Marine natural products as inhibitors of cystathionine beta-synthase activity

Article abstract of DOI:10.1016/j.bmcl.2015.01.013

A library consisting of characterized marine natural products as well as synthetic derivatives was screened for compounds capable of inhibiting the production of hydrogen sulfide (H₂S) by cystathionine beta-synthase (CBS). Eight hits were valid

Full text of DOI:10.1016/j.bmcl.2015.01.013

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1070–1072
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Marine natural products as inhibitors of cystathionine beta-synthase
activity
Megan K. Thorson a, Ryan M. Van Wagoner a, Mary Kay Harper a, Chris M. Ireland a, Tomas Majtan b,
Jan P. Kraus b, Amy M. Barrios a,
⇑
a Department of Medicinal Chemistry, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112, USA
b Department of Pediatrics, University of Colorado, Aurora, CO 80045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A library consisting of characterized marine natural products as well as synthetic derivatives was
screened for compounds capable of inhibiting the production of hydrogen sulfide (H2S) by cystathionine
beta-synthase (CBS). Eight hits were validated and shown to inhibit CBS activity with IC50 values ranging
from 83 to 187 lM. The majority of hits came from a series of synthetic polyandrocarpamine derivatives.
In addition, a modified fluorogenic probe for H2S detection with improved solubility in aqueous solutions
Received 13 August 2014
Revised 5 January 2015
Accepted 7 January 2015
Available online 14 January 2015
is reported.
Keywords:
Ó 2015 Elsevier Ltd. All rights reserved.
Hydrogen sulfide
Sulfur metabolism
Fluorogenic probe
Inhibitor screen
Cystathionine beta-synthase (CBS) is a type II fold pyridoxal-50-
phosphate (PLP) dependent enzyme that plays critical roles in sul-
fur metabolism. For example, CBS is responsible for regulating
homocysteine levels. Mutations in the gene that encodes for CBS
lead to homocystinuria, a genetic condition characterized by very
assay for CBS inhibitor screening.10 Despite some recent progress
in the field, there are only a few CBS inhibitors available,10–13
and only two that have routinely been used for inhibition of CBS
activity in vivo (aminooxyacetic acid, AOAA and hydroxylamine,
HA).11–13 Neither AOAA nor HA is selective for CBS over the other
major H2S-producing, PLP-dependent enzyme cystathionine
gamma-lyase (CGL)11 and there has been some controversy over
their inhibitory potency against CBS.12 Consequently there is a sig-
nificant need for the development of potent, selective CBS
inhibitors.
high plasma homocysteine levels (>50 lM compared to <10 lM
in the general population),1 lens dislocation, skeletal abnormalities
and vascular disease.1,2 In addition to its clear role in homocystinu-
ria, CBS plays a key role in the production of hydrogen sulfide
(H2S),
a recently recognized ‘gasotransmitter’ with biological
effects similar to, and often complementary to, those of nitric oxide
(NO).3–5 H2S acts as a vasodilator, has anti-inflammatory properties
and has been shown to be cardioprotective.6–8 While a possible
link between CBS-mediated homocysteine metabolism, H2S pro-
duction and cardiovascular health is intriguing, it has not been pos-
Natural products isolated from marine invertebrates have been
an exceptional source of chemical diversity and pharmaceutical
lead compounds.14 With the aim of streamlining the initial screen-
ing process while maximizing the chemical diversity that can be
sampled, our collaborators in the Ireland lab created a protocol
for fractionating marine invertebrate extracts.15,16 They also
selected a series of chemically diverse, well-characterized natural
products and synthetic derivatives for use in screening. In the work
reported here, we screened this subset of the ‘Marine Invertebrate
Compound Library’ consisting of 160 characterized marine natural
products and 80 purified synthetic derivatives, termed ‘MICL-240’,
for compounds capable of inhibiting CBS activity. From this library,
sible to obtain
a detailed understanding of these potential
connections because of a lack of chemical tools for monitoring
and manipulating the activity of CBS.9
We have previously shown that an H2S-selective, fluorogenic
probe can be used to monitor CBS activity and provides a facile
Abbreviations: CBS, cystathionine beta-synthase; CGL, cystathionine gamma-
lyase; H2S, hydrogen sulfide; PLP, pyridoxal-50-phosphate; AOAA, aminooxyacetic
acid; HA, hydroxylamine; MICL, Marine Invertebrate Compound Library; AzMC,
7-azido-4-methylcoumarin; AzCC, 7-azido-4-carbamoyl methyl coumarin.
we identified eight compounds with IC50 values below 200
(range: 83–187 M).
lM
l
Aromatic azides are now well-established H2S-reactive func-
tional groups, and the reduction of an aryl azide to an aryl amine
⇑
Corresponding author. Tel.: +1 801 581 3198.
E-mail address: amy.barrios@utah.edu (A.M. Barrios).
http://dx.doi.org/10.1016/j.bmcl.2015.01.013
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

M. K. Thorson et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1070–1072
1071
with concomitant increase in fluorescence has been exploited in
numerous H2S-selective fluorogenic probes.17,18 In previous work,
we and others have shown that 7-azido-4-methylcoumarin (AzMC)
undergoes a dramatic increase in fluorescence upon exposure to
H2S, with detection limits in the high nanomolar range
(Scheme 1).10,19 This probe has significant utility as an assay for
CBS and CGL activity, readily detecting the H2S produced by these
enzymes in a high-throughput format, facilitating enzyme inhibi-
tion screens.10 In the course of this work we noticed that, at higher
concentrations of probe or low concentrations of DMSO, the AzMC
probe would sometimes precipitate out of aqueous solution. This
has not been a problem in our screening efforts, but could be an
issue in cases where minimizing or eliminating DMSO is necessary.
Therefore, we created a modified version of the probe with a
carboxylate group rather than a methyl group at the 4-position
(Scheme 1) for increased solubility in aqueous solutions. The new
probe, 7-azido-4-carbamoylmethylcoumarin (AzCC) was synthe-
sized as follows: 3-aminophenol was protected with ethyl chloro-
formate prior to a Pechmann condensation reaction to form the
coumarin. After deprotection, the azide was formed from the
amine as shown in Scheme 1.10 The AzCC probe has an identical
response to H2S as the parent AzMC (Fig. 1), with the advantage
of increased solubility in aqueous solution. In addition, with the
carboxylate functionality, this probe could easily be conjugated
to another moiety if desired.
were subjected to a series of secondary screens. The validated hits,
with IC50 values, are shown in Figure 3.
For each compound, a dose–response curve was measured to
obtain the apparent IC50 values for inhibition of the activity of
full-length, wild-type CBS in the presence of 300 lM of the endog-
enous activator, S-adenosylmethionine. In addition, a series of con-
trol experiments was run to ensure that the hits did not interfere
directly or indirectly with the assay. Specifically, the lead com-
pounds shown in Figure 3 did not react with either the azidocoum-
arin probe or hydrogen sulfide to an appreciable extent (see
Supporting information). Interestingly, the majority of the hits
(hits A6, A9, B7 and C11 from the MNP2 sub-library) are synthetic
1
1
1
2
3
4
5
6
7
8
9
10 11
A
B
C
D
E
F
85.61597849
93.59758767
91.06346034
91.15800019
71.92830709
68.9305372
87.08681645
93.78909874
98.5277855
62.74310533
90.13383257
89.29926352
79.86854645
97.88946812
3
77.83871644
89.38199396
83.87802717
84.52014601
63.12792593
93.5929244
84.09802623
92.46135783
4
64.13564165
90.32492427
329.2917102
79.72779742
69.69782855
91.39575879
96.34210838
96.94820056
5
89.14895365
88.30552145
68.33035066
87.54010503
87.90535672
95.39681951
94.73929713
101.2562867
6
99.68645413
66.67203326
104.3928582
108.0366197
96.42801522
105.7737623
106.8368803
108.3122116
7
96.88740109
112.6142145
103.4360399
106.0172556
113.6787146
112.733458
106.2262571
103.9712026
109.5567261
118.8685553
108.5488728
117.1566301
9
122.1545607
119.4989384
113.4815299
106.6535613
111.1101704
81.5635213
90.6807939
81.83746342
110.9079686
109.9310695
115.0467191
117.8452714
111.5416573
115.337845
76.4825851
1
103.6874064
83.62124335
82.50787705
8
G
H
80.9138461
104.5592398
2
1
116.6444367
106.3927252
10 11
A
B
C
D
E
F
100.5573471
69.93266383
93.11732882
92.36423844
89.07300531
86.88263464
81.55806995
84.76810328
2
60.99608834
90.0810911
98.08242716
88.99675536
78.50270891
89.23357075
89.6663469
83.98432733
3
51.99363994
88.82380852
110.2968055
90.98332985
35.55990229
89.22682848
89.25048922
88.09254186
4
48.4656435
69.90332685
95.19352399
87.47566656
62.82152227
89.49854898
77.35060904
65.833938
49.63128892
61.79767932
101.2242739
94.63217697
96.72586323
104.4537456
69.83531477
96.99728049
6
76.45655461
56.39965962
100.6496093
108.0274181
109.2348714
97.85690145
96.90971445
86.17147243
7
98.67035759
94.55101999
104.523122
103.6700699
98.99625993
90.54486997
104.2049894
107.0533915
8
52.18996175
112.9871167
108.6101718
110.3642232
83.80752842
104.8809126
103.8298683
108.1001785
9
111.8531212
116.1208369
110.4572671
113.841922
113.5299169
111.0994543
99.40058817
112.1231206
71.59574526
109.698696
119.3851506
111.7990443
112.5920806
114.7931109
110.029723
112.1083321
With the aim of identifying novel CBS inhibitors, we screened
the MICL-240 library looking for compounds that significantly
decreased enzyme activity. As shown in Figure 2, several hits were
identified in this initial screen using 2
pounds capable of reducing the activity of CBS by more than 50%
were re-screened at 4 g and background corrected. The hits that
showed a concentration-dependent inhibition of enzyme activity
lg of each compound. Com-
G
H
5
10 11
l
A
B
C
D
E
F
88.6224448
93.69116835
103.2233328
80.68423491
88.93768889
67.82441909
75.95069478
73.33962539
78.39850718
93.26269171
91.49559349
80.92061478
76.84526087
89.13336491
82.59029247
96.05464439
90.2945773
85.14175583
81.38564302
90.56261386
83.68674652
89.73814423
67.71151172
71.38614432
65.58056169
88.57024801
95.42154252
81.2016987
89.31337667
72.62324318
89.65642756
93.87017797
74.16439663
73.51826879
87.73920222
87.4715138
94.57599802
78.07123559
98.45434531
82.88844058
87.84378501
95.47460839
93.56467176
81.48329285
94.24006969
90.9745968
98.3129602
89.35106158
99.54802251
123.1177211
98.67292476
56.7033534
88.88053333
95.81818822
101.1366276
95.96835476
116.0016102
91.83555644
104.5383301
94.51900369
108.2164151
103.6607118
106.5192816
69.47461495
116.5570382
87.4843184
102.5351688
104.420566
99.75641382
101.9612883
114.8890178
82.80631094
119.3719039
113.0881232
68.9902348
R
R
114.4238574
i) NaNO2
108.100221
1
H2SO4, 0oC
114.4053907
95.26082989
112.995761
ii) NaN3
G
H
H2N
O
O
N3
O
O
0
10
20
30
40
50
60
80
80
100
H2S
Initial Screen, 2µg
Scheme 1. The 7-azidocoumarin analogs can be readily synthesized from the 7-
aminocoumarin starting materials. H2S-mediated reduction regenerates the fluo-
rescent 7-aminocoumarin. When R=H, the probe is 7-azido-4-methylcoumarin
(AzMC). When R=COOH, the probe is 7-azido-4-carbamoylmethylcoumarin (AzCC).
Figure 2. Heat map showing the results from the initial library screen. Lighter
boxes represent strong inhibition of CBS activity and darker boxes represent weak
or no inhibition.
Figure 1. Hydrogen sulfide reduces both AzMC (10
(10 M, open circles) to the corresponding 7-aminocoumarins with a concomitant
linear increase in fluorescence.
lM, solid circles) and AzCC
l
Figure 3. Top validated hits from the MICL-240 marine natural product library with
IC50 values for inhibition of CBS activity.

1072
M. K. Thorson et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1070–1072
Figure 4. An analysis of structurally related compounds from MICL-240 lacking inhibitory activity against CBS provides insight into the SAR of the hit compounds.
compounds derived from polyandrocarpamines A and B, 2-amino-
imidazolone compounds isolated from the ascidian Polyandrocarpa
sp.20 The MICL-240 library contained 17 compounds from this
structural class in MNP plate 2, wells A2–B8. While several of these
compounds inhibited CBS activity in our initial screen (Fig. 2), only
5 showed activity in both of the secondary screens (Fig. S1). An
investigation of the structures of the validated hits compared to
compounds that did not inhibit CBS activity in the initial screen
provides some information about the structure–activity relation-
ship of these compounds. As shown in Figure 4, compounds con-
taining only the thiohydantoin or aromatic moiety are poor
inhibitors of CBS activity (compounds MNP2 A2, A7 and A10). In
addition, compounds MNP2-A8, MNP2-B6 and MNP2-B8, where
both hydroxyl groups are etherified and the thiohydantoin either
is methylated both at the 3 position and on the sulfur atom or
replaced by a hydantoin moiety do not show up as hits. Thiohydan-
toin methylation alone (compound MNP2-B7) does not prevent
enzyme inhibition while replacement of the thiohydantoin moiety
with a hydantoin moiety does (MNP2-B6, MNP2-B3).
CBS plays a key role in biological sulfur metabolism and its
importance in numerous physiological and pathological pathways
is not yet fully appreciated. One of the key challenges in under-
standing the biological roles of this enzyme is a lack of potent,
selective inhibitors that can be used to probe its activity in vivo.
In this work, we have identified a handful of novel scaffolds for
inhibiting CBS. The majority of the hits from our screen of the
MICL-240 library were synthetic compounds derived from the
polyandrocarpamines A and B, marine natural products isolated
from the ascidian Polyandrocarpa sp. These scaffolds may serve as
useful starting points for the development of potent and selective
CBS-targeted tools necessary for advancing the field.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.01.
013.
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Acknowledgements
This work was supported by the American Heart Association
grant 11GRNT7530023 to A.M.B. and award CA36622 from the
National Institutes of Health to C.M.I.