Toward Direct Protein S-Persulfidation: A Prodrug Approach That Directly Delivers Hydrogen Persulfide

Article abstract of DOI:10.1021/jacs.7b09795

A general strategy of delivering hydrogen persulfide (H₂S₂) is described herein. Esterase- and phosphatase-sensitive H₂S₂ prodrugs with tunable release rates have been synthesized. Their utility is validated in examining protein S-persulfidation. With this unique approach of directly delivering H₂S₂, our findings reaffirmed that S-persulfidation leads to decreased activity of glyceraldehyde 3-phosphate dehydrogenase. This new approach complements available prodrugs/donors that directly deliver a single species, including hydrogen sulfide, perthiol, and COS, and will be very useful as part of the toolbox for delineating the mechanisms of sulfur signaling.

Full text of DOI:10.1021/jacs.7b09795

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Communication
Toward Direct Protein S-Persulfidation: A Prodrug
Approach that Directly Delivers Hydrogen Persulfide
Bingchen Yu, Yueqin Zheng, Zhengnan Yuan, Shanshan Li, He Zhu, Ladie
Kimberly De La Cruz, Jun Zhang, Kaili Ji, Siming L. Wang, and Binghe Wang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09795 • Publication Date (Web): 06 Dec 2017
Downloaded from http://pubs.acs.org on December 6, 2017
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Toward Direct Protein S-Persulfidation: A Prodrug Approach that
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Bingchen Yu, Yueqin Zheng, * Zhengnan Yuan, Shanshan Li, He Zhu, Ladie Kimberly De La
Cruz, Jun Zhang, Kaili Ji, Siming Wang and Binghe Wang*
Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA
30303 United States
Supporting Information Placeholder
single or at least as a dominant species. Herein, we deꢀ
scribed our work in this regard.
ABSTRACT: A general strategy of delivering hydrogen
persulfide (H ) is described herein. Esteraseꢀ and phosꢀ
phataseꢀsensitive H prodrugs with tunable release rates
have been synthesized. Their utility is validated in examinꢀ
ing protein Sꢀpersulfidation. With this unique approach of
2 2
S
2
S
2
Scheme 1. The design concept of H S prodrugs.
2
2
directly delivering H
persulfidation leads to decreased activity of glyceraldehyde
ꢀphosphate dehydrogenase (GAPDH). This new approach
complements available prodrugs/donors that directly delivꢀ
er a single species including hydrogen sulfide, perthiol, and
COS, and will be very useful as part of the toolbox in delineꢀ
ating the mechanisms of sulfur signaling.
2 2
S , our findings reaffirmed that Sꢀ
3
2 2
The need of developing H S prodrugs derives from its
unstable nature.40 We used the “trimethyl lock”ꢀfacilitated
lactonization system as the caging group for prodrug prepaꢀ
ration (Scheme 1).41 Specifically, H
2 2
S is caged as two thiꢀ
olacid groups linked by a disulfide bond (Scheme 1). A
masked phenol hydroxyl group serves as a latent nucleoꢀ
phile for initiation of H S release through lactonization.
2 2
Hydrogen sulfide (H
2
S) plays roles in physiological and
Scheme 2. Mechanism of esterase-triggered H
release.
2
S
2
pathological processes and has promising therapeutic poꢀ
1ꢀ10
tential. Recent studies suggest that endogenous hydrogen
polysulfides (H , n≥2) and perthiol (RS H, n≥2) have simꢀ
ilar physiological effects as H S, but with greater potency in
For example, hydrogen polysulfides were
2
S
n
n
2
11ꢀ15
some cases.
2+
found to be able to induce Ca influx by activating transient
receptor potential (TRP)A1 channels in rat astrocytes and
are 320 times more potent than
16
H
2
S.
Protein Sꢀ
persulfidation (sometimes referred to as Sꢀsulfhydration), in
which the thiol group of cysteine (ꢀSH) in protein is conꢀ
verted to a perthiol group (ꢀSSH), has proven to be a major
2 2
An esteraseꢀsensitive H S prodrug, BW-HP-301 (301),
was prepared (Scheme 2). 301 is a colorless oil and stable
for days at room temperature and months at ꢀ20 °C. We
17ꢀ20
signaling pathway involving sulfur.
Many enzymes can
studied whether esterase would promote H
2 2
S release from
go through this process, leading to significant changes in
42
3
01 using a H fluorescence probe DSPꢀ3. Thus, 301
2 2
S
1
9,21,15,22,17,23
activity.
fidate” a thiol group. Therefore, either some other oxidation
reaction(s) needs to happen or the H S source contains sulꢀ
2
Chemically, H S cannot simply “persulꢀ
was incubated with porcine liver esterase (PLE) at 37 °C in
phosphateꢀbuffered saline (PBS, pH = 7.4) for 30 min to
fully consume 301; then 20 ꢀM of DSPꢀ3 was added. Strong
fluorescence was observed in experiments with PLE (Figure
2
1
5,24
fur species at higher oxidation states.
Thus, it is likely
that hydrogen polysulfide and/or perthiol derived from H
2
S
S
1
A), indicating the release of H
fluorescence was detected without PLE. Incubation with
00 ꢁM Na S led to negligible fluorescence intensity inꢀ
crease, showing the stability of DSPꢀ3 toward H S. To proꢀ
2 2
S . In contrast, negligible
25ꢀ28
are the actual dominant species in Sꢀpersulfidation.
H
2
11,12,29
and perthiol can be produced enzymatically.
The facts
2
2
that hydrogen polysulfide and perthiol can lead to protein Sꢀ
persulfidation and there are enzymatic pathways to produce
such species lend further support to the possibility that hyꢀ
drogen polysulfide and perthiol are the actual signaling
2
vide further direct evidence of H
bromobimane (mBB) to trap H
SSꢀmBB (Scheme 3 and Figure 1B). 68 ± 8 μM of mBBꢀ
SSꢀmBB was detected from 100 μM prodrug 301 in the
presence of 10 unit/mL of PLE. The less than 100% converꢀ
sion could be due to many reasons, including slow reaction
2
S
2
release, we used monoꢀ
2
S
2
in a stable form, mBBꢀ
43
29
molecules in certain processes.
Given the significant role of hydrogen polysulfide and
perthiol in protein Sꢀpersulfidation signaling, there is a
need to prepare prodrugs for generating hydrogen polysulꢀ
fide as single species without perturbing cellular redox
chemistry. Several labs have reported beautiful work in preꢀ
kinetics and stability issues for H
we also used the same method to trap H
cially available Na . About 71 ± 9 μM of mBBꢀSSꢀmBB was
detected in 100 μM of Na
that the prodrug and Na
2
S
2
. As a reference point,
2 2
S
from commerꢀ
2 2
S
3
0ꢀ34
35
31,36ꢀ39
paring prodrugs for H
2
S,
perthiol, and COS.
2
S
2
solution (Figure S2). The fact
What are missing in the toolbox are prodrugs for H
2 2
S as a
2 2
S gave the same results in both
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the DSPꢀ3 and mBB assays strongly suggest that the release
from the prodrug was nearly 100%. The efficiency of the
trapping reaction was most likely the reason causing
the less than 100% conversion to the trapped product, mBBꢀ
SSꢀmBB. It is important to noted that H concentration
from the addition of 100 μM Na started decreasing from
the moment of dissolution (Figure S2). However, with 100
μM 301, the H concentration gradually increased with
2 2
peak concentration of 10 ꢀM of H S was detected (Figure
2B) with a halfꢀlife of 28 min in the presence of 2 units/mL
ALP (Table S4).
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2 2
H S
2 2
To assess the utility of the prodrug in delivering H S in
biological systems, cells were coꢀtreated with 100 ꢁM 301
and PLE or 100 ꢁM 303 and ALP (Figure S9), respectively.
2 2
S
2 2
S
2 2
Strong fluorescence was observed, indicating H S release.
2 2
S
No obvious cytotoxicity was found at up to 100 ꢂM of the
prodrugs or lactone on H9c2 cells after 24 and 48 h incubaꢀ
tion (Figure S5,6).
the addition of 2 units/mL PLE and reached a peak concenꢀ
tration at 25 min.
Scheme 3. Direct detection of H
agent mBB.
2
S
2
by trapping
Scheme 4. Structures of 302 and 303
0
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0
O
O
O
O
O
P
O
O
OH
PLE
N
S
O
S
O
OH
O
O
S
+
N
N
S
HO
O
S
P
BW-HP-301
H
2
S
2
S
N
OH
N
Br
O
O
O
O
N
O
excess amount
O
BW-HP-302
BW-HP-303
monobromobimane (mBB)
mBB-SS-mBB
2 2
Figure 2. A) Qualitative assessment of H S release from 303 by DSPꢀ3. The
concentration of DSPꢀ3 is 20 ꢁM and ALP is 10 units/mL in PBS (1% MeOH).
n = 3. 1) 100 ꢁM 303 + ALP; 2) 100 ꢁM 303; 3) ALP; 4) ALP + 100 ꢁM
2 2 2 2
Na S . B) H S releasing profile form 40 ꢁM 301 and 302 with 1 unit/mL
PLE in PBS (2% DMSO) and 303 with 2 units/mL ALP at 37 °C in PBS (2%
MeOH). 20 ꢁM DSPꢀ3 was used. n = 3.
2 2
Figure 1. A) Qualitative detection of H S release from 301 by DSPꢀ3. 301
was incubated with 2 units/mL PLE in PBS (2% DMSO) for 30 min and then
DSPꢀ3 (20 ꢁM) was added. Data were acquired at 515 nm with excitation at
4
90 nm. n = 3. 1) 100 ꢁM 301 + PLE; 2) 100 ꢁM Na
ꢁM 301; 5) 200 ꢁM Na S + PLE. B) Direct detection of H
agent mBB from 301 in PBS (2% DMSO). n = 3.
2
S
2
+ PLE; 3) PLE; 4) 100
2
2 2
S
by trapping
One would expect the sustained concentration of H
2
S
2
to
Sꢀpersulfidation is a major sulfur signaling pathway.17,19
be dependent on the release rate from a prodrug since the
generation and consumption of thiol species in the biologiꢀ
cal system is a dynamic process. Thus, we wanted to prepare
prodrugs with varying release rates. Specifically, BW-HP-
We then examined the Sꢀpersulfidation efficiency of the
prodrugs on GAPDH using a tagꢀswitch assay.48 Incubation
with 100 ꢁM 301 and 10 units/mL PLE at 37℃led to a sigꢀ
nificant increase in GAPDH Sꢀpersulfidation level (Figure
302 (302) was synthesized (Scheme 4) and its H
ability was assessed (Figure S1). The H release profiles
2 2
S release
3
B and C, line 1) compared to the untreated group (line 5).
In contrast, incubation with 200 ꢁM H S (line 4) failed to
elevate GAPDH Sꢀpersulfidation level. Protein samples were
also treated with 100 ꢁM Na or 100 ꢁM H followed by
00 μM H S (line 2, 3) to mimic the two endogenous Sꢀ
persulfidation process (Figure S10).
ods each led to a significant increase of GAPDH Sꢀ
persulfidation level. Such results further affirm that H
itself is incapable of protein Sꢀpersulfidation. Previously
2 2
S
2
from 301 and 302 are shown in Figure 2B. From 40 ꢀM of
the prodrugs, 301 had a peak concentration of 15 ꢀM at
around 15 min, while 302 maintained a sustained concenꢀ
tration of about 3 ꢀM. The release profile of 302 was also
studied by using mBB. A plateau of 35 ± 7 μM of mBBꢀSSꢀ
mBB was detected from 100 μM of 302 in the presence of
10 units/mL PLE (Figure S2). For 100 ꢁM of the prodrug,
the halfꢀlife was determined to be 24 min for 301 and 172
min for 302 by HPLC (Table S4). The slower release rate of
2
S
2
2 2
O
1
2
15,48,49
These two methꢀ
2
S
50
H
2
S has been shown to abolish Sꢀpersulfidation by itself.
The roles that H S plays in Sꢀpersulfidation and signaling
2
3
02 was presumably due to the bulkier nature of the cycloꢀ
are dependent on ROS and cellular redox environment.
However, 301 was able to induce Sꢀpersulfidation indeꢀ
propanecarbonyl ester, which hinders esteraseꢀmediated
hydrolysis.
44
pendent of ROS. These H
tools to conduct Sꢀpersulfidation without perturbing the
redox balance. Even with data suggesting H being the
2 2
S donors are important research
In an effort to broaden the tunability of H
phosphataseꢀsensitive H prodrug, BW-HP-303 (303),
was synthesized (Scheme 4). Alkaline phosphatase (ALP) is
2 2
S release, a
S
2
2 2
S
2
dominant species released from 301, we can’t exclude the
45ꢀ47
possibility that other polysulfide derived from H S degraꢀ
widely used for prodrug activation.
dependent H release was examined by DSPꢀ3 (Figure
A). Incubation of 303 with 10 units/mL ALP led to strong
fluorescence, demonstrating H release. In the absence of
phosphatase, almost no H was detected, indicating the
chemical stability of the prodrug. A peak concentration of
5 ± 8 μM of mBBꢀSSꢀmBB was achieved from 100 μM 303
in the presence of 10 units/mL ALP. Such result is similar to
that of 100 μM 301 and Na , demonstrating the efficient
release from 303 (Figure S2). From 40 ꢀM 303, a
Phosphataseꢀ
2 2
2 2
S
dation may also play a role in Sꢀpersulfidation because of
the unstable nature of H
2
2 2
S .
S
2
It has been a subject of debate as to whether Sꢀ
persulfidation leads to increased or decreased activity of
2
2 2
S
17,23
GAPDH.
To address this point, GAPDH was treated with
the exact conditions that was used in the Sꢀpersulfidation
assay above. Then enzyme activity was determined. As
shown in Figured 3B and D, GAPDH treated with 301,
6
2 2
S
Na
2
S
2
, or H
2
S and H
2
O
2
together each showed elevated proꢀ
2 2
H S
2
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tein Sꢀpersulfidation levels and decreased enzyme activity
compared with untreated groups. Meanwhile, H S alone
failed to affect enzyme activity. GAPDH activity also showed
a concentration dependent decrease in response to 301 with
a half inhibition concentration of approximately 1 μM (Figꢀ
ure 3E). Maximal inhibition was achieved with 5 μM of the
AUTHOR INFORMATION
Author Contributions
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#
These authors contributed equally and the names are listed
alphabetically.
Corresponding Author
3
01 with 17 ± 1% catalytic activity remaining. Further
A
Binghe Wang: Wang@gsu.edu;
Yueqin Zheng: yzheng6@gsu.edu
Sulfhydration Induction
, BW-HP-301
2, Na
1
Protein Sulfhydration detection
by a tag-switch assay
GAPDH
SH
2 2
S
ACKNOWLEDGMENT
3
, H
,Na
, No treatment
2 2 2
O + Na S
GAPDH activity assay
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Partial financial support from the GSU Brains and Behavꢀ
iors Fellowship Program to BY is gratefully acknowledged.
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