Pancreatic Lipase Inhibitory Cyclohexapeptides from the Marine Sponge-Derived Fungus Aspergillus sp. 151304

Article abstract of DOI:10.1021/acs.jnatprod.0c00549

Three new cyclohexapeptides, petrosamides A-C (1-3), were isolated from the sponge-derived fungus Aspergillus sp. 151304. Their structures were elucidated by detailed 1D and 2D spectroscopic analyses, and the absolute configurations of the amino acid residues were determined by the advanced Marfey's method. These peptides displayed significant and dose-dependent pancreatic lipase (PL) inhibitory activities, with IC50 values of 7.6 ± 1.5, 1.8 ± 0.3, and 0.5 ± 0.1 μM, respectively. Further inhibition kinetics analyses showed that compound 3 inhibited PL in a noncompetitive manner, while molecular dynamics simulation revealed that it could bind to PL at the entrance of the catalytic pocket.

Full text of DOI:10.1021/acs.jnatprod.0c00549

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Pancreatic Lipase Inhibitory Cyclohexapeptides from the Marine
Sponge-Derived Fungus Aspergillus sp. 151304
Wei-Zhuo Tang,⊥ Jing-Tang Liu,⊥ Qing Hu, Rong-Jing He, Xiao-Qing Guan, Guang-Bo Ge, Hua Han,
Fan Yang,* and Hou-Wen Lin*
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ABSTRACT: Three new cyclohexapeptides, petrosamides A−C (1−3),
were isolated from the sponge-derived fungus Aspergillus sp. 151304.
Their structures were elucidated by detailed 1D and 2D spectroscopic
analyses, and the absolute configurations of the amino acid residues were
determined by the advanced Marfey’s method. These peptides displayed
significant and dose-dependent pancreatic lipase (PL) inhibitory
activities, with IC50 values of 7.6 1.5, 1.8 0.3, and 0.5 0.1 μM,
respectively. Further inhibition kinetics analyses showed that compound
3 inhibited PL in a noncompetitive manner, while molecular dynamics
simulation revealed that it could bind to PL at the entrance of the
catalytic pocket.
arine-derived fungi have emerged as a promising
reservoir of structurally diverse and biologically active
exploration led to the identification of three new cyclo-
hexapeptides, petrosamides A−C (1−3). Herein, the isolation
and structure elucidation of these compounds and their
pancreatic lipase (PL) inhibitory effects are described.
M
natural products for drug discovery.1−4 Remarkably, cyclic
peptides have attracted a great deal of attention in recent
decades and have been widely used as therapeutic agents due
to their large surface area, exceptional stability, low toxicity,
and easily automated chemical synthesis.5−7 Notably, several
representative members have reached the pharmaceutical
market, such as emodepsin8 and pasireotide,9 or are currently
under clinical evaluation, such RA10149510 and APL-2.11
Structurally, cyclic peptide scaffolds often consist exclusively of
natural amino acids or can be modified by methylation12 or
incorporation of a lipid;13 additionally, some compounds
include nonpeptide motifs.14 The cyclic peptides display a
wide range of biological activities, including cytotoxic,15,16
antimicrobial,17 anti-inflammatory,18 antimitotic,19 anti-infec-
tive,20 Na+/K+-ATPase inhibitory,21 and HIV cytopathic
inhibitory activities.22 Thus, the unique structures and diverse
functions of cyclic peptides render these peptides of
considerable interest to both chemists and biologists.
RESULTS AND DISCUSSION
■
Petrosamide A (1) was isolated as a white, amorphous powder.
The molecular formula of 1, C40H54N6O10, with 17 degrees of
unsaturation, was deduced from a positive HRESIMS ion at
1
m/z 779.3990 [M + H]+. The H NMR spectroscopic and
HSQC data of 1 displayed four deshielded NH proton signals
(δH 7.37, 7.11, 8.68, and 8.48) and six α-H protons (δH 4.16−
4.80), revealing a peptide structure bearing six residues. Two
intense singlets at δH 2.32 and 1.87, each integrating for three
protons, suggested the presence of one N-methyl group and
one acylated methyl group. Additionally, the signals observed
in the 13C NMR spectrum were attributed to six carbonyls (δC
168.8−173.4), six α-amino acid carbon resonances (δC 54.4−
62.0), and one oxygenated sp3 carbon (δC 69.6), thus
suggesting that 1 was a depsipeptide (Table 1). The detailed
analysis of the HSQC, COSY, and HMBC spectra allowed the
construction of six spin systems, consisting of one Pro, one
Tyr, one acetylated Thr, one N-methylated Tyr, and two Val
residues (Val1 and Val2). The position of the acetyl group was
In our previous endeavor to discover unique and biologically
active natural products from marine sources, we isolated and
identified a multitude of cyclic peptides occurring in both
marine sponges, e.g., phakellistatins 15−18 and fuscasins A−D
from Phakellia fusca23,24 and reniochalistatins A−E from
Reniochalina stalagmitis,25 and sponge-derived fungi, such as
oryzamides A−C from Nigrospora oryzae26 and a tetrapeptide
and sclerotiotide L from Aspergillus violaceofuscus.27 Encour-
aged by these findings, the sponge (Petrosia sp.)-derived fungus
Aspergillus sp. 151304 was targeted for the purpose of
continuing to explore these types of intriguing structures on
the basis of their cyclic peptide LC-MS profiles; this
Received: May 18, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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advanced Marfey’s analysis following UPLC-MS comparison
against available commercial standards.29 The results demon-
strated that all the amino residues in petrosamide A (1) were
in the L-configuration (Figure S11).
Petrosamide B (2) was obtained as a pale yellow, amorphous
powder that gave a [M + H]+ ion at m/z 793.4156 in the
HRESIMS, consistent with a molecular formula of
C41H56N6O10, which indicated 17 degrees of unsaturation.
The peptide nature of 2 was confirmed from the presence of
four NH proton signals (δH 6.80, 7.01, 8.39, and 8.70) and the
six α-H protons (δH 4.13−4.68) in the 1H NMR spectrum and
from the six amide/ester-type carbonyl carbons (δC 168.3−
173.5) in combination with six α-methine carbons (δC 54.1−
62.2) in the 13C NMR data. The detailed comparison of the
1D and 2D NMR data between 1 and 2 readily revealed an
overall similarity in the structures, with the exception that the
resonances of the acetyl group in 1 were replaced by the
resonances of a propionyl group in 2. This deduction was
supported by mass spectrometry and the observation of
HMBC correlations of H-2′ and H-3′ to C-1′ as well as a
COSY cross-peak between H-2′ and H-3′ (Figure S22). The
connection of the residues was accomplished by HMBC and
ROESY correlations in the same manner as 1, resulting in the
final sequence of 2 being assigned as cyclo-(Tyr-N-Me-Tyr-
Val2-Pro-Val1-N-Pr-Thr), which was further confirmed by the
corresponding ESIMS/MS fragments (Figure 2 and Figure
S1). The ΔδCβ‑Cγ value of 5.1 ppm of the Pro residue was
indicative of a trans geometry for the proline amide bond in
2.24,25,28 The absolute configurations of the amino acid
residues in 2 were determined as L-configurations (Figure
S21) by using the advanced Marfey’s method.29
Figure 1. Selected 2D NMR correlations of petrosamide A (1) and
petrosamide C (3).
Petrosamide C (3) was obtained as a white, amorphous
powder. The HRESIMS showed a positive protonated
molecule peak at m/z 765.3829 [M + H]+, establishing the
molecular formula of C39H52N6O10 with 17 degrees of
confirmed by HMBC correlations from Thr-H-2′ and Thr-NH
to Thr-C-1′ and by a ROESY correlation between Thr-NH and
Thr-H-2′, whereby the placement of the aminomethyl unit was
established by its HMBC correlation with the α-amino acid
carbon in Tyr. The connection of these amino acid residues
was determined using HMBC and ROESY analysis. HMBC
correlations from Tyr-NH (δH 8.48) to N-Me-Tyr-CO (δC
168.4), from N-Me-Tyr-Hα (δH 4.80) to Val2-CO (δC 171.5),
and from Val2-NH (δH 8.68) to Pro-CO (δC 173.4) provided
the sequence of four amino acids, Tyr-N-Me-Tyr-Val2-Pro.
ROESY correlations from the δ-proton of Pro (δH 3.71) to the
α-proton of Val1 (δH 4.32) and from the NH of Val1 (δH 7.11)
to the α-proton of N-Ac-Thr (δH 4.64) resulted in the final
series of the hexapeptide as Tyr-N-Me-Tyr-Val2-Pro-Val1-N-
Ac-Thr. Given the 17 degrees of unsaturation inherent in 1,
seven were revealed to be amide-type carbonyls. Two phenyl
rings in the Tyr amino moieties accounted for eight more
degrees of unsaturation, while one pyrrolidine ring in the Pro
amino residue possessed another site of unsaturation. The
remaining unsaturation equivalent required the presence of a
cyclic peptide structure. Finally, a key HMBC correlation from
N-Ac-Thr-Hβ (δH 5.11) to Tyr-CO (δC 170.1) established the
connectivity between the two amino residues N-Ac-Thr and
Tyr via an ester, thus completing the cyclohexapeptide
backbone. The above assignments were further supported by
the ESIMS/MS fragment ions at m/z 616, 473, 374, and 277,
corresponding to the successive loss of Tyr, N-Ac-Thr, Val, and
Pro (Figure 2 and Figure S1). A trans geometry for the proline
amide bond in 1 was evident from the ΔδCβ‑Cγ value of 5.0
ppm of the Pro residue.24,25,28 The absolute configurations of
the amino acid moieties in 1 were deciphered using the
1
unsaturation. The H NMR spectrum of 3 contained six α-H
protons at δH 3.47, 4.23, 4.26, 4.41, 4.46, and 4.62, with the
associated six α-methine 13C NMR resonances at δC 62.1, 55.6,
59.9, 53.5, 55.7, and 53.7, respectively, showing that it
exhibited the characteristic profile of a peptide-type structure.
A comparison of its 1D and 2D NMR spectra with those of 1
demonstrated that 3 possessed the same macrocyclic skeleton,
except for the absence of one methyl group substituted in the
NH of the Tyr residue, which was consistent with a 14 mass
unit less than that of 1. The linkages of six amino acid moieties,
consisting of Tyr (×2), N-Ac-Thr, Val (×2), and Pro residues,
were achieved using HMBC and ROESY analyses in a similar
way to those in 1 and were clarified as cyclo-(Tyr2-Tyr1-Val2-
Pro-Val1-N-Ac-Thr). The MS2 fragmentation patterns of 3
(Figure 2 and Figure S1) were in good accordance with the
structure as determined by the above 2D NMR analysis.
Meanwhile, the observation of the ΔδCβ‑Cγ value of the Pro
residue with 4.7 ppm indicated a trans geometry for the proline
amide bond in 3.24,25,28 Interestingly, by using the advanced
Marfey’s method,29 the Thr, Pro, and Tyr amino residues in 3
were determined to be in the L-configuration, whereas the two
Val residues in 3 comprised one L- and one D-configured
residue (Figure S32), providing two possible absolute
configurations for 3 (3a and 3b, Figure 3). The discrimination
between these two possibilities was completed by using
selected ROESY correlations. The ROESY correlations from
the β-proton of Pro to the β-proton of Tyr1 and from the α-
proton of Pro to the H-5 of Tyr1 agreed well with the 3b
B
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1
Table 1. H (600 MHz) and 13C (150 MHz) NMR Data for Petrosamides A−C (1−3) in DMSO-d6
petrosamide A (1)
position δC, type δH (J in Hz)
petrosamide B (2)
position δC, type δH (J in Hz)
petrosamide C (3)
position δC, type δH (J in Hz)
unit
unit
unit
N-Ac-L-
Thr
CO
168.8, C
N-Pr-L-
Thr
CO
169.1, C
N-Ac-L-
Thr
CO
167.7, C
α
54.4, CH 4.64, dd (9.5,
2.8)
69.6, CH 5.11, m
α
54.1, CH 4.64, dd (9.5,
2.6)
69.3, CH 5.10, dd (6.5,
2.6)
α
53.7, CH 4.62, m
68.1, CH 5.08, m
β
β
β
γ
15.8, CH3 1.01, d (6.5)
7.37, brs
170.2, C
γ
15.8, CH3 1.05, d (6.5)
6.80, d (9.5)
173.4, C
γ
14.3, CH3 0.98, d (6.6)
7.31, d (8.2)
168.8, C
NH
1′
NH
1′
NH
1′
2′
22.6, CH3 1.87, s
2′
28.4, CH2 2.08, m; 2.01, m
2′
22.0, CH3 1.94, s
3′
CO
α
9.4, CH3
170.1, C
0.96, t (7.5)
L-Val1
CO
α
170.2, C
L-Val1
L-Val1
CO
α
168.9, C
56.4, CH 4.32, m
29.4, CH 1.88, m
18.7, CH3 0.83, d (6.4)
18.8, CH3 0.82, d (6.4)
7.11, m
173.4, C
58.3, CH 4.47, dd (8.8,
4.3)
56.5, CH 4.27, m
29.1, CH 1.88, m
18.6, CH3 0.86, d (6.6)
18.7, CH3 0.83, d (6.6)
7.01, brs
173.5, C
58.2, CH 4.49, dd (8.8,
4.3)
55.7, CH 4.46, m
29.6, CH 2.06, m
18.5, CH3 0.90, d (6.8)
17.7, CH3 0.90, d (6.8)
7.62, brs
β
β
β
γ
γ
γ
δ
δ
δ
NH
CO
α
NH
CO
α
NH
CO
α
L-Pro
L-Pro
L-Pro
172.3, C
59.9, CH 4.26, m
β
29.1, CH2 2.13, m; 1.66, m
β
29.1, CH2 2.14, m; 1.67, m
β
28.6, CH2 2.11, m;
1.78, m
γ
24.1, CH2 1.76, m
γ
24.0, CH2 1.78, m
γ
23.9, CH2 1.86, m
δ
47.5, CH2 3.71, m; 3.58, m
δ
47.4, CH2 3.77, m; 3.59, m
δ
46.6, CH2 3.61, m;
3.54, m
170.3, C
62.1, CH 3.47, t (7.7)
28.1, CH 2.12, m
18.5, CH3 0.80, d (6.6)
18.8, CH3 0.70, d (6.7)
7.87, d (6.7)
L-Val2
CO
α
171.5, C
L-Val2
CO
α
171.2, C
D-Val2
CO
α
54.7, CH 4.16, m
28.8, CH 1.33, m
18.1, CH3 0.79, d (6.7)
19.9, CH3 0.72, d (6.7)
8.68, brs
54.6, CH 4.13, m
28.9, CH 1.39, m
18.0, CH3 0.80, d (6.7)
19.8, CH3 0.76, d (6.7)
8.70, brs
β
β
β
γ
γ
γ
δ
δ
δ
NH
CO
NH
CO
NH
CO
N-Me-L-
Tyr
168.4, C
N-Me-L-
Tyr
168.3, C
L-Tyr1
170.7, C
α
β
62.0, CH 4.80, t (6.3)
33.3, CH2 3.22, dd (14.3,
7.8)
α
β
62.2, CH 4.68, t (6.9)
33.3, CH2 3.32, m
α
β
53.5, CH 4.41, d (6.6)
36.3, CH2 2.96, m
2.50, dd (14.3,
6.3)
2.49, m
2.80, m
1
128.0, C
1
128.0, C
1
127.1, C
2, 5
3, 6
4
129.8, CH 6.94, d (8.1)
115.1, CH 6.61, d (7.6)
156.1, C
2, 5
3, 6
4
129.7, CH 6.93, d (8.2)
115.0, CH 6.63, d (8.2)
156.1, C
2, 5
3, 6
4
129.6, CH 7.00, d (7.9)
114.7, CH 6.67, d (8.1)
155.7, C
OH
9.23, brs
OH
9.23, brs
OH
NH
CO
α
8.81, brs
7.58, d (9.1)
N-Me 28.6, CH3 2.32, s
CO
α
N-Me 28.5, CH3 2.35, s
CO
α
L-Tyr
170.1, C
L-Tyr
169.8, C
L-Tyr2
169.2, C
55.0, CH 4.31, t (9.6)
36.4, CH2 2.84, d (10.7)
54.8, CH 4.39, m
36.3, CH2 2.90, dd (14.1,
4.3)
55.6, CH 4.23, m
35.6, CH2 2.83, m
β
β
β
2.66, t (12.5)
127.5, C
2.70, dd (14.1,
10.8)
1″
1″
127.4, C
1″
126.4, C
2″, 5″ 129.9, CH 6.86, d (8.0)
3″, 6″ 115.1, CH 6.59, d (7.6)
2″, 5″ 129.8, CH 6.89, d (8.2)
3″, 6″ 115.1, CH 6.62, d (8.2)
2″, 5″ 129.4, CH 7.00, d (7.9)
3″, 6″ 114.7, CH 6.67, d (8.1)
4″
155.9, C
4″
155.9, C
4″
155.5, C
OH
NH
9.26, brs
8.48, d (8.6)
OH
NH
9.23, brs
8.39, d (9.0)
OH
NH
8.87, brs
8.15, brs
conformer, wherein Val1 is L and Val2 is D (Figure 3). In
contrast, these long-range correlations were inconsistent with
the 3a conformer, wherein Val1 is D and Val2 is L. Consistent
with the results, ROESY correlations of the β-proton of Val1
and the OH of Tyr2 to the NH of Val1 were more reasonable in
the 3b arrangement. Therefore, the complete structure of
petrosamide C was concluded to be 3.
Structurally, these three new cycloheptapeptides incorporate
N-acylated-threonine amino residues into their peptide back-
bones in which compounds 1 and 3 both have N-acetylated-
C
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mechanism and inhibitory kinetics of compound 3 was
conducted by Lineweaver−Burk analysis. As illustrated in
Figure 4B, the Lineweaver−Burk plots indicated that 3 could
inhibit the PL-mediated 4-MUO hydrolysis in a non-
competitive inhibition manner, with the Ki value of 2.0 μM.
Subsequently, molecular docking simulations were performed
to mimic the interactions between compound 3 and PL. As
demonstrated in Figure 5, compound 3 could tightly bind to
PL at the entrance of the catalytic pocket, with the calculated
binding affinity of −7.2 kcal/mol. This finding suggested that 3
could affect the ingress of the substrate and the release of the
hydrolytic product by occupying the entrance of the catalytic
pocket of PL. Additionally, a detailed 2D analysis result
showed that 3 could create a strong interaction with PL via
hydrogen bonding and hydrophobic interactions (Figure S34).
Additionally, the cytotoxicities of 1−3 were evaluated
toward four human cancer cell lines, HepG2, MCF-7, HeLa,
and PC-9. These compounds exhibited no cytotoxicities
against the above cancer cell lines with IC50 values greater
than 20 μM, indicating their pharmacological potential with
low chance of toxicity.
EXPERIMENTAL SECTION
■
Figure 2. ESIMS/MS fragment ions (m/z) of petrosamides A−C (1−
3).
General Experimental Procedures. Optical rotations were
measured with a Rudolph Autopol VI polarimeter. UV spectra were
recorded on a Hitachi U-3010 spectrophotometer. NMR spectra were
collected on an Agilent 600 MHz NMR instrument, and chemical
shifts were referenced to the solvent peaks of DMSO-d6 (δH 2.50/δC
39.5). The HRESIMS and ESIMS/MS spectroscopic data were
acquired with a Waters Xevo G2-XS QTOF system equipped with a
Waters Xevo G2-XS QTOF spectrometer and a C18 Acquity UPLC
HSST 3 column (Waters, 2.1 × 100 mm, 1.8 μm). Column
chromatography (CC) was performed on silica gel 60 (200−300
mesh, Qingdao). The compounds were purified by a Waters 1525
separation module connected to a 2998 photodiode array detector
using a Waters XBridge C18 column (Waters, 10 × 250 mm, 5 μm).
TLC was conducted on silica gel HSGF254 plates (Qingdao). 1-
Fluoro-2,4-dinitrophenyl-5-L-/D-leucinamide (L-/D-FDLA) reagent
for Marfey’s method and all the amino acid standards were purchased
from Sigma-Aldrich Chemical Corporation.
threonine residues, whereas compound 2 possesses an N-
propionyl threonine unit. The PL inhibitory activities of
compounds 1−3 were evaluated. As shown in Figure 4A, 1, 2,
and 3 inhibited PL-mediated 4-methylumbelliferyl oleate (4-
MUO) hydrolysis in a dose-dependent manner, with IC50
values of 7.6 1.5, 1.8 0.3, and 0.5 0.1 μM, respectively.
These results revealed that the propionylation of the Thr unit
might be favorable for the biological properties, as indicated by
the comparison of the inhibitory activities of 1 and 2. The
inhibition potency of compound 3 was more potent than that
of a known, naturally occurring PL inhibitor, isoginkgetin.30
Encouragingly, further characterization of the inhibitory
Figure 3. Selected ROESY correlations of two possible absolute configurations of petrosamide C (3): (3a) Val1 as D and Val2 as L; (3b) Val1 as L
and Val2 as D.
D
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Figure 4. Inhibitory activities of petrosamides A−C (1−3) against PL. (A) Dose−inhibition curves of 1, 2, and 3 against PL-mediated 4-MUO
hydrolysis. (B) Left: Lineweaver−Burk plots of 3 against PL-mediated 4-MUO hydrolysis. Right: Corresponding second plot of PL inhibition by 3.
The second plot was drawn using the slope of the lines from the Lineweaver−Burk plot versus the concentration of 3. All the data are shown as
mean SD, n = 3.
10−80%, v/v) to give nine fractions (F1−F9). The third fraction (F3,
330 mg) was purified by silica gel CC eluted with CH2Cl2−MeOH
(30:1) to obtain four fractions (F3.1−F3.4). F3.3 (92 mg) was
purified by semipreparative reversed-phase HPLC with MeOH−H2O
(45:55, v/v) to yield petrosamide A (1, 2.2 mg, tR = 25.6 min, 2.0
mL/min), while F3.4 was purified by semipreparative reversed-phase
HPLC with MeOH−H2O (50:50, v/v) to yield petrosamide C (3, 2.1
mg, tR = 20.1 min, 2.0 mL/min).
Petrosamide A (1): white, amorphous powder; [α]2D5 −88 (c 0.5,
1
MeOH); UV (MeOH) λmax (log ε) 201 (4.69), 277 (3.61) nm; H
and 13C NMR data (DMSO-d6), Table 1; ESIMS/MS data, Figures 2
and S1; HRESIMS m/z 779.3990 [M + H]+ (calcd for C40H55N6O10,
Figure 5. Molecular docking results of 3 in PL. (A) 3D structure of
PL docked with 3. (B) Detailed binding mode of 3 with the residues
surrounding the catalytic pocket of PL.
779.3980).
Petrosamide B (2): pale yellow, amorphous powder; [α]2D5 −77 (c
0.5, MeOH); UV (MeOH) λmax (log ε) 202 (4.54), 275 (3.65) nm;
1H and 13C NMR data (DMSO-d6), Table 1; ESIMS/MS data,
Figures 2 and S1; HRESIMS m/z 793.4156 [M + H]+ (calcd for
C41H57N6O10, 793.4136).
Fungal Material. The strain Aspergillus sp. 151304 (GenBank No.
SUB7318612) was isolated from the inner tissue of a Petrosia sp.
marine sponge collected from Yongxing Island, China. The sample
was deposited at the Research Center for Marine Drugs, Renji
Hospital, Shanghai Jiao Tong University, Shanghai, China.
Petrosamide C (3): white, amorphous powder; [α]2D5 +24 (c 0.5,
MeOH); UV (MeOH) λmax (log ε) 204 (5.04), 223 (4.86), 277
(4.14) nm; 1H and 13C NMR data (DMSO-d6), Table 1; ESIMS/MS
data, Figures 2 and S1; HRESIMS m/z 765.3829 [M + H]+ (calcd for
C39H53N6O10, 765.3823).
Culture, Extraction, and Isolation. The fungus was initially
cultivated on potato dextrose agar (PDA) plates for 7 days at 28 °C.
Subsequently, the spores were directly inoculated into 250 mL
Erlenmeyer flasks containing 100 mL of seed medium (potato 5 g,
peptone 10 g, dextrose 15 g, and artificial seawater salts31 5 g in
purified water) and were grown on a rotary shaker (180 rpm) for 72 h
at 28 °C. The amplified fermentation was carried out in 50 × 2-L
Erlenmeyer flasks. Each flask contained of 80 g of rice, 0.5 g of
peptone, and 120 mL of distilled H2O, and 20 mL of seed culture was
transferred and incubated under static conditions at room temper-
ature for 40 days. The fermented substrate was repeatedly extracted
with EtOAc (3 × 10 L) to afford 15.0 g of organic extract after
removal of the organic solvent by evaporating under reduced pressure.
The extract was subjected to silica gel CC eluted with a gradient
CH2Cl2−MeOH system (100:1−0:1) to give seven fractions, A−G.
Fraction E (1.2 g) was further separated by CC over Sephadex LH-20
using MeOH as the eluent, affording three subfractions, E1−E3.
Subsequently, E1 (560 mg) was fractionated into nine major fractions
(E1.1−E1.9) through reversed-phased ODS CC (CH3CN−H2O, 10−
70%, v/v) separation. E1.3 (50 mg) was purified by semipreparative
reversed-phase HPLC with CH3CN−H2O (30:70, v/v) to provide
petrosamide B (2, 1.6 mg, tR = 28.2 min, 2.0 mL/min). Fraction F
(2.3 g) was subjected to reversed-phase ODS CC (CH3CN−H2O,
Absolute Configuration Assignments of Petrosamides A−C
(1−3). Compounds 1−3 (1 mg each) were hydrolyzed in 6 N HCl (1
mL) at 110 °C for 18 h. The reaction solutions were evaporated to
dryness, and the acid hydrolysates (suspended in 100 μL of H2O)
were supplemented with 40 μL of 1 M NaHCO3 following treatment
with 100 μL of L-FDLA (10 mg/mL in acetone) solution. The
mixture was stirred at 45 °C for 90 min, and the reaction was
quenched with the addition of 40 μL of 1 N HCl. Then, the residue
was dissolved in MeOH for subsequent analysis. Authentic amino acid
standards were treated with L-FDLA and D-FDLA as described above,
yielding the L-DLA and D-DLA standards. Marfey’s derivatives of 1−3
were subjected to UPLC-MS using a Waters HSS T3 column (2.1 ×
100 mm, 1.8 μm, 0.4 mL/min), and their retention times were
compared against those of the standards. The retention times of the
corresponding L-DLA-derivatized amino acids were 14.22 min (L-
Pro), 15.96 min (L-Val), 15.03 min (L-Tyr), 12.06 min (L-N-Me-Tyr),
and 11.95 min (L-Thr), whereas the retention times of the D-DLA-
derivatized amino acids were 15.82 min (D-Pro), 18.18 min (D-Val),
16.34 min (D-Tyr), 12.44 min (D-N-Me-Tyr), and 14.76 min (D-Thr).
Conformationally optimized stereostructures of two possible
configurations of petrosamide C (3) were analyzed using molecular
E
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modeling and selected ROESY experiment data. The lowest energy
conformers for 3a and 3b were obtained by ChemBioOffice software
(version 14.0), followed by an MM2 calculation to minimize the
conformation energy. Subsequently, the conformers were reoptimized
using density functional theory (DFT) calculations at the B3LYP/6-
31G(d,p) level in the gas phase by the Gaussian 09 program (Figure
S33).
China; orcid.org/0000-0003-2583-8603; Email: yang-
fan@sjtu.edu.cn
Hou-Wen Lin − Research Center for Marine Drugs, State Key
Laboratory of Oncogenes and Related Genes, Department of
Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiao
Tong University, Shanghai 200127, People’s Republic of
China; orcid.org/0000-0002-7097-0876;
Email: franklin67@126.com
Lipase Inhibition Assay. The lipase inhibitory activity of all the
compounds was determined using a previously described method.32
PL (type II, from porcine pancreas) and 4-MUO served as the
reaction enzyme and fluorogenic substrate, respectively. In brief, the
incubation mixture (200 μL) contained 2 μL of lipase (10 μg/mL),
194 μL of McIlvane buffer (0.1 M), 2 μL of DMSO or 2 μL of stock
solution of each test compound (10 μM). After the reaction mixtures
were incubated for 3 min at 37 °C, the hydrolytic reaction was
initiated by adding 4-MUO (10 μM). The final concentration of
DMSO was 2%. A multimode microplate reader (SpectraMax iD3,
Molecular Devices) was used to monitor the florescence signal of the
hydrolytic product (4-MU) at intervals of 1 min for a 40 min
incubation at an excitation wavelength of 340 nm and an emission
wavelength of 460 nm (gain = 500). The residual activities of PL were
determined as follows: the residual activity (%) = (the fluorescence
intensity of 4-MU in the presence of inhibitor)/the fluorescence
intensity of 4-MU in negative control (DMSO only) × 100%. All the
measurements were conducted in triplicate. Isoginkgetin and orlistat
(a synthetic marketed PL inhibitor) were used as the positive
controls, with IC50 values of 2.90 μM and 0.75 nM, respectively.30
Inhibition Kinetics Analyses. The inhibition kinetics of
compound 3 against PL were carefully investigated using a panel of
inhibition kinetic assays. The inhibition constant (Ki) was determined
by utilizing a panel of hydrolytic rates at various substrate
concentrations in the presence of different inhibitor concentrations.
The details for determination of the inhibition mode and the Ki value
were similar to previously reported methods.33
Molecular Docking Simulation. Molecular docking simulation
was conducted by AutoDock Vina (version 1.1.2).34 The crystal
structure of PL (PDB ID: 1ETH) was retrieved from the Protein Data
Bank (http://www.rcsb.org/pdb). The structure of 3 was generated in
ChemBioOffice (version 14.0), followed by an MM2 calculation to
minimize the conformation energy. Before the docking calculation,
crystal water was removed. The hydrogens were added to the
structure of PL in the hydrogen module, and Kollman united partial
charges were assigned. The size of the grid box was 80 Å × 80 Å × 80
Å, with the spacing of 0.375 Å, which encompassed the active pocket
of the pancreatic lipase structure. A Lamarckian genetic algorithm was
applied as a default search function. The docking pose that had the
lowest binding energy was represented as the most favorable binding
conformation.
Authors
Wei-Zhuo Tang − College of Biological and Environmental
Engineering, Changsha University, Changsha 410022, People’s
Republic of China; Research Center for Marine Drugs, State Key
Laboratory of Oncogenes and Related Genes, Department of
Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiao
Tong University, Shanghai 200127, People’s Republic of
China; orcid.org/0000-0003-4542-9460
Jing-Tang Liu − Research Center for Marine Drugs, State Key
Laboratory of Oncogenes and Related Genes, Department of
Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiao
Tong University, Shanghai 200127, People’s Republic of China
Qing Hu − Institute of Interdisciplinary Integrative Medicine
Research, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, People’s Republic of China
Rong-Jing He − Institute of Interdisciplinary Integrative
Medicine Research, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, People’s Republic of China
Xiao-Qing Guan − Institute of Interdisciplinary Integrative
Medicine Research, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, People’s Republic of China
Guang-Bo Ge − Institute of Interdisciplinary Integrative
Medicine Research, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, People’s Republic of China;
orcid.org/0000-0002-9670-4349
Hua Han − School of Medicine, Tongji University, Shanghai
200092, People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00549
Author Contributions
⊥W. Z. Tang and J. T. Liu contributed equally to this work.
Notes
The authors declare no competing financial interest.
Cytotoxicity Assay. The cytotoxic activities of compounds 1−3
against the four human cancer cell lines were tested as illustrated
formerly.35 Paclitaxel was used as the positive control.
ACKNOWLEDGMENTS
■
This research was funded by the National Key Research and
Development Program of China (2018YFC0310900,
2017YFC1700200), the National Natural Science Foundation
of China (81922070, 81973286, 81973393), the Shanghai
Rising-Star Program (19QA1405300), Shanghai Municipal
Health Commission Excellent Young Talent Program (No.
2018YQ28), Program of Shanghai Academic/Technology
Research Leader [18XD1403600], and Innovative Research
Team of High-Level Local Universities in Shanghai (SSMU-
ZLCX20180702).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00549.
■
sı
1D/2D NMR, HRESIMS, and UV spectra of com-
pounds 1−3; HRESIMS/MS chromatograms of the
advanced Marfey’s analysis for 1−3; docking result for 3
(PDF)
REFERENCES
■
AUTHOR INFORMATION
Corresponding Authors
Fan Yang − Research Center for Marine Drugs, State Key
Laboratory of Oncogenes and Related Genes, Department of
Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiao
Tong University, Shanghai 200127, People’s Republic of
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