Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge Theonella swinhoei That Inhibit Ras/Raf Interaction

Article abstract of DOI:10.1021/acs.jnatprod.0c00136

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d₆ due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC₅₀ values of 5.8 and 8.5 μM, respectively.

Full text of DOI:10.1021/acs.jnatprod.0c00136

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Article
Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge
Theonella swinhoei That Inhibit Ras/Raf Interaction
Chang-Kwon Kim, Dongdong Wang, Heidi R. Bokesch, Richard W. Fuller, Emily Smith,
Curtis J. Henrich, David E. Durrant, Deborah K. Morrison, Carole A. Bewley, and Kirk R. Gustafson*
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ABSTRACT: Two new cyclic depsipeptides named swinhopepto-
lides A (1) and B (2) have been isolated from the marine sponge
Theonella swinhoei cf. verrucosa, collected from Papua New Guinea.
They each contain 11 diverse amino acid residues and 13-carbon
polyketide moieties attached at the N-terminus. Compounds 1 and
2 each exist as two conformers in DMSO-d6 due to cis/trans
isomerism of the proline residue, and their structures were
successfully assigned by extensive NMR analyses complemented by
chemical degradation and derivatization studies. Swinhopeptolide
B (2) contains a previously undescribed 2,6,8-trimethyldeca-
(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant
inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 μM, respectively.
ponge-derived cyclic depsipeptides are widely recognized
for their unusual structural features and potent bio-
for ras and melanoma (60%), thyroid (39%), and ovarian
(12%) for raf.21,22 While a number of small-molecule inhibitors
targeting this pathway have been approved as drugs or are
currently undergoing clinical trials, response rates are highly
variable and therapeutic efficacy is often limited by the
development of resistance.23,24 Additional therapeutic options
for targeting dysfunction of Ras−Raf−MEK−ERK signaling
are clearly needed.
S
activities. The callipeltins, isolated from a Callipelta sp. marine
sponge, were the first representatives of a family of non-
ribosomal peptides that contain many unusual amino acids
including 3,4-dimethylglutamine and β-methoxytyrosine resi-
dues, along with diverse new polyketide moieties.1−5 Related
macrocyclic peptides include papuamides A−F from Theonella
mirabilis, T. swinhoei, and Melophlus sp.,6−8 mirabamides A−H
from Siliquariaspongia mirabilis and Stelletta clavosa,9,10
neamphamides A and B from Neamphius huxleyi and
Neamphius sp.,11−13 stellettapentins A and B from Stelletta
sp.,14 and stellatolides A−G from Ecionemia acervus.15 These
peptides exhibit potent biological properties including anti-
HIV, antifungal, and cytotoxic activities. Inhibition of HIV by
the papuamides involves blocking virus entry into target cells,16
while the antifungal and cytotoxic effects of these peptides are
mediated through specific binding to phosphatidylserine in cell
membranes.17,18
Based on the varied biological properties reported for these
metabolites and other cyclic depsipeptides, we tested a diverse
array of prefractionated marine sponge extracts in a cell-based
screen for materials that can impede the interaction between
Ras, an ocogenic GTPase, and Raf, a serine/threonine protein
kinase. These proteins are part of the Ras−Raf−MEK−ERK
signal transduction cascade, which is also described as the
mitogen-activated protein kinase (MAPK) pathway.19,20 Ras/
Raf signaling pathways are considered good potential
theapeutic targets for cancer treatment, as mutations in the
ras and raf genes are abundant in numerous cancer types
including pancreatic (95%), colorectal (45%), and lung (35%)
RESULTS AND DISCUSSION
■
In conjuction with an ongoing anticancer natural product
discovery effort at the National Cancer Institute,25 the extract
of a Papua New Guinea collection of the sponge Theonella
swinhoei cf. verrucosa was tested and showed potent activity in a
cell-based anti-Ras/Raf drug screen. Bioassay-guided fractio-
nation of the extract by sequential application of C18 flash
chromatography, Sephadex LH-20 separation, and C18 HPLC
provided two peptide-based metabolites. Herein, we report the
structures of two cyclic depsipeptides named swinhopeptolides
A (1) and B (2), which are comprised of numerous unusual
amino acid residues and N-linked aliphatic polyketide moieties.
Compound 2 contains a 2,6,8-trimethyldeca-(2E,4E,6E)-
trienoic acid (Tdta) moiety which is the first report of this
Received: February 6, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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A

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1
Table 1. H NMR (600 MHz) and 13C NMR Data (150 MHz) of Compound 1 in DMSO-d6
major conformer 1a
minor conformer 1b
δH (J in Hz)
units
position
δC, type
δH (J in Hz)
δC, type
Pro
1
2
3
171.7, C
59.2, CH
29.0, CH2
170.8, C
58.0, CH
28.4, CH2
a
4.43, m
4.19, m
2.16, m
2.19, m
1.89, m
1.78, m
4
5
24.4, CH2
46.6, CH2
1.87, (2H, m)
3.53, m
24.4, CH2
46.2, CH2
1.87, (2H, m)
3.65, m
3.41, m
3.53, m
β-OMeTyr
1
2
3
1′
2′, 6′
3′, 5′
4′
167.5, C
53.5, CH
82.9, CH
127.3, C
129.2, CH
114.8, CH
157.8, C
56.2, CH3
168.2, C
53.3, CH
83.6, CH
127.7, C
129.2, CH
114.7, CH
157.6, C
56.1, CH3
4.90, dd (9.2, 8.3)
4.30, d (9.2)
4.80, dd (9.2, 8.3)
4.15, d (9.2)
7.11, d (8.4)
6.71, d (8.4)
7.08, d (8.4)
6.67, d (8.4)
3-OMe
3.03, s
2.97, s
NH
8.23, d (8.3)
8.42, d (8.3)
N-MeThr
1
170.2, C
169.9, C
2
3
4
62.1, CH
62.5, CH
20.0, CH3
30.1, CH3
4.24, d (9.4)
3.69, m
0.39, d (5.8)
2.91, s
64.7, CH
62.6, CH
19.6, CH3
28.7, CH3
4.16, d (9.4)
3.67, m
0.52, d (5.8)
2.65, s
N-Me
Ala
1
2
3
NH
1
171.8, C
46.0, CH
16.2, CH3
172.5, C
43.6, CH
17.6, CH3
4.43, m
1.23, d (6.8)
7.18, m
4.81, m
1.16, d (6.8)
8.41, d (7.8)
Gly
168.0, C
168.7, C
2
42.5, CH2
4.02, m
3.35, m
8.84, br t (5.5)
41.3, CH2
3.86, m
3.52, m
7.93, m
NH
1
2
O-MeSer
170.2, C
55.9, CH
70.7, CH2
170.2, C
55.8, CH
70.3, CH2
4.27, m
3.91, m
4.26, m
3.90, m
3
3.63, m
3.66, m
3-Me
58.1, CH3
3.27, s
58.2, CH3
3.27, s
NH
8.10, br d (4.1)
8.10, br d (4.1)
3-OHLeu
1
2
3
4
5
6
NH
1
2
170.3, C
170.1, C
54.9, CH
76.8, CH
27.8, CH
18.8, CH3
18.8, CH3
52.8, CH
77.3, CH
28.3, CH
19.4, CH3
19.5, CH3
4.76, m
4.90, d (10.2)
1.96, m
0.72, d (6.6)
0.72, d (6.6)
8.24, br d (8.9)
4.75, m
5.41, d (10.3)
1.95, m
0.79, d (6.7)
0.79, d (6.7)
8.68, br d (8.9)
3,4-DiMeGln
170.2, C
56.3, CH
38.1, CH
40.8, CH
176.7, C
13.6, CH3
15.9, CH3
172.3, C
56.9, CH
35.1, CH
42.3, CH
178.1, C
15.1, CH3
14.1, CH3
4.31, dd (9.1, 6.6)
1.98, m
2.35, m
4.17, dd (9.1, 6.6)
2.10, m
2.56, m
3
4
5
3-Me
4-Me
NH
5-NH2
0.84, d (6.5)
1.03, d (6.8)
8.59, br d (6.6)
7.21, br s
0.97, d (6.5)
1.12, d (6.8)
8.08, d (6.6)
7.48, br s
6.95, br s
7.30, br s
Dab
1
2
3
4
168.7, C
53.9, CH
47.8, CH
14.4, CH3
168.3, C
54.1, CH
47.7, CH
14.6, CH3
4.57, t (6.6)
3.67, m
1.11, d (6.8)
4.58, t (6.6)
3.66, m
1.12, d (6.8)
B
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Table 1. continued
major conformer 1a
δH (J in Hz)
7.98, m
minor conformer 1b
δH (J in Hz)
units
position
δC, type
δC, type
NH
3-NH2
7.97, m
ND
b
ND
Aba
Ser
1
2
3
4
NH
1
2
165.0, C
129.9, C
128.7, CH
12.9, CH3
165.1, C
130.0, C
127.7, CH
12.9, CH3
6.42, q (6.8)
1.68, d (6.8)
9.18, br s
6.38, q (6.8)
1.68, d (6.8)
9.14, br s
170.0, C
54.7, CH
61.4, CH2
169.8, C
55.0, CH
61.6, CH2
4.37, m
3.85, m
4.36, m
3.84, m
3
3.64, m
3.63, m
NH
1
2
7.93, d (7.8)
7.92, d (7.8)
Dhtda
175.6, C
77.0, C
175.4, C
77.1, C
3
4
5
6
7
8
9
71.1, CH
126.4, CH
135.9, CH
130.8, C
137.8, CH
33.6, CH
29.8, CH2
4.64, d (10.2)
5.41, t (10.6)
6.03, d (12.0)
70.9, CH
126.5, CH
135.8, CH
130.8, C
137.7, CH
33.6, CH
29.8, CH2
4.64, d (10.2)
5.41, t (10.6)
6.03, d (12.0)
5.23, d (9.3)
2.32, m
1.35, m
5.23, d (9.3)
2.32, m
1.35, m
1.22, m
1.22, m
10
11.8, CH3
22.4, CH3
16.4, CH3
20.5, CH3
0.82, t (6.2)
1.13, s
1.75, s
11.8, CH3
22.5, CH3
16.4, CH3
20.5, CH3
0.82, t (6.2)
1.13, s
1.75, s
2-Me
6-Me
8-Me
0.93, d (6.5)
0.93, d (6.5)
a
b
Coupling constant not determined due to signal broadening or overlapped signals. Chemical shifts not determined.
aliphatic substituent among cyclic depsipetides. These new
sponge metabolites exhibited low micromolar activity in the
Ras/Raf inhibition assay.
(Figure 1). The sequence of Ser-Aba-Dab was assigned from
HMBC correlations between the Aba NH (δH 9.18)/Ser C-1
The molecular formula of swinhopeptolide A (1) was
deduced to be C66H103N13O21 by HRESIMS measurements in
conjunction with extensive NMR analyses. However, both the
1
13C and H NMR spectra of 1 showed a greater number of
signals than expected, based on molecular weight and
molecular formula considerations. Detailed examination of
the NMR data (Table 1) revealed that numerous signals were
paired with each other (1H NMR integration ratio of
approximately 3:2 in DMSO-d6), indicating a mixture of
conformational isomers. The presence of a large number of
exchangeable amide NH protons (δH 9.18−6.95) and carbonyl
1
carbons (δC 175.6−165.0) in the H and 13C NMR spectra of
1 indicated characteristic signals of a peptide. Detailed analysis
of 2D NMR (HSQC, COSY, TOCSY, HSQC-TOCSY,
HMBC, and ROESY) data led to the assignment of four
common amino acid residues consisting of proline (Pro),
alanine (Ala), glycine (Gly), and serine (Ser), together with
the following seven nonproteinogenic amino acid residues: β-
methoxytyrosine (β-OMeTyr), N-methylthreonine (N-
MeThr), O-methylserine (O-MeSer), 3-hydroxyleucine (3-
OHLeu), 3,4-dimethylglutamine (3,4-DiMeGln), 2,3-diamino-
butanoic acid (Dab), and 2-amino-2-butenoic acid (Aba). In
addition, a 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic
acid (Dhtda) moiety was established. NMR assignments for
the major and minor constituents 1a and 1b, respectively, are
listed in Table 1.
Figure 1. Key TOCSY, HSQC-TOCSY, HMBC, and ROESY
correlations for swinhopeptolide A (1).
(δC 170.0) and the Dab H-2 (δH 4.57)/Aba C-1 (δC 165.0)
and ROESY cross-peaks between the Aba NH/Ser H-2 (δH
4.37) and the Dab NH (δH 7.98). The Z configuration of Aba
was confirmed by a ROESY correlation between the Aba H3-4
(δH 1.68) and Aba NH (δH 9.18). The next sequence of Dab-
3,4-DiMeGln-3-OHLeu-O-MeSer-Gly-Ala-N-MeThr-β-OMe-
Tyr-Pro was very similar to the corresponding sequence of
papuamide A, the only difference being replacement of the C-
The amino acid sequence of 1 was assigned from a
combination of inter-residue ROESY and HMBC correlations
C
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analyzed by preparing both the L- and D-DLA derivatives, and
LC-MS comparison with appropriate amino acid standards
(Figure S16A−D). For the amino acids with multiple chiral
centers, L-Dab, D-3-OHLeu, L-N-MeThr, and 3,4-dimethyl-L-
glutamine, commercial standards were not available. Thus,
their DLA derivatives were compared by LC-MS with the
corresponding derivatives prepared from an authentic sample
of mirabamide A,9 which contains the same nonproteinogenic
amino acid residues. In addition to comparison of the LC-MS
retention time of the DLA derivatives, the presence of a
(2S,3S)-Dab residue in 1 was supported by the characteristic
large coupling constant of J2,3 = 6.6 Hz that was observed and
by a ROESY correlation between NH and H-4. In previous
NMR studies with synthetic Dab,27 the diastereomers of Dab
showed distinct differences, since the H-2/H-3 coupling
constant in the (2R,3S)-isomer is approximately 3.0 Hz,
while in the (2S,3S)-isomer it is 6.1−7.0 Hz, thus supporting
the (2S,3S)-Dab assignment (Figure S17A). Similarly, for D-3-
OHLeu, a characteristic large coupling constant J2,3 = 10.2 Hz
and a ROESY correlation between NH and H-4 supported the
absolute configuration assignment of C-3 as R (Figure S17B).
A large coupling constant J2,3 = 9.4 Hz in L-N-MeThr, in
conjunction with a ROESY correlation between H3-4 and
NHβ‑OMeTyr and the absence of a ROESY observed between the
N-Me group and H3-4 was consistent with an R configuration
at C-3 (Figure S17C). For the 3,4-dimethyl-L-glutamine
residue, a large coupling J2,3 = 9.1 Hz and the presence of
ROESY correlations between 3-Me/H-2 and 4-Me/NH
supported an absolute configuration assignment of S for C-3.
Comparison by LC-MS of the L-DLA derivative of 3,4-
DiMeGln derived from 1 with the same residue from
mirabamide A showed similar retention times. Thus, the
configuration was determined as (2S,3S,4R)-DiMeGln in 1
(Figure S17D), which is consistent with the configuration of all
the other DiMeGln residues that have been reported from
marine cyclicpeptides. Due to the decomposition of β-
OMeTyr during acid hydrolysis conditions, this residue of 1
was converted to β-OMeAsp via ozonolysis and oxidative
workup, prior to acid hydrolysis and Marfey’s derivatization.2
Then, LC-MS comparison with a mirabamide A sample that
was ozonized in a similar manner before hydrolysis and
Marfey’s analysis revealed (2R,3R) as the configuration of the
β-OMeTyr residue in 1 (Figure S18). The large coupling of J2,3
= 9.2 Hz observed for the β-OMeTyr residue supported this
assignment.12 This completed the absolute configuration
assignment for the amino acid-derived portion of swinhopep-
tolide A (1). The Dhtda polyketide moiety in 1 was unstable,
as it decomposed during acid hydrolysis, and also during
various attempts of direct derivatization, so the configuration
of this component could not be directly assigned. However,
papuamide B contains a Dhtda side chain and its absolute
configuration was assigned as 2S,3R,8R in a total synthesis
terminal homoproline residue with a proline in 1. The
connectivity of amino acids was also confirmed by both
HMBC and ROESY data, with the following correlations
observed: the 3,4-DiMeGln NH/Dab C-1, the O-MeSer H-2/
3-OHLeu C-1, the Gly H2-2/O-MeSer C-1, the Ala H-2/Gly
C-1, the N-MeThr N-Me/Ala C-1, and the Pro H2-5/β-
OMeTyr C-1 from the HMBC experiment; the Dab H-2/3,4-
DiMeGln NH, the 3,4-DiMeGln H-2/3-OHLeu NH, the 3-
OHLeu H-2/O-MeSer NH, the Gly NH/O-MeSer NH, the
Gly NH/Ala NH, the Ala H3-3/N-MeThr N-Me, the N-MeThr
H-2/β-OMeTyr NH, and the β-OMeTyr 3-OMe/Pro H2-4
from the ROESY data. The presence of an ester bond between
the carbonyl group of Pro and the hydroxy group of the 3-
OHLeu was suggested by the deshielded chemical shift of the
3-OHLeu oxymethine proton H-3 (δH 4.90) and confirmed by
the presence of an HMBC correlation between this proton and
the Pro carbonyl carbon C-1 (δC 171.7). The Dhtda moiety
was linked to the N-terminus of the serine residue by HMBC
correlations from the Ser H-2 (δH 4.37) and the Dhtda 2-Me
(δH 1.13) protons to the carbonyl (δC 175.6) of Dhtda (Figure
1). The C-4/C-5 olefin in the Dhtda residue of 1 was assigned
as Z based on J4,5 = 12.0 Hz, while the C-6/C-7 double bond
was assigned as E on the basis of ROESY correlations between
H-5/H-7 and 6-Me/8-Me. These double bond configurations
are the same as the Dhtda moieties of the papuamides6−8 and
mirabamides.9,10 Thus, the planar structure of swinhopeptolide
A (1) was elucidated.
8
1
study. Very close correspondence between the H and 13C
NMR data for the Dhtda residues in both swinhopeptolide A
(1) and papuamide B when measured in CD3OD (Table S2,
Supporting Information) suggests that they share the same
2S,3R,8R absolute configuration.
The molecular formula of 2 was deduced to be
C66H101N13O19 by HRESIMS and NMR analysis. The 1H
and 13C NMR spectra of 2 (Table S1) were very similar to
those of 1, except the C-2 and C-3 carbinol carbons of the
Dhtda moiety in 1 were replaced with a nonprotonated sp2
carbon (δC 128.7) and an sp2 methine (δC 134.4). This
The absolute configurations of the amino acid constituents
of 1 were determined by acid hydrolysis and application of the
advanced Marfey method.26 The amino acids with a single
stereogenic center, L-Pro, D-Ser, L-Ala, and D-O-MeSer, were
D
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suggested that the amide linked 2,3-dihydroxy-2,6,8-trimethyl-
deca-(4Z,6E)-dienoic acid (Dhtda) residue in 1 was replaced
with a 2,6,8-trimethyldeca-(2,4,6)-trienoic acid (Tdta) moiety
in 2. The more shielded chemical shift of C-4 in the Tdta
relative to C-4 in Dhtda (from δC 126.4 in 1 to δC 121.8 in 2)
and the deshielded nature of C-5 (from δC 135.9 to δC 142.9)
was consistent with a conjugated triene functionality in 2. This
assignment was confirmed by TOCSY correlations that
extended from H-3 (δH 7.00) to the Tdta H-5 (δH 6.56)
and by HMBC correlations from the 2-Me to C-1 (δC 169.3),
C-2 (δC 128.7), and C-3 (δC 134.4). The double bond
configurations at C-2/C-3, C-4/C-5, and C-6/C-7 were
assigned as E based on ROESY correlations between 2-Me/
H-4, H-4/6-Me, H-3/H-5, and H-5/H-7 (Figure 2).
as cyclosporine and rapamycin have been found to effectively
block specific protein−protein interactions. The finding that
swinhopeptolides A (1) and B (2) were active in the cell-based
BRET assay for Ras−Raf interactions suggests that they can
suppress or destabilize the Ras−Raf complex. Detailed
biochemical confirmation and evaluation of these effects is
warranted, but these initial results indicate that the
swinhopeptolides may represent a new structural class of
protein−protein interaction modulators.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 241 polarimeter, and UV spectra were
acquired in spectroscopy grade MeOH using a Varian Cary 50 UV−
vis spectrophotometer. NMR spectra were obtained with a Bruker
Avance III NMR spectrometer equipped with a 3 mm cryogenic
probe and operating at 600 MHz for 1H and 150 MHz for 13C.
Spectra were referenced to residual solvent signals at δH 2.50 and δC
39.5 (DMSO-d6), and HMBC experiments were optimized for nJCH
=
8.3 Hz. HRESIMS data were acquired on an Agilent Technology 6530
Accurate-mass Q-TOF LC/MS. HPLC separations were performed
on a Varian ProStar HPLC system using a Phenomenex Luna C18 (5
μ, 100 Å, 250 × 10 mm) column run with the indicated gradient.
Animal Material. Samples of the marine sponge Theonella
swinhoei cf. verrucosa were collected by scuba at a depth of 18 ft, in
Papua New Guinea in 1993, and kept frozen until extraction. The
collection was carried out by the Coral Reef Research Foundation
under contract with the National Products Branch, U.S. National
Cancer Institute. A voucher specimen (voucher ID # 0CDN1829)
was deposited at the Smithsonian Institution, Washington, D.C.
Extraction and Isolation. The frozen specimens of Theonella
swinhoei (194.5 g wet wt) were repeatedly extracted according to the
standard NCI methodology outlined in McCloud30 to give 50 g of an
aqueous extract. A 5.2 g aliquot of the extract was subjected to C18
reversed-phase flash column chromatography using a stepwise
gradient elution with 100% H2O (fraction A, 4.2 g), 2:1 H2O−
MeOH (fraction B, 267 mg), 1:2 H2O−MeOH (fraction C, 179 mg),
and 100% MeOH (fraction D, 90 mg). The active fraction D was
chromatographed on a Sephadex LH-20 column (25 × 510 mm),
using 1:1 MeOH−CH2Cl2 as eluent, to obtain 20 fractions. The
combined active fractions (13−14) were separated by semipreparative
reversed-phase HPLC (Phenomenex Luna C18 column, 10 mm ×
250 mm; 2.0 mL/min, MeCN−H2O, 50:50), yielding six peaks rich in
secondary metabolites. Purification of the subfractions (4 and 5) was
accomplished by analytical HPLC (Phenomenex Luna C18 column,
4.6 mm × 250 mm; 0.8 mL/min, CH3CN−H2O gradient (20:80−
100:0, containing 0.1% trifluoroacetic acid)) to yield compounds 1
(5.1 mg) and 2 (0.9 mg), respectively, as amorphous solids.
Figure 2. Key TOCSY, HMBC, and ROESY NMR correlations for
the Tdta moiety in 2.
Characteristic coupling constants J3,4 = 10.6 Hz and J4,5
=
15.7 Hz, were consistent with the assignment of 2,6,8-
trimethyldeca-(2E,4E,6E)-trienoic acid (Tdta). The absolute
configuration of 2 was assigned by LC-MS analysis of the D/L-
DLA derivatives of the acid hydrolysate and comparison of the
retention times with those of 1 and mirabamide A. This
established that all of the amino acid residues in
swinhopeptolide B (2) possessed identical configurations to
those in 1.
Swinhopeptolides A (1) and B (2) were tested in a cell-
based Bioluminescence Resonance Energy Transfer (BRET)
assay to assess their effects on Ras−Raf protein−protein
interactions. This assay employs the NanoLuc luciferase
attached to the full-length Raf protein as the energy donor
and a HaloTag fluorescent ligand on the full-length Ras protein
that acts as a fluorescent energy acceptor. When the two
proteins bind together, the two fluorescent reagents are
brought into close spatial proximity and an acceptor signal at
618 nm is generated, but in the absence of protein−protein
binding, the two reagents remain spatially separated and the
donor signal at 460 nm predominates. The ration between the
fluorescent signals at 460 and 618 nm is indicative of the
degree of Ras−Raf binding. Using this assay system,
swinhopeptolides A (1) and B (2) were effective inhibitors
of this protein−protein interaction with IC50 values of 5.8 and
8.5 μM, respectively (Supporting Information).
Swinhopeptolide A (1). Pale yellow, amorphous powder; [α]D25 −5
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.24), 229 (3.17),
276 (2.07) nm; IR (film) νmax 3300, 2936, 1740, 1670, 1453, 1204,
1137 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z
1436.7281 [M + Na]+ (calcd for C66H103N13O21Na, 1436.7288).
Swinhopeptolide B (2). White, amorphous powder; [α]2D5 −13 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.05), 228 (2.52), 274
(1.81) nm; IR (film) νmax 3300, 2948, 1743, 1679, 1447, 1208, 1141
1
cm−1; H and 13C NMR data, Table S1; HRESIMS m/z 1380.7416
[M + H]+ (calcd for C66H102N13O19, 1380.7420).
Acid Hydrolysis of Peptides and LC-MS Analysis of D/L-DLA
Derivatives.25 Compounds 1, 2, and mirabamide A (0.2 mg each)
were separately dissolved in degassed 6 N HCl (0.6 mL) and heated
in sealed glass vials at 110 °C for 17 h. The hydrolysates were
evaporated to dryness and dissolved in H2O (50 μL), and to each
sample, 20 μL of 1 N NaHCO3 and 100 μL of a 1% solution in
acetone of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA or D/
L-FDLA) were added. The mixtures were heated to 40 °C for 40 min
and then cooled to room temperature, neutralized with 2 N HCl (20
μL), and evaporated to dryness. The residues were dissolved in
CH3CN and then analyzed by LC-MS (Poroshell 120 EC-C18
Most efforts to suppress the Ras−Raf−MEK−ERK signal
transduction cascade have targeted the active sites of the
various signaling proteins. Much less attention has focused on
disrupting critical protein−protein interactions such as the
Ras−Raf complex. Small molecules that can effectively inhibit
protein−protein interactions are relatively rare, in large part
because protein−protein interaction domains are generally flat
and quite large, covering hundreds to thousands of square
nanometers.28,29 However, a number of natural products such
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Article
column, 4.6 × 150 mm, 1.0 mL/min, CH3CN−H2O gradient 5:95−
100:0, containing 0.1% formic acid in 60 min). An Agilent 6130
Quadrupole mass spectrometer was used for ESIMS detection
(positive and negative ion mode). DLA derivatives were detected
by absorption at 340 nm, and assignment was secured by ion-selective
monitoring. The retention times (tR) of the D/L-DLA mixtures (with
the L-DLA tR underlined) were as follows.
Swinhopeptolide A (1). L-Pro (23.10), D-Pro (24.85), m/z 408 [M
− H]−; L-Ser (20.88), D-Ser (21.41), m/z 400 [M + H]+; L-Ala
(22.96), D-Ala (25.47), m/z 384 [M + H]+; L-O-MeSer (22.50), D-O-
MeSer (25.43), m/z 414 [M + H]+; L-Dab (32.25), D-Dab (34.82)
(32.32, from mirabamide A), m/z 705 [M − H]− (bis DLA
derivative); L-3-OHLeu (23.10), D-3-OHLeu (26.91) (26.97, from
mirabamide A), m/z 440 [M − H]−; L-N-MeThr (21.31), D-N-MeThr
(22.81) (21.30, from mirabamide A), m/z 426 [M − H]−; 3,4-DiMe-
L-Gln (22.89), 3,4-DiMe-D-Gln (24.43) (22.25, from mirabamide A),
m/z 468 [M − H]−.
Swinhopeptolide B (2). L-Pro (23.11), D-Pro (24.87), m/z 408 [M
− H]−; L-Ser (20.90), D-Ser (21.45), m/z 398 [M − H]−; L-Ala
(22.97), D-Ala (25.49), m/z 382 [M − H]−; L-O-MeSer (22.54), D-O-
MeSer (25.48), m/z 412 [M − H]−; L-Dab (32.28), D-Dab (34.85),
m/z 705 [M − H]− (bis derivative); L-3-OHLeu (22.81), D-3-OHLeu
(24.31), m/z 440 [M − H]−; L-N-MeThr (21.29), D-N-MeThr
(22.81), m/z 426 [M − H]−; 3,4-DiMe-L-Gln (22.49), 3,4-DiMe-D-
Gln (25.51), m/z 468 [M − H]−.
Absolute Configuration of β-Methoxytyrosine. A stream of
ozone in O2 was bubbled through individual cooled solutions of
compounds 1, 2, and mirabamide A (0.2 mg each) in MeOH (2 mL)
at −78 °C for 5 h. Hydrogen peroxide (30%, 10 drops) was added,
and the reaction mixture was allowed to stand at 23 °C overnight. The
solvent was removed under a stream of N2, and the resultant
ozonolysis products were dissolved in degassed 6 N HCl (0.5 mL) in
an evacuated glass tube and heated at 160 °C for 16 h. The solvent
was removed in vacuo, and the resulting material was subjected to
Marfey’s derivatization as described above. The derivatized amino
acids were dissolved in CH3CN, and then, analysis of the DLA
mixtures (with the L-DLA tR underlined) was as follows: L-β-OMeAsp
(33.40), D-β-OMeAsp (34.71) for 1, L-β-OMeAsp (32.94), D-β-
OMeAsp (34.74) for 2 (34.62, from mirabamide A), m/z 458 [M +
H]+.
Authors
Chang-Kwon Kim − Molecular Targets Program, Center for
Cancer Research, National Cancer Institute, Frederick,
Maryland 21702-1201, United States
Dongdong Wang − Molecular Targets Program, Center for
Cancer Research, National Cancer Institute, Frederick,
Maryland 21702-1201, United States
Heidi R. Bokesch − Molecular Targets Program, Center for
Cancer Research, National Cancer Institute, Frederick,
Maryland 21702-1201, United States; Basic Science Program,
Leidos Biomedical Research, Inc., Frederick National Laboratory
for Cancer Research, Frederick, Maryland 21702-1201, United
States
Richard W. Fuller − Molecular Targets Program, Center for
Cancer Research, National Cancer Institute, Frederick,
Maryland 21702-1201, United States
Emily Smith − Molecular Targets Program, Center for Cancer
Research, National Cancer Institute, Frederick, Maryland
21702-1201, United States; Basic Science Program, Leidos
Biomedical Research, Inc., Frederick National Laboratory for
Cancer Research, Frederick, Maryland 21702-1201, United
States
Curtis J. Henrich − Molecular Targets Program, Center for
Cancer Research, National Cancer Institute, Frederick,
Maryland 21702-1201, United States; Basic Science Program,
Leidos Biomedical Research, Inc., Frederick National Laboratory
for Cancer Research, Frederick, Maryland 21702-1201, United
States
David E. Durrant − Laboratory of Cell and Developmental
Signaling, Center for Cancer Research, National Cancer
Institute, Frederick, Maryland 21702-1201, United States
Deborah K. Morrison − Laboratory of Cell and Developmental
Signaling, Center for Cancer Research, National Cancer
Institute, Frederick, Maryland 21702-1201, United States
Carole A. Bewley − Laboratory of Bioorganic Chemistry,
National Institute of Diabetes and Digestive and Kidney
Diseases, NIH, Bethesda, Maryland 20814, United States
Ras−Raf Assay. A modification of a live cell bioluminescence
resonance energy transfer (BRET) analysis system for assessing Ras−
Raf protein interactions was used to identify disrupters of Ras−Raf
binding in cells.31 Cells expressing Ras and Raf proteins fused to
BRET acceptor and donor domains, respectfully, were treated with
test samples at 10 μg/mL for 4 h followed by assessment of the BRET
signal. Acceptor, donor, and BRET signals were each monitored to
control for protein expression levels. The same assay system was used
for bioassay-guided fractionation and to estimate potency of the final
purified active components.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00136
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Grateful acknowledgement goes to the Natural Products
Support Group (NCI at Frederick) for extraction and A.
Wamiru for Ras/Raf assay support. This research was
supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research
and with federal funds from the National Cancer Institute,
National Institutes of Health, under contract
HHSN261200800001E. The content of this publication does
not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does mention of trade
names, commercial products, or organizations imply endorse-
ment by the U.S. Government.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00136.
Experimental procedures, additional figures, and full
spectroscopic data for all new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
REFERENCES
■
Kirk R. Gustafson − Molecular Targets Program, Center for
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0001-6821-4943; Phone: +1-301-846-5197;
Email: gustafki@mail.nih.gov; Fax: +1-301-846-6851
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