The synthesis and functional evaluation of a mitochondria-targeted hydrogen sulfide donor, (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)decyl) triphenylphosphonium bromide (AP39)

Article abstract of DOI:10.1039/c3md00323j

Synthesis and bioavailability of the endogenous gasomediator hydrogen sulfide (H₂S) is perturbed in many disease states, including those involving mitochondrial dysfunction. There is intense interest in developing pharmacological agents to generate H₂S. We have synthesised a novel H₂S donor molecule coupled to a mitochondria-targeting moiety (triphenylphosphonium; TPP⁺) and compared the effectiveness of the compound against a standard non-TPP⁺ containing H₂S donor (GYY4137) in the inhibition of oxidative stress-induced endothelial cell death. Our study suggests mitochondria-targeted H₂S donors are useful pharmacological tools to study the mitochondrial physiology of H₂S in health and disease.

Full text of DOI:10.1039/c3md00323j

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CONCISE ARTICLE
The synthesis and functional evaluation of a
mitochondria-targeted hydrogen sulfide donor,
(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)-
phenoxy)decyl)triphenylphosphonium bromide
(AP39)
Cite this: DOI: 10.1039/c3md00323j
Sophie Le Trionnaire,a Alexis Perry,b Bartosz Szczesny,c Csaba Szabo,c
a
a
b
*
Paul G. Winyard, Jacqueline L. Whatmore, Mark E. Wood
a
*
and Matthew Whiteman
Synthesis and bioavailability of the endogenous gasomediator hydrogen sulfide (H2S) is perturbed in many
disease states, including those involving mitochondrial dysfunction. There is intense interest in developing
pharmacological agents to generate H2S. We have synthesised a novel H2S donor molecule coupled to a
mitochondria-targeting moiety (triphenylphosphonium; TPP+) and compared the effectiveness of the
compound against a standard non-TPP+ containing H2S donor (GYY4137) in the inhibition of oxidative
stress-induced endothelial cell death. Our study suggests mitochondria-targeted H2S donors are useful
pharmacological tools to study the mitochondrial physiology of H2S in health and disease.
Received 26th October 2013
Accepted 5th April 2014
DOI: 10.1039/c3md00323j
www.rsc.org/medchemcomm
highlighted the therapeutic potential of pharmacological
strategies to increase H2S bioavailability in these pathologies.
1 Introduction
There is extensive evidence for a role of oxidative stress, and
the over-production of detrimental oxidants, in the vascular
endothelium of several human disease states including dia-
betes, obesity, hypertension and stroke.11 Oxidant species such
as peroxynitrite (ONOOꢀ) and hydrogen peroxide (H2O2), as well
as the products of biomolecule oxidation such as the lipid-
derived aldehyde 4-hydroxynonenal (4-HNE), are well known to
induce endothelial and mitochondrial dysfunction and cell
death.11
Mitochondria are key intracellular organelles determining
cell fate (survival and death) and they are a key source of (and
target for) detrimental oxidant production within cells.12 As
such, compounds that can ‘target’ mitochondria and prevent
or limit mitochondria-mediated oxidative stress have received
considerable attention. Lipophilic cations such as triphenyl-
phosphonium (TPP+) have been extensively used for this
purpose.13 TPP+ derivatives are extensively and rapidly taken
up by mitochondria in vivo, due to the large negative
membrane potential inside the mitochondria (Djm), resulting
in a high concentration of the TPP+-derivatives within the
mitochondria.13 The covalent attachment of the lipophilic
TPP+ cation has become established as a generic and robust
method for selectively delivering small, bioactive molecules to
mitochondria in vivo.13
Hydrogen sulde (H2S) is now convincingly established as the
third gasotransmitter,1–3 alongside the more familiar nitric
oxide (NO) and carbon monoxide (CO). It is enzymatically
produced from amino acids cysteine, homocysteine and cys-
tathionine by two pyridoxal-50-phosphate-dependent enzymes,
namely cystathionine-g-lyase (CSE) and cystathionine-b-syn-
thase (CBS).1–3 A third a-ketoglutarate and cysteine-dependent
enzyme, located in the mitochondria of cells, 3-mercaptopyr-
uvate sulfurtransferase (3-MST)4 has also been proposed to
generate H2S within the mitochondria.5
In the vascular system, H2S has been proposed as an endo-
thelium-derived vasodilator and genetic knock-out of CSE in
animals results in hypertension6 and accelerates atheroscle-
rosis.7 Similarly, decreased H2S biosynthesis and/or bioavail-
ability have been observed in various human disease states (and
animal disease models)1–3 including those with marked mito-
chondrial and endothelial dysfunction and oxidative stress such
as hypertension, diabetes, obesity,8 pre-eclampsia9 and aer
myocardial infarction.10 Collectively, these studies have
aUniversity of Exeter Medical School, St. Luke's Campus, Magdalen Road, Exeter EX1
2LU, England, UK. E-mail: m.whiteman@exeter.ac.uk; Tel: +44 (0)1392 722942
bBiosciences, College of Environmental and Life Sciences, University of Exeter, Geoffrey
Pope Building, Stocker Road, Exeter EX4 4QD, England, UK. E-mail: m.e.wood@exeter.
ac.uk; Tel: +44 (0)1392 723450
H2S donor compounds such as GYY4137 1 (Fig. 1)14 have
been shown to exert prominent vasodilatory activity in vitro14,15
and in hypertensive animals in vivo.9,14,15 GYY4137 1 also
cDepartment of Anesthesiology, University of Texas Medical Branch, Galveston, TX,
USA
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Fig. 1 H2S donors; GYY4137 1, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-
3-thione 2.
inhibited inammatory signalling in in vitro16–19 and in vivo
models of sepsis,18 arthritis,20 pre-eclampsia9 and atheroscle-
rosis.21 Furthermore, GYY4137 has also been shown to inhibit
oxidative stress-mediated cytotoxicity and loss of mitochondrial
function induced by H2O2, ONOOꢀ and 4-HNE.22 However,
GYY4137 is highly water soluble,14 limiting its cellular perme-
ability, and the above studies have all used high concentrations
of GYY4137 ($100 mM) for anti-inammatory, vasodilatory,
cytoprotective and mitochondrial protective effects to be seen.
The need for such high concentrations may further limit its
therapeutic potential and alternative strategies for generating
potent H2S donor molecules would be useful.
Dithiolethiones, such as 2, have been proposed as novel
species for H2S delivery (reviewed in ref. 23) (Fig. 1). Dithiole-
thione derivatives of non-steroidal anti-inammatory drugs (e.g.
naproxen, diclofenac, indomethacin) have been shown to
generate H2S in vivo, promote gastro-intestinal blood ow and
prevent gastric injury in vivo, and exert prominent anti-inam-
matory effects with marked additional pharmacological benet
over the parent NSAID alone.23–28
Scheme
1
Preparation of AP39 3. Reagents and conditions: (a)
triphenylphosphine (1.0 equiv.), CH3CN, reflux, 70 h; (b) 5-(4-
hydroxyphenyl)-3H-1,2-dithiole-3-thione 2 (1.0 equiv.), N,N-dicyclo-
hexylcarbodiimide (1.0 equiv.), 4-dimethylaminopyridine (0.05 equiv.),
CH2Cl2, room temp., 22 h (73% from 5).
cerebral microvascular endothelial cells (hCMEC/D3) (Fig. 2A).
Furthermore, exposure of bEnd.3 cells to AP39 3 (100 nM) for 1
hour resulted in an increase in the uorescence of an alternative
H2S uorophore AzMC, with signicant co-localisation of the
signal to mitochondria (Mitotracker) (Fig. 2B).
2.3 Cytotoxicity
Preliminary control experiments were conducted to conrm
that the addition of up to 5 mM AP39 and 2 mM GYY4137 for 24
h did not induce signicant cytotoxicity compared with vehicle
controls (alamarBlue®; data not shown). Fig. 3 shows concen-
tration-dependent (7–250 nM) inhibition of oxidative stress-
mediated toxicity in hCMEC/D3 cells by AP39 3 and GYY4137 1
induced by [A] SIN-1 (500 mM), [B] H2O2 (50 mM) and [C] 4-HNE
(10 mM). The concentration where 50% of cell viability was
recovered was estimated to be 25 nM (vs. SIN-1), 30 nM (vs.
H2O2) and >125 nM (vs. 4-HNE). In order to show that the
cytoprotective effects were due to AP39 3 and not the constituent
parts of the AP39 molecule, cells were incubated with 100 nM of
the H2S donor moiety (ADT-OH, 2), the phosphonium salt
containing the mitochondria-targeting motif 4 individually and
in combination, and then exposed the cells to SIN-1, H2O2 or 4-
HNE. Neither ADT-OH 2, compound 4 (TPP-decanoate) or 100
nM each of ADT-OH 2 with 4, induced signicant cytoprotection
against oxidative stress-mediated cytotoxicity (Fig. 3A–C) con-
rming that cytoprotection was due to AP39 3.
Therefore, with the above observations in mind, we have
synthesised a TPP+-derivative of dithiolethione 2 to generate a
novel mitochondria-targeted H2S donor molecule. We have then
evaluated its effects in a cellular model of oxidative stress and
compared its efficacy with the established standard H2S donor,
GYY4137 1.
2 Results
2.1 Synthesis
AP39 3 was prepared in an overall yield of 73% using the two-
step process shown in Scheme 1. Phosphonium salt 4 was
prepared from 10-bromodecanoic acid 5 and triphenylphos-
phine under reux conditions in acetonitrile33 and coupled,
using standard carbodiimide conditions, to 5-(4-hydroxy-
phenyl)-3H-1,2-dithiole-3-thione 2.26 Final purication of 3 was
achieved using ash chromatography on silica gel (eluting with
methanol), the product forming a crisp foam (which could be
handled as a solid) on removal of the solvent.
We next compared the efficacy of AP39 3 against an estab-
lished H2S donor compound GYY4137 1, which has previously
been shown to protect human mesenchymal progenitor cells
and articular chondrocytes from oxidative stress-mediated
2.2 Intracellular generation of H2S from AP39
Fig. 2 shows the generation of H2S from AP39 3, detected using toxicity.22 Fig. 3 also shows that when used at the same
two different uorescence probes selective for sulde, WSP-1 concentration as AP39 (7–250 nM), GYY4137 1 was not cyto-
(ref. 29 and 30) and 7-azido-4-methylcoumarin (AzMC).31,32 AP39 protective. However, as expected14–20 signicant inhibition of
induced a concentration-dependent increase in WSP-1 uores- oxidative stress-induced cell death was observed with much
cence, indicative of intracellular H2S generation29 in human higher concentrations (e.g. $100 mM) (Fig. 3D). From these
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Fig. 2 Intracellular generation of H2S from AP39. Endothelial cells
were treated with 7–250 nM AP39 for 1 hour and intracellular H2S was
detected in human hCMEC/D3 cells using WSP-1 [A]. Co-localisation
of mitochondria and H2S was visualised after 1 hour treatment of
murine bEnd.3 cells with AP39 (100 nM) by fluorescence microscopy
using MitoTracker Red (for mitochondria) and AzMC (for H2S) [B].
Images are representative images of three or more independent
experiments. Note the concentration-dependent increase in H2S
signal which, at least in part, co-localises within the mitochondrial
regions suggesting AP39 generated H2S mainly in mitochondria.
studies, concentrations of 100 nM AP39 and 100 mM GYY4137 1
were used for subsequent experiments.
Fig.
3
Concentration-dependent inhibition of oxidative-stress
induced cell death by AP39 and GYY4137. hCMEC/D3 cells were
incubated with increasing concentrations of AP39 3 and GYY4137 1 (7–
250 nM) for 1 hour and [A] SIN-1 (500 mM), [B] H2O2 (50 mM) and [C] 4-
HNE (10 mM) added. Higher concentrations of GYY4137 1 (10–500 mM)
2.4 Effects of H2S donors on mitochondrial membrane
potential (DJm), ATP and mitochondrial oxidant production
Fig. 4 shows AP39 3 (100 nM) and GYY4137 1 (100 mM) signi- were also examined [D]. Control experiments were also performed
using ADT-OH 2, compound 4, alone and in combination [A–C]. Cell
viability was assessed by alamarBlue assay after 24 hours. *p < 0.05,
**p < 0.01 and *** p < 0.001 H2S donors cf. oxidant treatment.
cantly inhibited oxidative stress-induced loss of mitochondrial
membrane potential (Fig. 4A) and inhibited mitochondrial
production of reactive oxygen species, detected using MitoSOX
Red (Fig. 4B). Incubation of cells with AP39 3 (100 nM) or
GYY417 1 (100 mM) alone had no signicant effect on mito-
chondrial membrane potential or mitochondrial oxidant
production (data not shown).
3 Experimental
3.1 General synthetic chemistry methods
Furthermore, AP39 (100 nM) signicantly inhibited mito-
chondrial oxidant production induced by rotenone (5 mM), an Starting materials and reagents were obtained from commercial
inhibitor of mitochondrial respiratory complex I (Fig. 5A) and suppliers (Sigma-Aldrich and Fisher Scientic) and were used
loss of ATP (Fig. 5B). GYY4137 also prevented rotenone-induced without further purication. Infrared spectra were recorded
mitochondrial toxicity, albeit when used at a nal concentration using a Perkin Elmer Spectrum BX FT-IR spectrometer. Only
of 100 mM.
major absorbances are quoted, using the abbreviations: (for
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Fig. 5 Inhibition of rotenone induced mitochondrial toxicity by H2S
donors. Human hCMEC/D3 endothelial cells were incubated with
AP39 3 (100 nM) or GYY4137 1 (100 mM) for 1 hour prior to the addition
of rotenone (5 mM). Mitochondrial oxidant production was assessed
using MitoSOX (5 mM) [A] and ATP levels determined using firefly
lantern luciferase [B]. ##p < 0.01 and ###p < 0.001 oxidant treated
cells cf. control; **p < 0.01, ***p < 0.001 H2S donors cf. oxidant
treatment.
Fig. 4 Inhibition of oxidative-stress induced cell toxicity by H2S
donors. Human hCMEC/D3 endothelial cells were incubated with
AP39 3 (100 nM) or GYY4137 1 (100 mM) for 1 hour prior to the addition
of SIN-1 (500 mM), H2O2 (50 mM) or 4-HNE (10 mM). After 24 hours,
mitochondrial membrane potential [A] was determined using TMRM
and mitochondrial oxidant production [B] determined using MitoSOX.
###p < 0.001 oxidant treated cells cf. control; *p < 0.05, **p < 0.01,
***p < 0.001 H2S donors cf. oxidant treatment.
staining with alkaline potassium permanganate solution as
appropriate. Silica gel with particle size 40–63 mm was used for
ash chromatography. Solvents and reagents were used as
supplied commercially by Sigma-Aldrich and Fisher Scientic
without additional purication.
¨
A Buchi R110 Rotavapor was used for the removal of solvents
intensity) w, weak; m, medium; and s, strong. Samples were
prepared as potassium bromide discs. 1H NMR spectra were
recorded at 300 MHz using a Bruker ACF300 spectrometer.
Chemical shis are quoted in ppm relative to tetramethylsilane,
the residual solvent peak being used for referencing purposes.
Coupling constants are quoted to the nearest 0.5 Hz with peak
under reduced pressure (ca. 15 mmHg).
3.2 (10-Oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)-
decyl)triphenylphosphonium bromide (AP39) 3
multiplicities for single resonances being labelled as: s, singlet; A solution of 10-bromodecanoic acid 5 (500 mg, 1.99 mmol) in
d, doublet; t, triplet; m, unresolved multiplet, AA0BB0, AA0BB0 acetonitrile (5 cm3) was added to a stirred solution of triphe-
pattern and br, broad. Where delineation of individual reso- nylphosphine (522 mg, 1.99 mmol) in acetonitrile (5 cm3) and
nances is not possible, the relevant peaks are grouped together the resulting mixture was heated at reux for 70 h.33 Aer
(over a chemical shi range) as complex.
cooling to room temperature and evaporation of the solvent in
31P NMR spectra were recorded at 121 MHz using a Bruker vacuo, the residue was triturated with toluene (2 ꢁ 10 cm3) and
ACF300 spectrometer with external automatic referencing.
dissolved in dichloromethane (30 cm3). 5-(4-Hydroxyphenyl)-
13C NMR spectra were recorded at 75 MHz using a Bruker 3H-1,2-dithiole-3-thione26 2 (456 mg, 2.02 mmol), N,N-dicyclo-
ACF300 spectrometer. Chemical shis are quoted in ppm rela- hexylcarbodiimide (431 mg, 2.09 mmol) and 4-dimethylami-
tive to tetramethylsilane, the solvent peak being used for nopyridine (12 mg, 0.10 mmol) were added and the resulting
referencing. Coupling constants are quoted to the nearest 1 Hz solution was stirred at room temperature for 22 h before
with peak multiplicities being labelled using (where appro- ltering through a cotton wool plug and evaporation of the
priate) the abbreviation: d, doublet.
ltrate in vacuo. A dichloromethane solution of the residue was
Mass spectra were recorded by the EPSRC National Mass loaded onto a silica gel ash chromatography column, which
Spectrometry Service Centre, Swansea, UK, in positive ion was subsequently eluted with ethyl acetate, followed by meth-
electrospray (ES+) mode.
anol. The methanol fractions were combined and evaporated
Analytical thin layer chromatography was carried out using in vacuo and a dichloromethane solution of the residue was
Merck Kieselgel 60 F254, coated on aluminium plates, with vis- ltered through a lter paper to remove residual silica gel
ualisation of spots by quenching of UV (254 nm) uorescence or before evaporation in vacuo to give the title compound 3 (1.05 g,
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73%) as a crisp, orange foam (found [M ꢀ Br]+ (ES+) 641.1766, loaded with 7-azido-4-methylcoumarin (AzMC, SIGMA
C
37H38O2PS3 requires 641.1766); nmax (KBr disc)/cmꢀ1 2925 (m), L511455)30,31 and MitoTracker Red CMXRos (Invitrogen M7512)
2852 (m), 1754 (m) (C]O), 1599 (w), 1521 (w), 1485 (s), 1437 (s), uorogenic dyes at 10 mM and 50 nM nal concentration,
1410 (m), 1317 (m), 1278 (m), 1185 (s), 1168 (s), 1112 (s), 1026 respectively, for 30 min. Next, 100 nM of AP39 3 was added with
(s), 996 (m), 896 (w), 839 (w), 747 (m), 722 (m), 690 (m), 535 (m) fresh media and cells were further incubated for 1 hour, washed
and 508 (m); 1H NMR (300 MHz, CDCl3) 7.95–7.65 (17H, m, aryl twice with PBS containing calcium and magnesium. 50 ml of PBS
C(2)H, aryl C(6)H and phenyl CH), 7.42 (1H, s, CHC]S), 7.23 was then added to each chamber and micro cover glass was
(2H, part of AA0BB0, J ¼ 8.5 Hz, aryl C(3)H and C(5)H), 3.85 (2H, attached. The dye's specic uorescence was visualized under
m, CH2P+), 2.58 (2H, t, J ¼ 7.5 Hz, CH2C]O), 1.80–1.55 (6H, m, 40ꢁ magnication using a Nikon eclipse 80i inverted micro-
3 ꢁ CH2) and 1.42–1.19 (8H, m, 4 ꢁ CH2); 31P NMR (121 MHz, scope with Photometric CoolSNAP HQ2 camera and NIS-
CDCl3) 25.7 (P+); 13C NMR (75 MHz, CDCl3) 214.9 (C]S), 183.7 Elements BR 3.10 soware. An additional sulde-specic
(CH]C–S), 171.7 (C]O), 153.6 (arylC–O), 135.8 (arylC–C]CH), uorophore, WSP-1 (Caymen, 11179-5)29,30 was used to
134.9 (phenylC–H), 133.6 (d, J ¼ 9 Hz, phenylC–H), 130.4 (d, J ¼ determine intracellular generation of H2S. In these experi-
13 Hz, phenylC–H), 128.9 (C]CH–C(S)), 128.1 (arylC–H), 122.9 ments, cells were seeded overnight in black 96 well microplates
(arylC–H), 118.3 (d, J ¼ 83 Hz, phenylC–P+), 34.2 (CH2C(O)), 30.3 (Nalgen Nunc International) and incubated at 37 ꢂC with 10 mM
(d, J ¼ 13 Hz, CH2), 29.0 (CH2), 28.9 (2 ꢁ CH2), 28.8 (CH2), 24.6 WSP-1 for 30 min.30 Aer this time, cells were washed with pre-
(CH2), 23.0 (CH2) and 22.5 (d, J ¼ 18 Hz, CH2P+).
warmed phenol-red free culture media and AP39 (10–250 nM)
added and WSP-1 uorescence determined (Ex465nm, Em515nm
)
3.3 Morpholin-4-ium-4 methoxyphenyl(morpholino)
phosphinodithioate (GYY4137)
using a Molecular Devices M2e microplate reader.
3.6 Cytotoxicity assays
GYY4137 was synthesised as previously described.14
To induce oxidative stress, hCMEC/D3 cells were exposed to
either H2O2 (100 mM), SIN-1 (500 mM) or the lipid aldehyde
product of lipid peroxidation in vivo, 4-hydroxynonenal (4-HNE;
10 mM) for 24 hours in the presence or absence of H2S donors
GYY4137 1 (100 mM), AP39 3 (100 nM), ADT-OH 2 (100 nM) or
compound 4 (100 nM). Aer this time cell viability, as an end
point for cytotoxicity, was assessed by alamarBlue® assay (AbD
Serotec; #BUF012) according to the manufacturer's instruc-
tions. Fluorescence was then determined using a Molecular
Devices M2e plate reader (uorescence; Ex540nm/Em612nm) as
described. Data are expressed as % of untreated or vehicle
treated cells (% viability).
3.4 Cell culture
Human cerebral microvascular endothelial cells (hCMEC/D3)
were isolated from human brain and kindly provided by Dr
Pierre-Olivier Couraud, University of Paris Descartes.34–36 The
bEnd.3 mouse microvascular endothelial cell line was
purchased from the American Type Culture Collection (ATTC
#CRL-2299, Manassas, VA).37,38
The vascular endothelium is a prime target for oxidative
stress in vivo and loss of endothelial function and cell death are
observed in cardiovascular diseases such as atherosclerosis,
hypertension and diabetes.39,40 Therefore we used these cells as
an in vitro model of cellular oxidative stress.
3.7 Assessment of mitochondrial membrane potential
(Dcm)
hCMEC/D3 cells were seeded on type I collagen (Sigma, rat
tail; dilution 1 : 30) and cultured in endothelial cell media MV2
(PromoCell; C-22221) with a supplement mix (PromoCell; C- Mitochondrial membrane potential (DJm) in whole hCMEC/D3
39226) diluted to give fetal bovine serum (2.5%), epidermal cells was measured by using the uorescent potentiometric
growth factor (1.25 ng mlꢀ1), basic broblast growth factor probe tetramethylrhodamine methyl ester (TMRM; Invitrogen).
(2.5 ng mlꢀ1), insulin-like growth factor (5 ng mlꢀ1), vascular TMRM is a membrane-permeable cationic uorophore that
endothelial growth factor 165 (0.125 ng mlꢀ1), ascorbate electrophoretically accumulates in mitochondria in response to
(0.25 mg mlꢀ1) and hydrocortisone (0.05 ng mlꢀ1). hCMEC/D3 their negative potential and decrease of TMRM uorescence is
cells constitutively express microvascular endothelial cell indicative of DJm disruption and mitochondrial and cellular
markers including von Willebrand factor, CD31 (PECAM-1) and toxicity. Cells were incubated with SIN-1 (500 mM), H2O2 (100
CD105 (endoglin) up to passage 35. Cells used in this current mM) or 4-HNE (10 mM) in the presence or absence of H2S donors
study were between passages 25–34. The commercial bEnd.3 for 18 h and aer this time, cells were incubated for 30 min with
mouse microvascular endothelial cell line was cultured as TMRM (200 nM), washed with warm (37 ꢂC) phosphate buffered
described.37,38 Murine bEnd.3 cells were maintained in DMEM saline (pH 7.4) and TMRM uorescence (Relative Fluorescence
containing glucose (1 g lꢀ1) supplemented with 10% fetal bovine Units; RFU) determined by uorimetry (Ex548nm/Em573nm).41,42
serum (10%), non-essential amino acids, (1%) penicillin (100
IU mlꢀ1) and streptomycin (100 mg mlꢀ1) at 37 ꢂC in 10% CO2.
3.8 ATP determination
hCMEC/D3 cells were seeded overnight on 24 well plates
(Greiner) containing 1.0 ml culture media and treated with the
3.5 Detection of hydrogen sulde in living endothelial cells
Cells were seeded in Lab-Tek II chamber cover glass (Nalgen respiratory chain inhibitor rotenone (5 mM), and the oxidants
Nunc International) and incubated overnight at 37 ꢂC and 10% SIN-1 (500 mM), H2O2 (100 mM) or 4-HNE (10 mM) in the presence
CO2 humidied incubator. Aer attachment, the cells were or absence of AP39 (100 nM) or GYY4137 (100 mM) for 18 hours.
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Aer this time, cells were washed twice with ice cold phosphate vascular endothelial cells.52 However, in each of these studies,
buffered saline (PBS) and lysed with 100 ml of ice-cold tri- the concentrations of H2S required for each of these studies are
chloroacetic acid (TCA; 10% w/v). 5 ml of TCA lysate was then oen vastly excessive (typically >1–200 mM) and well beyond that
added to 200 ml of arsenite buffer (containing 80 mM MgSO4, 10 seen in health or extreme pathology. Although the absolute level
mM KH2PO4 and 100 mM Na2HAsO4, mixed in a 1 : 1 : 1 ratio; of free H2S gas in the vasculature is not known, the most recent
pH 7.4) followed by 10 ml rey lantern extract (containing analytical techniques have suggested levels of free H2S are in the
luciferase; Sigma).41,42 Emitted light was recorded on a GloMax nM to low mM range, suggesting that many of the above cellular
Jr, (Promega) luminescence detector as described. ATP studies using NaSH or Na2S may not reect the endogenous
concentrations were determined using a freshly prepared effects of physiologically relevant concentrations of H2S found
standard curve and samples normalised to cellular protein in the vasculature.2
content using a commercial bicinchoninic acid-based kit
(Bio-Rad).
We recently showed RNAi-mediated knockdown of CSE
rendered mitochondria more sensitive to oxidative stress-
induced mitochondrial toxicity, an effect which was overcome
by the H2S donor GYY4137 1 (albeit $100 mM) suggesting that
endogenous H2S may function to preserve mitochondrial
integrity.22 This is supported by in vivo studies. For example, in a
rodent cardiac ischaemia-reperfusion model, both Na2S infu-
sion and selective cardiac CSE over-expression preserved mito-
chondrial ultrastructure, preserved ATP generation and
respiratory chain function in vivo,45 possibly via a mitochondrial
pathway involving the preservation of Bcl-2 signalling, inhibi-
tion of Bax, upregulation of PKC and opening of mitochondrial
3.9 Intracellular oxidant production
hCMEC/D3 cells were treated with oxidants and H2S donors
(GYY4137 1, 100 mM; AP39 3, 100 nM) as described above. The
production of mitochondria-derived reactive oxygen species was
determined by uorimetry using the mitochondria-targeted
oxidant probe MitoSOX Red™ (Invitrogen, 5 mM) and uores-
43
cence determined (RFU) Ex390/Em580nm
.
3.10 Statistical analysis
K
ATP-channels.10,52
All graphs are plotted with mean ꢃ SEM. In all cases, the mean
values were calculated from data taken from at least six separate
experiments, performed on separate days using freshly
prepared reagents. Where signicance testing was performed,
an independent t-test (Student's; two populations) was used;
*P < 0.1, **P < 0.05, ***P < 0.01.
Collectively, these in vitro and in vivo studies have strongly
suggested that mitochondria are key regulatory targets for
endogenous and exogenously applied H2S suggesting that
compounds which deliver H2S to mitochondria could be ther-
apeutically useful. To test this hypothesis in vitro, we exposed
microvascular endothelial cells to oxidative insult and exam-
ined cell viability and mitochondrial toxicity aer oxidative
insult using
dithiolethione (AP39, 3) and a well established but non-targeted
a
novel mitochondria-targeted TPP+-derived
4 Discussion
H2S has been proposed as a novel cytoprotective mediator.22,44–46 H2S donor molecule GYY4137 1. Concentrations of GYY4137
In isolated mesenchymal progenitor cells RNAi-mediated (100 mM) were chosen based on Fig. 3D and the cytoprotective
knockdown of CSE potentiated SIN-1, H2O2 and 4-HNE-medi- effects reported for this compound in a variety of cellular
ated mitochondrial toxicity, an effect reversed with GYY4137.22 models.14–22 In each assay (Fig. 3–5), AP39 3 signicantly
Genetic knockout of CSE renders endothelial6 and striatal inhibited oxidative stress induced toxicity at concentrations
Q11147 cells more sensitive to oxidative stress-induced toxicity more than 1000-fold lower than that of GYY4137 (e.g. 100 nM
and blood vessels more prone to atherosclerosis.21 However, it AP39 cf. 100 mM GYY4137). In contrast, incubation of cells with
should be noted that this is not seen in all models. For example, TPP+–decanoate (compound 4) which targets mitochondria, but
hepatocytes from CSEꢀ/ꢀ mice exposed to galactosamine/lipo- cannot generate H2S or the H2S donor moiety ADT-OH alone
polysaccharide were shown to be more sensitive to oxidative (e.g. lacking TPP+), did not prevent oxidant-induced cell death
injury than wild type animals,48 although it is unclear if the (Fig. 3A–C). Similarly, incubation of cells with compound 4 and
effects were mediated by H2S since CSE knock out did not ADT-OH 2 together also failed to show cytoprotective effects
signicantly decrease plasma or liver H2S levels and markedly indicating that the whole AP39 molecule (i.e. mitochondria-
increased plasma homocysteine levels.48 Nevertheless, in cell targeted H2S delivery) was required for cell preservation
culture experiments, H2S/HSꢀ generated from NaSH (albeit at (Fig. 3A–C). Whilst our study clearly shows inhibition of
$100 mM) has been shown to prevent detrimental cytotoxic oxidative stress-induced cellular toxicity and mitochondrial
effects and oxidative protein modication induced by oxidants preservation by AP39, it is not possible to determine from these
such as hydrogen peroxide, superoxide, hypochlorite and per- data whether mitochondrial protection was the cause or a
oxynitrite (reviewed in ref. 46). In neuronal cells, NaSH ($100 consequence of cytoprotection. Nevertheless, TPP+-derivatised
mM) also inhibited oxidative stress-mediated cell death aer H2S donor compounds such as AP39 may be useful tools in
treatment with b-amyloid49 or the mitochondrial toxin 1-methyl- studying mitochondria–H2S interactions.
4-phenylpyridinium (MPP+).50 Furthermore, H2S is also reported
Although further work is required to dene the precise bio-
to inhibit the expression and activity of NAD(P)H oxidase, a key molecular pathways by which AP39 3 prevents cellular injury,
source of extra-mitochondrial oxidant species51 and it upregu- the pathways for endogenous mitochondrial generation of H2S
lates the expression of the antioxidant enzyme thioredoxin-1 in have recently been uncovered.5,52–56 The mitochondrial enzyme
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3-MST generates low levels of H2S and 3-MST-derived H2S
concentrations in the nanomolar range were very recently
shown to contribute electrons to the Krebs cycle and enhance
mitochondrial electron transport and bioenergetics,5 an effect
blocked during oxidative stress54 but reversed with exogenous
H2S.54 Recently, CSE53 and CBS54,55 have been shown to trans-
locate from the cytosol in cells to the mitochondrial matrix in
response to hypoxic and calcium stress, to generate H2S within
the mitochondria, possibly acting as an electron acceptor in the
mitochondrial respiratory chain, resulting in increased cellular
ATP production and cell preservation.53–56 It is possible that
these effects were also mediated by AP39-derived H2S, contrib-
uting to the overall cytoprotective effects of this compound
since we observed co-localisation of H2S generation with mito-
chondria (Fig. 2B), preservation of ATP levels (Fig. 5B) and
mitochondrial Djm (Fig. 4A), and decreased mitochondrial
oxidant production (Fig. 4B and 5A). Therefore, it appears that
the endogenous enzymes responsible for H2S generation are
either located within or translocate to the mitochondria in
response to cellular stress and mitochondria-derived H2S
contributes to overall cellular bioenergetics and cellular health.
Since mitochondrial production of H2S is perturbed by oxidative
stress, whilst mitochondrial dysfunction and oxidative stress
are key pathological events in several human disease states
(including hypertension, stroke, neurodegenerative disease,
arthritis, hepatitis etc.; reviewed in ref. 1–3), it is likely that
mitochondrial H2S delivery may represent a novel approach to
study mitochondrial function in health and disease. This
potentially reveals a novel therapeutic strategy to treat (or limit)
mitochondrial dysfunction in human disease.
2 M. Whiteman, S. Le Trionnaire, M. Chopra, B. Fox and
J. Whatmore, Emerging role of hydrogen sulde in health
and disease: critical appraisal of biomarkers and
pharmacological tools, Clin. Sci., 2011, 121, 459–488.
3 M. S. Vandiver and S. H. Snyder, Hydrogen sulde: a
gasotransmitter of clinical relevance, J. Mol. Med., 2012, 90,
255–263.
4 N. Shibuya, M. Tanaka, M. Yoshida, Y. Ogasawara,
T. Togawa, K. Ishii and H. Kimura, 3-Mercaptopyruvate
sulfurtransferase produces hydrogen sulde and bound
sulfane sulfur in the brain, Antioxid. Redox Signaling, 2009,
11, 701–714.
´
´
5 K. Modis, C. Coletta, K. Erdelyi, A. Papapetropoulos and
C. Szabo, Intramitochondrial hydrogen sulde production
by
3-mercaptopyruvate
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6 G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng,
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H2S as a physiologic vasorelaxant: hypertension in mice
with deletion of cystathionine gamma-lyase, Science, 2008,
322, 587–590.
7 S. Mani, H. Li, A. Untereiner, L. Wu, G. Yang, R. C. Austin,
´
J. G. Dickhout, S. Lhotak, Q. H. Meng and R. Wang,
Decreased endogenous production of hydrogen sulde
accelerates atherosclerosis, Circulation, 2013, 25(127),
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8 M. Whiteman, K. M. Gooding, J. L. Whatmore, C. I. Ball,
D. Mawson, K. Skinner, J. E. Tooke and A. C. Shore,
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5 Conclusions
9 K. Wang, S. Ahmad, M. Cai, J. Rennie, T. Fujisawa, F. Crispi,
J. Baily, M. R. Miller, M. Cudmore, P. W. Hadoke, R. Wang,
Our current study shows that coupling H2S generating dithio-
lethione (AP39) to a moiety well characterised to enable selective
targeting of intracellular mitochondria (TPP+) greatly increased
the cytoprotective potency of H2S relative to a standard and non-
targeted H2S donor compound GYY4137. Selective pharmaco-
logical manipulation of mitochondrial H2S may represent a
novel approach to preserve mitochondrial function and prevent
oxidative stress-induced cellular injury in vivo.
´
E. Gratacos, I. A. Buhimschi, C. S. Buhimschi and
A. Ahmed, Dysregulation of hydrogen sulde producing
enzyme cystathionine-g-lyase contributes to maternal
hypertension and placental abnormalities in preeclampsia,
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10 M. Lavu, S. Bhushan and D. J. Lefer, Hydrogen sulde-
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medicine, Oxford University Press, 2007, ISBN-10: 019856869X.
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13 R. A. Smith, R. C. Hartley, H. M. Cocheme and M. P. Murphy,
Mitochondrial Pharmacology, Trends Pharmacol. Sci., 2012,
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Acknowledgements
The authors are grateful to the EPSRC National Mass Spec-
trometry Service Centre (Swansea, UK) for determination of
mass spectra and to Ms Srijana Rai for her technical support.
We are also grateful to the American Diabetes Association (#7-
12-BS-184) and the Medical Research Council (UK; G0800107)
for their generous research support.
14 L. Li, M. Whiteman, Y. Y. Guan, K. L. Neo, Y. Cheng,
S. W. Lee, Y. Zhao, R. Baskar, C. H. Tan and P. K. Moore,
Characterisation of
a
novel, water-soluble hydrogen
sulde-releasing molecule (GYY4137). New insights into
the biology of hydrogen sulde, Circulation, 2008, 117,
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