Ulleungdin, a Lasso Peptide with Cancer Cell Migration Inhibitory Activity Discovered by the Genome Mining Approach

Article abstract of DOI:10.1021/acs.jnatprod.8b00449

The advances of genomic sequence analyses and genome mining tools have enabled the exploration of untapped microbial natural products. Through genome mining studies to discover cryptic natural products, we found biosynthetic genes encoding a new lasso peptide in the genome sequence of a soil bacterium, Streptomyces sp. KCB13F003 isolated from Ulleung Island (a small volcanic island), Korea. The production and purification of the encoded peptide, named ulleungdin, were achieved by optimizing the culture conditions followed by LC-MS-targeted isolation. Structure elucidation was performed by NMR spectroscopic and MS spectrometric analyses and chemical means (Marfey's and GITC derivatizations), proving ulleungdin to be a new 15-mer class II lasso peptide with a threaded structure. Biological evaluation with the cell invasion assay and time-lapse cell tracking analysis revealed that ulleungdin has significant inhibitory activities against cancer cell invasion and migration.

Full text of DOI:10.1021/acs.jnatprod.8b00449

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Ulleungdin, a Lasso Peptide with Cancer Cell Migration Inhibitory
Activity Discovered by the Genome Mining Approach
Sangkeun Son,†,§ Mina Jang,†,‡,§ Byeongsan Lee,† Young-Soo Hong,†,‡ Sung-Kyun Ko,†,‡
Jae-Hyuk Jang,*,†,‡ and Jong Seog Ahn*,†,‡
†Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Korea
‡Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon
34141, Korea
S
* Supporting Information
ABSTRACT: The advances of genomic sequence analyses and genome mining
tools have enabled the exploration of untapped microbial natural products. Through
genome mining studies to discover cryptic natural products, we found biosynthetic
genes encoding a new lasso peptide in the genome sequence of a soil bacterium,
Streptomyces sp. KCB13F003 isolated from Ulleung Island (a small volcanic island),
Korea. The production and purification of the encoded peptide, named ulleungdin,
were achieved by optimizing the culture conditions followed by LC-MS-targeted
isolation. Structure elucidation was performed by NMR spectroscopic and MS
spectrometric analyses and chemical means (Marfey’s and GITC derivatizations),
proving ulleungdin to be a new 15-mer class II lasso peptide with a threaded
structure. Biological evaluation with the cell invasion assay and time-lapse cell
tracking analysis revealed that ulleungdin has significant inhibitory activities against
cancer cell invasion and migration.
he ribosomally synthesized and post-translationally
of a new lasso peptide from Streptomyces sp. KCB13F003
Tmodified peptides (RiPPs) are encoded as a propeptide isolated from a soil sample from Ulleung Island, Korea.
Ulleung Island, a small volcanic island situated 120 km off the
eastern coast of South Korea, has a distinctive ecosystem and
high biodiversity, as seen by the presence of many endemic
plants.7
that is then processed into the mature peptide enzymatically.
Lasso peptides, an intriguing family of RiPPs, are characterized
by their unique knot-like structure in which the C-terminal tail
threads through the N-terminal macrolactam.1 These topolog-
ically unique peptides show a wide range of bioactivities
ranging from antimicrobial to enzyme inhibition and receptor
antagonism, making them a promising family of natural
products for medicinal applications.2
With the progression of bacterial genome sequencing and
biosynthetic knowledge, the genome mining approach has
become a promising tool for identifying new bacterial
products. It has been demonstrated that a combination of
genome mining and pleiotropic or pathway-specific methods
for activating targeted cryptic gene clusters can rationally
produce new compounds.3 In the cases of the genome mining
approach aimed at the discovery of novel RiPPs, such as lasso
peptides, the precursor sequences and protease cleavage sites
obtained by in silico analysis of genome data allow prediction of
the structure of the mature compounds and facilitate LC-MS-
based detection and purification.4 Our group has continued the
exploration of novel natural products from bacteria inhabiting
diverse ecological niches in the Korean Peninsula and recently
discovered new chlorinated nonribosomal peptides by the
genome mining approach.5,6 A search for biosynthetic gene
clusters that have the potential to encode cryptic new
metabolites from several genome sequences of in-house
actinomycetes uncovered the cryptic biosynthetic gene cluster
Through evaluating a variety of culture media for strain
KCB13F003, conditions were developed to produce a
compound corresponding to the predicted lasso peptide as
shown by an LC-MS chromatogram of the culture extract.
Subsequently, ulleungdin, a new 15-mer class II lasso peptide
was isolated. Ulleungdin inhibited the invasion and migration
of human lung carcinoma A549 cells. Cancer cell invasion and
migration are the first step in tumor metastasis, the most
frequent cause of death for cancer patients, and compounds
inhibiting these cellular phenotypes could be used for the
development of anticancer agents and understanding of modes
of cancer cell metastasis.8 Genome mining and structural
analysis of ulleungdin based on NMR spectroscopic and MS
spectrometric data and a discussion of its biological activity are
presented here.
RESULTS AND DISCUSSION
■
In general, lasso peptides are biosynthesized by at least three
essential genes that are responsible for a precursor peptide
(gene A) and two maturation enzyme coding genes (gene B for
Received: June 4, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Gene cluster for the biosynthesis of ulleungdin. (a) Structure of the uld cluster. (b) Proposed functions of the genes in the uld cluster.
a cysteine protease and gene C for an ATP-dependent lactam
synthase).9 The core peptide sequence that includes the
primary sequence of the lasso peptides is transformed by
maturation enzymes to yield the final product. Analysis of the
genome of Streptomyces sp. KCB13F003, assembled in two
contigs with a total size of 9.27 Mb, presented 31 putative
biosynthetic gene clusters encoding a variety of secondary
metabolites including nonribosomal peptides, polyketides, and
RiPPs.10 Among them, one biosynthetic gene cluster,
designated uld, was of special interest because of its potential
to encode a lasso peptide (Figure 1). A BLASTP similarity
search for each gene in the uld cluster was then performed to
annotate their functions. The gene uldC (GenBank accession
number: MH277338) codes for an enzyme with significant
sequence similarity to McjC, responsible for the cyclization of
Figure 2. LC-MS screening for the detection of ulleungdin in CSP
culture extract. (a) HPLC profiling of the culture extract with UV-
microcin J25.11 The precursor peptide coding gene uldA
(MH277337) was found upstream of the gene uldC by a
photodiode array detection (scan UV 200−420 nm). The peak for
BLASTP similarity search showing high similarity to CptA, the
44-residue precursor peptide of chaxapeptin, and this allowed
the prediction of the positions for the cleavage at the TxG
motif and cyclization between Gly1 and Asp8.12 The primary
sequence of the lasso peptide encoded in the uld cluster was
therefore deduced to be GFIGWGKDIFGHYGG. The
expected molecular weight of the lasso peptide was calculated
to be 1592 Da considering macrolactam formation. Based on
the above information, the isolation of the pure lasso peptide
for detailed spectroscopic analysis and bioactivity tests was
carried out. The compound showing the predicted ion peak
was not found in any crude extracts or fractions that were
generated by previous chemical studies of strain KCB13F003.
Alteration of cultivation parameters is a proven approach to
effecting secondary metabolite production by actinomycetes.
Thus, to identify the encoded peptide in the uld gene cluster,
six media were evaluated for the production of the targeted
lasso peptide (Figure S1). Analyses of the extracts obtained
from cultures in a medium containing corn steep solids showed
a small peak with m/z 1591.3 for [M − H]− (Figure 2). The
scale-up fermentation, sequential solvent extraction, and the
reversed-phase chromatographic separation led to the isolation
of the pure peptide (1, 3.2 mg).
ulleungdin is colored in blue. (b) Targeted extracted-ion chromato-
gram for m/z 1591 [M − H]−.
terminal y-series ions y4−y7 (Figures 3a, S4, and S5). The
macrolactam portion of ulleungdin did not fragment under
standard MS conditions and was found to be resistant to
trypsin degradation, supporting the highly compact structure of
the peptide. Although DMSO-d6 has been commonly used as
an NMR solvent for lasso peptides,13 the measurement of the
NMR spectra of ulleungdin in DMSO-d6 was hampered due to
severe peak broadening. Ulleungdin was soluble in DMSO-d6,
C5D5N, CD3OD, CD3OH, and a CD3OD/CDCl3 mixture (v/
1
v, 1:1) but insoluble in CD3COCD3, D2O, and CDCl3. H
NMR data recorded in CD3OD or CD3OH showed broad but
rather distinguishable resonances, and these solvents were
chosen for comprehensive NMR assignments (Figure S3 and
Table S1). In the HSQC spectrum, numerous methylenes
resonating at δC/H 43.0−46.0/3.0−4.5 were present, consistent
with the high content of Gly in the core peptide. Identification
of each residue, especially Ile and Lys, was achieved primarily
based on the DQF-COSY and TOCSY correlations starting
with the amide NH protons (Figure 3b). The HMBC
correlations of one spin system containing a low-field NH
proton at δH 10.33 clearly revealed the Trp residue. Similarly, a
network of HMBC correlations from the ortho-coupled
aromatic protons at δH 6.77 (d, J = 7.7 Hz) to the neighboring
carbons including the oxygenated sp2 carbon at δC 157.7 was
indicative of the presence of Tyr. Analysis of the HSQC-
TOCSY spectrum led to the assignment of proton and carbon
Ulleungdin (1) was obtained as a white, amorphous powder.
Its molecular formula was established as C78H101N19O18 by
analysis of the HR-ESIMS data. To obtain spectrometric data
for the primary sequence, ulleungdin was first analyzed with
ESIMS/MS fragmentation. The resulting spectra showed
dehydrated N-terminal b-series fragment ions b8−b14 and C-
B
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Figure 3. Structure determination of ulleungdin. (a) ESIMS/MS data for ulleungdin (m/z 1591 [M − H]− and 797 [M + 2H]2+). (b) Structure of
ulleungdin (1) with key 2D NMR correlations used for determining the amino acid sequence.
resonances for benzene rings in the two Phe residues. Although
the low quality of the HMBC spectrum precluded the full
NMR assignment of the imidazole ring in the His residue, the
presence of His could be deduced by the remaining COSY
correlations from an amide proton to β-protons, together with
the observation of the deshielded sp2 methine signal at δC/H
134.9/8.39.
Analysis of a complete set of sequential NOEs between the
amide protons and α-protons, together with a few HMBC
signals for the amide carbonyls, enabled the residue order
(GFIGWGKDIFGHYGG) terminating in a carboxylic acid (δC
176.9) to be determined (Figure 3b). The strong NOE signals
of the amide NH of Gly1 (δH 8.76) to the β-protons of Asp8
(δH 3.40 and 2.73) provided direct evidence of a side-chain-to-
backbone covalent linkage, leading to the macrolactam
consisting of eight residues.
Although the highly challenging NMR assignments due to
the poor spectrum quality precluded NMR-based 3D structure
calculation of ulleungdin, intensive inspection of NMR spectra
enabled us to observe noticeable features of the signals which
provided evidence that it has a lasso structure.14 The NOESY
spectrum of ulleungdin showed the sequential and intraresidue
NOEs and those between the tail and the macrolactam (Tyr13-
Hα/Gly6-H2α; Tyr13-H2β/Ile3-H3γ2; His12-Hα/Trp5-Hα, Gly6-
NH; His12-NH/Trp5-Hα). The lasso fold was also rationalized
by a large 1H signal dispersion of the amide protons (δH 7.19−
8.90), which is a sign of rigid and defined structures.
Unthreaded lasso peptides show a narrower range for the
NH chemical shift values.15 In addition, the 1H NMR
spectrum had an unusual upfield signal for the secondary
methyl of Ile9 at δH 0.36, which is considerably different from
the Ile positioned in the peptides of nonthreaded topology (δH
∼0.9).16 The amide NH protons of the Lys7, Asp8, and Tyr13
residues were detected in the deuterated CD3OD solvent,
indicating that slow proton/deuterium exchange occurred.
Additionally, tandem mass spectrometry of ulleungdin using
collision-induced dissociation (CID) revealed that [M + 2H]2+
species of ulleungdin yielded the lasso-specific interlocked
product ion [(b8)*(y6)]2+ (Figure S5).17 The above character-
istic features of the threading residues established that
ulleungdin possesses the lasso fold with a threading tail
through the macrolactam, not a branched cyclic chain with a
free tail.
The absolute configurations of the α-carbons were
determined by advanced Marfey’s analysis with FDLA (1-
fluoro-2,4-dinitrophenyl-5-leucine amide).18 The acid hydro-
lysate of ulleungdin was derivatized with L- and D-FDLA, and
the products were analyzed by LC-MS, which enabled us to
confirm that all the amino acids present in ulleungdin are the L-
form. The absolute configuration of the additional stereocenter
of the L-Ile residue was established by LC-MS comparison of
the GITC derivative of the acid hydrolysate with those of
authentic L-Ile and L-allo-Ile.19 The LC-MS comparison,
together with the co-injection experiments, established that
ulleungdin contains L-Ile.
The sequence of ulleungdin is similar to that of the known
lasso peptide anantin (Figure 4), although it is shorter by two
residues and has two amino acid substitutions (K7G14 vs
N7S14).20 On the basis of a homology search using BLASTP
analysis, we proposed the functions for the genes in the uld
cluster (Figure 1). The gene uldA codes for the precursor
Figure 4. Sequence alignment of four lasso peptides, ulleungdin,
anantin, sungsanpin, and chaxapeptin. The producing strains of each
peptide are indicated on the right.
C
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Figure 5. Inhibitory effect of ulleungdin on the invasion of A549 cells. (a) Effect of ulleungdin on A549 cell viability. The cells were treated with
different concentrations of ulleungdin for 24 h. Cell viability was measured with the EZ-Cytox cell viability assay kit. Values are the mean SD of
three independent experiments. (b) Representative images of A549 cells treated with ulleungdin or DMSO in the Matrigel-coated upper chamber.
After a 24 h incubation, the invaded cells were stained with crystal violet and captured by a microscope at a 100× magnification. Scale bar indicates
100 μm.
Figure 6. Time-lapse cell tracking analysis of cellular movement. (a) The holographic records of cell movement are presented as individual colored
lines. A549 cells were treated with the indicated concentrations of ulleungdin and captured every 15 min for 12 h. (b) The average of migration
(μm), motility (μm), and motility speed (μm/h) was analyzed using the HoloStudio software and represented in the bar graph.
peptide of ulleungdin, and the three genes uldC, uldB1
(MH277339), and uldB2 (MH277340) are proposed to be
responsible for the maturation process (uldC for cyclization
and uldB1 and B2 for processing). The uld cluster contains
split B1 and B2 proteins, as seen in those for lariatin and
streptomonomicin.21 It has been documented that B1 and B2
are involved in recognition and cleavage of the precursor
peptide, respectively.22 While many of the lasso peptide
biosynthetic gene clusters commonly contain ABC transporters
in the same direction of transcription as the three essential
genes, the uld gene cluster instead features the adjacent MFS
transporter (orf1: MH277336) in the opposite direction. It was
also found that a putative N-acetyltransferase (orf 2:
MH277341) is encoded nearby the uldB2 gene. The first
acetylated lasso peptide albusnodin and its biosynthetic gene
cluster harboring an acetyltransferase gene were recently
reported,23 but acetylated ulleungdin was not detected in this
study. A further BLAST search revealed that the most similar
cluster with uld is present in the recently sequenced genome of
Streptomyces lydicus A02, an industrially used natamycin-
producing strain (Figure S22).24 The 16-mer core peptide of
the cluster in S. lydicus A02 (AJT67350.1) has an additional
Arg residue, together with the 15 residues identical to UldA.
Given the pathological significance of cell migration in
cancer metastasis,25 and the previous observed activity of other
lasso peptides,12,26 the inhibitory effects of ulleungdin on
cancer cell invasion and migration were investigated. Based on
the WST cell viability assay, ulleungdin showed no cytotoxicity
against human lung carcinoma A549 cells up to a
concentration of 100 μM (Figure 5a). The inhibitory effect
of ulleungdin on the invasion of A549 cells with the Transwell
chamber invasion assay was determined (Figure 5b). After
D
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to an ESI source that was connected to a Thermo Scientific Dionex
Ultimate 3000 Rapid Separation LC system using a Waters HSS T3
column (Waters, 2.1 × 150 mm, 2.5 μm). The typical operating
parameters were as follows: spray needle voltage, +5 kV; ion transfer
capillary temperature, 275 °C; nitrogen sheath gas, 35; and auxiliary
gas, 5 (arbitrary units). The CID MS/MS collision energy was 35 V,
and the isolation width was 1.0 Da.
treatment with 50 μM ulleungdin for 24 h, the rate of cells that
invaded through the basement membrane was significantly
decreased (48.4
5.7% inhibition). To further evaluate the
effect of ulleungdin on cell migration, time-lapse cell tracking
analysis was performed by a HoloMonitor M4 time-lapse
cytometer. The HoloMonitor M4 is a cell analyzer for adherent
cells to monitor movement patterns and motility by tracking of
individual cells.27 After treatment of ulleungdin, the cells were
tracked over a period of 12 h with 15 min intervals. The
acquired data were then computationally processed to create
holographic images over a range of focal distances. Compared
to the control groups, both the migration and motility of the
ulleungdin-treated cells were remarkably decreased in a dose-
dependent manner (Figure 6). To investigate the mechanism
of action of ulleungdin, the expression levels of cell migration
related proteins (MAP kinases, focal adhesion proteins, and
matrix metalloproteinases) were analyzed by Western blotting;
however, ulleungdin did not influence the expression or the
phosphorylation of the tested proteins (Figure S23).
Although a diverse class of secondary metabolites from
Streptomyces, such as migracins, migrastatin, rakicidin D, and
CPI-2081, have been reported to exhibit inhibitory activities
against cancer cell migration or invasion with low micromolar
potency,28 related bioactivities of lasso peptides have been
relatively underexplored. Previously, cancer cell invasion
inhibitory activities of lasso peptides have been reported for
two peptides, sungsanpin and chaxapeptin, which were isolated
from a marine-derived Streptomyces species and a hyperarid
soil-derived Streptomyces leeuwenhoekii, respectively.12,26 Ul-
leungdin and these two peptides all consist of 15 amino acids
and harbor an 8-mer macrolactam formed between Gly1 and
Asp8 (Figure 4). Under the cell invasion assay with A549 cells,
three lasso peptides exhibited similar potencies (48, 53, and
56% at 50 μM for ulleungdin, sungsanpin, and chaxapeptin,
respectively). While sungsanpin (GFGSKPIDSFGLSWL) and
chaxapeptin (GFGSKPLDSFGLNFF), particularly their ring
residues, share a high degree of sequence similarity (73.3% and
87.5%), ulleungdin has a relatively low similarity to these two
peptides (33.3%). It is possible that the five residues
commonly present in these three peptides might contribute
to their bioactivities. Considering the low similarity of
ulleungdin with the other two peptides, the bioevaluation
results also raised the possibility that the length of the amino
acid sequence or the size of the macrolactam ring could
correlate with the anti-invasion activities of lasso peptides.
The discovery of ulleungdin expands the diversity of the
structural and biological aspects of lasso peptides and also
demonstrates that a genome-guided strategy could be
successfully applied even for limited genomic information
from in-house actinomycetes.
Bacterial Material and Fermentation. The producing strain
KCB13F003 (GenBank accession number for the 16S rRNA gene
sequence: MH620707) was isolated and identified as previously
described.29 Briefly, the strain was obtained from a soil sample from
Ulleung Island, Republic of Korea, by dilution plating of a soil
suspension onto a selective medium containing humic acid. For
preparation of the inoculums, it was cultured in SY agar (10 g of
starch; 1 g of yeast extract; 1 g of tryptone; 17 g of agar; 1 L of
distilled water) at 28 °C until sporulation took place. The following
six media were used for the production studies: YMG (4 g of glucose;
10 g of malt extract broth; 4 g of yeast extract; 1 L of distilled water),
TSB (30 g of tryptic soy broth; 1 L of distilled water), MSM (30 g of
mannitol; 10 g of starch; 20 g of malt extract; 10 g of yeast extract; 1 L
of distilled water), PDB (24 g of potato dextrose broth; 1 L of distilled
water), CSP (4 g of corn steep solid; 10 g of starch; 5 g of
bactopeptone; 0.5 g of CaCO3; 1 L of distilled water), CLM (5 g of
lactose; 5 g of casamino acid; 5 g of malt extract; 1 L of distilled
water). Two agar plugs with a 5 mm diameter were aseptically
punched into the agar medium and transferred into a 1 L baffled
Erlenmeyer flask containing 250 mL of medium. The cultures were
grown at 28 °C for 5 days on a rotary shaker (125 rpm). Large-scale
culture of the strain was performed in the same manner using CSP
medium.
Extraction and Isolation. The 15 L of culture was harvested and
centrifuged at 5000 rpm for 25 min to yield the supernatant and a
mycelia cake. The supernatant was then extracted three times with an
equal volume of EtOAc. The mycelia cake was extracted with 1 L of
70% acetone three times and concentrated in vacuo to yield an
aqueous residue, which was further extracted with EtOAc. The
resulting EtOAc-soluble fractions obtained from the supernatant and
the mycelia were combined and dried in vacuo to yield 3.3 g of extract.
This was separated first by reversed-phase ODS flash chromatography
using a stepwise elution with MeOH/H2O (500 mL each of 20, 40,
60, 80, and 100% aqueous MeOH). The desired molecule was found
in the 100% MeOH by LC-MS analysis. Separation of this fraction
using a semipreparative ODS HPLC system (OptimaPak C18, 10 μm,
10 × 250 mm, 20−42% CH3CN/H2O containing 0.05% formic acid
over 30 min, 3 mL/min) yielded semipure ulleungdin (4.5 mg, tR =
20.8 min), which was further purified using a Cosmosil Cholester
column (Cosmosil Cholester, 5 μm, 10 × 250 mm, 15−40% CH3CN/
H2O containing 0.05% formic acid over 52 min, 3 mL/min) to yield
ulleungdin (3.2 mg, tR = 31.0 min).
Ulleungdin (1): white, amorphous powder; [α]2D1 +28.6 (c 0.05,
MeOH); UV (MeOH) λmax (log ε) 207 (4.7), 221 (4.6), 282 (3.8)
1
nm; H and 13C NMR see Table S1; HR-ESITOFMS m/z 796.8862
[M + 2H]2+ (calcd for C78H103N19O18, 796.8864).
Advanced Marfey’s Analysis. The absolute configurations of the
α-carbons present in ulleungdin were determined following acid
hydrolysis and FDLA derivatization. Approximately 0.1 mg of
ulleungdin was hydrolyzed with 6 M HCl (0.5 mL) at 100 °C for 1
h. The solution was evaporated, and the dried hydrolysate was
resuspended in 1 M NaHCO3 (0.1 mL). FDLA derivatizations were
performed with 1% L-FDLA (0.1 mL) in acetone at 40 °C for 1 h. The
reaction was quenched by the addition of 2 M HCl (20 μL), and then
the resulting mixture was diluted 2-fold with CH3CN and analyzed by
LC-MS. Derivatization with D-FDLA was also carried out in the same
manner described above. The FDLA-derivatized amino acids were
detected using ion extraction chromatograms. Retention times of the
L- and D-derivatives of the amino acids present in ulleungdin were as
follows: Gly 11.72, 11.72 m/z 368 [M − H]−; Phe 13.09, 14.24 m/z
458 [M − H]−; Ile 12.96, 14.63 m/z 424 [M − H]−; Trp 12.96, 13.65
m/z 497 [M − H]−; Lys 14.80, 15.24 m/z 733 [M − H]− (bis
derivative); Asp 10.96, 11.13 m/z 426 [M − H]−; His 12.83, 13.41
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1020 polarimeter. UV spectra were recorded
on an Optizen 2120 UV spectrophotometer. NMR spectra were
recorded on a Bruker Biospin Advance II 900 NMR spectrometer
1
(900 and 225 MHz for H and 13C NMR, respectively) at the Korea
Basic Science Institute (KBSI) in Ochang, Korea. HR-ESIMS data
were obtained on a Waters SYNAPT G2 Q-TOF mass spectrometer
at the KBSI in Ochang, Korea. Semipreparative HPLC was performed
on a Young Lin YL9100 HPLC system coupled with a Young Lin
YL9160 PDA detector. Octadecylsilyl (ODS) silica gel (Cosmosil, 75
μm) was used for reversed-phase flash chromatography. ESIMS/MS
data were obtained using a Thermo LTQ XL linear ion trap attached
E
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m/z 742 [M − H]− (bis derivative); Tyr 15.03, 15.77 m/z 768 [M −
AUTHOR INFORMATION
Corresponding Authors
*(J.-H. Jang) Tel: +82-43-240-6164. Fax: +82-43-240-6169. E-
mail: jangjh@kribb.re.kr.
*(J. S. Ahn) Tel: +82-43-240-6160. Fax: +82-43-240-6169. E-
mail: jsahn@kribb.re.kr.
ORCID
Young-Soo Hong: 0000-0001-7768-2239
Jae-Hyuk Jang: 0000-0002-4363-4252
Jong Seog Ahn: 0000-0001-5166-4358
■
H]− (bis derivative).
GITC Derivatization. Approximately 0.1 mg of ulleungdin was
subjected to acid hydrolysis as described above for Marfey’s analysis.
Six percent trimethylamine (0.2 mL) and 1% GITC in acetone (0.2
mL) were added to the acid hydrolysate and allowed to react at room
temperature for 20 min. After quenching by the addition of 5% acetic
acid (0.2 mL), the mixture was analyzed by LC-MS. The L-Ile
derivatized with GITC was eluted at 58.96 min. The retention times
of the GITC-derivatized L-Ile and L-allo-Ile prepared in the same
manner were 59.02 and 58.77 min, respectively. The co-injection
experiment of the GITC-derivatized sample and authentic L-Ile
showed a single peak at 59.08 min.
Cell Viability Assay. The human lung cancer cell line A549 was
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, 100 units/mL penicillin,
and 100 μg/mL streptomycin at 37 °C with 5% CO2 in a humidified
atmosphere. A549 cells (4 × 103 cells/well) were seeded in 96-well
cell culture plates overnight and treated in triplicate with various
concentrations of ulleungdin. After a 24 h incubation, 10 μL of EZ-
Cytox cell viability assay solution (Daeil Lab service) was directly
added and incubated at 37 °C for 2 h. The absorbance was measured
using a microplate reader at 450 nm. The cell viability was normalized
to the control group.
Transwell Chamber Invasion Assay. A Transwell permeable
support system with polycarbonate translucent inserts with a pore size
of 8.0 μm was used for the cell invasion assay. A549 cells (1 × 105
cells/well) were incubated in the Matrigel-coated upper chamber with
ulleungdin in 300 μL of serum-free medium for 24 h, whereas the
lower chamber was loaded with DMEM medium containing serum to
attract invasive cells. After removal of the noninvasive cells, the
invasive cells were fixed in 4% paraformaldehyde for 15 min, stained
with 0.2% crystal violet, and captured by a microscope at a 100×
magnification.
Author Contributions
§S. Son and M. Jang contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the International Joint Research
Project (ASIA-16-011) of the NST (National Research
Council of Science & Technology), Young Researcher
Program (NRF-2017R1C1B2002602) of the NRF (National
Research Foundation of Korea), and KRIBB Research
Initiative Program funded by the Ministry of Science ICT
(MSIT) of Republic of Korea. We thank the Korea Basic
Science Institute, Ochang, Korea, for providing the NMR and
HR-ESIMS data. We also appreciate Dr. H. W. Ryu (KRIBB)
for assistance with HR-ESIMS data acquisition.
REFERENCES
■
(1) (a) Metsker, M. L. Nat. Rev. Genet. 2010, 11, 31−46.
(b) Hegemann, J. D.; Zimmermann, M.; Xie, X.; Marahiel, M. A.
Acc. Chem. Res. 2015, 48, 1909−1919.
Time-Lapse Cell Tracking Analysis. A549 cells (6 × 103 cells/
100 μL) were seeded in μ-Slide I, ibiTreat (ibidi) and incubated for
the adherence of the cells. After 1 h, 500 μL of DMEM medium was
added into both reservoirs and incubated overnight. Ulleungdin with
the indicated concentrations was treated and directly transferred to
the HoloMonitor M4 time-lapse cytometer (Phase Holographic
Imaging) kept in a 37 °C incubator. Live cells were captured every 15
min for 12 h, and the migration was analyzed with the HoloStudio M4
software. Migration is the value indicating the shortest distance
between the starting point of the cell tracking and the current point.
Motility represents how far the cells have moved during a total
analysis time. Motility speed is the total cell movement per hour.
Western Blot Analysis. Cells were seeded in six-well plates (1.5
× 105 cells/well) for 16 h and treated with different concentrations of
ulleungdin. Following the 24 h treatment, cells were lysed in 2×
Laemmli sample buffer (Elpis Bio). Proteins were separated by SDS-
PAGE, transferred onto nitrocellulose membranes (Bio-Rad), and
probed with the following antibodies. ERK (#9102), p-ERK (#9101),
JNK (#9252), p38 (#9212), p-p38 (#4631), MMP-9 (#13667), and
MMP-2 (#13132) were obtained from Cell Signaling Technology,
Inc. p-JNK (sc-6254), FAK (sc-271126), p-FAK (sc-81493), paxillin
(sc-365379), p-paxillin (sc-365020), and β-actin (sc-47778) were
purchased from Santa Cruz Biotechnology. The immunoreactive
proteins were visualized with the SuperSignal West Pico chem-
iluminescent substrate or SuperSignal West Femto maximum
sensitivity substrate (Thermo-Fisher Scientific).
(2) (a) Knappe, T. A.; Manzenrieder, F.; Mas-Moruno, C.; Linne,
U.; Sasse, F.; Kessler, H.; Xie, X.; Marahiel, M. A. Angew. Chem., Int.
Ed. 2011, 50, 8714−8717. (b) Hegemann, J. D.; Simone, M. D.;
Zimmermann, M.; Knappe, T. A.; Xie, X.; Leva, F. S. D.; Marinelli, L.;
Novellino, E.; Zahler, S.; Kessler, H.; Marahiel, M. A. J. Med. Chem.
2014, 57, 5829−5834. (c) Metelev, M.; Tietz, J. I.; Melby, J. O.; Blair,
P. M.; Zhu, L.; Livnat, I.; Severinov, K.; Mitchell, D. A. Chem. Biol.
2015, 22, 241−250.
(3) (a) Ziemert, N.; Alanjary, M.; Weber, T. Nat. Prod. Rep. 2016,
33, 988−1005. (b) Harvey, A. L.; Edrada-Ebel, R.; Quinn, R. J. Nat.
Rev. Drug Discovery 2015, 14, 111−129. (c) Zerikly, M.; Challis, G. L.
ChemBioChem 2009, 10, 625−633.
(4) (a) Maksimov, M. O.; Pelczer, I.; Link, A. J. Proc. Natl. Acad. Sci.
U. S. A. 2012, 109, 15223−15228. (b) Skinnider, M. A.; Johnston, C.
W.; Edgar, R. E.; Dejong, C. A.; Merwin, N. J.; Rees, P. N.; Magarvey,
N. A. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E6343−E6351.
(c) Tietz, J. I.; Schwalen, C. J.; Patel, P. S.; Maxson, T.; Blair, P. M.;
Tai, H. C.; Zakai, U. I.; Mitchell, D. A. Nat. Chem. Biol. 2017, 13,
470−478.
(5) (a) Oh, M.; Jang, J.-H.; Choo, S.-J.; Kim, S.-O.; Kim, J. W.; Ko,
S.-K.; Soung, N.-K.; Lee, J.-S.; Kim, C.-J.; Oh, H.; Hong, Y.-S.; Ueki,
M.; Hirota, H.; Osada, H.; Kim, B. Y.; Ahn, J. S. Bioorg. Med. Chem.
Lett. 2014, 24, 1802−1804. (b) Son, S.; Ko, S.-K.; Jang, M.; Kim, J.
W.; Kim, G. S.; Lee, J. K.; Jeon, E. S.; Futamura, Y.; Ryoo, I.-J.; Lee, J.-
S.; Oh, H.; Hong, Y.-S.; Kim, B. Y.; Takahashi, S.; Osada, H.; Jang, J.-
H.; Ahn, J. S. Mar. Drugs 2016, 14, 72−83. (c) Son, S.; Ko, S.-K.;
Jang, M.; Lee, J. K.; Kwon, M. C.; Kang, D. H.; Ryoo, I.-J.; Lee, J.-S.;
Hong, Y.-S.; Kim, B. Y.; Jang, J.-H.; Ahn, J. S. J. Nat. Prod. 2017, 80,
1378−1386.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00449.
(6) Son, S.; Hong, Y.-S.; Jang, M.; Heo, K. T.; Lee, B.; Jang, J.-P.;
Kim, J.-W.; Ryoo, I.-J.; Kim, W.-G.; Ko, S.-K.; Kim, B. Y.; Jang, J.-H.;
Ahn, J. S. J. Nat. Prod. 2017, 80, 3025−3031.
(7) (a) Yu, J.-P.; Jin, S.-D.; Kim, W.-B.; Kang, J.-H.; Kim, I.-K.;
Kang, T.-H.; Han, S.-W.; Park, C.-Y.; Jeon, H.-B.; Choi, J.-T.; Paek,
Supplementary tables and figures for LC-MS, HR-
ESIMS, and NMR spectra (PDF)
F
DOI: 10.1021/acs.jnatprod.8b00449
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
W.-K. J. Asia-Pac. biodivers 2013, 6, 175−187. (b) Shin, H.; Oh, S.-H.;
Lim, Y.; Hyun, C.-W.; Cho, S.-H.; Kim, Y.-I.; Kim, Y.-D. J. J. Plant
Biol. 2014, 57, 174−185.
(b) Igarashi, Y.; Shimasaki, R.; Miyanaga, S.; Oku, N.; Onaka, H.;
Sakurai, H.; Saiki, I.; Kitani, S.; Nihira, T.; Wimonsiravude, W.;
Panbangred, W. J. Antibiot. 2010, 63, 563−565. (c) Singh, J. P.;
Tamang, S.; Rajamohanan, P. R.; Jima, N. C.; Chakraborty, G.;
Kundu, G. C.; Gaikwad, S. M.; Khan, M. I. J. Med. Chem. 2010, 53,
5121−5128. (d) Arai, Y.; Iinuma, H.; Ikeda, Y.; Igarashi, M.; Hatano,
M.; Kinoshita, N.; Ukaji, T.; Simizu, S.; Umezawa, K. J. Antibiot. 2013,
66, 225−230.
(8) (a) Yamaguchi, H.; Wyckoff, J.; Condeelis, J. Curr. Opin. Cell
Biol. 2005, 17, 559−564. (b) Clark, A. G.; Vignjevic, D. M. Curr.
Opin. Cell Biol. 2015, 36, 13−22.
(9) Maksimov, M. O.; Pan, S. J.; Link, A. J. Nat. Prod. Rep. 2012, 29,
996−1006.
(29) Son, S.; Ko, S.-K.; Jang, M.; Lee, J. K.; Ryoo, I.-J.; Lee, J.-S.;
Lee, K. H.; Soung, N.-K.; Oh, H.; Hong, Y.-S.; Kim, B. Y.; Jang, J.-H.;
Ahn, J. S. Org. Lett. 2015, 17, 4046−4049.
(10) Blin, K.; Wolf, T.; Chevrette, M. G.; Lu, X.; Schwalen, C. J.;
Kautsar, S. A.; Suarez Duran, H. G.; De Los Santos, E. L. C.; Kim, H.
U.; Nave, M.; Dickschat, J. S.; Mitchell, D. A.; Shelest, E.; Breitling,
R.; Takano, E.; Lee, S. Y.; Weber, T.; Medema, M. H. Nucleic Acids
Res. 2017, 45, W36−W41.
́
(11) Duquesne, S.; Destoumieux-Garzon, D.; Zirah, S.; Goulard, C.;
Peduzzi, J.; Rebuffat, S. Chem. Biol. 2007, 14, 793−803.
(12) Elsayed, S. S.; Trusch, F.; Deng, H.; Raab, A.; Prokes, I.;
Busarakam, K.; Asenjo, J. A.; Andrews, B. A.; van West, P.; Bull, A. T.;
Goodfellow, M.; Yi, Y.; Ebel, R.; Jaspars, M.; Rateb, M. E. J. Org.
Chem. 2015, 80, 10252−10260.
(13) Kersten, R. D.; Yang, Y.-L.; Xu, Y.; Cimermancic, P.; Nam, S.-J.;
Fenical, W.; Fischbach, M. A.; Moore, B. S.; Dorrestein, P. C. Nat.
Chem. Biol. 2011, 7, 794−802.
(14) (a) Wilson, K.-A.; Kalkum, M.; Ottesen, J.; Yuzenkova, J.;
Chait, B. T.; Landick, R.; Muir, T.; Severinov, K.; Darst, S. A. J. Am.
Chem. Soc. 2003, 125, 12475−12483. (b) Zimmermann, M.;
Hegemann, J. D.; Xie, X.; Marahiel, M. A. Chem. Biol. 2013, 20,
558−569.
(15) Xie, X. L.; Marahiel, M. A. ChemBioChem 2012, 13, 621−625.
(16) (a) Pavlaskova, K.; Nedved, J.; Kuzma, M.; Zabka, M.; Sulc, M.;
Sklenar, J.; Novak, P.; Benada, O.; Kofronova, O.; Hajduch, M.;
Derrick, P. J.; Lemr, K.; Jegorov, A.; Havlicek, V. J. Nat. Prod. 2010,
73, 1027−1032. (b) Kawahara, T.; Itoh, M.; Kozone, I.; Izumikawa,
M.; Sakata, N.; Tsuchida, T.; Shin-Ya, K. J. Antibiot. 2016, 69, 66−68.
(c) Jiao, W.-H.; Khalil, Z.; Dewapriya, P.; Salim, A. A.; Lin, H.-W.;
Capon, R. J. J. Nat. Prod. 2018, 81, 976−984.
(17) (a) Rosengren, K. J.; Clark, R. J.; Daly, N. L.; Goeransson, U.;
Jones, A.; Craik, D. J. J. Am. Chem. Soc. 2003, 125, 12464−12474.
(b) Jeanne Dit Fouque, K.; Lavanant, H.; Zirah, S.; Hegemann, J. D.;
Fage, C. D.; Marahiel, M. A.; Rebuffat, S.; Afonsoa, C. Analyst 2018,
143, 1157−1170.
(18) Harada, K.; Fujii, K.; Hayashi, K.; Suzuki, M.; Ikai, Y.; Oka, H.
Tetrahedron Lett. 1996, 37, 3001−3004.
(19) Hess, S.; Gustafson, K. R.; Milanowski, D. J.; Alvira, E.; Lipton,
M. A.; Pannell, L. K. J. Chromatogr. A 2004, 1035, 211−219.
(20) Wyss, D. F.; Lahm, H. W.; Manneberg, M.; Labhardt, A. M. J.
Antibiot. 1991, 44, 172−180.
(21) (a) Iwatsuki, M.; Tomoda, H.; Uchida, R.; Gouda, H.; Hirono,
S.; Omura, S. J. Am. Chem. Soc. 2006, 128, 7486−7491. (b) Metelev,
M.; Tietz, J. I.; Melby, J. O.; Blair, P. M.; Zhu, L.; Livnat, I.; Severinov,
K.; Mitchell, D. A. Chem. Biol. 2015, 22, 241−250.
(22) (a) Zhu, S.; Fage, C. D.; Hegemann, J. D.; Mielcarek, A.; Yan,
D.; Linne, U.; Marahiel, M. A. Sci. Rep. 2016, 6, 35604. (b) Cheung,
W. L.; Chen, M. Y.; Maksimov, M. O.; Link, A. J. ACS Cent. Sci. 2016,
2, 702−709.
(23) Zong, C.; Cheung-Lee, W. L.; Elashal, H. E.; Raj, M.; Link, A. J.
Chem. Commun. 2018, 54, 1339−1342.
(24) Wu, H.; Liu, W.; Shi, L.; Si, K.; Liu, T.; Dong, D.; Zhang, T.;
Zhao, J.; Liu, D.; Tian, Z.; Yue, Y.; Zhang, H.; Xuelian, B.; Liang, Y.
Sci. Rep. 2017, 7, 9114.
(25) Steeg, P. S. Nat. Rev. Cancer 2016, 16, 201−218.
(26) Um, S.; Kim, Y. J.; Kwon, H.; Wen, H.; Kim, S. H.; Kwon, H.
C.; Park, S.; Shin, J.; Oh, D. C. J. Nat. Prod. 2013, 76, 873−879.
(27) (a) Mann, C. J.; Yu, L.; Lo, C.-M.; Kim, M. K.; Rylander, C. G.;
Dave, D.; Akkin, T.; Milner, T. E.; Diller, K. R.; Welch, A. J. Opt.
Express 2005, 13, 8693−8698. (b) Sebesta, M.; Egelberg, P. J.;
Langberg, A.; Lindskov, J.-H.; Alm, K.; Janicke, B. Proc. SPIE 2016,
971813.
(28) (a) Nakae, K.; Yoshimoto, Y.; Sawa, T.; Homma, Y.; Hamada,
M.; Takeuchi, T.; Imoto, M. J. Antibiot. 2000, 53, 1130−1136.
G
DOI: 10.1021/acs.jnatprod.8b00449
J. Nat. Prod. XXXX, XXX, XXX−XXX