Delivery and Release of Small-Molecule Probes in Mitochondria Using Traceless Linkers

Article abstract of DOI:10.1021/jacs.7b04415

Mitochondria-penetrating peptides (MPPs) are specific targeting vectors for the localization of small molecules to the mitochondrial matrix. Mitochondrial targeting of small molecules has enabled the development of a number of potential therapeutics and c

Full text of DOI:10.1021/jacs.7b04415

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Communication
Delivery and Release of Small- Molecule Probes
in Mitochondria Using Traceless Linkers
Eric K. Lei, and Shana O. Kelley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b04415 • Publication Date (Web): 30 Jun 2017
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Delivery and Release of Small-Molecule Probes in Mitochondria
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†
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Eric K. Lei and Shana O. Kelley
†
§
Department of Biochemistry, Faculty of Medicine, Department of Chemistry, Faculty of Arts and Science, and
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontarꢀ
#
io M5S 3M2, Canada
Supporting Information Placeholder
2
chondria that is very difficult to penetrate.
ABSTRACT: Mitochondriaꢀpenetrating peptides (MPPs)
are specific targeting vectors for the localization of small
molecules to the mitochondrial matrix. Mitochondrial targetꢀ
ing of small molecules has enabled the development of a
number of potential therapeutics and chemical probes.
However, the need for covalent conjugation of small moleꢀ
cules to MPPs can negatively affect the activity of the apꢀ
pended cargo against its cellular target. Here, we describe
cleavable linkers designed for the traceless release of
chemical cargo from MPPs following mitochondrial transit.
The cleavage kinetics of a number of disulfides were invesꢀ
tigated using a fluorescent reporter system in order to optiꢀ
mize linker stability for mitochondrial release. The stability
of monoꢀ and disubstituted disulfides was determined to be
sufficient during transit through the cytosol while still allowꢀ
ing for release of the cargo within 24 hours. This linker sysꢀ
tem successfully released the compound Luminespib, an
HSP90 inhibitor, which was deactivated by direct MPP conꢀ
jugation. The releasable conjugate regenerated Luminespib
activity and induced mitochondrial phenotypes of HSP90
inhibition. This linker may prove useful in expanding the
repertoire of small molecules that can be used with mitoꢀ
chondrial targeting vectors.
Over the last several years, a variety of molecular delivꢀ
ery systems that can transport cargo into mitochondria have
been reported. We have developed mitochondria penetratꢀ
ing peptides (MPPs), which are mitochondrial localization
vectors that directly target small molecules to the mitochonꢀ
The use of MPPs for mitochondrial small
molecule targeting has proven useful for the development of
new probes for mitochondrial biology and investigating drug
2
3
ꢀ5
drial matrix.
6ꢀ
activities within the mitochondria with organellar specificity.
10
However, all of the MPP conjugates generated and studꢀ
ied to date feature covalent and uncleavable linkers, and
therefore the peptide remains attached to molecular cargo.
While this approach has produced several interesting comꢀ
pounds with drugꢀlike properties and significant levels of
activity for a variety of probes, the presence of the delivery
vehicle after transport to mitochondria is a limitation of
MPPs and many other mitochondrial delivery vectors. Sevꢀ
eral existing examples of cargo release in the mitochondria
have focused on taking advantage of enzymatic cleavage of
1
1, 12
a labile ester linker.
However, linkers that rely of enzyꢀ
matic cleavage are particularly sensitive to sterics around
the cleavage site. In addition, enzyme expression can vary
by cell type, environment, and metabolic status, which could
make cleavage kinetics inconsistent. Therefore, we hyꢀ
pothesized that a linker cleaved by endogenous chemical
agents could be more suitable for mitochondrial smallꢀ
molecule targeting and release.
The mitochondria of mammalian cells are important enerꢀ
gy producers and regulators of programmed cell death.
Their function is critical for cellular health, and dysregulation
of mitochondria has been connected with a variety of huꢀ
1
man diseases. The delivery of therapeutics or small molꢀ
We chose disulfides as a basis for a releasable linker beꢀ
cause reducing agents, particularly glutathione, are ubiquiꢀ
tous in the cell but relatively scarce in the external environꢀ
ecule probes to this cellular organelle is challenging, howꢀ
ever, because of the doubleꢀmembrane structure of mitoꢀ
Figure 1. Overview of linkers tested for mitochondrial delivery and release of molecular cargo. Reporter conjugates contained the
TAMRA fluorophore [Y] paired to a BHQꢀ2 quencher [X] through disulfide linkers. Compound 1 features an uncleavable linker, while comꢀ
pounds 2, 3, and 4 contained unsubstituted, monosubstituted, and disubstituted disulfides, respectively. Z represents the structure of the miꢀ
tochondriaꢀpenetrating peptide selected for this study. The arginines are incorporated into the peptide as unnatural dꢀamino acids, and the
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an uncleavable control (Compound 1).
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We investigated the in vitro cleavage of the compounds
to assess their relative stabilities (Figure 2A). The fluoresꢀ
cence of the compounds in buffered solution in the presꢀ
ence of dithiothreitol was monitored over a period of 2.5
hours. Compound 1 did not exhibit any increases in fluoꢀ
rescence, as expected from the inclusion of an uncleavable
linker in this conjugate. Compound 4, which included a
disubstituted carbon proximal to the disulfide, exhibited the
slowest cleavage kinetics. Compounds 2 and 3, bearing
unsubstituted and monosubstituted carbons next to the diꢀ
sulfide, respectively, exhibited faster cleavage kinetics, with
compound 2 being cleaved with 10 minutes and compound
3 requiring approximately 45 minutes.
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Figure 2. In vitro and in cellulo cleavage kinetics. (A) Fluoresꢀ
cence recovery of the reporter conjugates in PBS incubated with
0.5 mM DTT. (B) Fluorescence recovery of the indicated reporter
conjugates in K562 cells. K562s were treated with the correspondꢀ
ing reporter then lysed. Lysates were split and the fluorescence of
one sample was normalized to a second sample which was treated
for 10 minutes with 25 mM TCEP as a fully cleaved control.
When fluorescence recovery correlated with linker cleavꢀ
age was monitored in cellulo, a similar trend was observed
albeit with longer release times (Figure 2B). Cells were inꢀ
cubated with the compounds and the fluorescence of cell
lysates was monitored over 48 hours. Compound 2 exhibitꢀ
ed the fastest fluorescence recovery kinetics with saturation
being reached in about 20 hours. However, this compound
also exhibited measurable levels (10%) of cleavage directly
following treatment, suggesting rapid cytosolic cleavage
making it less attractive for mitochondrial delivery applicaꢀ
tions. The monoꢀ and disubstituted conjugates exhibited
lower levels of initial cleavage, and reached saturation over
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ment . Disulfides have been used successfully in a numꢀ
1
4, 15
ber of peptideꢀbased cytosolic delivery agents.
Howevꢀ
er, as relative concentrations of glutathione in the mitoꢀ
16
chondrial matrix and the cytoplasm are similar, the stabilꢀ
ity of a disulfideꢀbased linker as it passes through the cytoꢀ
plasm must be tested to identify a structure with an optimal
delivery and release profile.
A reporter system for linker stability (Figure 1) was develꢀ
oped by combining a MPPꢀconjugated fluorophore (Y) with
a fluorescence quencher (X) linked by a disulfide bond. The
selection of the MPP was based on prior studies identifying
peptides with high levels of cellular uptake and mitochondriꢀ
al localization, and low levels of cytotoxicity. Proximityꢀ
based quenching of the fluorescence occurs when the linker
is intact, but is disrupted upon disulfide cleavage, leading to
a fluorescence turnꢀon signal. We tested three linkers feaꢀ
turing thiols with differing levels of substitution that would
~
48 hours. Overall, cleavage times are slower in cells verꢀ
sus in buffered solution, likely reflecting the complexity of
the cellular environment.
The timeꢀdependence of linker cleavage in live cells was
also confirmed visually using fluorescence microscopy. In
experiments where all imaging conditions were held conꢀ
stant over the time course, all three disulfideꢀcontaining
reporters exhibited a time dependent increase in fluoresꢀ
cence over time as opposed to the uncleavable control
(Figure 3). These results indicate that all three of these
disulfides can be used for mitochondrial delivery, depending
on the desired cleavage kinetics. The monosubstituted
linker was prioritized as a platform for further development
as it had low preincubation cleavage while still releasing the
majority of its cargo within 24 hours.
1
7
modulate intracellular stability to identify a structure that
would maximize delivery of small molecule cargo. Three
reporter conjugates featuring the different linkages (Comꢀ
pounds 2-4, Figure 1) were synthesized and compared to
The extent of mitochondrial localization for the three disulꢀ
fideꢀlinked compounds was also assessed (Supplemental
Figure S1). When compared to a known mitochondrial stain
(mitotracker deep red), all three of the disulfideꢀlinked comꢀ
pounds exhibited high levels of colocalization. The extent of
colocalization was assessed quantitatively through the calꢀ
culation of Pearson’s correlation coefficients, and the values
were above 0.75 for all three conjugates.
To showcase the ability of this linker chemistry to release
cargo into the mitochondrial matrix, the HSP90 inhibitor
Luminespib was used as a test cargo. HSP90 inhibitors
have attracted intense pharmacological interest due to their
chemotherapeutic properties and their lack of toxicity to
18
nonꢀcancer cells.
A number of HSP90 inhibitors have
been developed in recent years targeting the cytoplasmic
HSP90 pools of cancer cells. Inhibition of cytoplasmic
HSP90 has been previously shown to cause arrest of canꢀ
cer cell growth by antagonizing the stabilizing effect of
HSP90 on signaling proteins involved in cancer cell growth
19
and survival.
However, induction of cell death by cytoꢀ
plasmic HSP90 inhibition has been found to be inconsistent,
with some compounds inducing cell death in some cell lines
Figure 3. Time-dependent images of reporter conjugates in
living cells. Fluorescence microscopy of cells treated with the
reporter conjugates over time. Image acquisition settings were
maintained between compounds and timepoints. Scale bar repreꢀ
sents 20 ꢁm.
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Figure 5. Mitochondrial involvement in mechanism of cyto-
toxicity for mitochondrially-targeted Luminespib. (A) Apoptoꢀ
sis visualized via Annexin V staining of K562 cells treated for 24
hours with 2.5 ꢁM of the compounds indicated. Cell populations
were gated with Annexin V+/Sytox redꢀ cells as early apoptotic,
and Annexin V+/Sytox red+ cells as late apoptotic. Necrotic cells,
defined as Annexin Vꢀ/Sytox red+ cells were excluded as levels
were negligible [<1%]. (B) Mitochondrial mass as measured by
incubation with Mitotracker Green FM staining of K562 cells treatꢀ
ed with 2.5 ꢁM of the indicated compounds for 24 hours. Cells
with Sytox red staining were gated against and excluded. (C)
Mitochondrial membrane depolarization of K562 cells as measꢀ
ured by TMRM staining treated with 2.5 ꢁM of the indicated comꢀ
pounds for 24 hours.
Figure 4. Application of linker strategy for Luminespib. (A)
Chemical structure of the releasable Luminespib MPP conjugate,
compound 5. (B) Chemical structure of the uncleavable Luꢀ
minespib conjugate, compound 6. (C) Chemical structure of parent
drug, Luminespib. (D) Mechanism of disulfide cleavage and linker
selfꢀimmolation. (E) Localization of a fluorescently labeled comꢀ
pound 5 (Supplemental Figure S4), with peptide fluorescence is
shown in the green channel and mitochondria labeled with Mitoꢀ
tracker Deep Red in the red channel. Insets are outlined by the
dashed boxes. The Pearson’s coefficient for this compound was
regeneration of Luminespib through selfꢀimmolation of the
thiolꢀcarbonate (Figure 4D).
minespib after linker cleavage was shown to occur rapidly
Supplemental Figure S2), and mitochondrial localization of
The regeneration of Luꢀ
(
0.92. Scale bar represents 20 ꢁm. (F) Toxicity of compound 5 at
a fluorescentlyꢀlabeled analogue was confirmed (Figure
4E).
different time points in K562 cells. (G) Toxicity of compound 6 at
different time points in K562 cells.
Leukemia cells treated with compound 5 exhibited a timeꢀ
dependent increase in cell toxicity over 48 hours (Figure 4F)
which was distinct from the growth inhibition induced by
Luminespib alone (Supplemental Figure S3). In contrast,
the uncleavable compound 6 exhibited low levels of toxicity
that remained static over time (Figure 4G). At two hours,
only a small amount of Luminespib would have been generꢀ
ated from compound 5, and the similarity between the toxꢀ
icity between the two peptides suggests that the effects
observed are from nonspecific toxicity of the peptides themꢀ
selves, rather than an effect from the released Luminespib.
Conversely, the timeꢀdependent toxicity observed only with
compound 5, and not the uncleavable compound 6, sugꢀ
gests that the difference in effects between the peptides is
due to the cleavage and regeneration of Luminespib. It is
noteworthy that the cells were washed after treatment with
the compounds.
20
and growth arrest in others. This has led to difficulties in
the clinical application of HSP90 inhibitors, especially as
21
single agents. Recent studies exploring HSP90 inhibitors
delivered to the mitochondrial matrix via cationic vectors
have suggested that inhibition of mitochondrial HSP90 and
TRAPꢀ1, a mitochondrial analogue, can more consistently
22, 23
and rapidly induce cell death via induction of apoptosis.
However, IC50 values for the best characterized mitochonꢀ
drial HSP90 inhibitors are relatively high (~ 10 µM), indicatꢀ
ing that cationic vectors may not lead to optimal efficacy.
We selected the HSP90 inhibitor Luminespib as a candidate
for our traceless linker approach. This compound has not
previously been tested for mitochondrial activity because
the functional groups that could be used for conjugation of a
24
delivery vector are also involved directly in protein binding.
In order establish that the mechanism of cytotoxicity of
mitochondriallyꢀtargeted Luminespib (5) was linked to mitoꢀ
chondrial effects, we monitored the mode of cell death, efꢀ
fects on mitochondrial mass, and mitochondrial depolarizaꢀ
tion. As controls, the parent compound Luminespib (Figure
4C, compound 7), and the empty disulfide vector (Suppleꢀ
mental Figure S5) were also tested. The cells were treated
Luminespib was conjugated to a mitochondriaꢀpenetrating
peptide via a monosubstituted disulfide as shown in Figure
4A (compound 5). A nonꢀcleavable analogue (compound 6)
was also generated as shown in Figure 4B. Cleavage of the
disulfide linker in compound 5 by glutathione in the mitoꢀ
chondrial matrix was designed to trigger the release and
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. Smith, R. A.; Hartley, R. C.; Murphy, M. P. Antioxid. Redox.
with 2.5 ꢁM of each compound. Compound 5 produced
significant populations of early and late apoptotic cells after
24 hours as visualized by Annexin V staining, as opposed to
the parent compound and the peptide controls which exhibꢀ
ited no increase when tested (Figure 5A). In addition, coꢀ
treatment of the parent compound with either the uncleavaꢀ
ble compound 6 or the empty vector did not induce apoptoꢀ
sis, indicating that the effects observed with mitochondrially
targeted Luminespib were not due to a nonspecific synerꢀ
gistic effect between the peptide and cytosolic HSP90 inhiꢀ
bition by Luminespib. The mitochondrial mass of cells
treated with the mitochondriallyꢀtargeted Luminespib (5)
exclusively exhibited an increase in mitochondrial mass at
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4 hours (Figure 5B). These results suggest a cleavage
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specific induction of mitochondrial swelling, an indicator of
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mitochondrial toxicity and mitochondrial dependent apoptoꢀ
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minespib (5) also exhibited mitochondrial depolarization,
suggesting compromised mitochondrial integrity (Figure
5C). In both experiments, no induction of mitochondrial
dysfunction was observed in any of the control compounds
or any compound at 2 hours (Supplemental Figure S6, S7),
suggesting the effects induced by mitochondriallyꢀtargeted
Luminespib (5) derive from free Luminespib generated in
the mitochondrial matrix and not from the vector itself.
1
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and enzymeꢀindependent fashion. This strategy can be
used to localize compounds to the mitochondria which have
critical functional groups that otherwise make them incomꢀ
patible with targeting vectors. We have also shown that the
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ASSOCIATED CONTENT
Supporting Information
23. Kang, B. H.; Plescia, J.; Song, H. Y.; Meli, M.; Colombo, G.;
Beebe, K.; Scroggins, B.; Neckers, L.; Altieri, D. C. J. Clin. Invest.
2009, 119 (3), 454ꢀ64.
Methods and materials, cleavage data, viability and mitoꢀ
chondrial health data, cleavage quantification.
2
4. Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.;
Cansfield, J. E.; Cheung, K. M.; Collins, I.; Davies, N. G.; Drysdale,
M. J.; Dymock, B.; Eccles, S. A.; Finch, H.; Fink, A.; Hayes, A.;
Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.; Lockie, A.;
Martins, V.; Massey, A.; Matthews, T. P.; McDonald, E.; Northfield,
C. J.; Pearl, L. H.; Prodromou, C.; Ray, S.; Raynaud, F. I.;
Roughley, S. D.; Sharp, S. Y.; Surgenor, A.; Walmsley, D. L.;
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AUTHOR INFORMATION
Corresponding Author
*shana.kelley@utoronto.ca
2
5. Desagher, S.; Martinou, J. C. Trends Cell. Biol. 2000, 10 (9),
Author Contributions
3
69ꢀ77.
The manuscript was written through contributions of all auꢀ
thors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the Ontario Research Fund, the Natural Sciences
and Engineering Council of Canada, and the Canadian Inꢀ
stitutes for Health Research for support of this work.
REFERENCES
1. Nunnari, J.; Suomalainen, A. Cell 2012, 148 (6), 1145ꢀ59.
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2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
ACS Paragon Plus Environment