Characterization of regio- and stereo-selective sulfation of bufadienolides: Exploring the mechanism and providing insight into the structure-sulfation relationship by experimentation and molecular docking analysis

Article abstract of DOI:10.1039/c5ra22153f

Bufadienolides are a major class of bioactive compounds derived from amphibian skin secretion. Recent studies demonstrate that bufadienolides have a promising role in targeted cancer chemotherapy. However, extensive metabolism and inactivation strongly restrict the clinical applications of bufadienolides. This study aimed to systematically characterize the sulfation of six representative bufadienolides (including bufalin, resibufogenin, cinobufagin, bufotalin, telocinobufagin and deacetylcinobufagin) in amphibian skin secretion and to provide insight into the structure-sulfation relationship by experimentation and molecular docking analysis with series of bufadienolides and derivatives. For all the six representative bufadienolides, one corresponding monosulfate was detected in the incubation mixtures. The sulfates were accurately identified as bufadienolides 3-O-sulfates by NMR and HPLC-MSⁿ techniques. Reaction phenotyping studies using human recombinant sulfotransferase (SULT) and liver S9 demonstrated that SULT2A1 mediated the formation of bufadienolide 3-O-sulfate with a high specific selectivity. Further kinetic evaluation demonstrated that deacetylcinobufagin could be used as a preferred probe of SULT2A1. The regio- and stereo-selective sulfation properties of SULT2A1 and the structural variation effects of bufadienolides were investigated by docking analysis, which revealed the significance of appropriate molecule orientation and hydrophobic interactions of motifs with SULT2A1 His99 residues. Additionally, significant differences between humans and animal species were observed in the sulfation of bufalin and resibufogenin. This study provided important data for elucidating the mechanisms of bufadienolide sulfation and leads to a better understanding of the bufadienolide-SULT interaction which can be further used in preclinical development and rational use of bufadienolides.

Full text of DOI:10.1039/c5ra22153f

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Characterization of regio- and stereo-selective
sulfation of bufadienolides: exploring the
mechanism and providing insight into the
structure–sulfation relationship by
Cite this: RSC Adv., 2016, 6, 5774
experimentation and molecular docking analysis†
Jing Ning,‡a Yonglei Cui,‡a Chao Wang,‡ac Peipei Dong,a Guangbo Ge,b
Xiangge Tian,a Jie Hou,a Xiaokui Huo,a Baojing Zhang,a Tonghui Mad
ac
*
and Xiaochi Ma
Bufadienolides are a major class of bioactive compounds derived from amphibian skin secretion. Recent
studies demonstrate that bufadienolides have a promising role in targeted cancer chemotherapy.
However, extensive metabolism and inactivation strongly restrict the clinical applications of
bufadienolides. This study aimed to systematically characterize the sulfation of six representative
bufadienolides (including bufalin, resibufogenin, cinobufagin, bufotalin, telocinobufagin and
deacetylcinobufagin) in amphibian skin secretion and to provide insight into the structure–sulfation
relationship by experimentation and molecular docking analysis with series of bufadienolides and
derivatives. For all the six representative bufadienolides, one corresponding monosulfate was detected in
the incubation mixtures. The sulfates were accurately identified as bufadienolides 3-O-sulfates by NMR
and HPLC-MSn techniques. Reaction phenotyping studies using human recombinant sulfotransferase
(SULT) and liver S9 demonstrated that SULT2A1 mediated the formation of bufadienolide 3-O-sulfate
with a high specific selectivity. Further kinetic evaluation demonstrated that deacetylcinobufagin could
be used as a preferred probe of SULT2A1. The regio- and stereo-selective sulfation properties of
SULT2A1 and the structural variation effects of bufadienolides were investigated by docking analysis,
which revealed the significance of appropriate molecule orientation and hydrophobic interactions of
motifs with SULT2A1 His99 residues. Additionally, significant differences between humans and animal
species were observed in the sulfation of bufalin and resibufogenin. This study provided important data
for elucidating the mechanisms of bufadienolide sulfation and leads to a better understanding of the
bufadienolide–SULT interaction which can be further used in preclinical development and rational use of
bufadienolides.
Received 22nd October 2015
Accepted 4th January 2016
DOI: 10.1039/c5ra22153f
www.rsc.org/advances
are a class of cardiac steroids rich in amphibian skin secre-
tion.1,2 They have a profound effect in increasing the contractile
force of the heart by inhibiting the enzyme Na+, K+–ATPase and
Introduction
Bufadienolides, characterized by the presence of
a six-
are widely prescribed for patients with cardiovascular disease in
Asia.3 For the last few years, bufadienolides have manifested
signicant potential applications in cancer therapies and may
represent a promising form of targeted cancer chemotherapy.4
It was recently evidenced that bufadienolides (including bufa-
lin, resibufogenin, cinobufagin and its analogues) are the
primary constituents responsible for the antitumor activity of
huachansu (cinobufacini injection) which is an intravenously
administered extract of toad venom which has been used for
cancer treatment for hundreds of years.5–7 Bufadienolides have
attracted the attention of scientists worldwide, leading to in-
depth investigations of their bioactivity and pharmacological
actions. These compounds have been found to be involved in
membered lactone (a-pyrone) ring located at position C17b,
aCollege of Pharmacy, Academy of Integrative Medicine, Dalian Medical University,
Dalian, China. E-mail: maxc1978@163.com; Fax: +86-411-86110419; Tel: +86-411-
86110419
bLaboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, China
cState Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China
dCollege of Basic Medical Science, Dalian Medical University, Dalian, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra22153f
‡ These authors contributed equally to this work.
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complex cell-signal transduction pathways and have the ability
to regulate cell growth, differentiation, apoptosis and glucose
metabolism in human tumors.8–10 Currently, the increasing
demands for the discovery and development of natural products
for cancer therapy have challenged scientists to expedite the
process for the drug ability assessment of bufadienolides.
However, collective evidences suggest that bufadienolides are
subjected to extensive metabolism in human and animals.11–14
The metabolism and pharmacokinetics study indicated that
bufadienolides were well-absorbed and rapidly eliminated from
systemic circulation,11–14 that the half-life is only 6 minutes in rat
aer intravenous administration of resibufogenin.14 Our
previous studies have demonstrated that CYP3A4 mediated
hydroxylation is involved in the metabolism of series of bufa-
dienolides in human.3,15,16 However, our latest study found that
bufadienolides were signicantly sulfated in human primary
hepatocytes, which indicated that sulfation was an important
route in their metabolism. Since the sulfates of bufadienolides
exhibited much less potency than the parent agent,17,18 the sul-
fation could be considered a clearance and inactivation pathway
of bufadienolides which play an important role in the modula-
tion of their exposure level and bioactive effects.
Understanding of sulfotransferases (SULT) substrate speci-
city is of great importance in predicting contribution of sulfa-
tion to substrate and metabolite disposition in vivo. Among the
identied human SULT enzymes, the major isoforms expressed
in adult liver are SULT1A1, SULT1A3, SULT1B1, SULT1E1, and
SULT2A1.19,20 Investigation on the interaction between steroids
and SULT demonstrated that SULT2A1 and SULT1E1 are
common SULT responsible for the sulfation of steroids,20 while
other isoform sometimes is involved in the sulfation reaction.
Bufadienolides possess typical steroid nucleus, while a-pyrone
substituent at C17 is an evidently characteristic that distinguish
from other steroids. It implied that the SULT bufadienolides
specicity may different from the knowledge has been acquired
and provide the ground for the optimization of bufadienolides.
However, there is thus far no investigation to uncover the
structural bases for the sulfation selectivity and structural vari-
ation on the catalytic efficacy of bufadienolides by SULT.
Fig. 1 Chemical structures of the main bufadienolides in amphibian
skin secretion.
incubated with human liver S9 (HLS9) in the presence of
30-phosphoadenosine-50-phosphosulfate (PAPS). The products
of the different bufadienolides were detectable and identied as
important metabolites in human hepatocytes. For all bufadie-
nolides, the m/z values for the [M ꢀ H]ꢀ of the metabolite
in human hepatic S9 increased 80 when compared with the
corresponding parent compounds (Fig. S1†). This indicated
that the metabolites were monosulfate conjugates of
bufadienolides.
Identication of the sulfate conjugates of bufadienolides
To determine the metabolic site of RB, the sulfate metabolites
of different bufadienolides were synthesized in vitro and
further identied by 1H and 13C-NMR spectra. The carbon
signals were assigned and are listed in Table S1.† For
example, compared with RB, a signicant upeld shi (Dd +
9.4) was observed at the C3 position, this was assigned
a sulfuric acid at the C3 position. Therefore, the sulfate
metabolite of RB was identied as an RB-3-sulfate. Similarly,
the metabolites of other bufadienolide were also identied
as 3-O-sulfates.
In this study, the objectives were to systematically characterize
of the sulfation of six representative bufadienolides in
amphibian skin secretion and to elucidate the structure–sulfa-
tion relationship by using series of bufadienolides and its
derivatives. This study will provide important data for elucidating
the mechanisms and effects of sulfation on bufadienolides
Assays by recombinant human SULT isoforms
metabolism and lead to a better understanding of the bufadie- The six representative bufadienolides were incubated with the
nolide–SULT interaction which can be further used in medicinal recombinant human SULT (rhSULT), including 1A1*1, 1A1*2,
chemistry to optimize the chemical structures of bufadienolides. 1A2, 1A3, 1B1, 1E1 and 2A1, to identify the SULT isoforms
And these ndings also may offer guidance for the rational use responsible for the conjugation reaction. At three substrate
and the preclinical development of bufadienolides.
concentrations, the sulfate conjugates were exclusively formed
by SULT2A1; no other metabolite was observed under the same
incubation conditions (Fig. 2). The sulfation rate of RB at the 1
mM substrate concentration was 2.60 nmol per min per mg
SULT2A1, which was higher than the other bufadienolides.
Among the six major bufadienolides, the sulfation rates of BF,
Results
Sulfation of the bufadienolides by human primary hepatocyte
and human liver S9
One metabolite was detected when the six bufadienolides, RB, CB and BFT signicantly decreased following increases in
including BF, RB, CB, BFT, TCB and DCB (Fig. 1), were substrate concentrations.
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that SULT2A1 was primarily responsible for the sulfation of
these bufadienolides in the human liver.
Additionally, it was evident that the sulfations of BF and RB
were catalyzed predominantly by SULT2A1 in liver S9 from
various animal species (Fig. S3†). These ndings suggested the
potential similarity in the metabolic enzymes for RB and BF
sulfations in liver S9 from different animal species.
Kinetics characterization
The kinetics analyses of the six representative bufadienolides in
amphibian skin secretion were performed in HLS9, recombi-
nant SULT2A1 and the combined recombinant SULT (Fig. 4).
The substrate inhibition model was tted to the kinetics prole
for the 3-O-sulfation of bufadienolides in HLS9 and SULT2A1.
The kinetics parameters of the sulfation of the six bufadieno-
lides were determined and are listed in Table 1.
When the kinetics parameters among the six bufadienolides
obtained in HLS9 and SULT2A1 and the combined recombinant
SULTs were compared, the Km and Vm were varied extensively.
In HLS9, it was found that RB showed the lowest Km (0.35 mM)
and the highest intrinsic clearance (628.6 mL per min per mg
protein), which was dozens-fold lower and hundreds-fold
higher than those of BFT (7.77 mM and 3.6 mL per min per mg
protein, respectively). The Km and Vmax values of TCB in HLS9
were slightly varied with those of RB; thereaer, the TCB
exhibited a relatively large intrinsic clearance among the six
bufadienolides. Additionally, SULT2A1 exhibited a medium
affinity and catalytic capacity toward CB, BF and DCB. The
affinity and intrinsic clearances were similar among all bufa-
dienolides obtained with SULT2A1, HLS9 and the combined
recombinant SULTs. This implied that SULT2A1 was primarily
responsible for catalysing the sulfation of bufadienolides in
human liver.
Fig. 2 Sulfation of the six bufadienolides by human recombinant
SULTs.
Chemical inhibition study
Seven selective inhibitors/substrates of major SULT isoforms
were used to screen the SULTs responsible for the formation of
bufadienolides sulfates in HLS9. Dehydroepiandrosterone
(DHEA), which is a selective substrate for SULT2A1, signicantly
inhibited the sulfation reaction of bufadienolides (Fig. 3) to less
than 10% activities. In contrast, the selective inhibitors/
substrates of other SULT isoform showed negligible inhibitory
effects (less than 20% inhibition, p < 0.05). To further determine
the primary role of SULT2A1 in the sulfation of the six bufa-
dienolides, we determined the inhibitory effects of DHEA with
different concentrations. The results showed that DHEA
exhibited a similar inhibitory prole toward SULT2A1 and
HLS9-mediated sulfations of the examined bufadienolides
(Fig. S2 and Table S2†). The collectively results strongly suggest
For other bufadienolide derivatives, our results showed that
the bufadienolides with 3-a-OH were not effectively catalyzed by
rhSULT2A1. The sulfate metabolite of OCB was undetectable
under the same incubation conditions as those treated with the
six representative bufadienolides. Additionally, the apparent Km
of rhSULT2A1-mediated sulfation was determined (Fig. S4 and
Table S3†). Although there were small differences in the
Fig. 3 Effects of selective SULT inhibitors/substrate on the formation
of bufadienolides sulfates in human liver S9.
Fig. 4 Kinetic plots of the six representative bufadienolides sulfation in HLS9 (A and B), rhSULT2A1 (C and D) and mixed isoforms (E and F).
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Table 1 Kinetic parameters of the six representative bufadienolides sulfation in HLS9, human recombinant SULT2A1, and the combined human
recombinant SULT
Compounds
BF
Enzymes
Vm (nmol per min per mg)
Km (mM)
Ksi (mM)
Vm/Km (mL per min per mg)
HLS9
SULT2A1
Mixed isoforms
HLS9
SULT2A1
Mixed isoforms
HLS9
SULT2A1
Mixed isoforms
HLS9
SULT2A1
Mixed isoforms
HLS9
SULT2A1
Mixed isoforms
HLS9
SULT2A1
0.0763 ꢁ 0.0056
6.15 ꢁ 1.03
3.19 ꢁ 0.50
6.63 ꢁ 1.49
6.18 ꢁ 2.72
0.353 ꢁ 0.071
0.451 ꢁ 0.163
0.676 ꢁ 0.242
1.45 ꢁ 0.21
2.95 ꢁ 0.62
2.57 ꢁ 0.48
7.77 ꢁ 1.72
21.9 ꢁ 12.5
6.58 ꢁ 1.40
1.69 ꢁ 0.24
0.482 ꢁ 0.086
1.15 ꢁ 0.18
8.97 ꢁ 1.35
4.50 ꢁ 0.58
3.53 ꢁ 0.78
36.9 ꢁ 5.1
23.8
5.86 ꢁ 1.38
3.42 ꢁ 1.61
19.0 ꢁ 3.6
927.6
194.2
628.6
10 822.2
5746.3
20.7
715.3
112.8
3.6
44.6
106.4
71.0
5041.7
1391.3
21.2
1360.0
439.1
1.20 ꢁ 0.42
RB
0.221 ꢁ 0.023
4.87 ꢁ 0.95
3.15 ꢁ 1.11
2.73 ꢁ 0.92
119.5 ꢁ 18.4
12.2 ꢁ 2.8
3.85 ꢁ 0.81
CB
0.030 ꢁ 0.002
2.11 ꢁ 0.27
0.297 ꢁ 0.032
0.028 ꢁ 0.003
0.982 ꢁ 0.454
0.705 ꢁ 0.102
0.123 ꢁ 0.011
2.42 ꢁ 0.10
20.8 ꢁ 4.2
BFT
TCB
DCB
79.2 ꢁ 17.0
8.00 ꢁ 4.31
15.5 ꢁ 3.1
125.4 ꢁ 17.9
128.2 ꢁ 20.0
29.0 ꢁ 5.1
1.60 ꢁ 0.11
0.193 ꢁ 0.015
6.12 ꢁ 0.31
1526.0 ꢁ 607.3
205.0 ꢁ 26.92
397.6 ꢁ 113.2
Mixed isoforms
1.55 ꢁ 0.12
structures of the bufadienolides, the Km varied up to a thou- studied, RLS9 exhibited the lowest affinity for BF and RB, and
sand-fold. CBFT exhibited the lowest Km (0.29 mM) and BFE the Km values were approximately 7-fold and 10-fold larger than
exhibited the largest Km (228 mM). Grouped by the Km values, those of HLS9, respectively. It was observed that the catalytic
CBFT, RB and TCB displayed markedly high affinities for activity of RaLS9 was higher than those of HLS9 and liver S9
SULT2A1; however, BFT, AB, GB, PBFE and BFE exhibited low from other mammals, which resulted in a relatively large
affinities for SULT2A1.
hepatic in vitro intrinsic clearance (Vm/Km) for the 3-O-sulfate of
BF and RB.
Correlation studies
The sulfation rates for DHEA and various bufadienolides were
determined using S9 from individual human liver (n ¼ 16).
Correlation analyses were conducted for the sulfations of DHEA
and bufadienolides. It found that the sulfation velocities of BF,
RB, CB, BFT, TCB and DCB were signicantly correlated with
DHEA sulfation velocities (Fig. S5†) with correlation coefficients
(r) of 0.954, 0.919, 0.941, 0.937, 0.973 and 0.956, respectively.
This evidence indicated that SULT2A1 played a dominant role
in the sulfation of bufadienolides in human livers.
Molecular docking studies
Molecular docking was carried out to explore the interaction
model between bufadienolide derivatives and SULT2A1. All the
bufadienolide derivatives (Fig. 1 and S4†) could dock into the
active pocket of SULT2A1. Assuming these analogues were
capable of binding with the enzyme in similar conformations,
we selected a reasonable bioactive mode from the top ve
Interspecies difference in bufadienolide sulfation
It is well-accepted that the choice of a suitable animal model
with similar metabolic mechanisms as humans is crucial for
results in drug trials because there are species-dependent
species-specic effects and toxicities.22 Comparing in vivo
pharmacological and pharmacokinetic in the present study, the
interspecies differences in the sulfations of RB and BF (i.e., the
compounds with the highest catalytic capacities and biological
activities) were studied using liver S9 obtain from monkey
(CyLS9), mini-pig (PLS9), dog (DLS9), rabbit (RaLS9), guinea pig
(GLS9), rat (RLS9) and mouse (MLS9).
The sulfate conjugates of the bufadienolides were analyzed
aer RB and BF were incubated with liver S9 from different
animals (except for DLS9 and MLS9). Kinetic studies were then
performed to compare the abilities to catalyze the sulfation of
Fig. 5 The Kinetic plots of BF (A and B) and RB (C and D) sulfation in
RB and BF (Fig. 5 and Table 2). Among the animal liver S9 liver S9 from monkey, mini-pig, rabbit, guinea pig and rat.
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Table 2 Kinetic parameters of BF and RB sulfation in liver S9 from monkey (CyLS9), mini-pig (PLS9), rabbit (RaLS9), guinea pig (GLS9) and rat
(RLS9)
Compounds
BF
Enzymes
Vm (pmol per min per mg)
Km (mM)
Ksi (mM)
Vm/Km (mL per min per mg)
CyLS9
PLS9
48.1 ꢁ 4.2
33.7 ꢁ 1.1
116.6 ꢁ 7.9
20.6 ꢁ 1.8
34.9 ꢁ 0.9
52.7 ꢁ 1.6
26.9 ꢁ 0.9
555.9 ꢁ 13.6
85.4 ꢁ 2.6
47.9 ꢁ 2.1
5.38 ꢁ 0.89
2.18 ꢁ 0.28
3.02 ꢁ 0.58
2.04 ꢁ 0.40
21.3 ꢁ 1.0
119.1 ꢁ 30.31
127.8 ꢁ 25.63
118.6 ꢁ 32.42
250.6 ꢁ 79.95
256.5 ꢁ 17.73
127.1 ꢁ 16.03
26.99 ꢁ 2.78
251.8 ꢁ 39.0
368.3 ꢁ 60.0
215.5 ꢁ 40.3
8.9
16.5
66.2
10.8
1.6
46.9
112.5
390.1
75.7
20.3
RaLS9
GLS9
RLS9
CyLS9
PLS9
RaLS9
GLS9
RLS9
RB
1.13 ꢁ 0.09
0.242 ꢁ 0.032
1.41 ꢁ 0.093
1.11 ꢁ 0.11
2.32 ꢁ 0.38
conformations ranked by their Hammett scores. The substrates explained by molecular docking study. It suggested that
in the 3-b-OH group formed hydrogen bonds with His99 appropriate orientations and the formation of hydrogen bonds
(Fig. 6A–L) which was the key site of SULT2A1 that interacting to key amino acid residues of the catalyst active site (His99)
with the classical substrate DHEA.21 However, for OCB, EBF, played important roles in the reaction (Fig. 6). The bufadieno-
ERB and EDCB, there were no hydrogen bonding interactions lides that could not form hydrogen bonding with His99 due to
between 3-a-OH and His99, as depicted in Fig. 6M–P. For the non-preferred orientations of the 3-OH site were not effi-
compounds with 3-a-OH sites, their 3-hydroxyl orientations caciously catalyzed by SULT2A1 (Fig. 6M–P). The results indi-
precluded the formation of hydrogen bonds with His99 cated that enclosing the 3-b-OH or introducing the chemical
(Fig. 6M–O). For OCB, it seemed that the presence of an alde- modications which could hinder the formation of hydrogen
hyde group at C19 interacting with the 3-OH site, which result in bonding with His99 may increase the metabolic stability of
a conformational rotation of the 3-OH site (Fig. 6P). The bufadienolides by reducing the sulfation. As reported previ-
chemscore was used to estimate the binding affinity between ously, two substrate-binding orientations have been identied
the bufadienolides and SULT2A1. The chemscore values for the for DHEA, including the primary, catalytic, orientation and the
different bufadienolide substrates are listed in Table S3.† The second alternative orentation.24 In comparison with the primary
chemscore values correlated with the experimental Km results. orientation, the second orientation penetrates further into the
For example, CBFT had the lowest chemscore value of ꢀ36.68, active site (Fig. S6†). Additionally, there is a 45ꢂ rotation of the
and the lowest Km for SULT2A1. BFE had the highest Km and DHEA out of the steroid plane with respect to the catalytic
chemscore values. When comparing the experimental potency, orientation. As shown in Fig. S7,† the binding modes of all the
we found that the binding affinities of GB, AB and BFT were bufadienolides were different from the primary catalytic orien-
correlated with the results obtained from the in vitro assays. tation of the DHEA according to the docking analysis. They
However, there were several exceptions; for example, DBT had located close to the DHEA molecule in the proposed alternative
a low affinity for SULT2A1, but the chemscore value was ꢀ35.18. orientation. The discrepancy may derive from the difference in
The disagreement between the molecular modeling results and structures of bufadienolides and DHEA. Considering that the
the in vitro results may be due to the complex interaction active site of SULT2A1 was hydrophobic in nature, the inclusion
mechanisms between substrates and enzyme.
of a-pyranone of bufadienolides may give rise to more hydro-
phobic interaction which leading to the difference between the
primary binding orientation of bufadienolides and DHEA.
The kinetic characterizations performed herein demon-
Discussion
Sulfate conjugation by cytosolic sulfotransferases is an impor- strated that the 3-O-sulfation of bufadienolides with marked
tant biotransformation reaction in the metabolism of drugs, variations in the apparent Km and intrinsic clearance values. In
hormones, neurotransmitters, and xenobiotic compounds.23 In comparison, RB and BFT showed the highest and lowest
the present study, 3-O-sulfation was found to be the common intrinsic clearance values, respectively, and varied by hundreds-
metabolic pathway of the six representative bufadienolides of fold. This was attributed to the evident differences of their
amphibian skin secretion in human liver. The catalytic enzyme affinities and maximum rates in the conjugation reactions. The
was identied as SULT2A1. Additionally, the 3-b-OH site was structure–sulfation relationship could be summarized as
identied as the corresponding sulfate conjugation site of follows: SULT2A1 shows stereo-selectivity for the 3-OH sites of
bufadienolides. Considering that bufadienolides with a 3-a-OH bufadienolides, but not the 3-a-OH sites. Hydrophilic group
were not effectively catalyzed by rhSULT2A1, it indicated that substitutions (such as hydroxyl substations at the C11, C14 and
the SULT2A1-mediated sulfate conjugation of bufadienolides C16 positions) typically decrease the binding affinities, with an
was a regio- and stereo-selective reaction. The complicated exception for hydroxyl substitutions at the C5 positions, which
interaction mode between bufadienolides and SULT2A1 were reversely increases the binding affinities (potentially attributed
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Fig. 6 The interaction binding modes and structural relationships of bufadienolide derivatives with SULT2A1. (A–P) The interaction binding mode
of (A) RB; (B) CB; (C) BF; (D) TCB; (E) DCB; (F) BFT; (G) CBFT; (H) DABT; (I) AB; (J) GB; (K) PBFE; (L) BFE; (M) EDCB; (N) EBF; (O) ERB; (P) OCB with
SULT2A1, where C, O, N and H atom were colored grey, red, blue and cyan, respectively. (Q) The relationships between chemical structures and
affinity toward SULT2A1.
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to the inclination to form additional hydrogen bonds that pig exhibited similar metabolic proles, kinetic behaviors and
further enhance the interaction between the bufadienolides intrinsic metabolic clearances of BF and RB sulfate conjugation
and active site, as shown in Fig. 6F). The aldehyde group at the with those of human. However, it found that the sulfate
C19 position prevents the sulfation processes. The OAc residue conjugates of the bufadienolides were undetectable when RB
at C16 decreased the affinity for SULT2A1. It should be noted and BF were incubated with liver S9 from dog and mouse. The
that the C5 position hydroxylation has been identied as the results reected a weak and decient capacity of the SULT2A1 in
major phase I metabolic reaction in our previous studies.3,15,16 the livers of dogs and mice, which was further conrmed by
Thus, the increased binding affinities due to hydroxyl substi- using endogenous substrate DHEA (data not shown). Addi-
tutions at C5 indicated a potent interplay between the phase I tionally, the variation on Km values for BF and RB sulfation
and phase II metabolic routes of bufadienolides. The above between HLS9 and RLS9 indicated the different catalytic
information could provide guidance for optimization the behavior of human and rat SULT in response to bufadienolide.
metabolic stability of bufadienolides.
These results implied that mouse, rat and dog models are not
Hitherto, the most commonly used probe substrate of suitable for simulating RB and BF sulfations in humans, and
SULT2A1 is endogenous steroid DHEA.19,25,26 However, it should the in vivo data obtained from the aforementioned animals
be noted that DHEA is not highly selective catalyzed by SULT2A1 should be cautiously utilized.
due to the participation of SULT1E1.27 In the present study,
SULT2A1 showed strong selectivity and specicity toward the
sulfation of the six studied bufadienolides. Although SULT2A1
Conclusions
and SULT1E1 are common sulfotransferases in the sulfation of The sulfation of bufadienolides was fully characterized in the
steroids,20 it had been reported that a large steric group at the D- present study. The conjugate metabolites of bioactive bufadie-
ring of steroids hindered the entrance of compounds into the nolides examined were identied as 3-O-sulfates in human
binding pocket of SULT1E1.28 Therefore, the high catalytic primary hepatocyte, and SULT2A1 was the specic isoform
selectivity may attribute to the characteristic structural feature responsible for the conjugation reaction with high regio- and
of a-pyrone attached at the C17 position. Additionally, from the stereo-selectivity. The interaction mode between bufadienolides
perspectives of enzyme activity quantication, our results showed and SULT2A1 was rst elucidated by model docking studies.
that DCB 3-O-sulfation displayed the weak substrate inhibition The results indicated the importance of appropriate orienta-
and relatively high catalysis rate. The high specic and appro- tions and hydrophobic interactions between the ligands and
priate kinetic behaviors all suggested that DCB could be a better His99 residue of SULT2A1. Moreover, the bufadienolides–sul-
probe for detection the activity of human SULT2A1.
fation relationship was elucidated and sulfation of DCB is
In humans, SULT2A1 is responsible for catalyzing the sul- veried to be a highly selective probe reaction for human
fation of hydroxysteroids, including DHEA, androgens and SULT2A1. Additionally, a signicant species-dependent varia-
pregnenolone, and bile acids.19,29 The SULT2A1-mediated sul- tion in sulfation pathway was also found. Our improved
fation of steroids has been reported to be an important process understanding of bufadienolides–SULT interaction will provide
for maintaining steroid hormone (such as deoxycorticosterone guidance for the rational use of bufadienolides in the clinic or
and dehydroepiandrosterone) levels.30 For example, the insight into the bufadienolides–sulfation relationship in
apparent Km of the SULT2A1-mediated sulfation of RB was 0.45 human.
mM, which was almost 5-fold lower than that of DHEA, indi-
cating the potential regulation of DHEA levels by bufadieno-
lides. Additionally, the docking of non-substrate molecule did
not result in a poor tting score in our study (Table S3†). These
Materials and methods
Materials
results indicated that these compounds could also enter into Bufalin (BF), resibufogenin (RB), cinobufagin (CB), bufotalin
the catalytic space of SULT2A1 and interact with the enzyme, (BFT), telocinobufagin (TCB), deacetylcinobufagin (DCB), cino-
but their orientations precluded the reaction. These all suggests bufotalin (CBFT), gamabufotalin (GB), deacetylbufotalin (DBT)
that the bufadienolide constituents may increase the level of were purchased from Shanghai Boyle Chemical Company
circulating unconjugated deoxycorticosterone and dehydroepi- (Shanghai, China). Other bufadienolides were isolated from
androsterone by inhibiting their conjugation. The potential amphibian skin secretion or obtained by biotransformation
for endocrine disruption by this mechanism or alterations to using microorganisms by the author (J. Ning) and unambigu-
the metabolism of drugs or other xenobiotics through the ously identied by NMR and MS techniques. Their purities were
inhibition of SULT2A1 requires further evaluation.
greater than 98% as determined by high-performance liquid
It is important to understand the differences of metabolism, chromatography with diode-array detection (HPLC/DAD). A
especially the prominent inactivated pathway in selecting the mixed pool of human liver S9 (HLS9), individual human liver S9,
appropriate animal model and in the interpretation of data and pooled liver S9 of male ICR/CD-1 mice (MLS9), male
from studies with animals. However, the data presented herein Sprague-Dawley rats (RLS9), male Dunkin-Hartley guinea pigs
and elsewhere suggest major inter-species differences in sulfa- (GLS9), male New Zealand rabbits (RaLS9), male Beagle dogs
tion and SULT enzyme proles.31 As a whole, the present qual- (DLS9), male Yucatan mini-pigs (PLS9) and male Cynomolgus
itative and quantitative interspecies study of BF and RB monkeys (CyLS9) were purchased from Research Institute for
sulfation showed that the monkey, mini-pig, rabbit and guinea Liver Diseases (RILD, Shanghai, China). Recombinant human
5780 | RSC Adv., 2016, 6, 5774–5783
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SULTs (rhSULT) were obtained from BD Gentest (Woburn, MA, shown in the d scale and were referenced to tetramethylsilane at
USA). Mixed, pooled human hepatocytes were purchased from d ¼ 0 ppm for the 1H and 13C spectra.
Celsis In vitro Technologies (Baltimore, MD, USA). Dehy-
droepiandrosterone (DHEA), 6-dichloro-p-nitrophenol (DCNP),
dithiothreitol (DTT), estrone, ibuprofen, quercetin, 30-phos-
phoadenosine-50-phosphosulfate (PAPS) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). All other reagents were
either of liquid chromatography (LC) grade or the highest grade
commercially available.
Recombinant SULT assay
The rhSULT (1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1E1, and 2A1) were
used to screen the involved isoform(s) in the sulfation of the six
bufadienolides in HLS9. Three substrate concentrations (1, 10
and 100 mM) were incubated with each of the rhSULT (0.02 to
0.2 mg mLꢀ1) at 37 ꢂC for 60 minutes. The incubations with
isoforms were carried out under standard assay procedures
described in the text.
Incubation system and analysis method
The standard incubation system for SULT reactions included
HLS9 or rhSULTs, DTT (8 mM), PAPS (1 mM), potassium
Kinetics characterization
phosphate buffer (50 mM, pH 7.4), MgCl2 (5 mM), and To estimate the kinetics parameters, the six compounds were
substrates in a nal volume of 200 mL. Aer pre-incubation at 37 incubated with HLS9, rhSULT2A1 and complex recombinants,
ꢂC for 3 minutes, the SULT reaction was initiated by adding which consisted of different proportions of rhSULT1A1*1,
PAPS and further incubated at 37 ꢂC in a water bath under 1A1*2, 1A2, 1B1, 1E1, and 2A1, respectively, according to their
constant shaking. The reaction was terminated by the addition hepatic contents in humans.19 The incubation conditions were
of ice-cold methanol (100 mL). The mixture was kept on ice until optimized to ensure that formation rates of metabolites were
ꢂ
pelleting at 20 000 g for 10 minutes at 4 C. Aliquots of super- in the linear range in relation to incubation time and protein
ꢂ
natants were stored at ꢀ20 C until analyses. Control incuba- concentration. The assays with HLS9 were conducted with
tions without PAPS, substrate or S9 were performed to ensure a differꢀen1 t protein concentration of 0.2 mg mLꢀ1 (BF), 0.06
that the metabolites produced were S9- and PAPS-dependent.
mg mL (RB), 0.4 mg mLꢀ1 (CB), 0.6 mg mLꢀ1 (BFT), 0.2
The Agilent 1200 HPLC system consisted of a quaternary mg mLꢀ1 (TCB), and 0.2 mg mLꢀ1 (DCB) for 30–60 min,
delivery system, a degasser, an auto-sampler and a UV-detector. respectively. The assays with SULT2A1 were conducted with
An Elite A SinoChrom ODS-BP (2.1 ꢃ 150 mm, 5 mm) analytical a differꢀen1 t protein concentration of 0.02 mg mLꢀ1 (BF), 0.006
column was used for quantication. The mobile phase con- mg mL (RB), 0.02 mg mLꢀ1 (CB), 0.06 mg mLꢀ1 (BFT), 0.006
sisted of an acetonitrile–0.1% formic acid aqueous solution at mg mLꢀ1 (TCB), and 0.02 mg mLꢀ1 (DCB) for 30–50 min,
a ow rate of 0.45 mL minꢀ1. An Applied Biosystems MDS Sciex respectively. The assays with complex isoforms were conducted
Qtrap 4500 Triple Quadrupole Mass Spectrometer (MS/MS) with a different protein concentration of 0.06 mg mLꢀ1 (BF),
equipped with an electrospray ionization (ESI) source was 0.02 mg mLꢀ1 (RB), 0.06 mg mLꢀ1 (CB), 0.2 mg mLꢀ1 (BFT), 0.04
used to analyze the target metabolites. The system was operated mg mLꢀ1 (TCB), and 0.06 mg mLꢀ1 (DCB) for 30–60 min,
in negative mode for BF–S (m/z 465.5–466.5), RB–S (m/z 462.5– respectively.
463.5), CB–S (m/z 520.7–521.7), BTF–S (m/z 522.4–523.4), TCB-S
To further explore the interaction between the bufadienolide
(m/z 480.8–481.8), and DCB–S (m/z 478.7–479.7). The negative derivatives and SULT2A1, kinetics analyses of other bufadie-
ion spray voltage and temperature were set at ꢀ4500 V and 600 nolide derivatives with classical structural features (Fig. S2†)
ꢂC, respectively. The curtain gas and collision-activated disso- were also carried out by using rhSULT2A1. The incubation
ciation gas were set at 10 psi and 5 psi, respectively; gas1 and conditions were optimized as described.
gas2 (nitrogen) were set at 30 L minꢀ1 and 45 L minꢀ1
respectively. The dwell times were 150 ms.
,
Chemical inhibition study
The six compounds were incubated with HLS9 in the presence
or absence of human SULT specic inhibitors or substrates,
including the SULT1A1 specic inhibitor mefenamic acid (0.5
mM),32 the SULT1E1 specic inhibitor ibuprofen (500 mM),
estrone (1 mM),32,33 the SULT2A1 substrate DHEA (10 mM) and
general inhibitors DCNP (10 mM) and quercetin (1 mM) (which
inhibit SULT1A1 and SULT1E1).34 To further assess the role of
SULT2A1, the inhibitory effects of DHEA (0–50 mM) toward the
sulfation of the six compounds catalyzed by HLS9 and SULT2A1
were investigated. The IC50 values were also determined.
Synthesis of metabolites and NMR spectroscopy
The sulfated metabolites of BF, RB, CB, BFT, TCB and DCB were
produced in vitro. Briey, the six compounds were each added
to the pyridine reaction mixture (2 mL), which contained
sulfuric acid (11.9 mg, 0.12 mM) and acetic anhydride (0.12
mM). Aer each mixture was stirred for 60 minutes at 60 ꢂC,
the reaction was quenched with 2 mL aqueous NH3 (25%). The
resultant residue was isolated and puried by reverse-phase-
column chromatography (methanol : H2O
purities of the six bufadienolides sulfate metabolites were
all above 98% as determined by HPLC-DAD analyses.
¼
1 : 2). The
Correlation study
All NMR experiments were performed on a Varian INOVA- The formation rates of the sulfate metabolites of the six
500 NMR spectrometer. 1H and 13C NMR spectra (500 MHz) compounds were compared with the rate of DHEA sulfation
were measured at room temperature (22 ꢂC). Chemical shis are using 16 individual HLS9 via linear regression analyses. The
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concentration of DHEA was 4 mM (near its Km). The six Professor of Liaoning Province, Dalian Outstanding Youth
compounds were incubated in HLS9 at protein concentrations Science and Technology Talent (2014J11JH132, 2015J12JH201),
of 0.02 mg mLꢀ1 (BF), 0.12 mg mLꢀ1 (RB), 0.4 mg mLꢀ1 (CB), 0.8 Project sponsored by Liaoning BaiQianWan Talents Program
mg mLꢀ1 (BFT), 0.6 mg mLꢀ1 (TCB) and 0.4 mg mLꢀ1 (DCB) for and Innovation Team of Dalian Medical University for nancial
30 minutes. Spearman's rank method was used to determine support.
the correlation analysis of HLS9 from the 16 individual donors.
When the r value was greater than or equal to 0.5 and the p value
was less than 0.05, the correlations were considered signicant.
1 J. B. Puschett, E. Agunanne and M. N. Uddin, Am. J. Kidney
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