Discovery of Amantamide, a Selective CXCR7 Agonist from Marine Cyanobacteria

Article abstract of DOI:10.1021/acs.orglett.9b00163

CXCR7 plays an emerging role in several physiological processes. A linear peptide, amantamide (1), was isolated from marine cyanobacteria, and the structure was determined by NMR and mass spectrometry. The total synthesis was achieved by solid-phase method. After screening two biological target libraries, 1 was identified as a selective CXCR7 agonist. The selective activation of CXCR7 by 1 could provide the basis for developing CXCR7-targeted therapeutics and deciphering the role of CXCR7 in different diseases.

Full text of DOI:10.1021/acs.orglett.9b00163

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Discovery of Amantamide, a Selective CXCR7 Agonist from Marine
Cyanobacteria
Xiao Liang,† Danmeng Luo,† Jia-Lei Yan,‡ Mohammad A. Rezaei,†,§ Lilibeth A. Salvador-Reyes,∥
Sarath P. Gunasekera,⊥ Chenglong Li,† Tao Ye,‡,# Valerie J. Paul,⊥ and Hendrik Luesch*,†
†Department of Medicinal Chemistry and Center for Natural Products, Drug Discovery and Development (CNPD3), University of
Florida, Gainesville, Florida 32610, United States
‡State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate
School, Xili, Nanshan District, Shenzhen 518055, China
§Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States
∥Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines
⊥Smithsonian Marine Station, Fort Pierce, Florida 34949, United States
#QianYan Pharmatech Limited, Shenzhen, 518172, China
S
* Supporting Information
ABSTRACT: CXCR7 plays an emerging role in several physio-
logical processes. A linear peptide, amantamide (1), was isolated
from marine cyanobacteria, and the structure was determined by
NMR and mass spectrometry. The total synthesis was achieved by
solid-phase method. After screening two biological target libraries,
1 was identified as a selective CXCR7 agonist. The selective
activation of CXCR7 by 1 could provide the basis for developing
CXCR7-targeted therapeutics and deciphering the role of CXCR7
in different diseases.
cyanobacteria, which might assist in illuminating the biological
he G protein-coupled receptors (GPCRs) are the largest
Tfamily of membrane receptors and represent the most role of CXCR7 and also serve as a template for the
development of therapeutic agents targeting CXCR7.
Marine cyanobacteria have been a valuable source for the
discovery of structurally diverse natural products as a result of
their prolific biosynthetic machinery. The diversity of the
secondary metabolites from marine cyanobacteria translates
into various biological activities.8 One important group of
these metabolites is ribosomal or nonribosomally synthesized
peptides or depsipeptides, which can function as GPCR
modulators and/or inhibitors of different proteases. For
instance, a group of modified linear peptides, brintonamides
A and D (Figure 1), were reported to be dual protease and
GPCR modulators.9 Now, we have isolated a linear peptide
amantamide (1, Figure 1) from two marine cyanobacterial
samples, which was then identified as a selective CXCR7
agonist.
commonly used signal transduction system in many different
species.1 GPCRs are the most intensively studied targets for
therapeutic agents since they are involved in diverse
pharmacological activities and they possess easily accessed
druggable sites at the cell surface.2,3 More than 100 GPCR-
targeted drugs have been approved by the Food and Drug
Administration (FDA), and even more agents are currently in
clinical trials.4 Therefore, as potential drug targets, GPCRs
present a promising biological matrix for the discovery of
therapeutics against various diseases. In addition to the
established role of GPCRs in inflammatory processes,
GPCRs are reported to function during tumor growth and
metastasis.5 For example, C-X-C chemokine receptor type 7
(CXCR7) is demonstrated to be a biological target in cancer
progression. CXCR7 promotes cell growth and metastasis in
various cancers, including lung, hepatocellular, colorectal, and
breast cancers.6 Moreover, CXCR7 expression is enhanced
during pathological inflammation and tumor development, and
emerging data suggest this receptor is an attractive therapeutic
target for autoimmune diseases and cancer.7 Herein, we
describe the isolation, structure elucidation, total synthesis,
preliminary biological investigation, and target elucidation
study of the first selective CXCR7 agonist from marine
One gray filamentous cyanobacteria sample (Oscilliator-
iales) was collected from Two Lover’s Point (Puntan dos
Amantes), Guam, and two natural products, amantelides A and
B, were previously isolated from that sample.10 Although
molecular methods are required for taxonomic character-
Received: January 14, 2019
© XXXX American Chemical Society
A
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Figure 2. ESI-MS/MS fragmentation pattern of amantamide (1).
acid standards. Of note, since 1 is a methyl ester and we used
MeOH during the isolation, we cannot fully exclude the
possibility that the corresponding carboxylic acid was esterified
during that process; however, the whole isolation was
conducted under neutral conditions where the esterification
is unlikely to have occurred.
Due to the limited amount of 1 obtained from isolation, the
total synthesis of 1 was developed to provide sufficient material
for subsequent biological studies. The synthesis of this
nonapeptide was accomplished by use of the standard Fmoc-
based solid-phase synthetic method (Scheme 1). Elongation of
the peptide chain was accomplished by stepwise coupling of
the required amino acids using O-(7-azabenzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU).
After coupling with butyric acid, the peptide was cleaved
from the resin mediated by TFA followed by esterification of
the resulting acid with tetramethylsilyldiazomethane to afford 1
Figure 1. Structure of amantamide (1) and related natural products.
ization, on the basis of microscopy this cyanobacterium may
fall within the genus Okeania.11 The nonpolar extract (EtOAc/
MeOH 1:1) was partitioned and then fractionated by silica
column chromatography, followed by reversed-phase HPLC to
[45% overall yield, white solid, [α]20 −9.0 (c 0.1, MeOH)].
D
Comparison of the 1H and 13C NMR spectrum of the synthetic
compound with that of the isolated natural product confirmed
the correct assignment of the structure (Figures S8 and S9).
Marine cyanobacteria are known to produce various
protease inhibitors. For example, tasiamide B was reported to
display selective inhibitory activity against BACE1, a potential
therapeutic target for Alzheimer’s disease,12 while tasiamide F
is a potent inhibitor of cathepsins D and E with greater
selectivity over BACE1.8 Considering the similarity of 1 with
these reported tasiamides as well as brintonamides (Figure 1)
and to probe its activity and selectivity, 1 was profiled against a
panel of 63 proteases in a dose−response format starting at 10
μM. As a result, 1 is identified to display modest inhibitory
activity against caspase 9 with IC50 value of 1.95 μM. For the
other proteases included in the screen, 1 either displayed little
inhibitory effect or exhibited IC50 values over 10 μM. The
screening results are shown in Figure S10.
yield amantamide (1) [white solid, [α]20 −9.0 (c 0.1,
D
MeOH)]}. Amantamide (1) was also isolated and purified
from another cyanobacterial sample, a similar gray filamentous
mat, collected from Anae Island, Guam, after an additional
hydrolysis−re-esterification process to get rid of a coeluting
impurity (Scheme S1). The HRESIMS of 1 in the positive
mode exhibited a [M + Na]+ peak at m/z 1102.6918,
suggesting a molecular formula of C57H93N9O11 with 16
degrees of unsaturation. The structure of 1 was determined
1
using a combination of 1D and 2D NMR techniques. The H
and 13C NMR spectra (Figures S1 and S2) indicated the
presence of several characteristic signals corresponding to α-
protons (∼δH 4−5 ppm), exchangeable amide protons (∼δH
7−8 ppm), three N-methyl groups (∼δH 2.9−3.1 ppm, ∼δC
29−31 ppm), and one O-methyl group (δH 3.71 ppm, δC 52.3
ppm), suggesting a peptidic structure. Examination of the 2D
NMR spectra (COSY, TOCSY, HSQC, HMBC, and NOESY)
of 1 in CDCl3 (Figures S3−7) revealed the presence of
proteinogenic amino acids Gly, Ile, Pro, Leu, and Val as well as
O-CH3-Pro, N-CH3-D-Phe, N-CH3-D-Val, N-CH3-L-Leu, and a
butyric acid (BA) residue. The sequence of these units was
confirmed by ESI-MS/MS fragmentation (Figure 2). To
establish the absolute configuration, a small portion of 1
(100 μg) was hydrolyzed using 6 N HCl (110 °C, 24 h), and
the hydrolysate was analyzed using chiral HPLC−MS. The
resulting residues revealed retention times corresponding to L-
Pro, N-CH3-D-Phe, Gly, L-Ile, N-CH3-D-Val, L-Pro, L-Leu, N-
CH3-L-Leu, and L-Val by comparison with authentic amino
As previously mentioned, we have recently shown that
marine cyanobacteria also produce peptidic GPCR modulators.
Thus, 1 was also profiled against a panel of 168 GPCR targets
in both agonist and antagonist mode at 10 μM final
concentration using PathHunter β-arrestin assays. The screen
identified CXCR7 to be the only GPCR target of 1 in agonist
mode, while 1 had no antagonistic effects on any of these
targets (Figures 3 and S11). Follow-up dose−response tests
indicate the EC50 of 1 against CXCR7 is 2.5 μM (Figure 3).
Recently, it has been reported that CXCR7 in human glioma
cells functions as a regulator to mediate cellular antiapoptotic
responses and exacerbate drug-induced apoptosis.13 CXCR7
activation is also reported to stimulate angiogenesis in vitro
and in vivo14 and promote alveolar repair and reduce fibrosis
B
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Scheme 1. Total Synthetic Route of Amantamide (1)
Figure 3. GPCR profiling of amantamide (1) using cell-based
PathHunter β-arrestin assays under agonist mode and antagonist
mode at 10 μM final concentration. Compound % activation or
inhibition is calculated relative to basal and max activity values
obtained through treatment of corresponding target ligand (100% at
EC80). (A) Profiling results shown in scatter plot. The hit identified
with >50% activation or inhibition is labeled. (B) Dose−response
curve of 1 against CXCR7 (EC50 2.5 μM). CXCL12, a CXCR7 ligand,
was used as positive control (EC50 0.017 μM). Data are presented as
mean SD (n = 2).
Figure 4. (A) Effects of amantamide (1) as well as CXCL12 (100 ng/
mL) and TC14012 (30 μM) as positive control on Erk1/2
phosphorylation (pErk1/2) are shown in Western blots, with total
Erk1/2 (tErk1/2) and GAPDH as loading controls. The correspond-
ing quantitative analysis of pErk1/2 expression is also shown. (B)
Transcript expression of VEGFA in A549 cells after 3 h treatment with
1 as well as positive controls CXCL12 (100 ng/mL) and TC14012
(30 μM). GAPDH was used as endogenous control. Data are
presented as mean SD, *P < 0.05, **P < 0.01, ****P < 0.0001,
compared to control using two-way ANOVA (n = 3).
after repetitive lung injury.15 Additionally, CXCR7 is
demonstrated to be a novel target for therapeutic vasculo-
genesis in patients with coronary artery disease and can
influence VEGFA expression in endothelial outgrowth cells,16
which altogether demonstrates the potential of CXCR7 as a
promising therapeutic target for drug discovery and pharmaco-
logical study.
To validate the cellular activity of amantamide (1) as a
CXCR7 agonist, the effect of amantamide (1) on Erk1/2
phosphorylation was monitored in A549 human lung cancer
cells, which is an important factor in the downstream pathway
of CXCR7 activation.16 The production of phosphorylated
Erk1/2 (pErk1/2) was significantly increased after 3 h in all
agonist treatment groups with an unchanged production of
total Erk1/2 (tErk1/2) (Figure 4A). Furthermore, the effect of
amantamide (1) on mRNA expression of the angiogenesis-
related gene VEGFA in A549 cells was measured after 3 h of
treatment. VEGFA is one of the most studied angiogenic
growth factors of the VEGF family, which promotes all steps in
the angiogenic cascade.17 As shown in Figure 4B, after
treatment with amantamide (1), the transcript level of
VEGFA in A549 cells was elevated greatly compared to
solvent (DMSO) control. Interestingly, we found that
CXCL12, the endogenous ligand of CXCR7 and also
CXCR4, induced transcript expression of VEGFA-like
amantamide (1), while TC14012 (Figure S12), a known
CXCR7 agonist and a CXCR4 antagonist, decreased VEGFA
expression instead. The involvement of CXCR4 might
contribute to such different results. In addition, the structural
difference of TC14012 with amantamide (1) may suggest
different modes of action for these agonists. Further
investigation is ongoing. It is noteworthy that when CXCR4
and CXCR7 are coexpressed, receptor heterodimers form as
much as receptor homodimers.18 Considering the confirmed
C
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expression of CXCR7 as well as CXCR4 in A549 cells,19,20 our
results indicate that amantamide (1) would be able to serve as
a selective agonist to help decipher the biological role of
CXCR7 in different disease models.
Information for details). However, ligand-displacement assays
and mutational studies would be necessary to validate the
binding mode of amantamide (1) with CXCR7.
In summary, we isolated amantamide (1), a new marine
cyanobacterial natural product, synthesized the compound, and
characterized it as a new CXCR7 agonist with confirmed
cellular activity. SAR studies to produce a diverse amantamide
analogue library and optimize the agonistic activity of
amantamide is ongoing. The selective activation of CXCR7
by 1 could provide the basis for developing CXCR7-targeted
therapeutics and deciphering the biological role of CXCR7
without interference with CXCR4.
To decipher the molecular interaction of amantamide (1)
against CXCR7, molecular modeling experiments were
performed. Brintonamide D was also identified as a CXCR7
agonist (EC50 3.97 μM), although it is a more potent CCR10
antagonist,9 so it was also included in the modeling process.
The sequence of CXCR7 protein was retrieved from UniProt
Knowledge Base with 362 residues.20 Due to lack of available
CXCR7 protein structure in the Protein Data Bank,21 we
developed a homology 3D model template for molecular
docking. Compared to a previous homology model published
by Montpas et al.,22 we expect our models be more reliable
because they possess much lower E values and the aligned
parts lie on a single segment (see Supporting Information for
details). The docking results (Figures 5 and S13) indicate the
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b00163.
Experimental procedures, spectral data, and biological
investigation results (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: luesch@cop.ufl.edu.
ORCID
Tao Ye: 0000-0002-2780-9761
Valerie J. Paul: 0000-0002-4691-1569
Hendrik Luesch: 0000-0002-4091-7492
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge financial support from the National Institutes
of Health, NCI Grant No. R01CA172310, the Debbie and
Sylvia DeSantis Chair professorship (H.L.), and Shenzhen
Peacock Plan (KQTD2015071714043444); M.A.R. acknowl-
edges Dr. Lydia E. Kavraki and Dr. Mark Moll (Department of
Computer Science, Rice University, Houston, TX) for
providing the DINC program for peptide docking.
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