant effect up to 10 M concentration. To confirm mitochondrial
localization of CPM3-GA, we labeled CMP3-GA with TMR (CPM3-
GATMR) and treated HeLa cells with CPM3-GATMR and MitoTracker
Green. Live-cell confocal microscopy of the treated cells showed co-
localization of CPM3-GATMR and MitoTracker Green, with a Pearson’s
correlation coefficient of 0.74 (Figure 5c). These data suggest that
CPM3 resulted in accumulation of GA inside the mitochondrial matrix,
thereby enhancing its anticancer activity. The higher IC50 value of
CPM3-GA (relative to GA) is presumably due to reduced binding
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201811940
COMMUNICATION
Figure 4. High-resolution fluorescence images showing the mitochondrial distribution of CPM3-anananTMR. (a) 3D-SIM reconstructed image of HeLa cells 48
h post-transfection with pGrx2-mito-roGFP (green) and treated with 2 M CPM3-anananTMR (red) for 2 h and 5 g/ml Hoechst 33342 dye (blue) for 15 min
prior to imaging. (b-d) Zoom-in images of the boxed areas from (a) showing detailed mitochondrial structures. Top panel, zoom-in images of the boxed areas
in (a); middle panel, localization of the mitochondrion marker (gray) within the stroma; and bottom panel, localization of CPM3-anananTMR (gray) at both the
periphery (which corresponds to the mitochondrial membrane) and the interior of mitochondria (which correspond to the cristae). Scale bars: 2 m.
affinity of CPM3-GA to Hsp90; conjugation of GA to CPM3 through a
a)
b)
c)
releasable linker[20] may produce a more potent anticancer agent. It is
also possible that CPMs have lower endocytic uptake and endosomal
escape efficiencies at lower concentrations.
The CPMs were tested for potential cytotoxicity. In MTT assays, up
to 10 M CPMs did not significantly affect the viability/proliferation of
HeLa cells (Figures S6a). At 100 M, CPM1-3 reduced the viability of
HeLa cells by 40-60%. We next tested CPM1-3 for potential effect on
mitochondrial membrane potential, a key indicator of cell health or
injury. Briefly, HeLa cells untreated or treated with CPM were labeled
1
1
20
00
with JC-10, a dye molecule which emits green fluorescence inside the
cytosol but red fluorescence inside the mitochondria.[21] The ratio of
CPM3
GA
80
60
40
20
0
green/red fluorescence (F520/F590) provides an indicator of the
mitochondrial membrane potential. Treatment of HeLa cells with a
known mitochondrial membrane depolarizer, p-trifluoromethoxy
CPM3-GA
[22]
carbonyl cyanide phenyl hydrazone (FCCP), caused retention of JC-
1
0 in the cytosol and dose-dependent increase of the F520/F590 ratio, to
0
2
[
4
6
8
10
up to 7-fold above the normal value at 20 M FCCP concentration
(Figure S6b,c). On the other hand, CPM1-3 did not perturb the
membrane potential at ≤10 M concentration. At 20 M, only CPM3
caused a small (2.5-fold) increase in the F520/F590 ratio. These results
indicate that the CPMs are relatively nontoxic to mammalian cells.
In conclusion, we have discovered a new family of non-peptidic
CPMs which are capable of highly efficient and specific delivery of
small molecules, peptides, and potentially other cargoes into the
mitochondrial matrix of mammalian cells. They are metabolically stable
(Figure S7) and appear to have minimal cytotoxicity to mammalian cells.
These CPMs should provide a powerful research tool for specific
delivery of chemical probes into the mitochondria. They may also be
applied to deliver therapeutic agents into the mitochondrial matrix for
potential treatment of mitochondrial diseases.
Compound], M
CPM3-GATMR
MitoGreen
Merge
R = 0.74
Figure 5. Delivery of Hsp90 inhibitor into the mitochondrial matrix by
CPM3. a) Structure of CPM3-GA. b) Effect of GA, CPM3, and CPM3-GA
on HeLa cell viability as measured by the MTT assay. c) Live-cell confocal
microscopic images of HeLa cells after 2 h treatment with 2 M CPM3-
TMR
GA
(red) and 15 min incubation with MitoTracker Green. R, Pearson’s
correlation coefficient.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201811940
COMMUNICATION
[
[
[
9] P. F. Chinnery, R. W. Taylor, K. Diekert, R. Lill, D. M. Turnbull,
R. N. Lightowlers, Gene Ther. 1999, 6, 1919.
10] P. K. Pathak, N. Kolishetti, S. Dhar, Wiley Interdiscip. Rev.
Nanomed. Nanobiotechnol. 2015, 7, 315.
11] a) Y. Yamada, H. Harashima, Biomaterials 2012, 33, 1589; b) V.
Weissig, G. G. D’Souza, V. P. Torchilin, J. Controlled Release
2001, 75, 401.
Acknowledgements
This work was supported by the National Institutes of Health
(
GM122459). S.A. and R.V.S. acknowledge the use of the facilities of
the Purdue University Bindley Bioscience Center, a core facility of the
NIH-funded Indiana Clinical and Translational Sciences Institute.
[
[
12] a) Z. Qian, T. Liu, Y.-Y. Liu, R. Briesewitz, A. M. Barrios, S. M.
Jhiang, D. Pei, ACS Chem. Biol. 2013, 8, 423; b) Z. Qian, J. R.
LaRochelle, B. Jiang, W. Lian, R. L. Hard, N. G. Selner, R.
Luechapanichkul, A. M. Barrios, D. Pei, Biochemistry 2014, 53,
Keywords: Cell-penetrating motif • cytosolic entry • drug
delivery • mitochondrial targeting
4
034.
[
[
1] L. Ernster, G. Schatz, J. Cell. Biol. 1981, 91, 227.
13] Z. Qian, A. Martyna, R. L. Hard, J. Wang, G. Appiah-Kubi, C.
2] For reviews on mitochondrial diseases, see: a) R. H. Malty, M.
Jessulat, K. Jin, G. Musso, J. Vlasblom, S. Phanse, Z. Zhang, and
M. Babu, J. Proteome Res. 2015, 14, 5; b) b) S. E. Calvo, V. K.
Mootha, Annu. Rev. Genomics Hum. Genet. 2010, 11, 25; c) P. L.
Toogood, Curr. Opin. Chem. Biol. 2008, 12, 457.
Coss, M. A. Phelps, J. S. Rossman, D. Pei, Biochemistry 2016, 55,
2
601.
[
[
14] Z. Qian, P. G. Dougherty, D. Pei, Chem. Commun. 2015, 51, 2162.
15] K. K. Maiti, W. S. Lee, T. Takeuchi, C. Watkins, M. Fretz, D. C.
Kim, S. Futaki, A. Jones, K. T. Kim, S. K. Chung, Angew. Chem.
Int. Ed. 2007, 46, 5880.
16] S. Norcross, K. J. Trull, J. Snaider, S. Doan, K. Tat, L. Huang, M.
Tantama, ACS Sens. 2017, 2, 1721.
17] S. Opstad, D. L. Wolfson, C. I. Øie, B. S. Ahluwalia,
Nanophotonics 2018, 7, 935.
18] M J. Drysdale, P. A. Brough, A. Massey, M. R. Jensen, J.
Schoepfer, Curr. Opin. Drug Discov. Devel. 2006, 9, 483.
19] B. H. Kang, J. Plescia, H. Y. Song, M. Meli, G. Colombo, K. Keebe,
B. Scroggins, L. Neckers, D. C. Altieri, J. Clin. Invest. 2009, 119,
[
[
3] M. P. Murphy, Biochim. Biophys. Acta 2008, 1777, 1028.
4] a) J. Fernꢀndez-Carneado, M. Van Gool, V. Martos, S. Castel, P.
Prados, J. de Mendoza, E. J. Giralt,J. Am. Chem. Soc. 2005, 127,
[
[
[
[
8
69. b) K. Zhao, G. M. Zhao, D. Wu, Y. Soong, A. V. Birk, P. W.
Schiller, H. H. Szeto, J. Biol. Chem. 2004, 279, 34682; c) P. Wipf,
J. Xiao, J. Jiang, N. A. Belikova, V. A. Tyurin, M. P. Fink, V. E.
Kagan, J. Am. Chem. Soc. 2005, 127, 12460.
[
[
[
5] J.-A. Chuah, T. Yoshizumi, Y. Kodama, K. Numata, Sci. Reports
2
015, 5, 7751.
6] S. R. Jean, M. Ahmed, E. K. Lei, S. P. Wisnovsky, S. O. Kelley,
Acc. Chem. Res. 2016, 49, 1893.
7] a) K. L. Horton, K. M. Stewart, S. B. Fonseca, Q. Guo, S. O. Kelley,
Chem. Biol. 2008, 15, 375; b) C. P. Cerrato, M. Pirisinu, E. N.
Vlachos, U. Langel, FASEB J. 2015, 29, 4589.
4
54.
[
[
[
20] E. K. Lei, S. O. Kelley, J. Am. Chem. Soc. 2017, 139, 9455.
21] C. Kirchhoff, H. Cypionka, Front. Microbiol. 2017, 8, 695.
22] S. W. Perry, J. P. Norman, J. Barbieri, E. B. Brown, H. A. Gelbard,
Biotechniques 2011, 50, 98.
[
8] A. T. Hoye, J. E. Davoren, P. Wipf, Acc. Chem. Res. 2008, 41, 87.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201811940
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Drug delivery: A non-peptidic cell-
penetrating motif is able to efficiently
and specifically deliver small-molecule
and peptidyl cargoes into the
George Appiah Kubi, Dr. Ziqing Qian,
Dr. S. Amiar, Ashweta Sahni, Prof. Dr.
R. V. Stahelin, and Prof. Dr. Dehua Pei*
mitochondrial matrix of mammalian
cells by crossing both plasma and
mitochondrial membranes.
Page No. – Page No.
Non-Peptidic Cell-Penetrating Motifs
for Mitochondrion-Specific Cargo
Delivery
Drug
Endosome
Mitochondrion
This article is protected by copyright. All rights reserved.