Leucinostatin Y: A Peptaibiotic Produced by the Entomoparasitic Fungus Purpureocillium lilacinum 40-H-28

Article abstract of DOI:10.1021/acs.jnatprod.8b00839

Leucinostatin Y, a new peptaibiotic, was isolated from the culture broth of the entomoparasitic fungus Purpureocillium lilacinum 40-H-28. The planar structure was elucidated by detailed analysis of its NMR and MS/MS data. The absolute configurations of the amino acids were partially determined by an advanced Marfey's method. The biological activities of leucinostatin Y were assessed using human pancreatic cancer cells, revealing the importance of the C-terminus of leucinostatins for preferential cytotoxicity to cancer cells under glucose-deprived conditions and inhibition of mitochondrial function.

Full text of DOI:10.1021/acs.jnatprod.8b00839

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Leucinostatin Y: A Peptaibiotic Produced by the Entomoparasitic
Fungus Purpureocillium lilacinum 40-H-28
Isao Momose,*,† Takefumi Onodera,† Hiroyasu Doi,† Hayamitsu Adachi,† Masatomi Iijima,†
Yohko Yamazaki,† Ryuichi Sawa,‡ Yumiko Kubota,‡ Masayuki Igarashi,‡ and Manabu Kawada†,‡
†Institute of Microbial Chemistry (BIKAKEN), Numazu, 18-24 Miyamoto, Numazu-shi, Shizuoka 410-0301, Japan
‡Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
S
* Supporting Information
ABSTRACT: Leucinostatin Y, a new peptaibiotic, was isolated from the
culture broth of the entomoparasitic fungus Purpureocillium lilacinum 40-
H-28. The planar structure was elucidated by detailed analysis of its NMR
and MS/MS data. The absolute configurations of the amino acids were
partially determined by an advanced Marfey’s method. The biological
activities of leucinostatin Y were assessed using human pancreatic cancer
cells, revealing the importance of the C-terminus of leucinostatins for
preferential cytotoxicity to cancer cells under glucose-deprived conditions
and inhibition of mitochondrial function.
olid tumors contain large areas that are nutrient- and
oxygen-deprived due to the immaturity and irregular
metabolites discovered in the period 2001−2010. We are
particularly interested in entomoparasitic fungi, including the
most well-known insect pathogenic fungus Cordyceps sinensis,
because they are rich sources of novel biologically active
substances with diverse structural architectures.13,14 Therefore,
we screened the metabolites of entomoparasitic fungi to
identify agents that preferentially reduce the survival of
nutrient-deprived cancer cells and identified a new peptai-
biotic15 named leucinostatin Y (2), which is produced by
Purpureocillium lilacinum 40-H-28 (Figure 1).
S
distribution of blood vessels therein.1,2 Certain cancer cells
have an inherent ability to tolerate harsh growth conditions,
such as nutrient and oxygen deprivation, and can survive for
prolonged periods of time.3 To survive in a nutrient-limited
environment, cancer cells utilize the PI3K-AKT-mTOR
pathway and the unfolded protein response.3−5 Kigamicins,
which are polycyclic xanthones isolated from the culture broth
of Amycolatopsis sp. ML630-mF1, inhibit activation of Akt and
exhibit preferential cytotoxicity to cancer cells under nutrient-
deprived conditions compared to those under nutrient-rich
conditions.6−8 Versipelostatin, a 17-membered macrocyclic
compound isolated from the culture broth of Streptomyces
versipellis 4083-SVS6, suppresses activation of the unfolded
protein response and shows highly selective cytotoxicity to
glucose-deprived cancer cells.9,10
A number of leucinostatins have been identified in previous
studies. Although the structures of leucinostatins have been
elucidated by tandem mass spectrometry (MS/MS), X-ray
diffraction, and chemical degradation,16−21 the H and 13C
1
NMR signals for leucinostatins have not been assigned.
1
Therefore, we assigned the H and 13C NMR spectra for
leucinostatins A (1) and Y (2). In this report, we describe the
isolation, structural elucidation (including NMR assignment),
and biological evaluation of 1 and 2.
Targeting nutrient-deprived cancer cells is an attractive
strategy for cancer therapy. Therefore, our research has
focused on screening cytotoxic agents that act preferentially
under nutrient starvation. In previous studies, we found that
inhibitors of mitochondrial functions such as leucinostatin A
(1, an inhibitor of mitochondrial ATPase) exhibit preferential
cytotoxicity against cancer cells adapted to nutrient-starved
conditions.11
Microbial metabolites are a rich and promising source of
drug candidates. In the early years of antibiotic studies,
approximately 60% of all bioactive microbial metabolites were
isolated from actinobacteria.12 However, the significance of
fungi as secondary metabolite producers has continuously
increased, accounting for 61% of all bioactive microbial
RESULTS AND DISCUSSION
■
The entomoparasitic fungus P. lilacinum 40-H-28 was isolated
from the synnema formed on Macroscytus japonensis Scott22
collected at Yokohama City, Kanagawa Prefecture, Japan. P.
lilacinum 40-H-28 was cultured in a liquid medium (16 L), and
the filtrate obtained from the cultured broth was applied to a
Diaion HP-20 column. The active fractions eluted with
Received: October 10, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Structures of leucinostatins A (1) and Y (2).
acetone were concentrated to give an oily material. The
resulting material was applied to a silica gel column and a
Sephadex LH-20 column, followed by HPLC to afford the new
peptaibiotic leucinostatin Y (2), together with the known
peptaibiotic leucinostatin A (1).
6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD), β-hydrox-
yleucine (HyLeu), three 2-aminoisobutyric acid (Aib), two
leucine (Leu), β-alanine (β-Ala), and alanine (Ala) residues in
2 (Figure 2). Thus, 2 possesses an Ala residue instead of the
N1,N1-dimethylpropane-1,2-diamine moiety of 1. The se-
quence of amino acid residues in 2 was established by the
HMBC spectrum, which reveals correlations between the
carbonyl carbon (δC 168.3) of the 4-methyl-2-hexenoic acid
moiety and the α-methine proton (δH 4.32) of MePro, the
carbonyl carbon (δC 175.9) of MePro and the ΝΗ proton (δH
8.55) of AHMOD, the carbonyl carbon (δC 176.6) of
AHMOD and the ΝΗ proton (δH 7.91) of HyLeu, the
carbonyl carbon (δC 174.1) of HyLeu and the ΝΗ proton (δH
7.82) of Aib1, the carbonyl carbon (δC 177.9) of Aib1 and the
ΝΗ proton (δH 7.54) of Leu1, the carbonyl carbon (δC 176.3)
of Leu1 and the ΝΗ proton (δH 7.77) of Leu2, the carbonyl
carbon (δC 175.8) of Leu2 and the ΝΗ proton (δH 7.76) of
Aib2, the carbonyl carbon (δC 176.0) of Aib2 and the ΝΗ
proton (δH 7.25) of Aib3, the carbonyl carbon (δC 177.4) of
Aib3 and the ΝΗ proton (δH 7.57) of β-Ala, and the carbonyl
carbon (δC 173.5) of β-Ala and the ΝΗ proton (δH 8.13) of
Ala.
In addition, high-resolution ESI tandem mass spectrometry
(MS/MS) analysis was performed to confirm the planar
structure of 2. The higher energy collisional dissociation
experiments for 2 gave most of the b-series product ions at m/z
194.1537, 222.1486, 435.2847, 546.3527, 649.4158, 762.4999,
875.5838, 960.6359, and 1045.6896 in positive ion mode
(Figures 3 and S10), allowing the determination of the
sequence of the eight N-terminal residues (but not the two C-
terminal residues). These product ions of 2 are barely
distinguishable from those of 1 (Figure S9), indicating that 1
and 2 have a common substructure from the 4-methyl-2-
hexenoic acid moiety to the Aib3 residue. Next, 2 was
subjected to HCD experiments in negative ion mode to verify
the C-terminal sequence (Figure S11). The product ions at m/
z 159.0772 and 88.0406 indicate the presence of β-Ala and Ala
at the C-terminus, suggesting that the structure of 2 contains
Leucinostatin A (1) was first isolated from the culture broth
of Penicillium lilacinum by Arai et al.23 Abe et al. reported the
total synthesis of 1 and revealed its correct structure in 2017.24
We obtained 1 as a colorless, amorphous solid. The molecular
formula of 1 was confirmed to be C62H111N11O13 by high-
resolution electrospray ionization mass spectrometry (HRE-
SIMS). Spectroscopic data of natural 1 are identical to those of
synthetic 1.24 1H and 13C NMR signals of natural and synthetic
1, however, have never been assigned to the appropriate
1
protons and carbons. Therefore, we assigned the H and 13C
NMR data of 1 using a similar approach to that for 2, which is
shown below (Table 1).
Leucinostatin Y (2) was obtained as a colorless, amorphous
solid. The UV spectrum of 2 exhibits absorption maxima at
205 and 223 nm and is very similar to that of 1.23 The
molecular formula of 2 was determined to be C60H104N10O15
based on HRESIMS and 1H and 13C NMR analysis, indicating
that 2 has two fewer carbon atoms, seven fewer hydrogens, one
fewer nitrogen, and two additional oxygen atoms than 1. The
planar structure of 2 was primarily elucidated using NMR
1
experiments. The assignments of the H and 13C NMR data
(Figures S1 and S2) for 2 are shown in Table 1. The 13C NMR
and DEPT data (Figure S3) along with the HSQC spectra
(Figure S4) revealed the presence of 60 carbons in 2, which
were categorized as 18 methyl, 11 methylene, 14 sp3 methine,
two sp2 methine, three N-substituted tertiary, and 12 carbonyl
carbons. The 1H NMR and HSQC spectra revealed the
presence of nine amide protons. The COSY spectrum shows
correlations between coupled protons (Figure S5), the HSQC-
TOCSY spectrum shows correlations between all protons
within a given spin system (Figure S6), and the HMBC
spectrum reveals long-range correlations between protons and
carbons in 2 (Figure S7). These data indicate the presence of
4-methyl-2-hexenoic acid, 4-methylproline (MePro), 2-amino-
B
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a
Table 1. NMR Spectroscopic Data for Leucinostatins A (1) and B (2) in CD3OH
leucinostatin A (1)
position δC, type δH (J in Hz)
leucinostatin Y (2)
δC, type δH (J in Hz)
168.3, C
leucinostatin A (1)
residue position δC, type δH (J in Hz)
leucinostatin Y (2)
residue
δC, type
176.3, C
δH (J in Hz)
ΔMHA
1
2
3
168.2, C
Leu1
1
2
3
176.9, C
120.9, CH 6.31, d (14.8) 120.8, CH 6.31, d (14.6)
154.4, CH 6.84, dd (8.0, 154.4, CH 6.82, dd (8.0,
56.0, CH 4.02, m
40.9, CH2 1.61, m
55.4, CH 4.06, m
40.7, CH2 1.62, m
14.8)
39.6, CH 2.33, m
30.0, CH2 1.45, m
14.6)
39.6, CH 2.32, m
30.0, CH2 1.43, m
12.1, CH3 0.91, t (8.0)
4
5
6
7
1
2
1.81, m
25.9, CH 1.82, m
1.82, m
26.0, CH 1.79, m
4
5
6
NH
1
b
12.1, CH3 0.91
21.6, CH3 0.88, d (6.0) 21.3, CH3 0.88, d (6.6)
b
19.5, CH3 1.09, d (6.6) 19.5, CH3 1.08, d (6.8)
176.1, C 175.9, C
64.6, CH 4.32, dd (6.4, 64.5, CH 4.32, dd (7.0,
23.3, CH3 0.94
7.60, d (6.2)
23.5, CH3 0.95, d (6.6)
b
MePro
7.54
Leu2
175.9, C
175.8, C
10.8)
39.3, CH2 1.58, m
34.7, CH 2.40, m
10.4)
39.2, CH2 1.54, m
34.6, CH 2.40, m
3
4
5
2
3
56.5, CH 4.01, m
40.7, CH2 1.68, m
1.81, m
55.4, CH 4.10, m
40.6, CH2 1.64, m
1.81, m
55.6, CH2 3.39, t (10.2) 55.6, CH2 3.36, t (9.8)
3.98, m
3.99, dd (8.2,
9.8)
4
25.8, CH 1.80, m
25.7, CH 1.79, m
6
1
2
3
16.8, CH3 1.15, d (6.6) 16.8, CH3 1.15, d (6.2)
5
6
NH
1
2
3
4
NH
1
2
22.2, CH3 0.89, d (6.0) 21.7, CH3 0.89, d (6.6)
b
AHMOD
176.9, C
176.6, C
23.1, CH3 0.94
7.81, d (5.8)
176.8, C
23.5, CH3 0.95, d (6.6)
7.77, d (4.8)
176.0, C
55.6, CH 4.20, m
37.3, CH2 1.50, m
1.94, m
55.6, CH 4.20, m
37.3, CH2 1.58, m
1.91, m
Aib2
Aib3
57.5, C
57.9, C
4
5
28.0, CH 1.79, m
45.1, CH2 1.39, m
1.48, m
28.1, CH 1.80, m
45.0, CH2 1.39, m
1.48, m
23.4, CH3 1.46, s
27.1, CH3 1.50, s
7.89, s
24.4, CH3 1.43, s
26.2, CH3 1.45, s
7.76, s
6
7
66.4, CH 4.15, m
66.5, CH 4.16, m
178.5, C
177.4, C
51.0, CH2 2.53, dd (4.4, 51.0, CH2 2.54, dd (4.4,
57.8, C
58.0, C
16.0)
16.0)
2.60, dd (7.8,
16.0)
2.60, dd (8.0,
16.0)
3
23.3, CH3 1.46, s
24.4, CH3 1.44, s
8
9
10
11
NH
1
212.5, C
37.5, CH2 2.49, m
7.7, CH3 1.01, t (7.4)
20.2, CH3 0.96, d (6.2) 20.1, CH3 0.97, d (6.0)
8.65, d (5.0) 8.55, d (5.0)
212.5, C
37.5, CH2 2.49, m
7.7, CH3 1.01, t (7.0)
4
NH
1
27.5, CH3 1.49, s
7.62, s
175.9, C
38.2, CH2 2.29, m
2.75, m
38.0, CH2 3.25, m
3.85, m
26.3, CH3 1.45, s
7.25, s
173.5, C
β-Ala
2
36.7, CH2 2.49, m
HyLeu
174.0, C
174.1, C
3
38.0, CH2 3.44, m
3.49, m
2
3
61.3, CH 4.17, m
76.3, CH 3.75, m
60.6, CH 4.19, m
76.9, CH 3.69, dd (4.4,
7.2)
NH
1
7.94, dd (5.0,
7.57, dd (6.0,
12.2)
8.0)
64.0, CH2 3.50, m
3.90, m
4
5
6
NH
1
2
3
4
NH
32.1, CH 1.72, m
32.2, CH 1.73, m
DMPD
17.7, CH3 1.02, d (6.4) 18.7, CH3 1.02, d (6.6)
b
19.9, CH3 0.92
7.88, d (7.0)
19.7, CH3 0.88, d (6.6)
7.91, d (6.4)
2
3
4
5
NH
1
2
3
NH
42.2, CH 4.49, m
18.6, CH3 1.24, d (6.6)
44.8, CH3 2.99, s
44.8, CH3 2.99, s
Aib1
178.1, C
177.9, C
59.4, C
23.4, CH3 1.48
27.2, CH3 1.53
58.1, C
b
b
b
23.7, CH3 1.49, s
27.1, CH3 1.50, s
7.82, s
7.62
Ala
176.5, C
7.97, s
49.6, CH 4.35, m
17.9, CH3 1.37, d (7.2)
8.13, d (6.4)
a
Abbreviations: ΔMHA, 4-methyl-2-hexenoic acid; MePro, 4-methylproline; AHMOD, 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid; HyLeu,
β-hydroxyLeucine; Aib, 2-aminoisobutyric acid; Leu, leucine; β-Ala, β-alanine; DMPD, N1,N1-dimethylpropane-1,2-diamine; Ala, alanine. Chemical
shifts of 1H (500 MHz) and 13C (125 MHz) NMR spectra of 1 were adjusted with solvent signal. Chemical shifts of 1H (600 MHz) and 13C (150
MHz) NMR spectra of 2 were adjusted with solvent signal. Multiplicity was not determined due to overlapping signals.
b
Ala instead of the N1,N1-dimethylpropane-1,2-diamine moiety
of the C-terminus in 1.
The absolute configurations of the constituent amino acids
in 2 were partially determined by an advanced Marfey’s
method. Compound 2 was hydrolyzed with 6 M HCl at 80 °C
for 12 h. The hydrolysate was derivatized with the advanced
Marfey reagents (i.e., the L- and D-forms of Nα-(5-fluoro-2,4-
dinitrophenyl)leucineamide [FDLA]) and analyzed using the
extracted ion chromatograms obtained by liquid chromatog-
raphy−mass spectrometry (LC-MS). The absolute config-
urations of the α-carbons of MePro, HyLeu, and Leu were
determined to be the L-form based on the principle that L-
FDLA derivatives of these L-amino acids are eluted faster than
the D-FDLA derivatives (Figure S13).25,26 In addition, L-
C
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Figure 2. Key correlations of 2 obtained by COSY, HSQC-TOCSY, and HMBC spectroscopies.
Figure 3. HRESIMS/MS fragmentation of 1 and 2. The numbers in black indicate the m/z valued observed in the positive mode. The numbers in
red indicate the m/z values observed in the negative mode.
MePro was revealed to be cis-4-methyl-L-proline by the
presence of a ROESY cross-peak between H-2 and H-4 of
MePro in 2 (Figures S8 and S14). AHMOD is converted to 4-
methyl-6-(2-oxobutyl)-2-piperidinecarboxylic acid (MOPA)
upon hydrolysis (Figure S15).16,27 The LC-MS analysis of
the FDLA-derivatized hydrolysate of 2 revealed a signal for
(2S)-MOPA, indicating the presence of (2S)-AHMOD in 2.
The absolute configurations of these amino acids in 2 are
identical to those in 1 (Figure S12). Regrettably, the absolute
configuration of Ala in 2 could not be determined by the
advanced Marfey’s method, because L-FDLA-L-Ala and the L-
FDLA-β-Ala have the same retention time (tR) in HPLC
analysis (L-FDLA-L-Ala, tR = 8.5 min; L-FDLA-D-Ala, tR = 10.5
min; L-FDLA-β-Ala, tR = 8.5 min). Therefore, the absolute
configuration of Ala in 2 was determined by analyzing
dipeptides obtained via coupling with Boc-L-Leu and Ala in
2. The results showed that the hydrolysate of 2 contained both
D-Ala and L-Ala as well as β-Ala (Figure S16). These results are
most likely an experimental artifact. It is possible that the
racemization of Ala during hydrolysis and/or the racemization
of Ala of the C-terminus in 2 during the purification process
might occur. Therefore, we isolated 2 without using formic
acid as an HPLC solvent additive and further hydrolyzed in 6
M HCl at 38 °C for 48 h. The hydrolysate was coupled with
Boc-L-Leu-OSu and analyzed by LC-MS. However, both D-Ala
and L-Ala were detected in the hydrolysate of 2. Thus, the
absolute configuration of Ala in 2 remains unclear. Taken
together, the structure of 2 was determined to be a new
peptaibiotic as shown in Figure 1.
The aldehyde derivative of 2 is thought to be a putative
precursor in the biosynthesis of 1.28 It seems most likely that 2
is generated by oxidation of the aldehyde derivative of 2.
Therefore, the absolute configuration of 2 is considered to be
identical to that of 1. Indeed, some of the amino acids in 2 are
shown to have the same absolute configuration as those in 1,
leading to the reasonable prediction that all the chiral centers
in 2 exhibit the same absolute configurations as those in 1.
Leucinostatins display a broad range of biological activities
against bacteria, fungi, plants, and cancer cells.23,29 Our
previous study has shown that a 24 h treatment with 1
D
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Figure 4. Effect 1 and 2 on the growth of human pancreatic PANC-1 cells and the central metabolism. (A) Preferential cytotoxicity to PANC-1
cells incubated with compounds in DMEM (+ Glucose, black) or GDM (− Glucose, red) for 24 h. Measurement of the oxygen consumption rate
(B) and the extracellular acidification rate (C) was performed in Seahorse assay medium supplied with 1 g/L glucose. Measurement of the oxygen
consumption rate (D) and the extracellular acidification rate (E) was performed in Seahorse assay medium with/without 1 g/L glucose.
selectively suppresses the growth of human pancreatic cancer
cells preferentially under nutrient-deprived conditions com-
pared with that under nutrient-sufficient conditions.11 In this
study, we revealed that a 24 h treatment with 1 shows
preferential cytotoxicity to human pancreatic cancer PANC-1
cells under glucose-limiting conditions as well as the
mitochondrial ATPase inhibitor oligomycin, whereas 2 does
not show any cytotoxicity in the presence or absence of glucose
(Figure 4A). These effects of 1 and 2 are exhibited not only in
PANC-1 cells but also in human pancreatic cancer BxPC-3,
PSN-1, and PK-8 cells (Figure S17). Meanwhile, a 72 h
treatment with 1 suppressed the growth of various cancer cells
under nutrient-sufficient conditions, while 2 was only slightly
active (Table S1). The antimicrobial activities of 1 and 2 were
E
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Figure 5. Effect of leucinostatin A (1) on glycolysis and oxidative phosphorylation (OXPHOS).
evaluated by the agar dilution method (Table S2); 1 was active
against Candida and Cryptococcus, while 2 showed much lower
activity against the microorganisms tested in this study.
Leucinostatin A (1) inhibits mitochondrial respiration by
inhibition of mitochondrial ATPase and by uncoupling
oxidative phosphorylation.30−33 To investigate the effect of 1
and 2 on the mitochondrial respiration of PANC-1 cells, we
evaluated the oxygen consumption rate as an indicator of
mitochondrial respiration using a Seahorse XFe96 analyzer.
The mitochondrial ATPase inhibitor oligomycin decreases the
oxygen consumption rate in PANC-1 cells (Figure 4B).
Conversely, the oxidative phosphorylation uncoupler p-
triflouromethoxyphenylhydrazone (FCCP) increases the oxy-
gen consumption rate. Treatment with 1 significantly decreases
the oxygen consumption rate like an oligomycin, indicating
that 1 acts as an inhibitor of mitochondrial ATPase in PANC-1
cells. In contrast, treatment with 2 has little effect on the
oxygen consumption rate. Next, we measured the extracellular
acidification rate as an indicator of glycolysis. Predictably, 1,
like oligomycin, increases the extracellular acidification rate,
whereas 2 has little influence on the extracellular acidification
rate (Figure 4C). These results indicate that the N1,N1-
dimethylpropane-1,2-diamine moiety in 1 plays an important
role in the inhibition of mitochondrial ATPase and preferential
cytotoxicity under glucose starvation. We also investigated the
relationship between the inhibition of mitochondrial function
and preferential cytotoxicity under glucose-limiting conditions.
The oxygen consumption rate is decreased by 0.6 μg/mL 1, in
the presence or absence of glucose (Figure 4D). Although 0.6
μg/mL 1 significantly increases the extracellular acidification
rate in the presence of glucose, 0.6 μg/mL 1 barely increases
the extracellular acidification rate in the absence of glucose
(Figure 4E). These data indicate that the suppression of
mitochondrial respiration by 1 under glucose-sufficient
conditions promotes ATP production through glycolysis.
However, 1 does not activate glycolysis under glucose-deprived
conditions. Therefore, glucose-sufficient cells can survive in the
presence of 1, whereas glucose-deprived cells cannot (Figure
5).
isolated from the synnema formed on M. japonensis Scott
collected in Japan and has been identified as the entomopar-
asitic fungus P. lilacinum 40-H-28. We found that P. lilacinum
strain 40-H-28 produces a new leucinostatin named
leucinostatin Y (2), together with leucinostatin A (1). The
1H and 13C NMR have been assigned for the first time.
Leucinostatins belong to a class of peptaibiotics that are
defined as fungal peptides containing α-monosubstituted α-
amino acids such as Aib, and 2 was found to be a peptaibiotic
comprising 10 α-amino acids (including unusual amino acids).
The spectroscopic data for 2 revealed that it has the same
structure as 1 but with the N1,N1-dimethylpropane-1,2-diamine
moiety at the C-terminus substituted with Ala. There are 24
known leucinostatin structures,21 all of which have a 1,2-
diaminopropane moiety at the C-terminus. Compound 2,
which lacks the 1,2-diaminopropane moiety, represents the
first leucinostatin of its type. In addition, 1 shows preferential
cytotoxicity under glucose-limiting conditions and inhibition of
mitochondrial respiration, whereas 2 does not show any
cytotoxicity or influence on mitochondrial function. Thus, the
N1,N1-dimethylpropane-1,2-diamine moiety of 1 plays an
important role in its biological activities. The druggability of
2 is poor because the pharmacological activity is weak.
However, information regarding the structure−activity rela-
tionship of 2 obtained in our study may be quite useful in the
development of new leucinostatin derivatives.
Recently, 1 was found to suppress the growth of prostate
cancer cells in a coculture system with prostate stromal cells
through reduction of insulin-like growth factor-I signaling.34,35
In the tumor microenvironment, tumor cells are exposed to
insufficient nutrient supply and are closely related to normal
cells such as stromal cells. Therefore, leucinostatins may be
promising anticancer agents that target tumor microenviron-
ments.
EXPERIMENTAL SECTION
■
General Experimental Procedures. NMR spectra were acquired
on a ECZ600R (JEOL, Tokyo, Japan) and an AVANCE III 500
(Bruker, Billerica, MA, USA). Chemical shifts are referenced to the
residual solvent peak in the NMR solvent. Infrared (IR) spectra were
recorded on an FT-710 Fourier-transform infrared spectrophotometer
(Horiba, Kyoto, Japan). UV absorption spectra were determined
In conclusion, we have discovered a new metabolite
produced by the fungal strain 40-H-28. This strain was
F
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Article
using a U-3210 spectrophotometer (Hitachi, Tokyo, Japan). Optical
rotation was measured using a 241 polarimeter (PerkinElmer,
Waltham, MA, USA). HRESIMS and MS/MS spectra were recorded
on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer
(Thermo Fisher Scientific, San Jose, CA, USA) and an LTQ Orbitrap
XL hybrid iontrap-Orbitrap mass spectrometer (Thermo Fisher
Scientific), respectively. LC-MS was performed by a Q Exactive
hybrid quadrupole-Orbitrap mass spectrometer with an UltiMate
3000 HPLC system (Thermo Fisher Scientific).
mL of 6 M HCl at 80 °C for 12 h. The reaction mixture was then
evaporated to dryness under reduced pressure, and the resulting
residue was dissolved in 120 μL of water. Subsequently, two vials were
each charged with 50 μL of the hydrolysate and 20 μL of 1 M
NaHCO3. Then, 50 μL of 10 mg/mL D-FDLA (Tokyo Chemical
Industry, Tokyo, Japan) in acetone was added to one of the two vials,
and an equivalent proportion of L-FDLA in acetone was added to the
other vial. The two reaction vials were stirred at 37 °C for 60 min.
Then, 20 μL of 1 M HCl was added to each vial, and the two reaction
mixtures were diluted 100-fold with 50% aqueous CH3CN. A 5 μL
aliquot of each diluent was injected into the LC-MS apparatus. LC-
MS analyses were performed under the following conditions: column,
Capcell Pak C18 UG120 (3 μm, 2.0 × 50 mm, Shiseido); flow rate,
0.3 mL/min; solvent gradient, 20−60% aqueous CH3CN containing
0.1% formic acid, linear gradient; mass spectrometer, positive ion;
detection, protonated molecules, m/z 424.1827 0.0013 for MePro,
m/z 442.1932 0.0013 for HyLeu, m/z 426.1983 0.0013 for Leu,
Isolation and Taxonomic Analysis of Strain 40-H-28. The
fungus strain 40-H-28 was isolated from the synnema formed on M.
japonensis Scott that was collected at Yokohama City, Kanagawa
Prefecture, Japan, on August 3, 2013. The entomoparasitic fungus
with synnemata from an imago of M. japonensis was first reported by
Kobayasi and designated as Isaria macroscyticola (Tutikamemusi-
take).22 The morphological characteristics of the I. macroscyticola are
similar to those of strain 40-H-28. However, strain 40-H-28 has
verticillate conidiophores and chains of ovoid conidia that are
characteristic of P. lilacinum (see graphical abstract, in which the
hyphae and conidia are stained with phenol cotton blue). Ebehard et
al. showed that P. cf. lilacinum infected Edessa rufomarginata
(hemiptera, pentotomidae) and formed synnemata.36 The total
genomic DNA was isolated from the cell body of the strain 40-H-
28 grown on potato dextrose agar (Becton, Dickinson and Company,
Sparks, MD, USA) at 25 °C. The ITS-5.8S gene sequence of strain
40-H-28 was found to be 100% identical to the registered sequences
of P. lilacinum found in the DDBJ/EMBL/GenBank databases. The
nucleotide sequence of the ITS-5.8S rDNA region of strain 40-H-28
was deposited in the DDBJ/EMBL/GenBank databases with the
accession number LC416799. Thus, the isolated strain 40-H-28 was
identified as P. lilacinum based on its morphological and phylogenetic
analysis.
Fermentation and Isolation of Leucinostatins. A slant culture
of P. lilacinum 40-H-28 was inoculated into a 500 mL Erlenmeyer
flask containing seed medium (100 mL) consisting of soybean meal
(2%) and mannitol (3%). The flask was incubated at 25 °C for 4 days
on a rotary shaker operating at 220 rpm. Aliquots (1 mL) of this seed
culture were transferred into 500 mL Erlenmeyer flasks containing
production medium (100 mL) having the same ingredients as the
seed medium. The flasks were cultured at 25 °C for 6 days on a rotary
shaker operating at 220 rpm.
The culture filtrate obtained from the fermented broth (16 L) was
applied to a Diaion HP-20 column (2 L, Mitsubishi Chemical, Tokyo,
Japan). After washing with 50% aqueous acetone, the fractions
containing the active compounds were eluted with acetone. The active
fractions were concentrated in vacuo, applied to a silica gel 60 column
(Merck, Darmstadt, Germany), and eluted with CHCl3−MeOH
(1:1). Then, the active fractions were concentrated under reduced
pressure to yield a brown material. The material was dissolved in
CHCl3−MeOH (1:1), applied to a Sephadex LH-20 column (GE
Healthcare, Little Chalfont, UK), and eluted with CHCl3−MeOH
(1:1). The fractions containing the leucinostatins were further
purified by preparative HPLC [Capcell Pak C18 UG-120 (5 μm 30
× 250 mm, Shiseido, Tokyo, Japan); flow rate, 20 mL/min; solvent
gradient, 5−95% aqueous CH3CN containing 0.1% formic acid, linear
gradient]. 1 and 2 were eluted at 28−30 min and 36−37 min,
respectively. These fractions were concentrated under reduced
pressure to give pure 1 (46.4 mg) and 2 (18.8 mg). Furthermore,
LC-MS analysis revealed that several leucinostatins in addition to 1
and 2 were present in the culture broth of P. lilacium 40-H-28 and the
MeOH extract of mycelia (Figure S18).
and m/z 508.2402
0.0015 for MOPA. Because AHMOD is
converted to MOPA upon hydrolysis, MOPA was detected instead of
AHMOD.
To determine the absolute configurations of Ala, the Boc-L-leucine
hydroxysuccinimide ester (Boc-L-Leu-OSu) was coupled with three
kinds of Ala (D-ALa, L-Ala, and β-Ala) to yield the dipeptides Boc-L-
Leu-D/L/β-Ala, respectively. Briefly, leucinostatins (0.1 mg) were
hydrolyzed with 0.5 mL of 6 M HCl at 38 °C for 48 h and the
reaction mixture was concentrated under reduced pressure to dryness.
Subsequently, to 80 μL of hydrolysate dissolved in 120 μL of water
were added 20 μL of 1 M NaHCO3 and 100 μL of Boc-L-Leu-OSu
(40 mg/mg, 1,4-dioxane). The reaction mixture was then stirred at 25
°C for 12 h, and 20 μL of 1 M HCl was added to neutralize. The
reaction mixture was diluted 200-fold with 50% aqueous CH3CN, and
5 μL of the diluent was injected into the LC-MS. The LC-MS analyses
were performed under the same conditions as those used in the
advanced Marfey’s method. The dipeptides were monitored using
extracted ion chromatograms for the cationized molecule, m/z
325.1734 0.0016 (M + Na)+.
Cell Culture. Human pancreatic cancer PANC-1, BxPC-3, and
PSN1 cells were obtained from the American Type Culture
Collection (Rockville, MD, USA), and PK-8 cells were obtained
from Riken Cell Bank (Ibaraki, Japan). Cells were grown at 37 °C
with 5% CO2 in Dulbecco’s modified Eagle medium (DMEM; Nissui,
Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS;
Cosmo Bio, Tokyo, Japan), 10 000 units/L penicillin G, and 10 mg/
mL streptomycin.
Glucose starvation was achieved by culturing cells in glucose-
deprived medium (GDM). The GDM composition was 265 mg/L
CaCl2·H2O, 400 mg/L KCl, 200 mg/L MgSO4·7H2O, 6400 mg/L
NaCl, 163 mg/L NaH2PO4·2H2O, 0.1 mg/L Fe(NO3)3·9H2O, MEM
vitamin solution (Invitrogen, Carlsbad, CA, USA), MEM amino acids
solution (Invitrogen), 292 mg/L L-glutamine, 42 mg/L L-serine, 30
mg/L L-glycine, 110 mg/L sodium pyruvate, 5 mg/L phenol red,
10 000 units/L penicillin G, 10 mg/L streptomycin, 25 mmol/L
HEPES buffer (pH 7.4), and 10% dialyzed FBS. The final pH was
adjusted to 7.4 with 10% NaHCO3.
Preferential Cytotoxicity in Glucose-Deprived Conditions.
Cells (2.5 × 104 cells/well) in 96-well plates were cultured in DMEM
for 24 h. The cells were then washed with phosphate-buffered saline
(−), and the medium was replaced with either fresh DMEM (+
glucose) or GDM (− glucose). Leucinostatins were then added to the
wells, and the cells were cultured for 24 h. Then, the medium was
replaced with DMEM containing 0.5 mg/mL 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) and
incubated at 37 °C for 4 h. After incubation, cells were supplemented
with 20% sodium dodecyl sulfate and incubated at 37 °C for 12 h.
Absorbance was measured at 570 nm using a spectrophotometer.
Measurement of Oxygen Consumption Rate and Extrac-
ellular Acidification Rate. Cellular mitochondrial function was
measured using a Seahorse XF Cell Mito stress test kit (Agilent
Technologies, Santa Clara, CA, USA). This kit can be used for
measuring key parameters of mitochondrial function (basal
Leucinostatin Y (2): colorless, amorphous solid; mp 107−111 °C;
[α]24D −24.0 (c 1.77, MeOH); UV (MeOH) λmax (log ε) 205 (4.45),
223 (sh 4.27) nm; IR (KBr) νmax 3332, 2960, 1658, 1537, 1441, 1387,
1365, 1293, 1223 cm−1; HRESIMS m/z 1205.7758 (calcd for
C60H105N10O15, 1205.7755).
Determination of Absolute Configurations of Amino Acids.
The absolute configurations of the α-carbons of MePro, AHMOD,
HyLeu, and Leu were determined by the advanced Marfey’s
method.25,26 The leucinostatins (0.1 mg) were hydrolyzed with 0.5
G
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Article
respiration, ATP production, proton leak, maximal respiration, and
nonmitochondrial respiration). The testing was performed in
accordance with the protocol provided in the supplier’s manual.
Briefly, 2 × 104 PANC-1 cells were plated onto XF cell culture plates
in DMEM and incubated at 37 °C with 5% CO2 overnight. Cells were
washed with assay medium (Seahorse XF base medium containing 1
g/L glucose, 1 mM sodium pyruvate, and 2 mM GlutaMax-1
(Thermo Fisher Scientific)) and replaced with assay medium. The
plate was placed at 37 °C in a CO2-free incubator for 30 min and then
loaded into a Seahorse XFe96 analyzer (Agilent Technologies). The
oxygen consumption rate and extracellular acidification rate were
monitored in real-time throughout the assay. Leucinostatins, 1 μM
oligomycin, 1 μM FCCP, and 0.5 μM rotenone plus 0.5 μM
antimycin were sequentially injected into each well. Data points
represent the mean rates of each measurement cycle, which consisted
of 2 min mixing time, followed by 3 min data acquisition (20 or 21
cycles in total).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00839.
■
S
Additional information (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-55-924-0601. Fax: +81-55-922-6888. E-mail:
imomose@bikaken.or.jp.
■
ORCID
(28) Wang, G.; Liu, Z.; Lin, R.; Li, E.; Mao, Z.; Ling, J.; Yang, Y.;
Yin, W. B.; Xie, B. PLoS Pathog. 2016, 12, No. e1005685.
(29) Lucero, A. H.; Ravizzini, A. R.; Vallejos, H. R. FEBS Lett. 1976,
68, 141−144.
Isao Momose: 0000-0002-9867-3881
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was financially supported by JSPS KAKENHI Grant
Number 15K01811. We thank Dr. H. Abe and Dr. T.
Watanabe (BIKAKEN) for providing synthetic leucinostatin A,
Dr. H. Hashizume (BIKAKEN) for determination of absolute
configuration of amino acids, Dr. T. Kudo (PRIMETECH)
and Dr. S. Atsumi (BIKAKEN) for the extracellular flux
analyzer-based analysis, Ms. Y. Shibuya (BIKAKEN) for
evaluation of the antimicrobial activity, and Ms. S. Kakuda
and Ms. K. Hitosugi (BIKAKEN) for technical assistance.
(34) Kawada, M.; Inoue, H.; Ohba, S.; Masuda, T.; Momose, I.;
Ikeda, D. Int. J. Cancer 2009, 126, 810−818.
(35) Abe, H.; Kawada, M.; Sakashita, C.; Watanabe, T.; Shibasaki,
M. Tetrahedron 2018, 74, 5129−5137.
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Christopher, Y.; Gonzalez, C.; Ramos, D.; Urbina, H.; Blackwell, M.
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DOI: 10.1021/acs.jnatprod.8b00839
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