Ammonium tetrathiomolybdate as a water-soluble and slow-release hydrogen sulfide donor

Article abstract of DOI:10.1016/j.bmcl.2016.02.005

Ammonium tetrathiomolybdate (TTM) was found to be a slow hydrogen sulfide (H₂S) releasing agent. Its H₂S generation capability in aqueous solutions was confirmed by UV–vis and fluorescence assays. TTM also showed H₂S-like cytoprotective effects in hydrogen peroxide (H₂O₂)-induced oxidative damage in HaCaT cells.

Full text of DOI:10.1016/j.bmcl.2016.02.005

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ammonium tetrathiomolybdate as a water-soluble and slow-release
hydrogen sulfide donor
a,y
b,y
b
a
b
a,
⇑
Shi Xu , Chun-Tao Yang , Fu-Hui Meng , Armando Pacheco , Li Chen , Ming Xian
a
Department of Chemistry, Washington State University, Pullman, WA 99164, United States
Department of Physiology, Guangzhou Medical University, Guangzhou 511436, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
2
Ammonium tetrathiomolybdate (TTM) was found to be a slow hydrogen sulfide (H S) releasing agent. Its
Received 5 January 2016
Revised 1 February 2016
Accepted 3 February 2016
Available online xxxx
2
H S generation capability in aqueous solutions was confirmed by UV–vis and fluorescence assays. TTM
2 2 2
also showed H S-like cytoprotective effects in hydrogen peroxide (H O )-induced oxidative damage in
HaCaT cells.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Ammonium tetrathiomolybdate
Hydrogen sulfide
Donor
Oxidative damage
Hydrogen sulfide (H
2
S) is newly recognized as a nitric oxide
suspected that certain sulfide-containing inorganic compounds
(
NO)-like gaseous transmitter that plays regulatory roles in many
might be able to serve as interesting H
2
S donors in aqueous solu-
1–5
physiological and pathological processes.
Endogenous produc-
tions. Like SNP, the release of H S from inorganic molecule-based
2
tion of H
2
S involves both enzymatic pathways (mediated by cys-
-lyase (CSE), and 3-
donors is likely to be the result of simple hydrolysis. As such, the
donors would be suitable for many biological studies. Herein we
wish to report the discovery of ammonium tetrathiomolybdate
tathionine b-synthase (CBS), cystathionine
mercaptopyruvate sulfurtransferase (3-MST)) and non-enzymatic
c
pathways (from the sulfane sulfur pools). Current knowledge
(TTM) as an effective inorganic H
TTM, with the formula (NH MoS
ing block in the chemistry of molybdenum.
copper chelator, TTM has been used therapeutically in the treat-
ment of copper toxicosis, especially for Wilson’s disease. It was
2
S donor.
strongly suggests that modulation of H
2
S could have potential
4
)
2
4
, is a commonly used build-
1
4,15
therapeutic values for certain disease states, including vasodila-
tion, anti-inflammation, anti-oxidation, and down regulation of
cellular metabolism under stress.1 Because of this, the search
As an excellent
–5
16
for compounds that can release H
activities of H S has become an attractive area in medicinal chem-
So far, a number of H S releasing compounds (also known
S donors), such as GYY4137, dithiothiones, N-mercapto-based
donors, persulfide-based donors, gem-dithiol-based donors, etc.,
2
S and mimic the beneficiary
previously noted that under strong acidic condition (5% H
2
SO
4
)
1
7
2
H S could be generated from TTM. We first wondered if TTM
2
istry.6
as H
–10
2
2
could produce H S under mild and biologically friendly conditions,
2
have been reported (Fig. 1).6
small organic molecules which can be triggered by certain biolog-
ically relevant or compatible reactions to release H S. Some of
On the other hand,
inorganic molecule-based donors, especially metal complexes,
which can slowly hydrolyze to release H S have not been reported.
Inspired by the fact that sodium nitroprusside (SNP), with the
–10
These compounds are normally
S
P
N
S
O
S
NH
2
O
S
2
them have shown promising bioactivities.6
–10
S
R
O
GYY4137
Dithiolethiones
2
O
S
SAc
SAc
O
AcHN
1
1–13
R
NHBu
formula Na
2
[Fe(CN)
5
NO], is a widely used NO donor,
we
S
N
H
Ph
S
R
R
O
O
⇑
Corresponding author. Tel.: +1 509 335 6073; fax: +1 509 335 8867.
E-mail address: mxian@wsu.edu (M. Xian).
These authors contributed equally to this work.
N-mercapto based donors
gem-Dithol-based donors
persulfide based donors
y
Figure 1. Representative H
2
S donors.
http://dx.doi.org/10.1016/j.bmcl.2016.02.005
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Xu, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.005

2
S. Xu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
especially under physiological pH. To this end, we measured H
release from TTM in four different pH buffers (5, 6, 7.4, and 8). Nor-
mally the release of H S from the donors can be determined by the
2
S
2
18
standard methylene blue (MB) method. However, a strong acidic
condition was involved in this method. As it is known that acidic
media would facilitate TTM hydrolysis, we envisioned that the
standard MB method was not appropriate. In this study, a zinc-sul-
fide precipitation based MB method was used.19 This method
should avoid the false positive signals caused by acid-promoted
TTM hydrolysis. Briefly, the solutions of 500
lM TTM were freshly
2
Figure 3. H S gas trapping set-up.
prepared in phosphate buffers under different pH. At different time
intervals, aliquots were taken to Eppendorf vials containing a mix-
ture of zinc acetate and NaOH solution. After 15 min, solid ZnS was
formed and collected by centrifugation. ZnS was then treated by a
MB cocktail. The resulted H
UV–vis measurements and calculated based on a calibration curve.
As shown in Figure 2, we observed immediate H S formation in all
of these TTM solutions. The level of H S in pH 5 was significantly
higher than the levels in other pH values. In all solutions we found
S concentrations were maintained in a stable level for a long
time (up to 15 h). These results indicate TTM is a stable and slow
release H S donor.
To further confirm the production of H
S gas trapping experiment was designed. As shown in Figure 3, a
solution of TTM was placed in a sealed glass vial. An Eppendorf vial
2
S concentrations were obtained by
2
2
H
2
2
2
S from TTM in buffers, a
H
2
2
0
containing a solution of WSP-5, a H
was also placed in the vial to trap the evaporated H
2
S specific fluorescent probe,
S from the TTM
2
solution. After incubation at 37 °C for 3 h, the trapping solution
was diluted and the fluorescence intensity (excitation at 502 nm,
emission at 525 nm) was measured. Both the positive and negative
controls (using the standard Na
also carried out using the same procedure for comparison. As
shown in Figure 4, TTM and Na S led to significant fluorescence
2
S solution or pure buffer) were
2
Figure 4. H S release of TTM detected by indirect fluorescent assay.
2
increases in the trapping solutions while the pure buffer did not
cause any detectable fluorescence increase. The pH dependence
One of the most well-studied biological functions of H
cytoprotective effects under oxidative stress. Being a H S donor,
TTM was expected to have similar effects. We then applied TTM
2
S is its
of H
was observed in pH 5 than in other pHs. These results further
demonstrated H S generation from TTM in buffers.
2
S release from TTM was also noted as higher fluorescence
2
2
2
Figure 2. H S release from TTM under different pHs.
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S. Xu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 5. Effects of TTM on H
under the treatment of 400 M H
concentrations of TTM for 1 h. (B) LDH release assay of cells treated with 400
in the absence or presence of 200 M TTM. Data were shown as the mean ± SE.
P < 0.01 versus Control group, P < 0.01 versus H alone group.
2
O
2
-induced cellular damage. (A) Cell viability assay
l
2 2
O
for 5 h in the absence or presence of various
lM
H
2
O
2
l
*
*
##
2 2
O
Figure 6. Effects of TTM on H
2
O
2
-induced MMP loss in HaCaT cells. Rh123 staining
followed by photofluorography for the observation of MMP. (A) Control cells, (B)
cells treated with 400 M H for 5 h, (C) cells treated with 400 M H
combined with pretreatment with 200 M TTM for 1 h. (D) Cells treated with TTM
l
2
O
2
l
2 2
O
in a cellular model of oxidative damage to study its cytoprotective
l
2
1,22
ability, using protocols established in our previous works.
shown in Figure 5A, hydrogen peroxide (H ) alone caused the
decrease of cell (HaCaT) viability due to oxidative damage. When
the cells were pre-treated with TTM (50, 100, 200 M), a dose-
dependent protective effect was observed. At a higher concentra-
tion (400 M), TTM had no protection, presumably because of
As
alone. (E) Quantitative analysis of the mean fluorescence intensity (MFI) of Rh123 in
2
O
2
group a–d using Image J software. Data were shown as the mean ± SE. **P < 0.01
versus Control group, *P < 0.05 versus H
2 2
O alone group.
l
cytoprotection against oxidative damage. These results should pro-
mote researchers to re-think the biological activities of TTM and
apply it to other studies.
l
the slight toxicity of TTM under high concentration. In addition,
lactate dehydrogenase (LDH) release assay was also carried out
to validate TTM’s cytoprotective effects. As shown in Figure 5B,
Acknowledgments
the exposure of cells to H
LDH release. However, LDH release was significantly reduced when
cells were pretreated with TTM (200 M).
2 2
O (400 lM) remarkably enhanced
This work was supported by the NIH
– United States
l
(
R01HL116571) and Natural Science Foundation of Guangdong
Mitochondria membrane potential (MMP) usually reflects
whether mitochondria are healthy, which further indicates if cells
are suffering from noxious damage. Rhodamine 123 (Rh123) stain-
ing followed by fluorescence photography can be used to observe
MMP. Figure 6A shows that under normal conditions the cells
had bright green fluorescence. When cells were treated with
Province in China (No. 2015A030313458).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.02.
H
2
O
2
(400
denced by decreased fluorescence, suggesting that H
cells. However, preconditioning with 200 M TTM greatly impeded
l
M), a dramatic MMP loss was noted (Fig. 6B), evi-
0
05.
2 2
O
damaged
l
References and notes
MMP loss by preserving mitochondrial functions (Fig. 6C). TTM
alone did not significantly alter MMP (Fig. 6D). These results con-
1
2
.
.
Li, L.; Moore, P. K. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169.
Fukuto, J. M.; Carrington, S. J.; Tantillo, D. J.; Harrison, J. G.; Ignarro, L. J.;
Freeman, B. A.; Chen, A.; Wink, D. A. Chem. Res. Toxicol. 2012, 25, 769.
firmed that TTM could exhibit H
oxidative injury in cells.
2
S-like cellular protection against
In conclusion, our results demonstrate that ammonium
tetrathiomolybdate (TTM) is an effective H S donor in aqueous buf-
fers. TTM’s release of H S is pH-dependent. Under neutral pH, the
release is a slow but sustained process. TTM can exhibit H S-like
3. Vandiver, M. S.; Snyder, S. H. J. Mol. Med. 2012, 90, 255.
4.
5.
6.
Wang, R. Physiol. Rev. 2012, 92, 791.
Kolluru, G. K.; Shen, X.; Bir, S. C.; Kevil, C. G. Nitric Oxide 2013, 35, 5.
Zhao, Y.; Pacheco, A.; Xian, M. Medicinal Chemistry: Insights into the Development
2
2
2
of novel H S Donors. Part VIII, p 365–388; In Chemistry, Biochemistry and
2
Please cite this article in press as: Xu, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.005

4
S. Xu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Pharmacology of Hydrogen Sulfide, Handbook of Experimental Pharmacology;
15. Prabhu, K. R.; Devan, N.; Chandrasekaran, S. Synlett 2002, 1762.
16. Brewer, G. J. Matallomics 2009, 1, 199.
17. Wang, H. W.; Skeldon, P.; Thompson, G. E.; Wood, G. C. J. Mater. Sci. 1997, 32,
497.
18. Abdalla, M. A.; Fogg, A. G.; Burgess, C. Analyst 1982, 107, 213.
19. Ang, A. D.; Konigstorfer, A.; Giles, G. I.; Bhatia, M. Adv. Biol. Chem. 2012, 2, 360.
20. Peng, B.; Chen, W.; Liu, C.; Rosser, E. W.; Pacheco, A.; Zhao, Y.; Aguilar, H. C.;
Xian, M. Chem. Eur. J. 2014, 20, 1010.
Moore, P. K.; Whiteman M., Eds.; Springer: Vol. 230, 2015.
Zhao, Y.; Biggs, T. D.; Xian, M. Chem. Commun. 2014, 11788.
Wallace, J. L. Trends Pharmacol. Sci. 2007, 28, 501.
Kashfi, K. Antioxid. Redox. Signal. 2014, 20, 831.
0. Wallace, J. L.; Wang, R. Nat. Rev. Drug. Disc. 2015, 14, 329.
1. Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. Chem. Rev.
7
8
9
1
1
.
.
.
2
002, 102, 1091.
1
1
2. Butler, A. R.; Megson, I. L. Chem. Rev. 2002, 102, 1155.
3. Hottinger, D. G.; Beebe, D. S.; Kozhimannil, T.; Prielipp, R. C.; Belani, K. G. J.
Anaesth. Clin. Pharmacol. 2014, 30, 462.
21. Zhao, Y.; Yang, C.; Organ, C.; Li, Z.; Bhushan, S.; Otsuka, H.; Pacheco, A.; Kang, J.;
Aguilar, H. C.; Lefer, D. J.; Xian, M. J. Med. Chem. 2015, 58, 7501.
22. Park, C.-M.; Zhao, Y.; Zhu, Z.; Pacheco, A.; Peng, B.; Devarie-Baez, N.; Bagdon, P.;
Zhang, H.; Xian, M. Mol. BioSyst. 2013, 9, 2430.
1
4. Gunasundari, T.; Chandrasekaran, S. J. Org. Chem. 2010, 75, 6685.
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