Structural Diversity and Anticancer Activity of Marine-Derived Elastase Inhibitors: Key Features and Mechanisms Mediating the Antimetastatic Effects in Invasive Breast Cancer

Article abstract of DOI:10.1002/cbic.201700627

Three new 3-amino-6-hydroxy-2-piperidone (Ahp)-containing cyclic depsipeptides, named loggerpeptins A–C (1–3), along with molassamide (4), were discovered from a marine cyanobacterium, extending the structural diversity of this prevalent scaffold of cyanobacterial serine protease inhibitors. Molassamide, which contains a 2-amino-butenoic (Abu) unit in the cyclic core, was the most potent and selective analogue against human neutrophil elastase (HNE). Given the growing evidence supporting the role of HNE in breast cancer progression and metastasis, we assessed the cellular effects of compounds 3 and 4 in the context of targeting invasive breast cancer. Both compounds inhibited cleavage of the elastase substrate CD40 in biochemical assays; however, only 4 exhibited significant cellular activity. As CD40 and other receptor proteolytic processing culminates in NFκB activation, we assessed the effects of 4 on the expression of target genes, including ICAM-1. ICAM-1 is also a direct target of elastase and, in our studies, compound 4 attenuated both elastase-induced ICAM-1 gene expression and ICAM-1 proteolytic processing by elastase, revealing a potential dual effect on migration through modulation of gene expression and proteolytic processing. Molassamide also specifically inhibited the elastase-mediated migration of highly invasive triple-negative breast cancer cells.

Full text of DOI:10.1002/cbic.201700627

Accepted Article
Title: Structural Diversity and Anticancer Activity of Marine-Derived
Elastase Inhibitors: Key Features and Mechanisms Mediating the
Antimetastatic Effects in Invasive Breast Cancer
Authors: Fatma H Al-Awadhi, Valerie J Paul, and Hendrik Luesch
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201700627
Link to VoR: http://dx.doi.org/10.1002/cbic.201700627
A Journal of
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10.1002/cbic.201700627
ChemBioChem
ACCEPTED ARTICLE
Structural Diversity and Anticancer Activity of Marine-Derived Elastase Inhibitors:
Key Features and Mechanisms Mediating the Antimetastatic Effects in Invasive
Breast Cancer
[a] [b],[c]
,
[d]
a],[b]
Fatma H. Al-Awadhi
, Valerie J. Paul and Hendrik Luesch*,[
[a]
[b]
Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development (CNPD3),
[c]
University of Florida, 1345 Center Drive, Gainesville, Florida 32610, United States. Department of Pharmaceutical
[d]
Chemistry, Faculty of Pharmacy, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. Smithsonian Marine
Station, Fort Pierce, 701 Seaway Drive, Fort Pierce, Florida 34949, United States.
* Corresponding author. E-mail: luesch@cop.ufl.edu
Abstract
Three
new
3-amino-6-hydroxy-2-piperidone
(Ahp)-containing
cyclic
depsipeptides named loggerpeptins A‒C (1‒3) along with molassamide (4)
were discovered from a marine cyanobacterium, extending the structural
diversity of this prevalent scaffold of cyanobacterial serine protease inhibitors.
Molassamide (4), containing the 2-amino-butenoic (Abu) unit in the cyclic core,
was the most potent and selective analogue against human neutrophil elastase
(HNE). Given the growing evidence supporting the role of HNE in breast cancer
progression and metastasis, we assessed the cellular effects of compounds 3
and 4 in the context of targeting invasive breast cancer. Both compounds
inhibited the cleavage of the elastase substrate CD40 in biochemical assays;
however, only 4 exhibited significant cellular activity. Since CD40 and other
receptor proteolytic processing culminates in NFKB activation, we assessed the
effects on the expression of target genes, including ICAM-1. ICAM-1 is also a
direct target of elastase, and in our studies compound 4 attenuated both
elastase-induced ICAM-1 gene expression and ICAM-1 proteolytic processing
by elastase, revealing a potential dual effect on migration through modulation
of gene expression and proteolytic processing. Molassamide (4) specifically
inhibited the elastase-mediated migration of highly invasive triple negative
breast cancer cells.
Keywords
Breast cancer, elastase, marine cyanobacteria, metastasis, protease inhibitors
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Introduction
Cyanobacterial serine protease inhibitors, composed of 19-membered ring
cyclodepsipeptides bearing the modified glutamic acid residue 3-amino-6-hydroxy-2-
piperidone (Ahp), are among the most predominant secondary metabolites isolated from
marine cyanobacteria.[1,2] More than 100 members of this class have been discovered, all
of which are characterized by the conserved backbone and a variable side chain,
exhibiting inhibitory activities against the proteases elastase, chymotrypsin, and
trypsin.[1,3] Some of these secondary metabolites contain the 2-amino-2-butenoic acid
(Abu) moiety incorporated within the macrocycle, which contributes to enhanced potency
towards elastase inhibition.[4] Lyngbyastatins 4–10[4,5] and symplostatins 5–10[6] are
examples of compounds bearing the Abu residue and considered among the most potent
marine derived elastase inhibitors. The structural basis towards elastase inhibition was
demonstrated in prior studies via SAR and X-ray cocrystallization studies, highlighting the
importance of the Ahp unit and N-Me-Tyr in the cyclic core, as well as a polar functionality
in the pendant side chain for the greatest activity.[6–8]
Human neutrophil elastase (HNE) is a serine protease with broad substrate
specificity, which hydrolyzes elastin and other extracellular matrix proteins such as
fibronectin, type IV collagen, and proteoglycans.[9] HNE has been shown to be implicated
in several diseases such as asthma, chronic obstructive pulmonary disease (COPD), and
cancer.[10,11] In the context of cancer, HNE has been shown to play a role in tumor
progression leading to metastasis and is associated with poor prognosis and relapse in
cancer patients.[12] The prognostic importance of HNE in cancer is attributed to its ability
to degrade the extracellular matrix as well as cleave substrates involved in tumor
promoting pathways such as migration, cell cycle regulation, and inflammation.[13]
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Sivelestat is the only clinically approved elastase inhibitor on the market used in
Japan and South Korea for the treatment of acute lung injury associated with systemic
inflammatory response syndrome;[14–16] however, due to its marginal clinical effects, its
approval has been stalled in Europe and United States.[17] Therefore, developing new
small molecule inhibitors of elastase is of great importance for the therapeutic
management of elastase-implicated pathogenesis.
We previously investigated the cellular effects of Ahp-containing cyanobacterial
cyclodepsipeptides on bronchial epithelial cells for application to treat inflammatory
conditions in respiratory diseases and subsequently also reported a total synthesis
strategy to enable further biological studies and catalyze the development of these
compounds.[6,18] Others have also developed and reported synthetic approaches of
tailored Ahp-cyclodepsipeptide supporting the facts that this scaffold is a promising
starting structure for the generation of customized serine protease inhibitors.[19,20]
However, there are no rigorous studies describing the cellular effects of these compounds
in cancer. Herein, we describe the isolation and structure determination of new members
of this family of serine protease inhibitors and report the evaluation of their inhibitory
effects on downstream cellular substrates of elastase that are implicated in breast cancer
progression.
Results and Discussion
Isolation and structure elucidation
Samples of the cyanobacterium DRTO-73 were collected from Loggerhead Key,
Florida on May 21, 2013. The freeze-dried sample was extracted directly with the non-
polar solvents EtOAc:MeOH (1:1) three times. The extract (17.4 g) was then partitioned
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between hexanes and 80% MeOH in H2O. The 80% MeOH in H2O fraction was
evaporated and further partitioned between n-BuOH and H2O. The n-BuOH fraction (2.1
g) was concentrated in vacuo and later subjected to silica gel chromatography using a
step gradient system starting the elution with CH2Cl2 followed by increasing i-PrOH in
CH2Cl2, and finally with MeOH. The purification of 50% i-PrOH/DCM silica gel fraction by
reversed-phase HPLC resulted in the isolation of several related analogues (1‒4), named
loggerpeptins A‒C (1‒3), in addition to the known compound molassamide (4)[21]
(Scheme) as colorless amorphous solids.
HRESIMS analysis of loggerpeptin A (1) in the positive mode revealed an adduct
ion peak at m/z of 1015.5087 [M + Na]+ suggesting a molecular formula of C50H72N8O13.
The 1H NMR spectrum suggests the presence of a tertiary amide (N-Me) singlet (δH 2.72,
δC 30.69) and six secondary amide NH doublets (δH 7.03, 7.40, 7.7, 7.81, 8.01, 8.37).
The 2D NMR data revealed the presence of valine, N-methyltyrosine, phenylalanine, 3-
amino-6 hydroxy-2-piperidone (Ahp), two threonine units, alanine, and a butanoic acid
(Ba) moiety (Table S1). The sequence of the amino acids was determined with the aid of
ROESY data to be: Ba, Ala, Thr-2, Thr-1, Leu, Ahp, Phe, N-Me-Tyr, Val. The structure
shows similarity in the core structure to the known compound kempopeptin A.[22]
However, it differs in the side chain where Ala and Ba moieties replace the Pro and Ac
units present in kempopeptin A.[22]
HRESIMS of loggerpeptin B (2) in the positive mode exhibited an adduct ion peak
at m/z of 1156.5901 [M + Na]+ suggesting a molecular formula of C57H83N9O15. The
combination of both 1D and 2D NMR data suggested similar core structure to compound
1 with additional Ala and Bu units extended from Thr-2 (Table S2).
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The HRESIMS of loggerpeptin C (3) in the positive mode revealed an adduct ion
peak at m/z of 997.4978 [M + Na]+ suggesting a molecular formula of C50H70N8O12. NMR
analysis revealed the presence of a methine quartet (δH 6.48, δC 129.8) which showed a
COSY correlation to a Me doublet (δH 1.63, δC 12.91). The latter showed an HMBC
correlation to a quaternary sp2 carbon (δC 131.0) and a carbonyl (δC 164.7) and a ROESY
correlation to a broad NH singlet (δH 9.26). Those signals are characteristic of an Abu unit
(Table S3). The NH (δH 9.26) of the Abu unit showed a ROESY correlation to the alpha
proton of alanine. Also, the methine quartet (δH 6.48, δC 129.8) of the Abu showed a
ROESY correlation to the NH (δH 7.05) of Thr-1. Therefore, the Abu unit was found to
replace Thr-2 that was present in compound 1. The sequence of the amino acids was
determined with the aid of ROESY and HMBC data to be: Ba, Ala, Abu, Thr-1, Leu, Ahp,
Phe, N-Me-Tyr, Val.
HRESIMS of compound 4 in the positive mode exhibited an adduct ion peak at m/z
of 985.4618 [M + Na]+ suggesting a molecular formula of C48H66N8O13 consistent with that
of molassamide, a cyanobacterial compound isolated from the cyanobacterium Dichothrix
utahensis.[21] Analysis of 1D and 2D NMR data suggested the presence of an Abu unit,
which replaced the Leu moiety present in the three other structures of loggerpeptins A‒
C (1‒3) (Table S4; Figure 1). This was confirmed by ROESY correlations between the
CH quartet (δH 6.56, δC 132.01) of the Abu and the alpha proton of Ahp (δH 3.83, δC 48.43)
and also the ROESY peak between the Me (δH 1.51, δC 13.08) of Abu and the alpha
proton of Thr-1 (δH 4.60, δC 55.47). This structure resembles lyngbyastatin 7 with respect
to the cyclic part of the molecule but differs in the side chain.[4]
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yr
al
V
-T
e
-M
N
la
A
-2
hr
T
Ba
-1
hr
T
O
O
O
O
HO
O
H
N
N
H
N
N
H
N
N
O
O
H
O
HN
H
O
HO
N
O
HO
Phe
u
e
L
p
h
A
Loggerpeptin A (1)
O
O
O
O
HO
O
H
N
N
H
N
N
H
N
N
O
O
H
O
O
HN
H
O
N
la
A
O
O
HO
HN
O
Ba
Loggerpeptin B (2)
u
b
A
O
O
O
HO
O
H
N
N
H
N
N
N
N
O
O
H
H
O
O
HN
H
O
N
O
HO
Loggerpeptin C (3)
O
O
O
HO
O
H
N
N
H
N
N
H
N
N
O
O
H
O
O
HN
H
O
HO
N
O
bu
A
HO
Molassamide (4)
Scheme. Structures of loggerpeptins A‒C (1‒3) and molassamide (4) isolated from a
marine cyanobacterium.
The absolute configuration of the stereocenters in the four compounds (1‒4) was
established by enantioselective HPLC-MS analysis of the acid hydrolyzates, which
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showed retention times corresponding to L-Thr, L-Ala, L-Val, L-Leu, L-Phe, and N-Me-L-
Tyr. In order to establish the absolute configuration of the Ahp unit, CrO3 oxidation was
carried out which resulted in the liberation of glutamic acid. The reaction product was then
subjected to acid hydrolysis, which was subsequently subjected to enantioselective
HPLC-MS analysis liberating L-Glu, thus establishing the configuration of the Ahp unit as
3S. The relative configuration of the Ahp unit was established based on ROESY
correlations within the Ahp unit and proton-proton coupling constants which were
observed in kempopeptin A and other related Ahp-containing cyanobacterial secondary
metabolites (3S,6R). The geometry of the Abu unit was established based on examination
of the ROESY spectrum, which revealed a correlation between the CH (δH 6.48) of the
Abu and NH of the Thr-1, indicating E configuration.
Compounds 1–4 represent the classical diversity of the compounds with some
notable novelties. Loggerpeptin C (3) is the first compound of this class with an Abu unit
in the side chain, while molassamide (4) with the Abu moiety in the cyclic core closely
resembles the most potent elastase inhibitors, including lyngbyastatin 7.[4]
Inhibition of Serine Proteases In Vitro
The similarity of loggerpeptins A‒C (1‒3) and molassamide (4) in terms of
structure, characterized by the presence of a 19-membered cyclic hexadepsipeptide core
containing the modified glutamic acid residue Ahp, suggested that they would have serine
protease inhibitory activities with selectivity towards chymotrypsin and elastase. The
antiproteolytic activities of 1‒4 were tested against bovine pancreatic chymotrypsin,
porcine pancreatic elastase, and human neutrophil elastase (HNE) (Figure 1). All
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compounds inhibited the enzymes tested with IC50 values in the sub-micromolarrange
(Table 1).
Figure 1. Dose response curves for loggerpeptins A‒C (1‒3), molassamide (4) and
sivelestat against serine proteases: A) Bovine pancreatic chymotrypsin, B) Porcine
pancreatic elastase, and C) Human neutrophil elastase. The dose-response is presented
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as % fold inhibition against solvent control (DMSO). Data is presented as mean ± SD,
n=3.
Compounds 1‒4 exhibited similar IC50 values against bovine chymotrypsin, which
were comparable to the reported IC50 value of kempopeptin A against the same enzyme
(IC50 0.3 µM).[22]
Table 1. IC50 values of loggerpeptins A‒C (1‒3), molassamide (4) and sivelestat against
serine proteases
Bovine Pancreatic Porcine Pancreatic Human Neutrophil
Compound
Chymotrypsin
0.24 ± 0.01 µM
0.22 ± 0.02 µM
0.35 ± 0.02 µM
0.24 ± 0.01 µM
NDa
Elastase
Elastase
Loggerpeptin A (1)
Loggerpeptin B (2)
Loggerpeptin C (3)
Molassamide (4)
Sivelestat
0.24 ± 0.01 µM
0.28 ± 0.01 µM
0.54 ± 0.13 µM
0.05 ± 0.01 µM
3.55 ± 0.14 µM
0.29 ± 0.04 µM
0.89 ± 0.09 µM
0.62 ± 0.38 µM
0.11 ± 0.01 µM
0.06 ± 0.01 µM
aNot determined; Data are presented as the mean ± SD, n=3.
In the porcine pancreatic elastase inhibition assay, loggerpeptins A and B (1 and
2) inhibited elastase with comparable IC50 values, whereas loggerpeptin C (3) was 2-fold
less potent than 1 and 2. Molassamide (4) was the most potent analogue with IC50 value
of 50 nM. The differences in activity highlight the importance of the residues adjacent to
the Ahp unit on its N-terminal side, which confer selectivity towards serine proteases. It
has been shown through previous crystallographic and SAR data that the presence of a
hydrophobic residue (Leu) between Ahp and Thr-1 confers selectivity towards elastase
and chymotrypsin.[3,7] Also it has been reported that the presence of the Abu unit in that
position enhances the potency towards elastase, which was the case for compound 4.[4]
Interestingly compounds 1‒3, and 4 were found to be ~12 fold and ~70 fold more potent
against porcine elastase compared to sivelestat (IC50 3.55 µM), respectively (Table 1).
When the antiproteolytic activities of 1‒4 were evaluated against the disease
relevant human neutrophil elastase (HNE), loggerpeptins B (2) and C (3) were 3 and 2-
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fold less potent than 1, respectively. Also, molassamide (4) was the most potent analogue
(IC50 110 nM). Compared to sivelestat (IC50 0.06 µM), compounds 1‒4 were found to be
~2‒15 fold less potent in inhibiting HNE (Table 1). As the IC50 value for a covalent inhibitor
would vary depending on the pre-incubation time with the enzyme, we also obtained the
progress curve of HNE treated with sivelestat without pre-incubation step (Figure S1).
Regarding the mode of inhibition of the Ahp-containing depsipeptides, prior studies and
X-ray cocrystallization data of another Ahp containing elastase inhibitor, lyngbyastatin 7,
did not show covalent bond formation between the Abu moiety and HNE.[6,20]
In all subsequent biological and molecular studies, we focused on loggerpeptin C
(3) which was isolated in higher yield, compared to loggerpeptins A (1) and B (2), to allow
rigorous biological evaluation, and on molassamide (4) as it was the most potent
analogue.
Molecular Docking
Consistent with prior studies,[7,8,23] the differences in the antiproteolytic activity between
loggerpeptin C (3) and molassamide (4) against porcine and HNE was rationalized by
molecular docking experiments. The docking was carried out using Autodock Vina[24] and
the published crystal structures of porcine (4GVU)[23] and human (3Q76)[25] neutrophil
elastases were used. Both compounds where found to exhibit comparable binding within
the binding pocket of the enzyme (Figures 2A and B). In compounds 3 and 4, the Leu/Abu
and the N-terminal amino acid residues (Thr-1, Thr-2/Abu, and Ala) were found to occupy
the subsites S1 through S4 of elastase, respectively. At the S1 subsite of elastase, the
Leu in 3 and Abu in 4 occupy the oxanion hole of the enzyme. These compounds exploit
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the same binding pattern previously reported in several other elastase-inhibitor
complexes, such as scyptolin, FR901277, and lyngbyastatin 7.[6,8,26] Also, the orientation
of the macrocycle in these compounds was comparable as demonstrated in the overlay
of the docked structures of molassamide (4) and lyngbyastatin 7 (Figure 2C). In the
docked structures of molassamide (4) in both receptors, the Abu moiety at the S1 subsite
was found to be in close proximity to the Ser residues (Ser 203 and Ser 222), potentially
resulting in previously reported CH/π interactions,[6,26] hence, contributing to the
enhanced potency compared to loggerpeptin C (3) bearing Leu moiety at the same
position (Figures 2C and D). Additionally, the Abu moiety was found to be in close
proximity to polar residues including Ser 222, Thr 221, Asp 202, and Gln 200, suggesting
potential H-bonding interactions (Figures 2C and D). This is consistent with the previously
reported co-crystal structure of lyngbyastatin 7 with porcine pancreatic elastase, where
the Abu moiety was found in close distance to Ser 203 contributing to a non-bonded CH/π
interaction.[6] Additionally, several H-bonding interactions were also reported between the
Abu of lyngbyastatin 7 and Gly 201, Ser 222, and indirectly to Thr 44.[6]
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Figure 2. Docked structures of A) loggerpeptin C (3) and B) molassamide (4) into HNE
(PDB: 3Q76). Overlay of docked structures of C) molassamide (4; blue) and lyngbyastatin
7 (yellow) and D) loggerpeptin C (3; pink) and molassamide (4; blue) into porcine
pancreatic elastase (PDB: 4GVU). The residues of the elastase enzyme in close proximity
to the Abu unit are displayed in stick representation (green).
Inhibition of Cleavage of CD40 by Human Neutrophil Elastase In Vitro
CD40 is a 44 KDa membrane protein and a member of tumor necrosis factor (TNF)
receptor superfamily and known to be expressed in breast carcinomas.[27,28] CD40 was
previously identified, in other studies, to be an extracellular substrate of elastase.[29] We
investigated the ability of HNE to cleave CD40 using CD40-IgG chimera (CD40ch)
because it contains the human CD40 N-terminal residues (21 to 193), including the
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predicted elastase cleavage sites, fused to human IgG1 fragment as an elastase
substrate.[29] CD40ch was incubated with or without HNE (molar ratio of elastase to CD40
of 1:10, respectively) at 37 ⁰C for 1.5 h and the resulting proteins were electrophoretically
separated on SDS-PAGE under reducing conditions followed by silver staining. The
incubation with HNE resulted in the cleavage of CD40ch into several fragments compared
to the incubation of CD40ch alone (60 kDa) which remained unprocessed (Figure 3, lanes
1 and 2). A previous study predicted, through in silico structural analysis and modeling of
elastase interactions with CD40, the potential elastase cleavage sites within CD40 to be
at Val 91, Ala 115, and Val 138.[29] Also, this was further confirmed by in vitro CD40
processing assay and subsequent Edman degradation analysis of cleaved products,
which revealed Val 138 to be the primary cleavage site.[29] In support of previous results,
our data revealed higher molecular weight fragments, potentially corresponding to a
sequence in the linker site and within the IgG sequence near the C-terminus, as well as
a lower molecular weight fragment (~14 kDa) closely correlated with the previously
established Val 138 at the N-terminus of the protein.
To assess the effects of representative members of our protease inhibitors on the
elastase-mediated cleavage of CD40, loggerpeptin C (3) and molassamide (4) were pre-
incubated with HNE at different concentrations (0.001
of CD40ch to the reaction.
̶ 100 µM) for 2 h before the addition
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Figure 3. The effects of the inhibitors (3 and 4) on the proteolytic processing of CD40 by
HNE. Incubation of HNE with CD40ch (molar ratio 1:10) in 0.1 M Tris-NaCl buffer, pH 7.5
at 4 °C for 2 h in the presence and absence of different concentrations of the elastase
inhibitors 3 (A) and 4 (B) followed by the addition of CD40ch and incubation for 1.5 h at
37 °C. Fragments were separated on SDS-PAGE under reducing conditions and silver
stained. Note: Intensity differences across panels are attributed to differentially staining
and exposure times.
The presence of the inhibitors (3 and 4) resulted in a complete inhibition of CD40ch
cleavage, where the ~60 kDa band corresponding to the full length was evident, up to 10
and 1 µM, respectively, with 4 being 10 fold more potent (Figures 3A and B). However, at
1‒0.1 µM of 3 and 0.1 µM of 4, HNE resulted in partial cleavage of CD40 into several
fragments; whereas, at lower concentrations, complete cleavage of CD40 was noted
(Figures 3A and B). Our conclusions were based on qualitative assessments of the bands
in the gel, as slight differences in the intensities between the bands due to the staining
and development process might hinder accurate quantitative assessments.
The Effect of Elastase Inhibition on the Cleavage of CD40 in MDA-MB-231
In order to choose a cell line to further investigate the cellular effects of our elastase
inhibitors, we assessed the level of expression of HNE and its extracellular (CD40)
substrate by Western blot analysis of lysates harvested from a panel of breast cancer cell
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lines (Figure S2). Consistent with other studies,[13,30] we did not detect NE protein in any
of the six breast cancer cell lines investigated. Also, the lack of NE mRNA in breast cancer
cells (MCF7, T47D, HER18, MDA-MN-231, MDA-MB-453) was previously confirmed by
RT-PCR.[31] However, immunohistochemical analysis of breast cancer tissues confirmed
the presence of NE in tumor associated neutrophils (TAN) within the tumor
microenvironment.[31] Hence, it has been proposed that within the tumor
microenvironment, TAN are the primary source of NE in breast tumors.[31] On the other
hand, our data showed that CD40 was detected in all breast cancer cell lines tested
(Figure S2). As MDA-MB-231 cell line is known to exhibit the highest level of NE
uptake,[30] and because its substrate CD40 was detected in MDA-MB-231 lysates, we
selected this cell line to further investigate the cellular effects of loggerpeptins C (3; a
representative of the newly isolated inhibitors) and molassamide (4; the most potent
analogue).
We examined the ability of 3 and 4 to inhibit HNE processing of CD40 within a
cellular context using MDA-MB-231 breast cancer cell line. Because we did not detect
HNE protein in MDA-MB-231 lysates, we added purified recombinant HNE (100 nM) to
the culture medium in the presence of different concentrations of compounds 3 and 4.
Following 24 h incubation, cell lysates were harvested and analyzed by Western blot
using anti-CD40 antibody that detects the N-terminal fragment of the protein (Figures 4A
and B). Our data showed a dose-dependent reduction in the level of processed N-terminal
fragment of the CD40 protein in the presence of 4 compared to HNE alone, with strong
inhibition (~70%) at 10 µM and virtually complete inhibition achieved at 50 µM. The results
were further confirmed by densitometric analysis of the bands (Figure 4D). However,
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loggerpeptin C (3) lacked significant activity in this cellular context at 10 µM and only
possessed marginal activity at 50 µM, demonstrating the importance of the Abu moiety
adjacent to the Ahp unit in the cyclic core, consistent with the enzyme assays. To rule out
the potential effect of cell viability on the expression level of CD40, the effects of HNE
and molassamide (4) on the viability of MDA-MB-231 cells were examined. There was no
reduction in cell viability by HNE and 4 (Figure S3).
Figure 4. The effects of compounds 3 and 4 on the cleavage of CD40 by HNE in MDA-
MB-231 cells A, B) MDA-MB-231 cells were seeded in 12-well plates and following 24 h
the medium was replaced with serum free medium containing different concentrations of
3 (A) and 4 (B) or solvent control in the presence of 100 nM HNE. Following 24 h
incubation, lysates were harvested and analyzed by Western blot. C,D) Densitometric
analysis of CD40 and actin bands in panels A and B.
The Effect of Elastase Inhibition on NFKB Target Gene Expression
Transcription factor NFKB is known to play critical roles in the development, progression,
proliferation, and survival of breast cancer cells.[32,33] Prior studies have shown that
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activated CD40 stimulates the recruitment of TNF receptor-associated factors (TRAF1
and TRAF2: positive regulators, TRAF3: negative regulator of NFKB) to the intracellular
domain of CD40.[27,34] The elastase-cleaved CD40 signaling is mediated through the
binding and interactions of TRAF1 and 2 within the intracellular domain of CD40 resulting
in nuclear translocation, upregulation of NFKB transcriptional activity, and increase in the
expression of NFKB target genes. We investigated the effects of HNE on the expression
levels of several NFKB target genes known to be involved in promoting breast cancer cells
migration (ICAM-1, CXCR4, CD44).[29,35–37] MDA-MB-231 cells were treated with 100 nM
HNE for different time points and gene expression was assessed by RT-qPCR. Our data
showed 2-fold increase in the expression levels of the Intracellular Adhesion Molecule-1
(ICAM-1) after 12 and 24 h in response to HNE (Figure S4) and 1.6-fold increase for
CXCR4 after 6 h, while CD44 transcript levels were unaffected in our experiments. ICAM-
1 is a cell surface glycoprotein known to be highly expressed in breast cancer tissues and
involved in the progression of metastasis.[35] We examined, therefore, the ability of the
most potent analogue, molassamide (4), to downregulate elastase-induced ICAM-1
expression in MDA-MB-231 cells (Figure 5A). The results revealed dose-dependent
decrease in the expression levels of ICAM-1 in response to 4, suggesting a potential
involvement of the ligand-activated full-length CD40 in the negative regulation of NFKB
transcription activity and subsequently downregulation of NFKB target gene expression.
In the absence of HNE, no significant change in the expression levels was noted in
response to 4 (Figure 5A).
Although, in this study, the effects of elastase inhibition on NFKB target gene expression
were evaluated using mainly compound 4, we previously assessed and reported the
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effects of sivelestat and other members of the Ahp-containing cyclodepsipeptides with
Abu in the cyclic core (symplostatin 5 and lyngbyastatin 7), using epithelial lung airway
model system.[6,18] The effects in both cellular systems appear to be similar and therefore
our data support potential applications of this class of elastase inhibitors in not only
inflammatory conditions but also in cancer.
Figure 5. The effects of elastase inhibition on ICAM-1gene expression and cleavage. A)
Effects of molassamide (4) on ICAM-1 gene expression in the presence and absence of
100 nM HNE. B) Effects of molassamide (4) on the levels of sICAM-1 in the presence
(grey bars) and absence (white bars) of 100 nM HNE. MDA-MB-231 cells seeded in 6-
well plates, incubated overnight then starved for additional 24 h. The medium was then
replaced with serum free DMEM containing different concentrations of 4 and 100 nM
HNE. After 12 h, RNA was extracted and the medium was collected. The transcript levels
and the levels of sICAM-1 were then assessed by RT-qPCR and ICAM-1 AlphaLisa,
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respectively. The asterisks denote significance of P < 0.05 relative to solvent control co-
treated with HNE using two-tailed unpaired t test (** denotes P ≤ 0.01, *** denotes P ≤
0.001, and **** denotes P ≤ 0.0001).
The Effect of Elastase Inhibition on ICAM-1 Cleavage
Disruption in cell-cell adhesion is known to play a critical role in breast cancer metastasis.
The cell-surface receptor, ICAM-1, is a regulator of cell-cell adhesion and affected by
elastase, being cleaved to generate a soluble form (sICAM-1) that is secreted into the
medium.[38] Prior studies have shown the important role of ICAM-1 in mediating tumor cell
adhesion and metastasis.[35] To determine the effects of HNE on the shedding of ICAM-
1 from the cell surface, the levels of sICAM-1 in culture medium prepared from HNE-
treated MDA-MB-231 cells were quantified by AlphaLisa. A significant increase (3.6-fold)
in the level of sICAM-1 was noted upon treatment with 100 nM HNE for 12 h (Figure 5B).
Next, we investigated the ability of molassamide (4) to attenuate the HNE-induced
shedding of ICAM-1. Co-treatment with (4) for 12 h resulted in a dose-depended reduction
(65% and 35% reduction at 50 and 10 µM, respectively) in the levels of sICAM-1 up to 10
µM (Figure 5B). On the other hand, 4 had no effects on the levels of sICAM-1 in the
absence of HNE (Figure 5B), further validating the direct role of HNE as a sheddase that
proteolytically processes the membrane-bound ICAM-1 to generate and release sICAM-
1 into the medium. The effects of sivelestat and the Ahp-containing elastase inhibitor,
symplostatin 5, on sICAM-1 levels have been previously reported; we had found these
compounds to attenuate the elastase-mediated proteolytic processing of the adhesion
protein ICAM-1 in bronchial epithelial cells.[6] Again, our similar findings in cancer cells
support the potential of this class of elastase inhibitors in the context of cancer.
Morphological Changes of MDA-MB-231 cells Induced by HNE
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Upon treatment with 100 nM HNE, a morphological change of MDA-MB-231 cells was
observed after 2 h when the cells started rounding. This morphological change was not
due to cell death as assessed by MTT. The HNE-induced cell rounding was prevented by
co-treatment with compound 4 up to 10 µM (Figures 6, S5). Also, no morphological
changes were observed upon treatment with 4 only (without HNE) up to 50 µM (Figure
S5). This data further supports the previously reported effects of sivelestat and
symplostatin 5,[6] suggesting a direct involvement of HNE in mediating the morphological
changes, possibly by affecting adhesion molecules, components of the extracellular
matrix, and potentially other unknown factors/mechanisms which remain to be
investigated.
Figure 6. The effects of molassamide (4) on HNE-induced morphological changes in
MDA-MB-231 cells. MDA-MB-231 cells were treated with 4 for 3 h in the presence of 100
nM HNE. Scale bar 200 µm.
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The Effect of Elastase Inhibition on the Migration of MDA-MB-231 cells
Elastase is known to promote tumor cell migration, and its inhibition in breast cancer cells
was shown to limit their migratory properties.[39] We therefore assessed the effects of
HNE on the migration of the highly invasive triple negative breast cancer cell line, MDA-
MB-231. Our data showed a dose-dependent increase in cell migration in response to
HNE, with the highest effect achieved at 100 nM (Figure 7A). The action of 4 on ICAM-1
gene expression as well as its effects as sheddase inhibitor prompted us to investigate
the effects of the elastase inhibitors, loggerpeptin C (3) and molassamide (4), on the
migration of MDA-MB-231 cells. The migration assay was carried out in the presence of
100 nM HNE and different concentrations of loggerpeptin C (3), molassamide (4), or
solvent control. While loggerpeptin C (3) had no effect on migration (Figure 7B),
consistent with the lack of potent cellular effects on CD40 cleavage, molassamide (4) at
10 µM inhibited the migration of MDA-MB-231 cells (Figure 7C). This result is in
agreement with a prior study where downregulation of ICAM-1 was shown to attenuate
the metastatic ability of breast cancer cells,[35] although molassamide (4) has additional
elastase-related effects on shedding and gene expression (see above). Additionally, we
examined the effects of 4 in the absence of HNE. Under these conditions, molassamide
(4) did not exhibit any migration inhibitory activity suggesting that it specifically inhibits the
elastase-induced migration but not basal migration that is mediated by other factors
(Figure 7D).
Although the effects of sivelestat on cancer progression were not evaluated in this study,
its role in restricting cancer cells proliferation and migration were previously reported.[40–
42] One study demonstrated the ability of sivelestat to inhibit the NE-induced migration of
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TE-13 esophageal cancer cell line.[42] Additionally, sivelestat was reported to inhibit the
migration of neutrophils in HUVEC model.[43] The migration inhibitory ability of other NE
inhibitors was also reported.[39,44,45] Epigallocatechin-3-gallate (EGCG) was shown to
reverse HNE-induced migration in A549 cancer cells while caffeic acid phenethyl ester
was reported to inhibit HNE-induced migration of PANC-1.[44,45] In breast cancer, the
effects of elafin, a HNE inhibitor, on cancer growth and progression were reported, thus
further supporting the role of elastase in mediating cancer progression and metastasis.[39]
Since these compounds also have the ability to inhibit bovine chymotrypsin (Table 1), we
assessed the effects on the chymotrypsin-like activity of purified 20S human proteasome.
Compound 4 did not inhibit the chymotrypsin-like activity of proteasomes in vitro
compared to the positive control, epoxomicin (Figure S6), excluding that any of the
biological effects are mediated by the proteasome, consistent with both the lack of effects
on cell viability (Figure S3) and our previous results demonstrating the lack of inhibitory
effects on human chymotrypsin.[6]
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Figure 7. The effects of elastase inhibition on the migration of MDA-MB-231 cells. A) The
effect of HNE on the migration of MDA-MB-231 cells. The cells were incubated with
different concentrations of HNE for 48 h. B, C) The effects of loggerpeptin C (3) and
molassamide (4) on the migration of MDA-MB-231 cells. MDA-MB-231 cells were
incubated for 48 h with 100 nM HNE and different concentrations of 3 (B) and 4 (C). D)
The effect of molassamide (4) on the migration of MDA-MB-231 cells in the absence of
HNE. The effect on migration was compared to the solvent control. The asterisks denote
significance of P < 0.05 relative to solvent control using two-tailed unpaired t test (**
denotes P ≤ 0.01).
Conclusions
Exploring a cyanobacterial sample collected from Loggerhead Key in Florida led
to the discovery of four structurally diverse cyclodepsipeptide protease inhibitors:
loggerpeptins A‒C (1‒3) and molassamide (4), adding to the growing family of Ahp-
containing secondary metabolites.[3–5,22,46] The Abu unit in the cyclic core (as in 4)
appeared to be critical for the elastase selectivity and cellular activity. We have shown
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that 4 inhibited the migration of the highly invasive MDA-MB-231 breast cancer cells,
which could be attributed to multiple on-target effects. Molassamide (4) inhibited the
cleavage of the extracellular substrate of elastase, CD40. We also demonstrated the
effect of elastase inhibition on NFKB target genes, one of which is ICAM-1. Our studies
revealed that 4 attenuated both ICAM-1 cleavage and downregulated elastase-induced
ICAM-1 gene expression. Additionally, molassamide (4) demonstrated the ability to
reverse the morphological changes induced by HNE in MDA-MB-231 cells, warranting
further investigation. Given the established role of HNE in cancer progression,
understanding the molecular mechanisms for elastase inhibition is critical for the design
and development of better inhibitors with improved potency and selectivity profiles, which
might have therapeutic potential in cancers that are characterized by high levels of
elastase in the tumor microenvironment.
Experimental Section
General Experimental Procedures. The optical rotation was measured using a Perkin-
1
Elmer 341 polarimeter. The H and 2D NMR spectra for compound 2 were obtained in
DMSO-d6 using Agilent VNMRS-600 MHz, a 5-mm cold probe spectrometer, and Bruker
600 MHz Avance II Spectrometer (for compounds 1, 3, and 4), using a 5-mm cryogenic
probe for indirect detection (Bruker Cryoprobe TXI). The spectra were referenced using
the residual solvent signal [δH/C 2.5/39.5 (DMSO-d6)]. The HRESIMS data were obtained
in the positive mode using Agilent LC-TOF mass spectrometer equipped with APCI/ESI
multimode ion source detector. LCMS data were obtained using API 3200 (Applied
Biosystems) equipped with an HPLC system (Shimadzu).
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Biological Material. Samples of the cyanobacteriumDRTO-73 were collected from
Loggerhead Key, Florida on May 21, 2013. This filamentous cyanobacterium forms dense
cushion-like colonies and has thin filaments with cells only 2.5-3.5 μm in width and 5-7
μm in length, which corresponds microscopically with Leptolyngbya sp. The genus
Leptolyngbya is polyphyletic and in need of taxonomic revision. This cyanobacterium is
pictured in the taxonomic guide to marine algae by Littler and Littler[39]. Voucher
specimens are retained at the Smithsonian Marine Station at Ft. Pierce.
Extraction and Isolation. The freeze-dried material was subjected to extraction with
EtOAc‒MeOH (1:1). The resulting extract was then partitioned between hexanes and
80% MeOH in H2O. The methanolic phase was evaporated to dryness and further
partitioned between n-BuOH and H2O. The n-BuOH layer was concentrated and
subjected to silica gel chromatography using CH2Cl2 and increasing gradients of i-PrOH
(2%, 4%, 6%, 10%, 20%, 50%), 100% i-PrOH, 50% i-PrOH in MeOH, and finally with
100% MeOH. The fraction that was eluted with 50% i-PrOH‒DCM was then purified by
reversed-phase HPLC [column, Synergi Hydro 4u-RP, 250 × 10.0 mm; flow rate, 2.0
mL/min; PDA detection 200−800 nm] using a linear MeOH−H2O gradient (70−100%
MeOH over 20 min, 100% MeOH for 10 min) to afford compound 4 (6 mg), compound 1
(4 mg), compound 3 (2.6 mg), compound 2 (2.3 mg) at tR 18.8 min, 19.4 min, 20.2 min,
and 21.1 min, respectively. Those fractions were subjected to further purification using
reversed-phase HPLC [column, luna 5u Phenyl-Hexyl, 250 × 10.0 mm; flow rate, 2.0
mL/min; PDA detection 200‒800 nm] using a linear MeOH−H2O gradient (70‒100%
MeOH over 10 min, 100% MeOH for 15 min) to afford compound 4 (3.5 mg), compound
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1 (0.5 mg), compound 3 (1 mg), compound 2 (0.3 mg) at tR 11.4 min, 11.5 min, 12.2 min,
and 12.7 min, respectively.
Loggerpeptin A (1): Colorless amorphous solid; [α]20D ‒24.0 (c 0.03, MeOH);
NMR data, 1H NMR, COSY, TOCSY, HSQC, HMBC, ROESY in DMSO-d6, see Table S1
and Supporting Information; HRESIMS m/z 1015.5087 [M + Na]+ (calcd for
C50H72N8O13Na, 1015.5117).
Loggerpeptin B (2): Colorless amorphous solid; [α]20D ‒47.3 (c 0.03, MeOH);
NMR data, 1H NMR, COSY, TOCSY, HSQC, HMBC, ROESY in DMSO-d6, see Table S2
and Supporting Information; HRESIMS m/z 1156.5901 [M + Na]+ (calcd for
C57H83N9O15Na, 1156.5906).
Loggerpeptin C (3): Colorless amorphous solid; [α]20D ‒29.5 (c 0.06, MeOH);
1
NMR data, H NMR, COSY, HSQC, HMBC, ROESY in DMSO-d6, see Table S3 and
Supporting Information; HRESIMS m/z 997.4978 [M + Na]+ (calcd for C50H70N8O12Na,
997.5011).
Molassamide (4): Colorless amorphous solid; [α]20D ‒18.4 (c 0.24, MeOH); NMR
data, 1H NMR, COSY, HSQC, HMBC, ROESY in DMSO-d6, see Table S4 and Supporting
Information; HRESIMS m/z 985.4618 [M + Na]+ (calcd for C48H66N8O13Na, 985.4647).
This data is identical to that previously reported for molassamide.[21]
Absolute Configuration. A sample of compounds 1‒4 (100 µg each) was hydrolyzed
with 6 N HCl (0.5 mL) at 110 ºC for 24 hr. The hydrolyzate was concentrated to dryness,
reconstituted in 100 µL of H2O, and then analyzed by chiral HPLC [column: Chirobiotic
TAG (250 × 4.6 mm), Supelco; solvent: MeOH/10 mM NH4OAc (40:60, pH 5.51); flow rate
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0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. L-Thr, L-Ala, L-Val, L-
Leu, L-Phe, and N-Me-L-Tyr eluted at tR 7.2, 8.2, 8.1, 9.0, 11.7, and 17.6 min, respectively.
The retention times (tR , min; MRM ion pair, parent→product) of the authentic amino acids
were as follows: L-Thr (7.2; 120→74), L-allo-Thr (7.7), D-Thr (8.5), D-allo-Thr (11.3), L-Ala
(8.2; 90→44), D-Ala (14.2), L-Val (8.1; 118→72), D-Val (14.4), L-Leu (9.0; 132→86), D-Leu
(18.7), L-Phe (11.7; 166→103), D-Phe (17.4), N-Me-L-Tyr (17.6; 196→77), N-Me-D-Tyr
(33.0). The compound-dependent MS parameters were as follows: Thr: DP 30, EP 5, CE
14, CXP 3; Ala: DP 26, EP 3, CE 19, CXP 3; Val: DP 31, EP 4, CE 14, CXP 2; Leu: DP
32, EP 7, CE 17, CXP 3; Phe: DP 31, EP 5, CE 37, CXP 2; N-Me-Tyr: DP 32, EP 3, CE
40, CXP 2. The source and gas-dependent MS parameters were as follows: CUR 50,
CAD medium, IS 5500, TEM 750, GS1 65, GS2 65.
To establish the absolute configuration of the Ahp unit, a sample of compound 4
(300 µg) was dissolved in 300 µL glacial AcOH. The solution was added to 2 mg of CrO3
and kept stirring at room temperature for 5 h. The reaction mixture was then applied onto
C18 SPE cartridge eluting with H2O first then MeOH. The oxidized material eluted with
MeOH was hydrolyzed with 6 N HCl (0.5 mL) at 110 ºC for 24 h. The hydrolyzate was
concentrated to dryness, reconstituted in 100 µL of H2O, and then analyzed by chiral
HPLC [column: Chirobiotic TAG (250 × 4.6 mm), Supelco; solvent: MeOH/10 mM
NH4OAc (40:60, pH 5.51); flow rate 0.5 mL/min; detection by ESIMS in negative ion mode
(MRM scan)]. L-Glu was eluted at tR 6.4 min. The retention times (tR , min; MRM ion pair,
parent→product) of the authentic amino acids were as follows: L-Glu (6.4; 146→102), D-
Glu (7.6). The compound-dependent MS parameters were as follows: DP -20, EP -10,
CE -14, CXP -3. The source and gas-dependent MS parameters were as follows: CUR
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30, CAD medium, IS -4500, TEM 750, GS1 65, GS2 65. The relative configuration was
assigned based on ROESY correlations.
Molecular Docking. The docking experiments were carried out using Autodock Vina.[24]
The published crystal structures used were 4GVU and 3Q76.[23,25] The 3D structures of 3
and 4 were minimized using Chem3D Pro 12.0 software (Cambridge Corporation). The
receptors were prepared, using Pymol and Autodock tools, by removing H2O molecules
and adding polar hydrogens. Autodock tools software was also used to generate receptor
grid. The interactions of the docked structures into the receptor were analyzed using
Pymol.
In Vitro Protease Inhibition Assays. The inhibition of chymotrypsin was assessed using
α-chymotrypsin from bovine pancreas (Sigma, C4129). The assay buffer was 50 mM Tris-
HCl, 100 mM NaCl, and 1 mM CaCl2 (pH 7.8). The assay buffer (39 µL), enzyme solution
(100 µL/ml in assay buffer, 10 µL), and various concentrations (100 µM final
concentration, half log dilutions) of test compounds (1 µL, dissolved in DMSO) were pre-
incubated at room temperature for 30 min before the addition of substrate solution (50 µL
of 1.5 mM N-succinyl-Gly-Gly-Phe-p-nitroanilide in assay buffer). The absorbance was
then measured at 405 nm every 30 s for 30 min using SpectraMax M5 plate reader
(Molecular Devices). The protease inhibitor phenylmethanesulfonyl fluoride (PMSF,
Sigma) was used as a positive control. The enzyme activity, expressed as a percentage,
of each well was calculated based on the initial slope of each progress curve compared
to that of the solvent control.
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The elastase inhibition assay was carried out using porcine pancreatic elastase
(Elastin Products Company, Owensville, MO) which was dissolved in assay buffer [1M
Tris-HCl (pH 8.0)] to give a concentration of 75 µg/mL. In brief, the assay buffer (79 µL),
elastase solution (75 µg/mL in assay buffer, 5 µL), and various concentrations (100 µM
final concentration, half log dilutions) of test compounds (1 µL, dissolved in DMSO) were
pre-incubated at room temperature for 15 min in a 96-well microtiter plate. Following
incubation, 15 µL of substrate solution (2 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide in
assay buffer) was added to each well. The reaction was monitored by measuring the
absorbance at 405 nm every 30 s for 30 min using SpectraMax M5 plate reader
(Molecular Devices). Sivelestat was used as a positive control.
The elastase assay was also carried out using human neutrophil elastase (Elastin
Products Company, Owensville, MO) which was dissolved in assay buffer [0.1 M Tris-
NaCl (pH 7.5)] to give a concentration of 100 µg/mL. In brief, the assay buffer (79 µL),
elastase solution (100 µg/mL in assay buffer, 5 µL), and various concentrations (100 µM
final concentration, half log dilutions) of test compounds (1 µL, dissolved in DMSO) were
pre-incubated at room temperature for 15 min in a 96-well microtiter plate. Following the
incubation period, 15 µL of substrate solution (2 mM N-(OMe-succinyl)-Ala-Ala-Pro-Val-
p-nitroanilide in assay buffer) was added to each well. The reaction was monitored by
measuring the absorbance at 405 nm every 30 s for 30 min using SpectraMax M5 plate
reader (Molecular Devices). Sivelestat was used as a positive control. In both assays, the
enzyme activity, expressed as a percentage, of each well was calculated based on the
initial slope of each progress curve compared to that of the solvent control.
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