Cyclic Cystine-Bridged Peptides from the Marine Sponge Clathria basilana Induce Apoptosis in Tumor Cells and Depolarize the Bacterial Cytoplasmic Membrane

Article abstract of DOI:10.1021/acs.jnatprod.7b00477

Investigation of the sponge Clathria basilana collected in Indonesia afforded five new peptides, including microcionamides C (1) and D (2), gombamides B (4), C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-N-styrylbutanamide (7), along with 11 known compounds, among them microcionamide A (3). The structures of the new compounds were elucidated by one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of the constituent amino acid residues in 1-7 were determined by Marfey's analysis. Microcionamides A, C, and D (1-3) showed in vitro cytotoxicity against lymphoma (Ramos) and leukemia cell lines (HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian carcinoma cell line (A2780) with IC₅₀ values ranging from 0.45 to 28 μM. Mechanistic studies showed that compounds 1-3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1 and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition, microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal inhibitory concentrations between 6.2 and 12 μM. Mechanistic studies indicate dissipation of the bacterial membrane potential.

Full text of DOI:10.1021/acs.jnatprod.7b00477

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX
pubs.acs.org/jnp
Cyclic Cystine-Bridged Peptides from the Marine Sponge
Clathria basilana Induce Apoptosis in Tumor Cells and Depolarize the
Bacterial Cytoplasmic Membrane
†
,‡
§
⊥
⊥
∥
Amin Mokhles i†, Fabian Stuhldreier, Katharina W. Wex, Anne Berscheid, Rudolf Hartmann,
▽
○
▽
Nidja Rehberg, Parichat Sureechatchaiyan, Chaidir Chaidir, Matthias U. Kassack,
†
⊥
§
§
,†
Rainer Kalscheuer, Heike Bro
̈
̈
tz-Oesterhelt, Sebastian Wesselborg, Bjorn Stork, Georgios Daletos,*
,
†
and Peter Proksch*
†
Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, Universitat
̈
sstraße 1, D-40225 Du
̈
‡
Department of Marine Biology, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran
§
Institute of Molecular Medicine I, Medical Faculty, Heinrich Heine University, Universitat
̈
sstraße 1, D-40225 Du
Department of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine,
University of Tubingen, Auf der Morgenstelle 28/E8, 72076 Tubingen, Germany
lich, Wilhelm-Johnenstrasse, 52428 Ju
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Universitatsstraße 1,
D-40225 Dusseldorf, Germany
Center for Pharmaceutical and Medical Technology, Agency for the Assessment and Application Technology,
0340 Jakarta, Indonesia
S Supporting Information
̈
sseldorf, Germany
⊥
̈
̈
∥
Institute of Complex Systems: Strukturbiochemie, Forschungszentrum Ju
̈
̈
lich, Germany
▽
̈
̈
○
1
*
ABSTRACT: Investigation of the sponge Clathria basilana
collected in Indonesia afforded five new peptides, including
microcionamides C (1) and D (2), gombamides B (4),
C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-
N-styrylbutanamide (7), along with 11 known compounds,
among them microcionamide A (3). The structures of the new
compounds were elucidated by one- and two-dimensional NMR
spectroscopy as well as by high-resolution mass spectrometry.
The absolute configurations of the constituent amino acid
residues in 1−7 were determined by Marfey’s analysis.
Microcionamides A, C, and D (1−3) showed in vitro cytotoxicity
against lymphoma (Ramos) and leukemia cell lines (HL-60,
Nomo-1, Jurkat J16), as well as against a human ovarian carci-
noma cell line (A2780) with IC50 values ranging from 0.45 to
2
8 μM. Mechanistic studies showed that compounds 1−3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1
and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition,
microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal
inhibitory concentrations between 6.2 and 12 μM. Mechanistic studies indicate dissipation of the bacterial membrane potential.
arine sponges represent a prolific source of struc-
turally unique peptides possessing diverse bioactivities,
The genus Clathria (order Poecilosclerida, family Micro-
cionidae) includes more than 800 species that are widely
5
M
primarily as antibiotic, anticancer, and neuroprotective agents,
thus suggesting their potential value for the development of leads
distributed in the shallow waters of tropical and temperate
regions. Chemical investigation of sponges belonging to this
genus has provided a diverse array of secondary metabolites,
1
in drug discovery. Prominent examples of bioactive sponge-
derived peptides include koshikamide A and calyxamide A
6
,7
8
9,10
11,12
such as alkaloids, carotenoids, steroids, and peptides.
1
reported from Theonella sp. and Discodermia calyx, respectively,
The latter class of compounds includes microcionamides A and B
both showing strong cytotoxicity toward P388 murine leukemia
as well as gombamide A from Clathria abietina and Clathria
2
,3
cells, as well as the diaminoacrilic acid-containing cyclic
peptides callyaerins A and B possessing inhibitory activity against
Received: June 4, 2017
4
Mycobacterium tuberculosis.
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00477
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Journal of Natural Products
Article
1
13
Table 1. H (600 MHz), C (150 MHz), HMBC, and ROESY NMR Data (DMSO-d , δ in ppm) of Microcionamide C (1)
6
a
b
Uunit
position
δ , type
δH (J in Hz)
HMBC
ROESY
C
E-PEA
NH
10.02, d (10.0)
Cys -α, E-PEA-β
1
α
122.8, CH
112.5, CH
1:136.0, C
7.35, dd (10.0, 14.8)
6.26, d (14.8)
Cys -CO, E-PEA-β, E-PEA-1
1
β
E-PEA-α, E-PEA-1, E-PEA-2/6
Ile -γ′
2
aromatic
2:124.9, CH
3:128.5, CH
4:126.1, CH
5:128.5, CH
6:124.9, CH
7.32, br dd (7.4, 1.4)
7.28, br dd (7.4, 7.1)
7.16, tt (7.1, 1.4)
E-PEA-β, E-PEA-1, E-PEA-6, E-PEA-4
E-PEA-1, E-PEA-5, E-PEA-4
E-PEA-2/6, E-PEA-3/5
7.28, br dd (7.4, 7.1)
7.32, br dd (7.4, 1.4)
8.15, d (7.8)
E-PEA-1, E-PEA-3, E-PEA-4
E-PEA-β, E-PEA-1, E-PEA-2, E-PEA-4
Cys1
Ile2
NH
CO
α
Ile -CO
2
Ile -α
2
167.2, C
52.6, CH
40.7, CH2
c
4.34
Cys -CO, Cys -β, Ile -CO
E-PEA-α, E-PEA-NH
1
1
2
β
3.19, br t (13.2); 3.30, dd (13.2, 4.7)
7.99, br s
Cys -CO, Cys -α
1 1
NH
CO
α
170.4, C
57.7, CH
34.6, CH
24.5, CH2
15.4, CH3
11.0, CH3
3.96, t (7.6)
1.88, m
Ile -CO, Ile -CO, Ile -β, Ile -γ, Ile -γ′
Cys -NH
2
3
2
2
2
1
β
Ile -α
2
γ
1.51, m; 1.06, m
0.86, d (6.9)
Ile -α
2
Ile -α
3
γ′
δ
Ile -α
2
c
0.81
Ile3
Ile4
Ile5
NH
CO
α
7.75, br s
171.2, C
57.6, CH
36.2, CH
24.5, CH2
15.1, CH3
11.2, CH3
4.04, br s
Ile -γ, Ile -γ′
Ile -γ
3
3
2
β
1.82, m
Ile -γ, Ile -γ′, Ile -δ, Ile -CO
3
3
3
3
γ
1.20, m; 1.54, m
0.93, d (6.8)
Ile -α
3
γ′
δ
Ile -α
3
c
0.87
c
NH
CO
α
7.34
d
n.d.
c
56.7, CH
35.9, CH
23.5, CH2
15.6, CH3
11.1, CH3
4.33
Ile -γ, Ile -γ′
Ile -β
5
4
4
β
2.00, br s
γ
1.16, m; 1.35, m
Ile -α, Ile -γ′, Ile -δ
4
4
4
c
γ′
δ
0.89
Ile -α, Ile -γ
4
4
c
0.82
Ile -β
4
NH
CO
α
8.53, d (9.8)
Cys -CO
Cys -α
6
6
170.4, C
57.8, CH
36.2, CH
23.9, CH2
15.2, CH3
10.7, CH3
4.19, dd (9.8, 6.9)
1.84, m
Ile -CO, Cys -CO, Ile -β, Ile -γ, Ile -γ′
5
6
5
5
5
β
Ile -α, Ile -γ′
Ile -α
4
5
5
γ
1.40, m; 1.10, m
Ile -α
5
c
γ′
δ
0.84
Ile -α, Ile -β
5
5
c
0.80
Cys6
Ile7
NH
CO
α
8.83, d (8.1)
Cys -β
Ile -α, Ile -β, Ile -γ
6
7
7
7
168.9, C
52.3, CH
40.1, CH2
4.62, br ddd (10.3, 8.1, 4.6)
3.11, br dd (12.8, 10.3); 3.39, dd (12.8, 4.6)
8.10, br d (5.4)
Cys -CO, Ile -CO, Cys -β
Ile -NH
5
6
7
6
β
Cys -CO, Cys -α
6 6
NH2
CO
α
Ile -CO, Ile -α, Ile -β
7
Ile -α, Ile -β, Ile -γ′
7
7
7
7
7
167.8, C
56.0, CH
36.2, CH
23.4, CH2
14.1, CH3
10.8, CH3
3.66, br t (5.4)
1.79, m
Ile -CO, Ile -γ, Ile -γ′
Cys -NH
7
7
7
6
β
Ile -CO, Ile -δ
7
Cys -NH
7
6
γ
1.10, m; 1.47, m
Ile -α
7
Cys -NH
6
c
γ′
δ
0.89
Ile -α
7
c
0.83
a
b
c
d
Data extracted from HSQC and HMBC spectra. Sequential NOEs. Signal overlap prevents determination of couplings. n.d.: not detected.
gombawuiensis, respectively, all featuring a disulfide linkage that is
rarely found in sponge-derived metabolites.
During our ongoing research for bioactive metabolites from
marine sponges, we investigated a specimen of the marine
sponge Clathria basilana, which was collected at Ambon Island,
Indonesia. Notably, the MeOH extract completely inhibited the
growth of the murine lymphoma L5178Y cell line at a
concentration of 10 μg/mL. Bioactivity-guided isolation afforded
five new peptides (1, 2, 4−6) and one new amide derivative (7),
in addition to one known peptide (3) and 10 known, structurally
unrelated aromatic compounds. In this study, the structure elu-
cidation of the new compounds as well as results of cytotoxicity
11,12
B
DOI: 10.1021/acs.jnatprod.7b00477
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Journal of Natural Products
Article
and antibacterial assays, followed by mechanistic studies, are
reported.
supported by the HMBC correlations from Ile -NH and Ile -Hα
5
5
to Cys -CO, as well as by the ROESY correlations between Ile -
6
4
Hα and Ile -Hβ and between Ile -NH and Cys -Hα (Figure 1).
5
5
6
RESULTS AND DISCUSSION
The latter cysteine moiety (Cys ) was linked to Ile based on the
■
6 7
HMBC cross-peak from Cys -Hα to Ile -CO (δ 167.8) and the
The MeOH extract of C. basilana was subjected to liquid−liquid
fractionation to yield n-hexane, EtOAc, and n-BuOH fractions.
Subsequent column chromatography of the EtOAc or n-BuOH
fraction followed by semipreparative reversed-phase HPLC afforded
five new peptides (1, 2, 4−6) and a new amide derivative (7).
Compound 1 was obtained as a white, amorphous solid. Its
molecular formula was determined to be C H N O S on the
6
7
C
ROESY correlation between Cys -NH and Ile -Hα, thus leading
6
7
to the overall peptide structure E-PEA-Cys -Ile -Ile -Ile -Ile -
1
2
3
4
5
Cys -Ile . Additionally, the cyclic nature of 1 was evident from
6
7
the disulfide linkage of the two cysteine residues that formed a
cystine moiety, thus accounting for the last degree of unsatu-
ration based on the molecular formula (C44H N O S ). The
4
4
72
8
7 2
72 8 7 2
+
basis of the prominent ion peak at m/z 889.5033 [M + H] in the
HRESIMS spectrum, accounting for 13 degrees of unsaturation.
In the H NMR spectrum, measured in DMSO-d , the peptidic
assignment was further supported by HRESIMS/MS, which+
showed the fragmentation ions at m/z 770.4297 for [M − E-PEA]
1
+
and at m/z 657.3454 for [M − (E-PEA+Ile
)] , originating from
7
6
7
nature of 1 was evident from the presence of NH signals in the
amide region (δH 7.34−10.02) and characteristic signals of
subsequent cleavage of the E-PEA and Ile units, both located
at the linear parts of the peptide (Figure S1-7). Hence, compound 1
was identified as a new natural product, and the name
microcionamide C was proposed given the structural relationship
between this compound and the known compounds micro-
α-amino protons (δ 3.66−4.62), as well as from a cluster of
H
primary and secondary methyl groups in the aliphatic region
(
δH 0.81−2.00). Detailed analysis of the 2D NMR (HSQC,
11
TOCSY, ROESY, and HMBC) spectra of 1 allowed the
assignment of seven spin systems, including those of five
isoleucine (Ile) and two cysteine (Cys) residues (Table 1).
The remaining signals included those of a monosubstituted
cionamides A (3) and B.
phenyl ring [δ 7.32 (2H, dd, J = 7.4, 1.4 Hz), 7.28 (2H, dd,
H
J = 7.4, 7.1 Hz), and 7.16 (1H, tt, J = 7.1, 1.4 Hz)] and two
olefinic protons at δH 6.26 (PEA-Hβ) and 7.35 (PEA-Hα),
suggesting the presence of a 2-phenylethen-1-amine (PEA) unit,
as supported by the HMBC correlations from PEA-Hα to
PEA-C1 (δ 136.0) and from PEA-Hβ to PEA-C2/6 (δ 124.9),
C
C
as well as by the TOCSY correlations of both PEA-Hα and
PEA-Hβ with PEA-NH. The configuration of the double bond of
PEA was deduced as E on the basis of the large coupling constant
3
(
J = 14.8 Hz) measured for the respective protons.
The connectivity of the amino acid residues and of E-PEA was
established by key HMBC and ROESY correlations (Figure 1).
Compound 2 was isolated as a white, amorphous solid.
The molecular formula of 2 was established as C H N O S by
44
72
8
7 2
1
HRESIMS, identical to that found for 1. Moreover, the H NMR
data of 2 were almost superimposable to those of 1, the only
notable difference being the resonances and coupling constants
of the olefinic protons at δ 5.74 (1H, d, J = 10.0 Hz) and 6.72
H
(
(
(
1H, t, J = 10.0 Hz), suggesting a (Z)-2-phenylethen-1-amine
Z-PEA) unit in 2 instead of a E-PEA unit, as in the case of 1
Table S2-1). Likewise, HRESIMS/MS of 2 showed fragmenta-
Figure 1. Key HMBC, ROESY, and TOCSY correlations of 1 and 4.
Accordingly, the HMBC correlations from E-PEA-Hα to
Cys -CO (δ 167.2), from Cys -NH to Ile -CO (δ 170.4),
tion ions originating from cleavage of the Z-PEA and Ile units
(Figure S2-9). Hence, the structure of 2 was assigned as the Z
isomer of 1 and was named microcionamide D.
7
1
C
1
2
C
and from Ile -Hα to Ile -CO (δ 171.2), along with the ROESY
2
3
C
correlations between Cys -Hα and E-PEA-NH and between
Compound 4 was obtained as a white, amorphous solid.
The molecular formula of 4 was deduced to be C H N O S
9 2
based on the prominent ion peak at m/z 1002.4572 [M + H] in
1
Ile -Hα and Cys -NH, suggested the peptide substructure
2
1
50 67
9
+
E-PEA-Cys -Ile -Ile . Moreover, the sequence Ile -Ile -Cys was
1
2
3
4
5
6
C
DOI: 10.1021/acs.jnatprod.7b00477
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Journal of Natural Products
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1
13
Table 2. H (600 MHz), C (150 MHz), HMBC, and ROESY NMR Data (DMSO-d , δ in ppm) of Gombamide B (4)
6
a
unit
position
δC, type
δH (J in Hz)
HMBC
ROESY
E-PEA
NH
10.32, d (10.0)
Cys -NH, Cys -α
1
1
α
123.0, CH
113.2, CH
1: 136.2, C
7.36, dd (14.7, 10.0)
6.33, d (14.7)
Cys -CO, E-PEA-1
1
β
E-PEA-NH, E-PEA-α, E-PEA-2/6, E-PEA-1
E-PEA-NH
aromatic
2: 125.3, CH
3: 128.6, CH
4: 126.4, CH
5: 128.6, CH
6: 125.3, CH
7.37, br d (7.7)
7.30, br t (7.7)
7.17, br t (7.7)
7.30, br t (7.7)
7.37, br d (7.7)
7.72, d (8.6)
E-PEA-β, E-PEA-1, E-PEA-6, E-PEA-4
E-PEA-1, E-PEA-5
E-PEA-2/6
E-PEA-1, E-PEA-3
E-PEA-β, E-PEA-1, E-PEA-2
Phe-CO, Cys -α, Cys -β
Cys1
Phe
NH
E-PEA-NH, Phe-α
E-PEA-NH
1
1
CO
167.8, C
51.6, CH
42.1, CH2
α
4.64, ddd (12.0, 8.6, 3.5)
2.93, dd (14.4, 12.0); 3.15, dd (14.4, 3.5)
8.97, d (8.8)
Cys -CO, Cys -β, Phe-CO
1 1
β
Cys -CO, Cys -α
1 1
NH
Phe-α, Phe-β, Pro -CO
Pro -α
3
3
CO
170.8, C
α
53.5, CH
34.0, CH2
1: 138.3, C
4.81, m
Phe-CO, Phe-β, Phe-1, Pro -CO
Cys -NH
3
1
β
2.99, m; 3.02, m
Phe-CO, Phe-α, Phe-1, Phe-2/6
aromatic
2: 129.3, CH
3: 128.1, CH
4: 126.2, CH
5: 128.1, CH
6: 129.3, CH
7.43, br d (7.5)
7.29, br t (7.5)
7.20, br t (7.5)
7.29, br t (7.5)
7.43, br d (7.5)
Phe-β, Phe-6, Phe-4
Phe-1, Phe-5
Phe-2/6
Phe-1, Phe-3
Phe-β, Phe-2, Phe-4
Pro3
Pro4
CO
α
171.4, C
60.3, CH
31.2, CH2
20.1, CH2
45.9, CH2
169.3, C
4.36, d (7.8)
Pro -CO, Pro -CO, Pro -β, Pro -γ, Pro -δ
Pro -α, Pro -β, Phe-NH
3
4
3
3
3
4
4
β
1.73, m; 1.88, m
1.42, m; 0.55, m
3.29, m; 3.26, m
Pro -CO, Pro -α, Pro -γ
3
3
3
γ
Pro -α
3
δ
Pro -α, Pro -β
3 3
CO
α
59.2, CH
30.2, CH2
21.9, CH2
46.3, CH2
4.84, dd (9.5, 2.0)
1.87, m; 2.22, m
1.76, m
Pro -β, Pro -γ, Pro -δ
Pro -α, Cys -α
4
4
4
3
5
β
Pro -CO, Pro -α, Pro -δ
Pro -α
4
4
4
3
γ
Pro -α
4
δ
3.34, m; 3.48, m
8.01, d (10.0)
Pro -β, Pro -γ
4 4
Cys5
Leu
NH
CO
α
Leu-CO, Cys -α, Cys -β
Leu-α
5
5
167.9, C
48.4, CH
38.0, CH2
4.56, td (10.0, 5.2)
Leu-CO, Cys -CO, Cys -β
Pro -α
5
5
4
β
2.61, br dd (12.0, 10.0); 3.22, br dd (12.0, 5.2)
8.05, d (9.2)
Cys -α, Cys -CO
5
5
NH
CO
α
Leu-α, Leu-β, Ile-CO
Ile-α, Ile-β
170.5, C
50.5, CH
41.2, CH2
24.0, CH
21.3, CH3
23.3, CH3
4.49, ddd (9.2, 8.0, 7.7)
1.58, m
Leu-CO, Leu-β, Leu-γ, Ile-CO
Cys -NH
5
β
Leu-CO, Leu-α, Leu-γ, Leu-δ, Leu-δ′
Leu-δ, Leu-δ′
Leu-β, Leu-γ, Leu-δ′
Leu-β, Leu-γ, Leu-δ
γ
1.63, m
δ
0.86, d (6.3)
0.89, d (6.4)
8.07, d (8.8)
δ′
NH
CO
α
Ile
Glp-CO, Ile-α, Ile-β
Glp-α, Glp-NH
170.7, C
57.0, CH
35.5, CH
24.2, CH2
15.4, CH3
10.4, CH3
4.16, t (8.8)
1.73, m
Ile-CO, Ile-β, Ile-γ, Ile-γ′, Glp-CO
Ile-CO, Ile-α, Ile-γ
Leu-NH
β
γ
1.02, m; 1.42, m
0.75, d (6.7)
0.72, t (7.4)
7.80, s
Ile-α, Ile-β, Ile-δ, Ile-γ′
Ile-α, Ile-β, Ile-γ
γ′
δ
Ile-β, Ile-γ
Glp
NH
CO
CO′
α
Glp-CO′, Glp-α, Glp-β, Glp-γ
Ile-NH
Ile-NH
172.4, C
177.4, C
55.1, CH
25.3, CH2
29.0, CH2
4.10, dd (8.7, 4.0)
1.78, m; 2.21, m
2.09, m; 2.07, m
Glp-CO, Glp-CO′, Glp-β, Glp-γ
Glp-CO, Glp-CO′
Glp-CO′, Glp-α, Glp-β
β
γ
a
Sequential NOEs.
D
DOI: 10.1021/acs.jnatprod.7b00477
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Journal of Natural Products
Article
the HRESIMS spectrum, accounting for 22 degrees of unsatu-
Pro -Cys -Leu-Pro -Pro -Cys -Glp. This assignment was also
6
1
3
4
5
ration. The planar peptidic structure of 4 was distinguished by
evident by the ROESY cross-peaks between Cys -NH/Leu-NH,
1
the abundance of the amide (NH) (δ 7.72−10.32) and α-amino
Leu-NH/Pro -Hα, and Cys -NH/Glp-Hα. The remaining two
H
3
5
(
δ 4.10−4.84) protons, as well as by the cluster of primary and
NH protons at δ 6.90 (1H, s) and 7.27 (1H, s) were attributed
H
H
1
secondary methyl groups in the aliphatic region of the H NMR
spectrum (Table 2). Analysis of the COSY and TOCSY NMR
spectra revealed the spin systems of seven amino acid residues,
including Pro (2 equiv), Cys (2 equiv), Phe, Leu, and Ile,
together with the E-PEA unit and the unusual amino acid residue
pyroglutamic acid (Glp). The presence of the latter was con-
firmed by key HMBC correlations from Glp-Hα to Glp-CO′
to a terminal carboxamide group, as supported by their HMBC
correlations to Pro -CO and Pro -Cα (Figure 2, Table 3). Finally,
6
6
(
δ 177.4) and Glp-Cγ (δ 29.0), from Glp-NH to Glp-CO′ and
C
C
Glp-Cα (δ 55.1), and from Glp-Hβ to Glp-CO (δ 172.4).
C
C
In analogy to 1 and 2, the linkages and assignments of the amino
acid residues in 4 were established by HMBC and ROESY NMR
data (Table 2). Accordingly, the connection between E-PEA and
Cys was deduced by the HMBC correlation from E-PEA-Hα to
1
Cys -CO (δ 167.8) as well as the ROESY cross-peak between
1
C
E-PEA-NH and Cys -Hα. Moreover, the HMBC correlations
1
from Cys -Hα and Cys -NH to Phe-CO (δ 170.8), from
1
1
C
Phe-Hα and Phe-NH to Pro -CO (δ 171.4), and from Pro -Hα
3
C
3
to Pro -CO (δ 169.3) disclosed the peptide fragment E-PEA-
4
C
Cys -Phe-Pro -Pro (Figure 1). This was further corroborated
1
3
4
Figure 2. Key HMBC, ROESY, and COSY correlations of 6 and 7.
by the ROESY cross-peaks between Cys -NH/Phe-Hα,
1
Phe-NH/Pro -Hα, and Pro -Hα/Pro -Hα. Likewise, the HMBC
3
3
4
cyclization via a disulfide bond between the two Cys residues was
suggested to satisfy the remaining element of unsaturation in the
structure of 6. This assignment was further corroborated by the
correlations from Cys -Hα to Leu-CO (δ 170.5), from Leu-Hα to
5
C
Ile-CO (δ 170.7), as well as from Ile-Hα to Glp-CO (δ 172.4),
C
C
+
in addition to the ROESY correlations between Pro -Hα/Cys -
4
5
fragment ions at m/z 623.2317 [M − Pro -NH ] and 595.2367
6
2
+
Hα, Cys -NH/Leu-Hα, Leu-NH/Ile-Hα, and Ile-NH/Glp-Hα
5
[M − (Pro -NH + CO)] , which were observed in the
6
2
extended this fragment, leading to the overall peptide sequence
HRESIMS/MS spectrum of 6 (Figure S6-8). Accordingly, com-
pound 6 was identified as a new natural product and was given
the name gombamide D.
E-PEA-Cys -Phe-Pro -Pro -Cys -Leu-Ile-Glp. Finally, a disulfide
1
3
4
5
bond between the two Cys residues was evident, consistent with
the 22 elements of unsaturation required by the molecular for-
mula. The structure of 4 was further corroborated by the frag-
Compound 7 was isolated as a white, amorphous solid. The
molecular formula of 7 was determined as C H N O based on
13
18
2
+
ment ions at m/z 665.2566 [M − Leu-Ile-Glp] and 883.3826
HRESIMS analysis, consistent with six degrees of unsaturation.
Detailed analysis of the 2D NMR (COSY and HMBC) spectra of
7 allowed the assignment of an unusual amide consisting of
Val and E-PEA residues, connected through an amide linkage, as
supported by the HMBC correlation from E-PEA-Hα to Val-CO
+
[
M − E-PEA] in the HRESIMS/MS spectrum, originating from
cleavage of the linear peptide sequence Leu-Ile-Glp and the PEA
unit, respectively (Figure S4-9). Hence, compound 4 was iden-
tified as a new natural product and was named gombamide B,
based on the structural relationship with the known compound
(δ 167.2) (Figure 2, Table 4). Thus, compound 7 was assigned
C
1
2
gombamide A.
as (E)-2-amino-3-methyl-N-styrylbutanamide, which is a new
natural product.
Compound 5 was isolated as a white, amorphous solid.
The molecular formula of 5 was deduced to be C H N O S by
5
0
67
9
9
2
The remaining compounds were identified on the basis of
NMR, HRESIMS, and specific rotation data analysis, as well as by
HRESIMS analysis, as in the case of 4. In analogy to 1 and 2,
1
11
the H NMR spectrum of 5 was similar to that of 4, the only
comparison with published data as microcionamide A (3),
difference being the Z-configuration of the ethylene protons in
six indole derivatives, namely, 1H-indole-3-carbaldehyde,
1H-indole-3-carboxylic acid, 6-bromo-1H-indole-3-carbalde-
hyde, 6-bromo-1H-indole-3-carboxylic acid, methyl 6-bromo-
1H-indole-3-carboxylate, and ethyl 6-bromo-1H-indole-3-car-
the PEA moiety instead of the E-configuration, as deduced by
3
their vicinal coupling constant ( J
= 9.7 Hz; Table S5-1). In a
Hα,Hβ
similar manner to 4, HRESIMS/MS of 5 showed MS fragment
+
+
13,14
15
ions of [M − Z-PEA] and [M − Leu-Ile-Glp] (Figure S5-7).
Thus, compound 5 was characterized as a new natural product
and was named gombamide C.
boxylate,
along with 7-bromo-4(1H)-quinolinone, the
δ-lactam derivative (3-(2-(4-hydroxyphenyl)-2-oxoethyl)-5,6-
16
17
dihydropyridin-2(1H)-one), 2-deoxythymidine, and 4-hydrox-
ybenzoic acid (Figure S9-1).
Compound 6 was isolated as a white, amorphous solid.
The molecular formula of 6 was determined as C H N O S by
The absolute configuration of each amino acid residue was
determined by Marfey’s method following acid hydrolysis
3
2
48
8
8 2
HRESIMS, consistent with 13 degrees of unsaturation. Detailed
interpretation of the COSY and HMBC NMR spectra of 6
established the presence of six amino acid residues, including
Pro (3 equiv), Cys (2 equiv), and Leu, along with a Glp unit.
As described for peptides 1, 2, 4, and 5, key HMBC correlations
from Pro -Hα to Cys -CO (δ 168.1), from Cys -NH to Leu-CO
18
(6 N HCl) of the isolated peptides 1−7 (0.5 mg each). Com-
parison of the resulting derivatives with those of standard amino
acids by HPLC led to the assignment of the L-configuration for all
amino acid residues (Table S8-1). Moreover, in an attempt to
improve the HPLC resolution between L/D-Ile and L/D-allo-Ile
6
1
C
1
(
δ 170.9), from Leu-NH to Pro -CO (δ 171.0), from Pro -Hα
residues, a C HPLC RP-column was successfully employed,
C
3
C
3
4
to Pro -CO (δC 169.6), and from Cys -NH to Glp-CO
instead of the commonly used C column, in analogy to the C
Marfey’s method, revealing the presence of only L-Ile residues
4
5
18
3
19
(δC 171.5) established the overall amino acid sequence of 6 as
E
DOI: 10.1021/acs.jnatprod.7b00477
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Journal of Natural Products
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1
13
Table 3. H (600 MHz), C (150 MHz), HMBC, and ROESY NMR Data (DMSO-d , δ in ppm) of Gombamide D (6)
6
a
unit
position
δC, type
δH (J in Hz)
HMBC
ROESY
Pro6
CONH2
6.90, s; 7.27, s
Pro -CO, Pro -α
Pro -α, Cys -β
6
6
6
1
CO
α
173.4, C
59.9, CH
29.3, CH2
24.3, CH2
46.6, CH2
4.16, dd (8.8, 3.5)
1.80, m; 2.02, m
1.87, m
Pro -CO, Cys -CO, Pro -β, Pro -γ, Pro -δ
6 1 6 6 6
β
Pro -CO, Pro -α, Pro -γ, Pro -δ
6 6 6 6
γ
Pro -α, Pro -β, Pro -δ
6 6 6
b
δ
3.52 ; 3.63, ddd (9.7, 7.4, 4.5)
Pro -α, Pro -β
Cys -α
6
6
1
Cys1
Leu
NH
CO
α
7.87, d (8.3)
Leu-CO, Cys -β, Cys -α
Leu-α, Leu-NH
1
1
168.1, C
49.2, CH
41.3, CH2
4.75, ddd (11.6, 8.3, 3.2)
2.78, dd (14.6, 11.6); 3.12, dd (14.6, 3.2)
9.17, d (9.0)
Cys -CO, Cys -β
Pro -δ
1
1
6
β
Cys -CO, Cys -α
1 1
NH
CO
α
Leu-α, Pro -CO
Cys -NH, Pro -α, Pro -δ
3
1
3
3
170.9, C
b
50.1, CH
37.7, CH2
24.2, CH
21.4, CH3
23.1, CH3
171.0, C
4.53
Leu-CO, Leu-β, Leu-γ
Cys -NH
1
β
1.37, m; 1.71, m
1.42, m
Leu-CO, Leu-α, Leu-γ, Leu-δ, Leu-δ′
Leu-α, Leu-δ, Leu-δ′
Leu-β, Leu-γ, Leu-δ′
γ
δ
0.79, d (6.4)
0.88, d (6.4)
δ′
CO
Leu-β, Leu-γ, Leu-δ
Pro3
Pro4
b
α
60.6, CH
31.2, CH2
21.2, CH2
45.9, CH2
169.6, C
4.52
Pro -CO, Pro -CO, Pro -β, Pro -γ, Pro -δ
Leu-NH, Pro -α
3
4
3
3
3
4
β
2.08, m; 2.21, m
1.56, m; 1.91, m
Pro -CO, Pro -α, Pro -γ, Pro -δ
3
3
3
3
γ
Pro -α
3
b
δ
3.27, br t (10.4); 3.43
Pro -β, Pro -γ
Leu-NH
3
3
CO
α
59.4, CH
30.4, CH2
21.8, CH2
46.5, CH2
4.91, dd (8.7, 2.5)
2.30, m; 1.94, m
1.73, m; 1.79, m
Pro -β, Pro -γ, Pro -δ
Pro -α, Cys -α
4
4
4
3
5
β
Pro -CO, Pro -α, Pro -δ
4
4
4
γ
b
δ
3.50 ; 3.35, dt (11.7, 7.7)
Pro -β
4
Cys5
Glp
NH
CO
α
8.54, d (9.7)
Glp-CO, Cys -α
Glp-α, Glp-NH
5
168.2, C
48.4, CH
37.5, CH2
4.58, ddd (11.1, 9.7, 5.2)
2.62, dd (12.4, 11.1); 3.23, dd (12.4, 5.2)
7.67, s
Cys -CO, Glp-CO, Cys -β
Pro -α
5
5
4
β
Cys -α, Cys -CO
5
5
NH
CO
CO′
α
Glp-CO′, Glp-α, Glp-β, Glp-γ
Cys -NH
5
171.5, C
177.6, C
54.9, CH
25.1, CH2
28.9, CH2
4.02, br dd (9.0, 2.6)
2.07, m; 2.15, td (9.2, 3.4)
2.00, m; 2.24, m
Glp-CO′, Glp-β
Glp-CO, Glp-CO′, Glp-γ
Glp-CO′, Glp-α
Cys -NH
5
β
γ
a
b
Sequential NOEs. Signal overlap prevents determination of couplings.
as constituents of the respective peptides (1−5) (Table S8-2).
Moreover, the ROESY correlations between Pro -Hα/Pro -Hα
respective E-PEA analogues 1 and 4 during extraction and/or
isolation (e.g., upon exposure to natural light), as previously
3
4
11
and Pro -Hα/Cys -Hα (Figure 1) in 4 and 5 suggested a cis
suggested for 3 and microcionamide B. Compounds 4 and 5
are structurally related to the known peptide gombamide A from
the sponge Clathria gombawuiensis, the latter compound bearing
pyroglutamic acid (Glp) and para-hydroxystyrylamide units
instead of the linear peptide chain Leu-Ile-Glp and PEA,
4
5
configuration for the amide peptide bonds, which was further
corroborated by the large Pro-Cβ and Pro-Cγ shift difference
values (Δδ
≈ 8−11 ppm). Similarly, in the case of 6 a cis
Cβ‑Cγ
peptide bond was deduced between Pro -Pro and Pro -Cys ,
3
4
4
5
12
whereas the ROESY correlation between Cys -Hα and Pro -Hδ
respectively. Interestingly, Glp is rarely reported from marine
invertebrates. In addition to the aforementioned gombamide A,
other examples of peptides bearing this unit include didemnins
1
6
in addition to the carbon resonances of Cβ and Cγ in Pro6
(
29.3 and 24.3 ppm, respectively) suggested a trans peptide bond
4
,20
21
between Pro -Cys .
from the tunicate Trididemnum solidum, as well as asteropsin A
6
1
22,23
Microcionamides C (1) and D (2) are stereoisomers differing
in the E/Z double-bond configuration of the PEA moiety, as
previously observed for the known analogues microcionamides A
and two pyroglutamyl dipeptides from Asteropus sp. sponges.
The intramolecular cyclization of N-terminal glutamine residues
into Glp is of special interest, as it protects peptides from
degradation by the action of exopeptidase enzymes or even
enables them to adopt the right conformation for binding to their
1
1
(
3) and B. The only difference between the structures of 1 and
compared to 3 and microcionamide B is the replacement of the
terminal Val residue in the latter compounds by Ile. In analogy to
and 2, compounds 4 and 5 were found to be E/Z stereoisomers.
2
24
receptors. Gombamide D (6) possesses a similar cyclic part to
4 and 5, the only difference being that the Phe residue is
substituted by Leu, as in the case of the cyclic thiopeptides
eudistomides A and B, reported from a Fijian ascidian of the
1
It should be noted, however, that the Z-PEA derivatives 2 and 5
were detected as the minor components in the crude extract,
and thus these compounds may be artifacts arising from the
25
genus Eudistoma. The promiscuous occurrence of these
F
DOI: 10.1021/acs.jnatprod.7b00477
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1
13
Table 4. H (600 MHz), C (150 MHz), and HMBC NMR
caspase-3 substrate PARP after treatment with 1, 2, and 3 (same
concentrations as in Figure 3A). In line with the results of
the caspase-3 activity assay, all compounds induced cleavage of
PARP within 4 to 8 h, indicating again activation of caspase-3
and thus execution of apoptosis. To prove caspase dependency
of the observed PARP cleavage, we additionally performed
co-incubation with the pan-caspase inhibitor QVD (Figure 3B).
QVD completely blocked cleavage of PARP after treatment
with 1, 2, and 3, demonstrating a causative role of caspases and
thereby occurrence of apoptosis.
Data (MeOH-d , δ in ppm) of (E)-2-Amino-3-methyl-N-
styrylbutanamide (7)
4
unit
position
δc, type
δH (J in Hz)
HMBC
E-PEA
α
β
122.8, CH
116.4, CH
7.46, d (14.7)
6.32, d (14.7)
Val-CO, E-PEA-1
E-PEA-2/6
aromatic 1: 137.2, C
2
: 126.6, CH 7.35, dt (8.3,
.5)
E-PEA-β, E-PEA-6,
E-PEA-4
1
3
: 129.8, CH 7.29, br dd (8.3, E-PEA-1, E-PEA-5
.3)
Autophagy is a major intracellular degradation route respon-
sible for the lysosome-mediated breakdown of soluble cytosolic
components and therefore plays a crucial role in maintaining
7
4
: 128.1, CH 7.19, tt (7.3, 1.5) E-PEA-2/6
5
: 129.8, CH 7.29, br dd (8.3, E-PEA-1, E-PEA-3
26
cellular homeostasis. Cancer cells in particular suffer from
7
.3)
: 126.6, CH 7.35, dt (8.3,
.5)
metabolic stress and nutrient deprivation due to fast prolifer-
ation. Thus, autophagy can support cancer cells to overcome
microenvironmental stress by allowing them to recycle dysfunc-
tional or unessential components. Autophagy thereby acts as a
mechanism of cell survival. Accordingly, inhibition of auto-
phagy is a promising therapeutic target for anticancer chemo-
6
E-PEA-β, E-PEA-2,
E-PEA-4
1
Val
CO
α
167.2, C
59.9, CH
31.6, CH
17.8, CH3
18.9, CH3
3.69, d (5.8)
2.24, m
Val-CO, Val-γ, Val-γ′
Val-γ, Val-γ′
β
γ
1.07, d (6.9)
1.10, d (6.9)
Val-β, Val-α
27,28
therapy.
In order to investigate potential inhibitory or stimu-
γ′
Val-β, Val-α
lating effects of 1−3 with regard to autophagy, we used murine
embryonic fibroblast (MEF) cells stably expressing mCitrine-
hLC3B and measured lysosomal degradation of mCitrine-
hLC3B under starvation conditions and after treatment with
compounds 1−3 or alternatively with the known autophagy
inhibitor bafilomycin A1 via flow cytometry. LC3 is an essential
component of autophagosomes (double-membraned key
structures of autophagy), which deliver engulfed cytoplasmic
components to lysosomes and get degraded by the lysosomal
system during autophagy. Thus, breakdown of LC3 is a suitable
indicator of autophagic processes. Incubation with 10 μM com-
pound 1 or 2 distinctly blocked starvation-induced degradation
of LC3, indicating inhibition of autophagy by these compounds
peptide analogues in different phyla of marine invertebrates
argues for symbiont microorganisms as true producers.
Cytotoxicity of the isolated metabolites (1−7 and known
compounds) was investigated using different tumor cell
lines. Microcionamides A, C, and D (1−3) showed in vitro
cytotoxicity against lymphoma (Ramos) and leukemia cell lines
29
(
HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian
carcinoma cell line (A2780) with IC50 values ranging from
.45 to 28 μM (Figures S10-1 and 3, Table 5). The remaining
0
Table 5. Cytotoxicity of Compounds 1−3 after Incubation for
4 h Reported as IC (μM)
2
5
0
(
Figure 4). Compound 3 on the other hand was inactive in this
compd
A 2780
Ramos
Jurkat J16
Nomo-1
HL-60
experiment, indicating the relevance of the terminal Ile for the
autophagy-inhibitory effect. In order to determine whether the
observed inhibition of autophagy by compounds 1 and 2 is
caspase-dependent, we additionally performed experiments with
cotreatment of QVD. Because QVD counteracted compound
1-related inhibition of autophagy, this inhibitory effect was at
least partially due to apoptotic processes. In contrast, the inhib-
itory effect mediated by compound 2 was completely unaffected
by cotreatment with QVD, indicating independence of apoptosis.
Finding new antibacterial agents unaffected by available resis-
tance determinants is of paramount importance in light of increasing
bacterial resistance development. Thus, we were also interested
in the antibacterial potential of the compounds (Table S10-2).
Compounds 1 and 3 showed antibacterial activity against the
Gram-positive bacterial species Enterococcus faecium and Staph-
ylococcus aureus in the low μM range. For both compounds, the
minimal inhibitory concentrations (defined as the lowest con-
centrations completely preventing visible bacterial growth) were
6.2 μM for S. aureus and 12 μM for E. faecium, respectively
(Table S10-2). Mycobacterium tuberculosis as well as the Gram-
negative species Klebsiella pneumoniae, Enterobacter aerogenes,
Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter
baumannii were not inhibited up to 100 μM. Compounds 2
and 5 were not investigated due to compound limitation. The
remaining compounds showed no antibacterial activity.
1
2
3
0.45
0.53
2.6
1.4
1.9
5.9
0.81
1.3
1.4
2.4
10
1.9
2.5
28
4.6
isolated compounds proved to be inactive in concentrations up
to 10 μM.
Due to their strong cytotoxic activity, we next addressed the
question of whether 1−3 cause apoptotic cell death. Activation of
caspase-3 was measured employing two different methods: first
by immunoblotting for the cleavage of the caspase-3 substrate
poly(ADP-ribose) polymerase-1 (PARP-1) and second by mea-
suring the fluorescence of the profluorescent caspase-3 substrate
Ac-DEVD-AMC. Activated caspase-3 cleaves the latter substrate
between aspartate (D) and 7-amino-4-methylcoumarin (AMC),
hence releasing the fluorogenic AMC, which can be subsequently
detected with a spectrofluorometer. Because cysteine-dependent
aspartate-directed proteases (caspases) in general and caspase-3
as an effector caspase in particular are central players of apoptotic
processes, activation of caspase-3 indicates induction of apo-
ptosis. As shown in Figure 3A, compounds 1−3 induced the acti-
vation of caspases in Jurkat and Ramos cells with rapid kinetics
within 4 h and reached a maximum after 6 to 7 h, indicating
a fast induction of apoptotic processes. However, compound 3
induced caspase activation only at 10 μM, whereas in contrast to
1
and 2 it displayed no effect at 1 μM (data not shown), which is
In order to investigate the mechanism of bacterial growth
inhibition, we employed as a first step a panel of bacterial reporter
strains that we constructed in the background of the Gram-positive
in accordance with the higher IC . To confirm activation of
caspase-3 via a further end point, we detected cleavage of the
5
0
G
DOI: 10.1021/acs.jnatprod.7b00477
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Figure 3. Compounds 1−3 rapidly induce apoptosis in human lymphoma and leukemia cell lines. (A) The kinetics of caspase-3 activation in Jurkat J16
acute T cell leukemia; left panel) and Ramos (Burkitt’s lymphoma; right panel) cells after treatment with indicated concentrations of compounds 1−3
(
were compared to those of staurosporine (STS, 2.5 μM), a well-established inducer of apoptosis. Caspase-3 activity was measured by the rate of cleavage
of the profluorescent caspase-3 substrate Ac-DEVD-AMC. Relative caspase-3 activity in DMSO (0.1% v/v)-treated control cells was set to 1. Data
shown are the mean ± SD from a representative experiment performed in triplicate. (B) Ramos cells were treated with compounds 1 (1 μM), 2 (1 μM),
or 3 (10 μM) in the absence or presence of the caspase inhibitor Q-VD-OPh (QVD, 10 μM) for the indicated duration between 2 and 8 h. After the
incubation period cleavage of PARP was determined by Western blot analysis. The expression of β-actin was determined as a protein loading control.
Shown is the result of a representative blot. STS (2.5 μM) served as the positive control for caspase-dependent cleavage of PARP.
previously found and validated to react specifically to distur-
30
bances of certain metabolic pathways or cell structures. Pre-
vious whole genome mRNA profiling of B. subtilis after treatment
with a broad range of antibiotic classes had identified these
promoters as particularly responsive to DNA, RNA, protein, or cell
envelope damage. Compounds 1 and 3 showed a specific
induction of the cell envelope stress sensing promoters liaI and
ypuA (Figure 5). While ypuA is known to respond broadly to
disturbances at the bacterial cell envelope, liaI showed in previous
studies a particularly strong reaction to compounds that interfere
31
with cycling of undecaprenyl precursors in cell wall synthesis.
With the aim to further explore the effects of the micro-
cionamides on the bacterial cell envelope, we determined effects
of compounds 1 and 3 on the membrane potential of S. aureus.
The membrane potential is an electrical gradient across the
bacterial cytoplasmic membrane, with a surplus of positive charge
outside, that bacteria establish in the course of respiration. The
membrane potential is essential for ATP generation by the F F -
O
1
Figure 4. Compounds 1 and 2 impair amino acid starvation-induced
autophagy. MEFs stably expressing mCitrine-hLC3B were cultivated in
full medium or starvation medium (EBSS) with 10 μM compounds 1, 2,
ATPase and for active transport processes across the cytoplasmic
membrane. For measurement of the membrane potential the
fluorescent dye DiOC (3) was used, which enters bacterial cells
2
or 3 for 4 h. Ten nM bafilomycin A (Baf. A1) served as the positive
1
to some extent and emits green fluorescence. The higher the
membrane potential, the more dye molecules accumulate in the
cells and aggregation triggers a red-shift of fluorescence emission.
Addition of compounds 1 and 3 efficiently dissipated the mem-
brane potential of S. aureus, as indicated by the strong loss of
red fluorescence (Figure 6). The effect occurred already at sub-
micromolar concentrations of 1 and 3, the latter compound
being slightly more potent. Consequently, microcionamides
A (3) and C (1) seem to kill bacteria by energy depletion.
Notably, while some energy depleting agents induce the liaI
promoter, e.g. the lantibiotic nisin, which uses the undecaprenyl
control for inhibition of autophagy. Total cellular mCitrine-hLC3B
signals were analyzed by flow cytometry. The median of fluorescence
intensity was plotted in a bar diagram. Values are normalized to DMSO
(
0.1% v/v)-treated cells cultivated in full medium (1.0) and represent
mean ± SD of 3 independent analyses. * = p ≤ 0.05, ** = p ≤ 0.01
Student’s t test, two-sample assuming unequal variances). Dashed lines
(
represent median mCitrine fluorescence intensity of cells cotreated with
caspase inhibitor Q-VD-OPh (10 μM) performed in triplicate.
model organism Bacillus subtilis. These strains express the reporter
gene β-galactosidase from five selected promoters that were
H
DOI: 10.1021/acs.jnatprod.7b00477
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Journal of Natural Products
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Figure 5. Reporter gene assay with compounds 1 and 3 for orienting
mode of action studies. Compounds 1 and 3 strongly induced the liaI
promoter, and a weak induction was detected for the ypuA promoter,
indicating that these compounds cause cell envelope stress. The other
three promoters indicating DNA damage (yorB), RNA damage (helD),
or translation arrest (bmrC) were not induced. The five different
promoters showed a strong induction when treated with the respective
positive controls: bacitracin (BAC), vancomycin (VAN), ciprofloxacin
(
CIP), rifampicin (RIF), and chloramphenicol (CHL).
precursor lipid II as a docking molecule to form pores within
32
the bacterial cytoplasmic membrane, many energy-depleting
agents, such as ionophores, do not effectively induce liaI. Whether
or not the strong liaI signal triggered by the microcionamides
indicates that bacterial cells sense an interference with their
undecaprenyl precursor cycle will require further studies.
In summary, microcionamides A, B, and C (1−3) exhibited
cytotoxicity on different human cancer cell lines via both induc-
tion of apoptosis and inhibition of starvation-induced autophagy.
Furthermore, microcionamides A (3) and C (1) showed a
significant inhibitory effect on Gram-positive bacteria, which is
probably correlated with their abilities to depolarize bacterial
cytoplasmic membranes. It is noteworthy that microcionamide
C (1) showed increased cytotoxic activity over microcionamide
A (3) by a factor of 5 or more on different eukaryotic cell lines,
while 3 was more potent against Gram-positive bacteria
compared to 1. Even though the difference in the MIC between
Figure 6. Effect of microcionamides A (3) and C (1) on the membrane
potential of Staphylococcus aureus NCTC 8325. A prominent loss of red
fluorescence indicative of strong membrane potential dissipation occurs
already at 0.8 μM of 1 or 0.2 μM of 3. The protonophore carbonyl
cyanide m-chlorophenyl hydrazone (CCCP) at 5 μM served as a
positive control, and DMSO was the negative control. Samples were
analyzed after 30 and 60 min of exposure to compounds. The experi-
ment was performed two times on two different days with independent
bacterial cultures. Results for both biological replicates are shown in the
graphs. Error bars indicate the standard error of the mean.
experiment was measured with a sweep width of 7000 Hz in F2 and
2
6400 Hz in F1. Two thousand data points were collected in F2, and 512
data points in F1 with quadrature detection in both dimensions. The
sweep width for the HC-HMBC experiments was set to 7000 Hz in F2
and 27900 Hz in F1. Two thousand data points in F2 and 512
data points in F1 were collected with quadrature detection in F2.
The chemical shifts were referenced to the residual solvent peaks at
1
and 3 against S. aureus was smaller than a whole titration step in
the MIC assay, a larger zone of inhibition of 3 was detected on
agar (Figure 5), and lower concentrations of 3 were able to cause
a disruption of the membrane potential in comparison to 1
1
δH 3.31(MeOH-d ) and 2.50 (DMSO-d ) for H and at δC 49.0
4
6
13
(MeOH-d ) and 39.5 (DMSO-d ) for C NMR. Low-resolution ESI
4
6
mass spectra were recorded on a Thermoquest Finnigan LCQ Deca
connected to an Agilent 1100 series LC. High-resolution mass
measurements were obtained on an LTQ Orbitrap Velos Pro (Thermo
Scientific). Solvents were distilled prior to use, and spectral grade
solvents were used for spectroscopic measurements. HPLC analysis was
performed using a Dionex UltiMate3400 SD coupled to a photodiode
array detector (DAD3000RS) with detection wavelengths at 235, 254,
280, and 340 nm. The separation column (125 × 4 mm,
L × i.d) was prefilled with Eurospher 100-10, C18 (Knauer, Germany).
HPLC analysis of L/D-Ile and L/D-allo-Ile following Marfey’s
derivatization was carried out on the EC 250/4.6 Nucleosil 120-5,
(
Figure 6). Conversely, gombamides B (4) and D (6) proved to
be inactive against both human and bacterial cells.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
■
measured with a JASCO P-2000 polarimeter. UV data were recorded
1
13
on a PerkinElmer Lambda 25 UV/vis spectrometer. H, C, and
D NMR spectra (HH-ROESY, HH-TOCSY, HC-HSQC, and
2
HC-HMBC) were recorded with standard pulse sequences on a Bruker
AVIII HD 600 spectrometer equipped with a QXI/QCI cryoprobe.
The sweep width for the homonuclear experiments (HH-ROESY,
HH-TOCSY) was 7000 Hz in F2 and F1. Two thousand data points
were collected in F2, and 512 data points in F1 with quadrature
detection in both dimensions. The mixing time for the HH-ROESY
experiment was 450 ms with a spin lock field of 5000 Hz and 80ms for
the HH-TOCSY with a spin lock field of 9500 Hz. The HC-HSQC
C (Macherey-Nagel) separation column. Column chromatography was
4
performed using Sephadex LH-20 or reversed-phase silica (RP C ) as
1
8
stationary phase. Semipreparative purification was accomplished
on a Merck Hitachi system (pump L7100 and UV detector L7400;
Eurospher 100 C , 300 × 8 mm, L × i.d; Knauer) with a flow rate of
18
5.0 mL/min. Thin-layer chromatography (TLC) was performed using
I
DOI: 10.1021/acs.jnatprod.7b00477
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
13
precoated silica gel 60 F254 plates (Merck) followed by detection under
UV at 254 nm or after spraying the plates with anisaldehyde reagent.
Sponge Material. The sponge was collected by scuba at Ambon,
Indonesia. The sponge was identified as Clathria basilana by Dr. Nicole
de Voogd (Naturalis Biodiversity Center, Leiden, The Netherlands),
and a voucher specimen was deposited at the Naturalis Biodiversity
Center, Leiden, The Netherlands (reference number RMNH POR
285 (3.22); H (600 MHz) and C (150 MHz) NMR data (MeOH-d ),
4
+
Table 4; HRESIMS m/z 219.1490 [M + H] (calcd for C H N O,
1
3
19
2
219.1492).
Marfey’s Analysis. For acid hydrolysis of the isolated peptides
(1−7), 0.5 mg of each was treated separately with 2 mL of 6 N HCl and
heated at 110 °C for 24 h. The resulting solutions were concentrated,
with consecutive addition of H O (5 mL each) to ensure complete
2
8
636). The sponge was preserved in a mixture of EtOH and H O
elimination of HCl. Accordingly, the mixture of 50 μL of each acid
hydrolysate, 100 μL of FDNPL (1% N-(5-fluoro-2,4-dinitrophenyl)-L-
2
(
70:30) and stored in a −20 °C freezer prior to extraction.
Extraction and Isolation. The thawed sponge material (820 g wet)
leucinamide in acetone), and 20 μL of 1 M NaHCO was heated at
3
was cut into small pieces and exhaustively extracted with MeOH
3 × 2L) at room temperature (rt). The extracts were combined and
40 °C for 1 h with frequent mixing. After cooling, 10 μL of 2 N HCl was
added into the reaction solution. Subsequently, the derivatized product
was concentrated to dryness and prepared for HPLC analysis by
dissolving in 1000 μL of MeOH. The same procedure was followed for
L- and D-amino acid standards. HPLC (C ) analysis of the derivatized
(
concentrated under vacuum to yield 6.5 g of crude extract. Subsequent
liquid−liquid partitioning afforded n-hexane, EtOAc, n-BuOH, and
aqueous fractions at amounts of 420 mg, 700 mg, 540 mg, and 4.9 g,
respectively. The EtOAc fraction was further purified by column
chromatography on Sephadex LH-20 (MeOH as mobile phase),
followed by reversed-phase vacuum liquid chromatography (RP-VLC)
18
amino acids was performed by comparing their retention times with
those of standards [gradient (MeOH, 0.1% HCOOH in H O): 0 min
2
(10% MeOH); 5 min (10% MeOH); 35 min (100% MeOH); 45 min
(100% MeOH); 25 °C, 1 mL/min] (Table S8-1). For improved
resolution, HPLC analysis of the derivatized L/D-Ile and L/D-allo-Ile
and semipreparative HPLC in a gradient system of H O/MeOH,
2
respectively, to yield 1 (7.7 mg), 2 (2 mg), 3 (1 mg), 4 (4.5 mg),
5
(1 mg), 7 (0.8 mg), 1H-indole-3-carbaldehyde (0.6 mg), 1H-indole-3-
carboxylic acid (0.5 mg), 6-bromo-1H-indole-3-carbaldehyde (1.2 mg),
-bromo-1H-indole-3-carboxylic acid (1 mg), methyl 6-bromo-1H-
indole-3-carboxylate (1 mg), ethyl 6-bromo-1H-indole-3-carboxylate
1.7 mg), 7-bromo-4(1H)-quinolinone (1 mg), (3-(2-(4-hydroxyphen-
residues was performed on a C analytical column [gradient (MeOH,
4
0.1% HCOOH in H O): 0% to 60% (MeOH) for 160 min; 25 °C,
2
1
8,33
6
1 mL/min] (Table S8-2).
Eukaryotic Cell Lines and Reagents. Acute T cell leukemia cells
(Jurkat J16, no. ACC- 282), Burkitt‘s lymphoma B lymphocytes
(Ramos, no. ACC-603), acute promyelocytic leukemia cells (HL-60, no.
ACC-3), and acute myeloid leukemia cells (Nomo-1, no. ACC-542)
were obtained from the German Collection of Microorganisms and
Cell Cultures (DSMZ). Wild-type murine embryonic fibroblasts
(
yl)-2-oxoethyl)-5,6-dihydropyridin-2(1H)-one) (2.2 mg), 2-deoxythymi-
dine (2.5 mg), and 4-hydroxybenzoic acid (4 mg). The n-BuOH fraction
was subjected to column chromatography on Sephadex LH-20 (MeOH as
mobile phase), followed by RP-VLC and semipreparative HPLCto yield6
3
4
(
5.6 mg).
(kindly provided by Xiaodong Wang) expressing mCitrine-hLC3B
were generated by retroviral gene transfer using pMSCVpuro/mCitrine-
hLC3B. To generate pMSCVpuro/mCitrine-hLC3B, full-length human
MAP1LC3B cDNA was cloned into pMSCVpuro/mCitrine vector
Microcionamide C (1): white, amorphous solid; [α]20 −56
D
(
c 0.1, MeOH); UV (λmax, MeOH) (log ε) 201 (3.72), 287 (3.09);
1
13
H (600 MHz) and C (150 MHz) NMR data (DMSO-d ), Table 1;
6
+
HRESIMS m/z 889.5033 [M + H] (calcd for C H N O S ,
(kindly provided by Michael Engelke, University of Go
lines were grown at 37 °C under humidified air supplemented with 5%
CO in RPMI 1640 (HL-60, Jurkat J16, Nomo-1, Ramos) or DMEM
̈
ttingen). All cell
4
4
73
8
7 2
+
8
89.5038); HRESIMS/MS m/z 770.4297 [M − E-PEA] (calcd for
+
C H N O S , 770.4303); 657.3454 [M − (E-PEA + Ile )] (calcd for
C H N O S , 657.3463).
2
36
64
7
7
2
7
(mCitrine-hLC3B-MEF) containing 10% fetal calf serum, 1% HEPES,
120 IU/mL penicillin, and 120 μg/mL streptomycin. The broad-range
caspase inhibitor N-(2-quinolyl)-L-valyl-L-aspartyl-(2,6-difluorophe-
noxy) methyl ketone [(QVD) #SML0063], the autophagy inhibitor
bafilomycin A1 [(Baf. A1) #B1793], and the kinase inhibitor stauro-
sporine [(STS) #S5921], used as positive control for induction of
apoptosis, were obtained from Sigma-Aldrich. The profluorescent
caspase-3 substrate Ac-DEVD-AMC was purchased from Biomol
(#ABD-13402). Human ovarian carcinoma (A2780) cells were obtained
from ECACC (Salisbury, Wiltshire, UK) and cultivated in RPMI-1640
medium supplemented with 10% fetal bovine serum, 120 μg/mL strepto-
mycin, and 120 U/mL penicillin. Cells were grown at 37 °C in a humid-
ified atmosphere containing 5% CO2.
3
0
52
6
6 2
2
0
Microcionamide D (2): white, amorphous solid; [α]
−40
D
(
c 0.1, MeOH); UV (λ , MeOH) (log ε) 210 (3.8), 271 (4.01);
max
1
13
H (600 MHz) and C (150 MHz) NMR data (DMSO-d ), Table S2-1;
6
+
HRESIMS m/z 889.5034 [M + H] (calcd for C H N O S ,
4
4
73
8
7 2
+
8
89.5038); HRESIMS/MS m/z 770.4294 [M − Z-PEA] (calcd for
+
C H N O S , 770.4294); 657.3451 [M − (Z-PEA + Ile )] (calcd for
3
6
64
7
7
2
7
+
C H N O S , 657.3451); 776.4184 [M − Ile ] (calcd for
3
0
52
6
6
2
7
C H N O S , 776.4184).
3
8
60
7
6 2
20
Gombamide B (4): white, amorphous solid; [α]
+28 (c 0.1,
D
1
MeOH); UV (λ , MeOH) (log ε) 212 (4.1), 290 (4.56); H (600
MHz) and C (150 MHz) NMR data (DMSO-d ), Table 2; HRESIMS
m/z 1002.4572 [M + H] (calcd for C H N O S , 1002.4576);
max
13
6
+
5
0
68
9
9 2
+
HRESIMS/MS m/z 883.3826 [M − E-PEA] (calcd for C H N O S ,
Determination of Eukaryotic Cell Viability. HL-60, Jurkat J16,
4
2
59
8
9 2
+
5
8
6
83.3826); 665.2566 [M − Leu-Ile-Glp] (calcd for C H N O S ,
Nomo-1, and Ramos cells were seeded at a density of 5 × 10 cells/mL
3
3
41
6
5 2
+
65.2566); 546.1833 [M − (E-PEA + Leu-Ile-Glp)] (calcd for
and incubated with different concentrations of compounds 1, 2, and 3
for 24 h. Cells treated with DMSO (0.1% v/v) for 24 h were used as the
negative control. After the incubation period MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Calbiochem #475989)
was added to the cells to a final concentration of 1 mg/mL, and the cells
were incubated further for 60 min and then centrifuged at 600 rcf for
5 min. The medium was aspirated, and 100 μL of DMSO was added to
each well to extract the formazan product from the cells. After 25 min of
incubation on a shaker at rt, the absorbances at 650 nm (reference
wavelength) and 570 nm (test wavelength) were measured using
a multiplate reader (Synergy Mx, BioTek). Viability and IC50 values
(IC50 = half-maximal inhibitory concentration) were calculated
using Prism 6 (GraphPad Software). Relative viability in DMSO
(0.1% v/v)-treated control cells was set to 100%. A2780 cells were
plated into 96-well microtiter plates (approximately 9000 cells/well)
(Sarstedt) and preincubated with growth medium overnight. Then, cells
were incubated with increasing concentrations of test compounds
for 72 h. After 72 h, 25 μL of a solution of MTT was added to each
well. After approximately 10 min, formazan crystals were formed, and
C H N O S , 546.1833).
2
5
32
5
5 2
20
Gombamide C (5): white, amorphous solid; [α]
+16 (c 0.1,
D
1
MeOH); UV(λ , MeOH) (log ε) 214 (4.01), 288 (4.4); H (600 MHz)
and C (150 MHz) NMR data (DMSO-d ), Table S5-1; HRESIMS m/z
max
13
6
+
1
002.4576 [M + H] (calcd for C H N O S , 1002.4576); HRESIMS/
50 68 9 9 2
+
MS m/z 883.3826 [M − Z-PEA] (calcd for C H N O S , 883.3841);
4
2
59
8
9 2
+
6
5
5
65.2566 [M − Leu-Ile-Glp] (calcd for C H N O S , 665.2566);
33
41
6
5 2
+
46.1833 [M − (Z-PEA + Leu-Ile-Glp)] (calcd for C H N O S ,
25
32
5
5 2
46.1839).
Gombamide D (6): white, amorphous solid; [α]20 −6 (c 0.1,
D
1
MeOH); UV(λ , MeOH) (log ε) 210 (3.8), 271 (4.01); H (600 MHz)
max
and 13C (150 MHz) NMR data (DMSO-d ), Table 3; HRESIMS m/z
6
+
7
37.3107 [M + H] (calcd for C H N O S , 737.3109); HRESIMS/MS
3
2
49
8
8 2
+
m/z 623.2317 [M − Pro -NH ] (calcd for C H N O S , 623.2317);
6
2
27 39
6
7 2
+
5
5
95.2367 [M − (Pro -NH + CO)] (calcd for C H N O S ,
6
2
26 39
6
6 2
95.2367).
E)-2-Amino-3-methyl-N-styrylbutanamide (7): white, amorphous
(
20
solid; [α] −38 (c 0.1, MeOH); UV (λmax, MeOH) (log ε) 220 (3.07),
D
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J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
medium was removed. Formazan crystals were then dissolved in 75 μL
of DMSO. Absorptions were measured at 544 nm (test wavelength) and
150 min using a multiplate reader (Synergy Mx, BioTek). Caspase
activity was determined as the slope of the resulting linear regressions.
Data points shown are the mean of triplicates; error bars = SD. Values
are normalized to DMSO (0.1% v/v)-treated cells (fold change = 1.00).
FACS-Based Analysis of Autophagy. The FACS-based analysis of
autophagy was adapted from a protocol previously described by Shvets
et al. MEF cells stably expressing mCitrine-hLC3B were cultured in
the indicated medium for 4 h with bafilomycin A1 (10 nM) and
compound 1, 2, or 3 (10 μM), harvested with 0.05% trypsin-EDTA, and
washed once with PBS. Subsequently, the intensity of mCitrine
fluorescence was analyzed by flow cytometry using FACSDiva software.
Reduction of mCitrine-hLC3B compared to medium control indicates
autophagy induction. Baf. A1 served as positive control for inhibition of
autophagy. Co-treatment with QVD was performed to exclude caspase-
dependent processes.
690 nm (reference wavelength) using the BMG FLUOstar (BMG
Labtechnologies, Offenburg, Germany). Absorption of the reference
wavelength was subtracted from the absorption of the test wavelength.
Determination of Antibacterial Activity. MIC values for
bacterial strains were determined in cation-adjusted Mueller-Hinton
broth by the broth microdilution method according to the recom-
3
8
3
5
mendations of the Clinical and Laboratory Standards Institute (CLSI).
For preparation of the inoculum the direct colony suspension method
was used. Briefly, serial 2-fold dilutions of test compounds were
prepared in microtiter plates and seeded with a final bacterial inoculum
5
of 5 × 10 colony forming units per mL (CFU/mL). After 16 to 20 h at
3
7 °C and ambient air, the minimal inhibitory concentration was read as
the lowest compound concentration preventing visible bacterial growth.
The strain panel included antibiotic-susceptible CLSI quality control
strains obtained from the American Type Culture Collection as
indicated by the ATCC strain label. E. faecium BM 4147-1 is a clinical
Bacterial Reporter Gene Assay. For mode of action studies, five
bacterial reporter strains were constructed in the genetic background of
B. subtilis 1S34, each carrying one of the following promoters fused to
the β-galactosidase reporter gene and inserted into the chromosomal
amyE locus. Induction of the yorB promoter indicates DNA damage,
the helD promoter (synonym yvgS) senses RNA damage, and the
bmrC promoter senses translation arrest. Induction of the ypuA or liaI
(synonym yvqI) promoters indicates cell envelope stress. Promoter
regions were chosen according to a previous publication by Urban
36
isolate cured of its vancomycin resistance plasmid, and A. baumannii
0
9987 is a clinical isolate from the University of Bonn, Germany.
Cells of M. tuberculosis H37Rv were grown aerobically in Middle-
brook 7H9 medium supplemented with 10% (v/v) ADS enrichment
5%, w/v, bovine serum albumin fraction V; 2%, w/v, glucose; 0.85%,
(
w/v, sodium chloride), 0.5% (v/v) glycerol, and 0.05% (v/v) tyloxapol
at 37 °C. For the determination of MIC against M. tuberculosis, bacteria
3
0
et al. For the agar-based reporter gene assay 20 mL of lysogeny broth
(LB) containing spectinomycin (50 μg/mL) were inoculated with 500 μL
of an overnight culture of B. subtilis 1S34 and grown to the stationary
were precultured until log-phase (OD6
= 0.5−1) and then seeded at
00nm
5
1
× 10 cells per well in a total volume of 100 μL in 96-well round-
7
bottom microtiter plates containing 2-fold serially diluted compounds at
phase. Cells were adjusted to a total cell number of 3 × 10 CFU/mL in
a concentration range of 100−0.78 μM. Microplates were incubated at
50 mL of LB soft agar (0.7% agar) containing 150 μg/mL of 5-bromo-4-
chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and poured into a
square Petri dish. After solidification, test compounds (20 nmol) were
spotted onto the agar plates. Paper discs (Oxoid) containing the
following reference antibiotics were used as positive controls: bacitracin
(10 μg), chloramphenicol (10 μg), ciprofloxacin (5 μg), rifampicin
(30 μg), and vancomycin (30 μg). Plates were analyzed for promoter
induction after incubation at 30 °C for 14 to 18 h. In the case of the bmrC
promoter, soft agar was prepared from Belitzky minimal medium, and
3
7 °C for 5 days. Afterward, 10 μL/well of a 100 μg/mL resazurin
solution was added and incubated at ambient temperature for a further
16 h. Then cells were fixed for 30 min after formalin addition (5%, v/v,
final concentration). For viability determination, fluorescence was quan-
tified using a microplate reader (excitation 540 nm, emission 590 nm).
Percentage of growth was calculated relative to rifampicin-treated
(
0% growth) and DMSO-treated (100% growth) controls.
Western Blot Analysis. Ramos cells were treated for the indicated
3
9
the plates were incubated at 37 °C.
durations with 1 μM of 1, 1 μM of 2, or 10 μM of 3, respectively.
Co-incubation with the caspase inhibitor QVD at a concentration of
Measurement of Bacterial Membrane Potential. Determina-
tion of membrane potential changes upon antibiotic treatment was
performed using the membrane potential-sensitive fluorescent dye
10 μM was used as proof of caspase dependency of the observed effects.
Subsequently, cells were pelletized at 600 rcf at 4 °C for 5 min, washed
with phosphate-buffered saline (PBS), and frozen in liquid nitrogen.
The cells were lysed in ice-cold lysis buffer [20 mM Tris-HCl, pH 7.5,
3
,3′-diethyloxacarbocyanine iodide (DiOC (3), Molecular Probes).
2
S. aureus NCTC 8325 was grown to the exponential phase in LB
medium, harvested, and resuspended to an optical density at 600 nm
1
50 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, 10 mM NaF, 2.5 mM
(
OD600) of 0.5 in PBS. Cells were incubated with 30 μM DiOC (3)
Na P O , 10 μM Na MoO , 1 mM Na VO , protease inhibitor cocktail
2
4
2
7
2
4
3
4
for 15 min and subsequently treated with compounds 1 and 3 at
different concentrations for 30 min. The protonophore carbonyl cyanide
m-chlorophenyl hydrazone (Sigma-Aldrich) at a concentration of 5 μM
was used as a positive control and DMSO as a negative control. Fluo-
rescence was measured 30 and 60 min after addition of the respective
compound at an excitation wavelength of 485 nm and two emission
wavelengths, 530 nm (green) as well as 630 nm (red), using a microplate
reader (TECANInfinite M200). Increased membrane potential promotes
intracellular dye accumulation, self-association of which causes a red-shift
of the fluorescence emission signal.
(
Sigma #P2714)]. The lysates were cleared from cell debris by cen-
trifugation at 11 000 rcf at 4 °C for 15 min, and the total protein
concentration was measured by Bradford assay and adjusted to equal
concentrations. After loading with Laemmli buffer and heating to 95 °C
for 5 min, 25 μg of the protein extract was separated by SDS-PAGE
[
10% tris-glycine polyacrylamide gel (v/v)] and transferred to a PVDF
membrane by Western blotting according to the standard protocol.
Analysis of proteins of interest was performed using primary mouse
antibodies to poly(ADP-ribose) polymerase-1 (Enzo Life Sciences
#
BML-SA250) or β-actin (Sigma-Aldrich #A5316) and IRDye800-
conjugated secondary antibodies (LI-COR Biosciences #926-32210/11).
Signals were detected with an infrared imaging system.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.7b00477.
■
Caspase-3 Activity Assay. Caspase activity was analyzed as
*
S
37
previously described. Briefly, Jurkat J16 cells and Ramos cells were
6
seeded at a density of 1 × 10 cells/mL in 96-well microtiter plates and
incubated with different concentrations of 1, 2, and 3 for the indicated
times. Cells treated with DMSO (0.1% v/v) were used as negative con-
trol. After the incubation period, cells were harvested by centrifugation
at 600 rcf at 4 °C and lysed by incubation with ice-cold lysis buffer
1
13
H NMR spectra of 1−7; C NMR spectra of 4, 6, and 7;
2
2
D NMR and HRESIMS spectra of the new compounds 1,
, and 4−7; HRESIMS/MS spectra of 1, 2, and 4−6;
(
20 mM HEPES, 84 mM KCl, 10 mM MgCl , 200 μM EDTA, 200 μM
1
13
2
tables of H, C, and 2D NMR data for 2 and 5; HPLC
analysis of the acid hydrolysates of 1−7 using Marfey’s
method; structures of known compounds; cytotoxicity
effects of 1−3 on human lymphoma and leukemia cell
lines, measured by MTT assay; table of antibacterial
EGTA, 0.5% NP-40, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 5 μg/mL
aprotinin)) for 10 min. After addition of 150 μL of reaction buffer
(
50 mM HEPES, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 2 mM
CaCl , 13.35 mM DTT, 70 μM DEVD-AMC) per well, fluorescence
2
(
Ex 360 nm, Em 450 nm) was measured at 37 °C over a time course of
K
DOI: 10.1021/acs.jnatprod.7b00477
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
activity of 1−7 and known compounds reported as MIC
(13) Cardellina, J. H.; Nigh, D.; VanWagenen, B. C. J. Nat. Prod. 1986,
9, 1065−1067.
4
(
(
PDF)
14) Rasmussen, T.; Jensen, J.; Anthoni, U.; Christophersen, C.;
Nielsen, P. H. J. Nat. Prod. 1993, 56, 1553−1558.
(15) AlTarabeen, M.; Hassan Aly, A.; Perez Hemphill, C.; Catalina, F.;
Rasheed, M.; Wray, V.; Proksch, P. Z. Naturforsch., C: J. Biosci. 2015, 70,
75−78.
AUTHOR INFORMATION
■
Corresponding Authors
Tel: ++492118114163. E-mail: georgios.daletos@uni-
duesseldorf.de.
E-mail: proksch@uni-duesseldorf.de.
*
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ACKNOWLEDGMENTS
■
Support of this project to P.P., H.B.-O., M.U.K., R.K., and A.B. by
the BMBF (project BALIPEND) and to K.W.W. by the German
Center for Infection Research is gratefully acknowledged. We
thank Dr. N. de Voogd (Leiden, Naturalis Biodiversity Center,
Leiden, The Netherlands) for identification of the sponge.
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Murray, T. F.; Ha, N. C.; Hamann, M. T.; Jung, J. H. Biochim. Biophys.
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Karsli-Uzunbas, G.; Kamphorst, J. J.; Chen, G.; Lemons, J. M. S.;
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and Mr. Straetener for expert technical assistance. The authors
wish to thank Dr. E. Ferdinandus (University Pattimura, Ambon,
Indonesia) and Prof. Dr. S. Wiryowidagdo (University Hassanu-
din, Makassar, Indonesia) for their support and help during sponge
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