Prevention of mitochondrial membrane permeabilization and pancreatic β-cell death by an enantioenriched, macrocyclic small molecule

Article abstract of DOI:10.1002/ejoc.201301769

Mitochondria produce the majority of cellular energy through the process of oxidative phosphorylation and play a central role in regulating the functionality and survival of eukaryotic cells. Under physiological stress, mitochondrial membrane permeabilization results in the release of apoptogenic material such as cytochrome c in the cytoplasm, which thereby initiates caspase activation and the consequent cell death. In our present study, we screened a series of compounds for their ability to inhibit mitochondrial membrane permeabilization and to prevent cytochrome c release during the endoplasmic reticulum stress in cultured pancreatic β-cells. Three benzofuran-based macrocyclic small molecules, that is, 2.4c, c104, and c108, were found to restore the depolarization of mitochondrial membrane potential and to prevent the release of cytochrome c from mitochondria. Interestingly, the acyclic precursor of 2.4c (i.e., 2.3c) did not show any effect, whereas the macrocyclic derivative obtained by utilizing ring-closing metathesis as the "stitching technology" led to this function. The macrocyclic architecture seems to play a crucial role in presenting various functional moieties in the right orientation to observe this effect. A series of compounds for their ability to inhibit mitochondrial membrane permeabilization and to prevent cytochrome c release during endoplasmic reticulum stress in cultured pancreatic β-cells is screened. Three benzofuran-based macrocyclic small molecules were found to restore the depolarization of the mitochondrial membrane potential and to prevent the release of cytochrome c from mitochondria. MPTP = Mitochondrial Permeability Transition Pore. Copyright

Full text of DOI:10.1002/ejoc.201301769

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201301769
Prevention of Mitochondrial Membrane Permeabilization and Pancreatic
β-Cell Death by an Enantioenriched, Macrocyclic Small Molecule
Ravikumar Jimmidi,[a][‡] Govardhan K. Shroff,[a][‡] M. Satyanarayana,[a] B. Ramesh Reddy,[a]
Jahnavi Kapireddy,[a] Mithila A. Sawant,[b] Sandhya L. Sitaswad,[b] Prabhat Arya,*[a] and
Prasenjit Mitra*[a]
Keywords: Natural products / Macrocycles / Mitochondria / Cell death / Diabetes
Mitochondria produce the majority of cellular energy
through the process of oxidative phosphorylation and play
a central role in regulating the functionality and survival of
eukaryotic cells. Under physiological stress, mitochondrial
membrane permeabilization results in the release of
apoptogenic material such as cytochrome c in the cytoplasm,
which thereby initiates caspase activation and the conse-
quent cell death. In our present study, we screened a series
of compounds for their ability to inhibit mitochondrial mem-
brane permeabilization and to prevent cytochrome c release
during the endoplasmic reticulum stress in cultured pancre-
atic β-cells. Three benzofuran-based macrocyclic small mole-
cules, that is, 2.4c, c104, and c108, were found to restore the
depolarization of mitochondrial membrane potential and to
prevent the release of cytochrome c from mitochondria. Inter-
estingly, the acyclic precursor of 2.4c (i.e., 2.3c) did not show
any effect, whereas the macrocyclic derivative obtained by
utilizing ring-closing metathesis as the “stitching technol-
ogy” led to this function. The macrocyclic architecture seems
to play a crucial role in presenting various functional moie-
ties in the right orientation to observe this effect.
(SERCA) pump inhibitor thapsigargin (note: structure not
shown). Thapsigargin treatment causes the depolarization
of the mitochondrial inner membrane potential and com-
promises cell survival.[7] To prevent this depolarization, we
utilized a small-molecule toolbox having 18 enantio-
enriched, benzofuran-derived compounds with 12-mem-
bered macrocyclic rings that are rich in 3D architectures
and that can be considered members of the broad family of
natural-product-inspired compounds. Our data reveals that
enantioenriched benzofuran-based macrocycles having a
12-membered ring, which we synthesized, prevent the depo-
larization of the mitochondrial membrane potential and in-
hibit apoptogenic cytochrome c release from mitochondria
in cultured pancreatic β-cells.
Introduction
Mitochondria play an essential role in pancreatic β-cell
homeostasis through their involvement in the modulation
of stimulus-coupled insulin secretion[1] and in the regulation
of cell survival.[2,3] The permeabilization of mitochondrial
membranes under the influence of various cytokines results
in the release of cytochrome c, which is known to activate
the caspase cascade.[4] Most importantly, during chronic en-
doplasmic reticulum (ER) stress, the accumulation of un-
folded proteins in the ER results in leakage of calcium from
the ER, which leads to calcium overload in the mito-
chondria[5,6] and the consequent opening of the mito-
chondrial permeability transition pore. The latter process
plays a decisive role in the depolarization of the mito-
chondrial membrane potential and programmed cell death.
In our present study, we induced chronic ER stress in
cultured BRIN-BD11 pancreatic β-cells by treating them
with the sarcoendoplasmic reticulum Ca2+ ATPase
Results and Discussion
There is growing interest in accessing small molecules
that are inspired by bioactive natural products having 3D
[a] Dr. Reddy’s Institute of Life Sciences (DRILS), University of architectures to explore their biological functions.[8] They
Hyderabad Campus
could have either multiple rings or macrocyclic architec-
Gachibowli, Hyderabad 500046, India
tures. The latter is quite interesting,[9] because there are nu-
E-mail: prabhata@drils.org
prasenjitm@drils.org
www.prabhatarya.org
merous examples of complex macrocyclic natural products
exhibiting a wide range of biological properties.[10] Owing
to several advantages that are associated with macrocyclic
rings, there is also growing interest[9,11] in developing modu-
lar synthesis methods that allow a diverse chemical toolbox
having different types of macrocyclic architectures to be ob-
[b] National Centre of Cell Science (NCCS), University of Pune
Campus,
Ganeshkhind, Pune 411007, India
[‡] R. J. and G. K. S. both contributed equally to this work
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301769
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P. Arya, P. Mitra et al.
SHORT COMMUNICATION
tained. Some of these advantages[10] include (1) preorgani-
Shown in Scheme 2 is our plan to obtain a 12-membered
zation, (2) enhanced cell permeation properties, and (3) the macrocyclic ring that utilizes benzofuran-based trans-β-
possibility of having numerous binding interactions, a prop- amino ester 2.1. Compound 2.2, a derivative of an N-pro-
erty that could be highly relevant to search for small mole- tected amino acid, was prepared (see Supporting Infor-
cule modulators of protein–protein[12] and other types of mation). In each case, following the derivatization of –OH
biomacromolecular (e.g., DNA/RNA–protein)[13] interac- as –OAllyl, the sample was submitted to ring-closing
tions.
Earlier, we reported an enantioenriched synthesis of “stitching technology”. All macrocyclic compounds that
benzofuran-based, 1,2-trans-β-amino acid 1.1 were synthesized are stable at room temperature.
(Scheme 1).[14] In this article, with the objective to explore
In our next approach, we were interested in accessing a
metathesis[15] (RCM, Grubbs 2nd generation catalyst) as the
further the additional large-ring chemical space, we report different type of macrocyclic architecture, that is, 3.2
here a modular approach that allowed us to incorporate (Scheme 3), that would arise from coupling the amino acid
two different types of 12-membered macrocyclic rings onto moiety to the primary hydroxy side of the benzofuran scaf-
the benzofuran scaffold (Scheme 1; see 1.2/1.2a and 1.3/ fold. To achieve this, we synthesized 3.1 as the N-protected
1.3a). The presence of an amino acid moiety within the chiral amine-based benzofuran derivative. This was further
macrocyclic architecture is an attractive feature to introduce coupled with the N-allyl derivative of the N-protected
a diverse array of chiral side chains having a variety of po- amino acid that produced the starting material required for
larities.
the Grubbs RCM-based stitching technology. Our last ap-
Scheme 1. Proposed synthesis of four different types of benzofuran-derived macrocyclic compounds, that is, 1.2/1.2a and 1.3/1.3a, from
enantioenriched 1.1. MEM = (2-methoxyethoxy)methyl.
Scheme 2. Synthesis of 12-membered macrocyclic derivative 2.4, which is derived from N-functionalized 1,2-trans-amino alcohol 2.3.
EDC = 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide,.
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Prevention of Mitochondrial Membrane Permeabilization
Scheme 3. Macrocycle 3.2 from N-functionalized, enantioenriched, 1,2-trans-amino alcohol 3.1.
proach involved the synthesis of 3.2 (Scheme 3; see also tected chiral amine derivative of benzofuran, 5.1 was easily
Supporting Information) that would arise from coupling prepared. This compound has the coupled protected amino
the amino acid moiety to the primary hydroxy side of the acid moiety on the primary amine and contains three diver-
benzofuran scaffold. The detailed synthesis plan is shown sity sites (R1, R2, and R3). Once subjected to –NAlloc re-
in the Supporting Information.
moval, it was then derivatized through amide coupling, fol-
In another plan (Scheme 4; see also Supporting Infor- lowed by bis(allylation) needed for the RCM technology.
mation), the goal was to replace the primary –OH group This approach was attempted with three cases, and macro-
by an –NH2 group to allow the incorporation of three di- cyclic product 5.2 was easily obtained as a single isomer,
versity sites into the 12-membered ring. To achieve this syn- although the olefin geometry remains to be assigned. The
thesis, 4.1 as the starting material was utilized. This pro- detailed synthesis methods are provided in the Supporting
duced 4.2 in several easy steps that included (1) primary Information.
–NH2 protection as an –NTeoc group {Teoc = [2-(trimeth-
ylsilyl)ethoxy]carbonyl}, (2) –NAlloc removal (Alloc = all-
yloxycarbonyl), (3) reductive alkylation (R3 as the diversity
point), (4) coupling with the protected amino acid (to intro-
duce two diversity sites as R1 and R2). Finally, 4.2 was sub-
jectedto–NTeocremoval, amidation, whichfurtheruponbis-
(allylation) produced the key precursor for the RCM-based
stitching technology. Once again, the RCM approach
worked well, and 4.3 as the macrocyclic derivative was ob-
tained as a mixture of two olefinic compounds. The details
of the synthesis plan are provided in the Supporting Infor-
mation.
Scheme 5. Synthesis of macrocycle 5.2 from N-functionalized, chi-
ral 1,2-trans diamine 5.1 (see Supporting Information).
Prevention of Thapsigargin-Induced Mitochondrial
Depolarization
Benzofuran-based macrocycles reported in this study
showed remarkable activity to prevent the depolarization of
the mitochondrial membrane potential (MMP) under thap-
sigargin-induced ER stress. Figure 1a shows the temporal
effect of thapsigargin on the depolarization of the MMP.
As the data reveals, a 36 h treatment of thapsigargin at a
concentration of 5.0 μm caused a 10-fold reduction in the
Scheme 4. 12-Membered macrocycle 4.3 from 1,2-trans chiral di-
mitochondrial membrane potential. To prevent this depo-
amine 4.1 (see Supporting Information).
larization of the MMP, we screened a library of benzo-
As described earlier, a similar approach that utilized 5.1 furan-based compounds to study their efficacy to rescue the
(Scheme 5) as a starting material led to the synthesis of phenotype (Figure 1b,c). Compound 2.4c was found to pre-
macrocyclic derivative 5.2. Using the orthogonally pro- vent the depolarization of the MMP induced on thapsigar-
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P. Arya, P. Mitra et al.
SHORT COMMUNICATION
gin treatment in cultured pancreatic β-cells. Compound 2.4c moiety in 2.4c is replaced by leucine to obtain c108. A com-
possessing a 12-membered macrocyclic ring and having an parative account of the efficacy of all these macrocycles to
N-(4-nitrobenzoyl)valine amino acid moiety fused to the prevent thapsigargin-induced depolarization of the MMP is
benzofuran scaffold showed the highest activity for the pre- shown in Figure 1. Compounds 2.4c, c104, and c108
vention of thapsigargin-induced depolarization of the mito- showed comparable efficacy in the prevention of thapsigar-
chondrial membrane potential. To validate the structural gin-induced depolarization of the MMP at a concentration
features of this compound, we further synthesized two more of 10.0 μm. Interestingly, the acyclic precursor of 2.4c (i.e.
related macrocyclic compounds, that is, c104 and c108. In 2.3c) did not show any effect. In addition, replacement of
c104, the N-(4-nitrobenzoyl)valine is replaced by an N-benz- the N-benzoylvaline amino acid moiety with an N-benzoyl-
oylvaline unit (i.e., no NO2 group), whereas the amino acid (phenylalanine) (i.e., 2.4b) group dramatically reduced the
Figure 1. Attenuation of thapsigargin-induced mitochondrial depolarization: (a) Temporal depolarization of the mitochondrial membrane
potential (ΔΨm) in pancreatic β-cells. (b and c) Screening potential of compounds that prevent the depolarization of the MMP. (d) Dose–
response curve for the rescue of thapsigargin-induced depolarization of ΔΨm by 2.4c. (Note: for detailed structural information of all
compounds tested, see Supporting Information).
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Prevention of Mitochondrial Membrane Permeabilization
Figure 2. Prevention of cytochrome c release from thapsigargin-treated cells upon treatment with 2.4c. Confocal microscopy images of
BRIN-BD11 cells immune-labeled with primary monoclonal mouse anti-cytochrome c antibody (green) depicting release of cytochrome c
from the mitochondria. Mitochondria are labeled by Mito-tracker Red (red), and the nucleus is visualized by DAPI (blue) staining.
Yellow indicates co-localization of cytochrome c with Mito-tracker Red in control and 2.4c-treated cells in mitochondria. Diffused staining
in thapsigargin-treated cells shows the release of cytochrome c from the mitochondria.
activity. A dose–response curve for the prevention of the of mitochondrial membrane permeabilization is yet to be
depolarization of the MMP by 2.4c is shown in Figure 1d, determined.
and the EC50 of the response was found to be 9.04 μm (Fig-
ure 1c).
Supporting Information (see footnote on the first page of this arti-
cle): Experimental details, characterization data, and copies of the
1H and 13C NMR spectra of all key intermediates and final prod-
ucts.
Prevention of Cytochrome C Release
Acknowledgments
In the next step, we evaluated the distribution of cyto-
chrome c in untreated, thapsigargin-treated, and 2.4c-
treated cells. As Figure 2a reveals, in normal cells, there was
a complete overlap of cytochrome c staining with Mito-
Tracker Red; this indicated its presence in the mito-
chondria. Treatment with thapsigargin for 18 h caused a
marked loss of Mito-Tracker Red and a concomitant re-
lease of cytochrome c into the cytoplasm (Figure 2b), which
was totally prevented with the use of 2.4c (Figure 2c). The
data suggest the role of 2.4c in preventing the release of
cytochrome c from the mitochondria, which is known to
activate cell death in pancreatic β-cells.
This work was supported by the Department of Science and Tech-
nology (DST) grant (SR/S1/OC-30/2010) to P. M. and P. A.; R. J.
and G. K. S. thank the Council of Scientific and Industrial Re-
search (CSIR) and the Indian Council of Medical Research
(ICMR) funding agencies for the award of PhD fellowships. An
earlier part of this work was performed by M. S. during his post-
doctoral tenure with P. A. at the National Research Council of
Canada (NRCC). Don Leek and Malgosia Darozewska are
thanked for the analytical services provided at the NRCC. We also
thank the analytical team at Dr. Reddy’s Institute of Life Sciences
for providing an excellent HPLC–MS and NMR support. Thanks
are due to Dr. Niyaz Ahmed and Ms. Pittu Sandhya, University of
Hyderabad, for allowing us to perform flow-cytometry experiments
in their laboratory.
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Received: November 26, 2013
Published Online: January 17, 2014
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