Intracellular location matters: Rationalization of the anti-inflammatory activity of a manganese(ii) superoxide dismutase mimic complex

Article abstract of DOI:10.1039/d0cc03398g

A conjugate of a Mn-based superoxide dismutase mimic with a Re-based multimodal probe 1 was studied in a cellular model of oxidative stress. Its speciation was investigated using Re and Mn X-fluorescence. Interestingly, 1 shows a distribution different fr

Full text of DOI:10.1039/d0cc03398g

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Bernard, E. Quévrain, M. Zoumpoulaki, S. Iriart, C. Lung-Soong, B. Lai, K. Medjoubi, L. Henry, S. Nagarajan,
F. Poyer, A. Scheitler, I. Ivanovic-Burmazovic, S. Marco, A. somogyi, P. Seksik, N. Delsuc and C. Policar,
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DOI: 10.1039/D0CC03398G
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Intracellular location matters: rationalization of the anti-
inflammatory activity of a manganese (II) superoxide dismutase
mimic complex
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
DOI: 10.1039/x0xx00000x
Emilie Mathieu, Anne-Sophie Bernard, Elodie Quévrain, Martha Zoumpoulaki, Sébastien Iriart,
a
b
c
a
d,e
Caroline Lung-Soong, Barry Lai, Kadda Medjoubi, Lucas Henry, Sounderya Nagarajan, Florent
d,e
f
f
d,e
c
Poyer, Andreas Scheitler, Ivana Ivanović-Burmazović, Sergio Marco, Andrea Somogyi,
g
a
a
Philippe Seksik, Nicolas Delsuc, and Clotilde Policar*
A conjugate of a Mn-based superoxide dismutase mimic with a Re- expressed in an inactive form.19, 20 SOD mimics are able to
based multimodal probe 1 was studied in a cellular model of substitute for SODs in SOD-deficient organisms, protect against
oxidative stress. Its speciation was investigated using Re and Mn radiation and ischemia-reperfusion injuries, and limit
X-fluorescence. Interestingly, 1 shows a distribution different from inflammation and other pathological processes associated with
5
-8, 11, 21
its unconjugated analogue but
a
similar concentration in oxidative stress.
Most of the metal complexes studied
mitochondria and a similar bioactivity.
in the literature for their bioactivity are used as anticancer or
antibacterial agents, thus most bioanalyses focus on their
toxicity towards cancer cells or micro-organisms. In contrast,
SOD-mimics are meant to restore normal functioning of cells
by limiting oxidative stress. As such, specific non-routine
A new class of metallo-drugs with catalytic activity and
inspired from metalloenzymes are emerging: they are of
interest as they may allow decreasing the administered doses
thus reducing potential side effects, toxicity and treatment
4, 22, 23
strategies are required to evaluate their bioactivity.
We
costs.1 Superoxide dismutase mimics, SOD mimics,
-3
4-13
belong
have recently set up several cellular models of oxidative
to this family of catalytic drugs. They reproduce the catalytic
activity of superoxide dismutases, metalloproteins involving
Cu-Zn, Mn, Fe, Ni or Cu at the active site that protect the cell
against oxidative stress by catalysis of superoxide
dismutation.1
4, 22-24
stress,
inflammation can be induced,
the bioactivity of SOD mimics.
one of which consists of epithelial cells in which
25, 26
which can be used to probe
22
A key question for
rationalizing the bioactivity of a molecule is its intracellular
concentration. The actual quantity in cells, and not the
concentration of incubation, is an important consideration for
4, 15
Their use in a biological context raises new challenges. Indeed,
parameters other than intrinsic catalytic activity are essential
for good efficacy: cell penetration, speciation, and intracellular
2, 22, 27
rationalizing the activity.
In addition, cells are
compartmented, so bioactivity depends on the sub-cellular
distribution. In the case of protection against oxidative stress,
mitochondria are believed to be a fragile site that requires
1
6
location(s) and concentration(s). It is therefore important to
investigate the activity of SOD mimics directly in the cellular
environment under oxidative stress, which is defined as an
imbalance between the production of reactive oxygen species
28, 29
specific protection.
In the present work, we have designed a new Mn-SOD mimic 1
1
7, 18
(ROS) and endogenous antioxidants.
Some pathologies,
(
Fig. 1) that was obtained by conjugation of a Re-tris carbonyl
such as inflammatory bowel diseases (IBD) or amyotrophic
lateral sclerosis (ALS), have been associated with impairment
of SOD, either because the enzyme is under-expressed or
30-33
probe that can be used for several imaging modalities,
31
including X-Ray fluorescence, to the parent Mn-SOD mimic 2.
This enabled us to image Mn and Re directly, in order to
correlate distribution of Mn with that of the Re-tagged ligand,
providing an insight into the stability of the SOD mimic in cells.
We have studied the bioavailability and intracellular
distribution of 1. Its activity in cells was determined and
compared with the non-conjugated parent complex 2.
Surprisingly, the new conjugate 1 showed a markedly different
intracellular distribution and accumulation, while displaying
overall similar bioactivity to the unconjugated analogue 2.
Interestingly, the concentration of 1 and 2 are similar at the
mitochondria, which is recognized to be the fragile organelle
to be protected under oxidative stress.
a. Laboratoire des biomolécules, LBM, Département de chimie, Ecole normale
supérieure, PSL University, Sorbonne université, CNRS, 75005 Paris, France. E-
mail: clotilde.policar@ens.fr
X-ray Science Division, Argonne National Laboratory, Argonne, Illinois 60439,
b.
United States.
c.
Synchrotron SOLEIL, BP 48, Saint-Aubin, 91192 Gif sur Yvette, France
Institut Curie, Research Center, PSL University, Chemistry, Modelling and Imaging
d.
for Biology (CMIB), Bât 110-112, Centre Universitaire, F-910405 Orsay, France
INSERM U 1196, CNRS UMR 9187, Paris-Saclay University, Paris-Sud University,
e.
University Centre, Orsay, France
Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg,
f.
Egerlandstr. 1, 91058 Erlangen, Germany
Sorbonne Université, Ecole normale supérieure, PSL University, CNRS, INSERM,
g.
AP-HP, Laboratoire des biomolécules, LBM 75012 Paris, France
†Electronic Supplementary Information (ESI) available: experimental procedures,
supplementary figures and tables. See DOI: 10.1039/x0xx00000x
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Journal Name
This study is in line with other works meant to better inflammatory reaction associated with oxidative stress in
View Article Online
2
2, 25, 26
DOI: 10.1039/D0CC03398G
understand the bioactivity of SOD mimics, including works on cells.
Prior to the study of the cellular bioactivity of the
on the application of SOD complex, we characterized the bioavailability of the complexes
or on the distribution and speciation of in HT29-MD2, by studying the accumulation and subcellular
3
4-36
mechanisms of SOD mimics,
7
, 10, 13, 37, 38
mimics,
these compounds.1
6, 22, 39, 40
location of 1. The quantification of the total manganese
The multimodal probe was synthesized with a pendant content was determined by electron paramagnetic resonance
aldehyde moiety meant for conjugation with the ligand L2 via a (EPR) titration in HT29-MD2 cells after a 6-hour incubation
reductive amination coupling (ESI §5). The effect of with 1 and acid treatment to release Mn from all coordination
2
2
functionalization on the lipophilicity of the L2 ligand was sites. We also compared the Mn-content in a fraction
studied using analytical HPLC (ESI §6), showing that coupling enriched in mitochondria using inductively coupled plasma
with the probe increased the retention time of L1 compared to mass spectrometry (ICP-MS), which provides a higher
L2 on the reversed-phase C18 column, and thus the sensitivity than EPR (ESI, §8). The quantification indicates that
4
1
lipophilicity of the molecule (Fig. S5). The conjugation did not intracellular overall accumulation is higher for 1 than for 2,
modify the fluorescence nor the IR properties of the probe (Fig. 2A.a., C vs. B) whereas the Mn-content in the
(Fig. S1 and S2). L1 was purified by HPLC and titrated by UV- mitochondrial fraction was not significantly different for cells
visible spectrophotometry (ESI §6-7). Metalation with MnCl
2
incubated with 1 or 2 (Fig. 2A.b., C vs. B).
leads to the formation of 1 (ESI for characterization §7).
Cells incubated with 1 were studied by IR and X-ray
fluorescence microspectroscopies. To avoid possible re-
localization upon chemical fixation and drying, we have used a
cryofixation-lyophilisation
procedure,
described. Fourier transform infrared spectromicroscopy
FTIR-SM) showed the two IR-bands (A1 and E) of the Re-tris-
as
previously
2
2
(
carbonyl core in cryofixed and freeze-dried HT29-MD2 cells
incubated with 1 (ESI §8 and Fig. S9).
Figure 1. Structures of 1, 2, L1, and L2.
Cyclic voltammetry experiments at different scan rates were
performed in HEPES buffer (0.1 M, pH 7.5) (Fig. S3). A quasi-
reversible system was obtained with ∆E
The apparent standard potential of 1 is of 243 mV vs. SCE
saturated calomel electrode), slightly higher than for 2 (196
p
= 90 mV at 50 mV/s.
(
4
2
mV vs. SCE). A similar difference in potential has been
previously described for similar functionalization of L1, and
other complexes containing amine based-ligands,
4
3
Figure 2. A. Quantification of total Mn content in cell lysates (a), or mitochondria-
enriched fractions (b). HT29-MD2 cells were incubated for 6 hours under different
4
4, 45
associated with the weaker basicity of a tertiary amine. conditions: A: medium only; B: 2 (100 µM); C: 1 (100 µM, 0.02%DMSO). a: Total Mn
content was determined in acid-digested cell lysates by titration using EPR (ESI). Data
represent mean ± SEM for 4 independent experiments. (*) p < 0.05, (**) p < 0.01 vs. A.
b: Total Mn content was determined in mitochondria-enriched fractions by ICP-MS.
Importantly, this value remains in the range appropriate for
4
superoxide dismutation. The dissociation constants (K
d
) were
-8
determined spectrophotometrically to be 9 ± 4 10 and 7 ± 3
Mitochondria were isolated using
a mitochondria isolation kit (Thermo Fisher
-7
1
0 at pH7.5 for 1 and 2 respectively (see Fig. S4 and ESI §7),
Scientific). Data represent mean ± SEM for 7 independent experiments.
2
2, 43, 46
which is similar to previous measurements for 2.
These B. IL8 secretion in HT29-MD2 cells. Intestinal epithelial cells HT29-MD2 were incubated
-1
for 7 hours under different conditions indicated in the figure. LPS (0.1 µg.mL ) was
added at the end of the first hour. Data represent means ± SEM for 7-10 independent
experiments. (*) p < 0.05, (**) p < 0.01 vs. B.
association constants mean that in the incubation conditions
100 µM), 97% of L1 and 92% of L2 are coordinated.
(
The intrinsic SOD activity, or activity outside of any cellular
context, was determined by the McCord-Fridovich assay (Table
The intracellular distribution of 1 was further studied by SR-
6
S1) and provided as a kMcCF (ESI §3). The kMcCF of 1 is 6.4 x 10
XRF. With this technique, heavy elements can be mapped in a
-1 -1
M .s , similar to the constant previously reported for 2 (7.0 x
0 M .s ). The SOD-like activity of 1 was also investigated
48-50
single cell with good spatial resolution (ca. 300 nm).
Figure
6
-1 -1 42
1
3
shows the distribution of phosphorus (P), manganese (Mn),
with a high excess of superoxide using a direct stopped-flow
measurement combined with fast diode-array UV-vis
zinc (Zn) and rhenium (Re) in a single cryofixed HT29-MD2 cell.
The mapping of P and Zn (K-lines) reveals the nucleus
detection.3
6, 43, 47
The kcat value was 6.46 x 10 M .s in MOPS
6
-1 -1
51
location. Since Re is an ultratrace element in biological
buffer (60 mM, ionic strength 150 mM, pH 7.8) is consistent
with the kMcCF, confirming that coupling with the probe does
not change the intrinsic SOD activity of the SOD mimic.
The bioactivity was investigated in a cellular model of oxidative
stress. Briefly, in this model, human intestinal epithelial cells
HT29-MD2 are activated with bacterial lipopolysaccharide
52
samples, the Re signal observed (L-lines, see ESI) can be
assigned to the presence of the ligand L1 or of the complex.
Note that the Mn map corresponds to that of 1 and
endogenous Mn, the latter being less abundant (see Fig. 2A).
As can be seen from the overlay of Mn and Re maps, part of
the Mn map overlaps with that of the Re, suggesting that in
these areas (white Fig. 2B, Fig. S14 and Fig. S15) complex 1 can
(LPS), a component of bacteria cell-membrane, to generate an
2
| J. Name., 2012, 00, 1-3
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still be intact. Moreover, it shows that 1 is not homogeneously 10 µM. They both had a weak pro-inflammatory effect when
View Article Online
DOI: 10.1039/D0CC0339 82 +
G
distributed, but has a perinuclear distribution with a punctate used on non-activated cells at 100 or 50 µM (Fig. S7). The Zn -
2
2
appearance. This is a striking difference from the homogenous complexes were used as redox inactive analogues of 2 and 1
distribution of 2 in cells and from the distribution of Mn in cells and displayed no effect on IL8 production on LPS-activated, or
2
2
incubated with MnCl
2
.
In addition, cells were co-incubated non-activated cells (Fig. S7). Note that DMSO, which is used to
with a mitochondrial marker, Mitotracker deep red, before solubilize 1 and was present in the cell culture medium at a
cryofixation and freeze-drying, and fluorescence images of final concentration of 0.02% (v/v), showed no effect on IL8
2
stained cells were recorded before X-fluorescence analysis production (Fig. S7). Finally, MnCl also does not exhibit any
(Fig.S12 and S13). Overlay of the Mitotracker signal with the anti-inflammatory activity (Fig. S7) Altogether these data
Re map (Fig. S13) showed areas where the two signals overlap. demonstrate that 1 has an anti-inflammatory activity similar to
This revealed that 1 is localized in perinuclear organelles 2 in LPS-activated cells. This anti-inflammatory activity is
including mitochondria, but not exclusively: 1 was clearly also associated with their redox properties, as the zinc-analogues
localized in organelles other than mitochondria. The more have shown no effect (Fig. S7).
hydrophobic nature of L1 could explain the accumulation of 1 The mitochondrial manganese SOD (MnSOD) is overexpressed
in membrane-rich organelles.
Finally, X-fluorescence spectroscopy can provide quantitative the cell against oxidative stress.
in cells activated with LPS (Fig. S8) as a feedback response of
2
2, 53-55
1 efficiently decreases
information on the amount of heavy elements in cells. Using MnSOD overexpression at 100 and 50 µM in LPS activated
the appropriate standards, we were able to determine the cells, with no detectable effect at 10 µM, which correlates well
amount of Mn and other elements (P, S, K, Fe) in cells (Fig. with the IL8 and COX2 data (Fig. S7-S8). Its ability to limit
S11). The average concentration of Mn for cells incubated with MnSOD overexpression in LPS-activated cells can be
2
22
1
is 6.8 ± 0.7 ng/cm , which is in the same range as other Mn- interpreted as an efficient anti-superoxide effect.
2
39, 40
complexes (5 to 60 ng/cm ) described in literature.
Interestingly, like 2, 1 is able to complement SOD and to
protect mitochondria from oxidative damage. The conjugation
to the Re-tris carbonyl probe did not change the overall
biological activity of the complex, although it changed
drastically its location.
Anti-inflammatory and antioxidant effects of 2 and 1 are in a
similar range, as demonstrated by IL8, COX2, and MnSOD
expression levels: both complexes are efficient in cells and can
reach their target(s). In addition, 2 and 1 are able to
complement the mitochondrial MnSOD under stress
conditions. Surprisingly, 1 and 2 have a similar bioactivity,
while showing a different overall concentration and a marked
different cellular distribution, but a similar mitochondrial Mn-
content. Finally, this similar mitochondrial Mn-content
Figure 3. Elemental distribution of P, K, Mn, Re, and Zn in a HT29-MD2 cell incubated bioactivity associated with a similar bio-activity makes sense as
with 1. The phosphorus (P), and zinc (Zn, K-lines) maps are used to identify the nucleus
mitochondria are believed to require specific protection under
area. The overlay (top right) corresponds to the Mn (magenta) and Re (L-lines) (green)
maps. The regions corresponding to an overlap of both elements are displayed in
white. Intestinal epithelial cells HT29-MD2 were incubated for 2 hours with 1 (100 µM,
2
8, 29
oxidative stress.
This stresses the fact that bioactivities are
compartmentalized: “more in cells” does not track with
0
.02%DMSO) before cryofixation and freeze-drying. Images were recorded on the 2-ID- “better bioactivity”. We propose that context-dependent
D beamline of APS synchrotron (excitation at 12.0 keV; integration time, 2 s/pixel; pixel bioactivity is a key factor for the efficacy of these new classes
size, 200 nm). Scale bar, 3 µm.
of metallodrugs. More generally, to rationalize bioactivities,
correlations are required, not with the incubation
The anti-inflammatory activity of 1 was investigated in HT29-
concentration or overall intracellular location, but ideally with
MD2 activated by LPS, used as a cellular model of oxidative
the concentration at a specific location.
stress.22 Two markers of inflammation, interleukin 8 (IL8)
secretion (Fig. 2B, and S7) and cyclooxygenase 2 (COX2)
expression (Fig. S8), were evaluated. These two markers are
Conflicts of interest
overexpressed in LPS-activated HT29-MD2 cells, which is Philippe Seksik declares consulting fees from Takeda, Abbvie,
2
2
consistent with the generation of an inflammatory reaction.
Merck-MSD and Biocodex, grants from Biocodex, sponsored
1
and 2 were assayed at 100, 50, and 10 µM incubation travel from Merck-MSD and Takeda. These COI are not related
concentrations at which no toxicity was observed, with or with the present work.
without LPS (Fig. S6). Interestingly, both compounds exert a
similar anti-inflammatory activity in LPS-activated cells at 100
and 50 µM as can be seen from the decrease in IL8 secretion
Acknowledgements
We thank Ecole Normale Supérieure Paris Saclay for E. Mathieu’s
PhD fellowship, TGE RENARD (FR 3443, CNRS) and UMR8601 and S.
Lajnef for access to the EPR spectrometer for the EPR experiments,
(Fig. 2B) and COX2 expression (Fig. S8) compared to LPS-
activated cells. No effect of the compounds was observed at
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Journal Name
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3. A.-S. Bernard, C. Giroud, H. Y. V. Ching, A. Meunier,VieVw. AArmticbleikOen,linCe.
Dr. J.L. Guerquin-Kern, Dr. S. Trepout and Dr. T.-D. Wu, for useful
discussions and help in cryofixed samples sample preparation, Dr. F.
Lambert for help in synthesis, Z. Gueroui for access and help with
Amatore, M. Guille Collignon, F. Lemaître and C. Policar, Dalton Trans.,
2
2
DOI: 10.1039/D0CC03398G
012, 41, 6399-6403.
4. A. Vincent, J. R. Fores, E. Tauziet, E. Quévrain, A. Dancs, A. Conte-Daban,
the fluorescence microscope at ENS and S. Clède for help in the A.-S. Bernard, P. Pelupessy, K. Coulibaly, P. Seksik, C. Hureau, K. Selmeczi, C.
recording of fluorescence images, B. Goetz for help with ICP-MS, Pr. Policar and N. Delsuc, Chem. Commun., 2020, 56, 399-402.
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