Coibamide A, a potent antiproliferative cyclic depsipeptide from the panamanian marine cyanobacterium Leptolyngbya sp.

Article abstract of DOI:10.1021/ja801383f

Coibamide A (1) is a new, potent antiproliferative depsipeptide which was isolated from a marine Leptolyngbya cyanobacterium collected from the Coiba National Park, Panama. The planar structure of 1 was elucidated by a combination of NMR spectroscopy and mass spectrometry. Exhaustive 1D and 2D NMR spectroscopy included natural abundance ¹⁵N and variable temperature experiments; mass spectrometry included TOF-ESI-MSⁿ and FT-MSⁿ experiments. Chemical degradation followed by chiral HPLC- and GC-MS analyses was used to assign the absolute configuration of 1. This highly methylated cyclized depsipeptide exhibited an unprecedented selectivity profile in the NCI 60 cancer cell line panel and appears to act via a novel mechanism. Copyright

Full text of DOI:10.1021/ja801383f

Published on Web 04/29/2008
Coibamide A, a Potent Antiproliferative Cyclic Depsipeptide from the
Panamanian Marine Cyanobacterium Leptolyngbya sp.
Rebecca A. Medina,† Douglas E. Goeger,† Patrice Hills,‡ Susan L. Mooberry,‡ Nelson Huang,§
Luz I. Romero,| Eduardo Ortega-Barr´ıa,| William H. Gerwick, and Kerry L. McPhail*,†
College of Pharmacy, Oregon State UniVersity, CorVallis, Oregon 97331, Southwest Foundation for Biomedical
Research, San Antonio, Texas 78227, Chemical and Screening Sciences, Wyeth Research, Cambridge, Massachusetts
02140, Instituto de InVestigaciones Cient´ıficas AVanzados y SeVicios de Alta Tecnolog´ıa, Clayton, Panama 5, Republic of
Panama, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, UniVersity
of California San Diego, La Jolla, California 92093
Received February 27, 2008; E-mail: kerry.mcphail@oregonstate.edu
Marine organisms continue to yield a diverse array of biologically
active molecules, a remarkable number of which are peptide-based
cancer cell toxins of putative microbial symbiont biogenesis.1 Develop-
ment of these as anticancer drugs has met with some success:2 ascidian-
derived dihydrodidemnin B (aplidin) has orphan drug status for the
treatment of multiple myeloma and acute lymphoblastic leukemia;
green algal isolate kahalalide F and TZT-1027, a synthetic analogue
of the cyanobacterial metabolite dolastatin 10, reached phase II clinical
trials. Other important cyanobacterial peptide leads include the
cryptophycins and curacin A,3 and these organisms continue to produce
a wealth of anticancer lead compounds.4 The high degree of N-
methylation of many of these cyanobacterial peptides may improve
their druggability since N-methylation has been shown to improve
pharmacological parameters such as lipophilicity, proteolytic stability,
and duration of action, properties for which regular peptides are
notoriously poor and which limits their bioavailability.5
In the context of our International Cooperative Biodiversity Groups
program (ICBG) based in Panama, which focuses on drug discovery,
biodiversity conservation, and sustainable economic growth, we have
isolated a potent cancer cell toxin with an unprecedented selectivity
profile in the NCI 60 cell line panel. This cyanobacterial depsipeptide,
named coibamide A in tribute to its discovery from the UNESCO
World Heritage Site of Coiba National Park,6 highlights the importance
of conserving pristine, unexplored repositories of diverse marine
organisms.
human lung tumor cells, and was also active against malaria,
leishmaniasis, and trypanosomal tropical disease parasites. This VLC
fraction was separated by reversed-phase C18 solid phase extraction
and isocratic HPLC to yield the optically active colorless oil ([R]D
-54.1) coibamide A (1, 6.3 mg).
The molecular composition of 1 was established as C65H110O16N10
from FT-MS data ([M + H]+ m/z 1287.8156, ∆ -2.4 mmu). The
peptidic nature of 1 was evident from its complex 1H NMR spectra in
all solvents (CDCl3, C6D6, DMSO, C5D5N). However, N-methyl
conformations were minimized in CDCl3 which showed numerous
R-proton multiplets (4.75-6.02), overlapped methyl doublets (δ
0.75-1.15), mutually coupled aromatic proton doublets (δ 7.14, 6.76),
a broad 2H amide proton signal (6.65), and deshielded singlets
integrating to 12 methyl groups attached to heteroatoms (δ 2.34-3.77).
The 13C NMR spectra for 1 in CDCl3 and C6D6 featured an
indeterminate number of resonances, with numerous ester/amide
carbonyl 13C signals, due to localized symmetry (O-Me-Tyr, N,N-
diMe-Val), steric constraints (N-Me-Thr), signal overlap and multiple
conformations. These data suggested a high degree of N- and
O-methylation, an observation supported by standard amino acid
analysis which yielded only one alanine and one O-methyl tyrosine
residue. These two amino acids, one hydroxy acid and eight N-
methylated residues were assigned from 2D experiments (CDCl3)
including COSY, TOCSY, multiplicity-edited HSQC, HSQC-TOCSY,
HMBC, H2BC7 and 1H-15N gHMBC.
Elucidation of seven of the eight N-methylated residues began
with HMBC correlations from each N-methyl singlet to the
corresponding R-carbon, the side-chain spin systems of which were
delineated by TOCSY experiments to give N-methylalanine, two
N-methylleucines, N-methylisoleucine, two N,O-dimethylserines,
and an N,N-dimethylvaline residue. The latter terminal residue was
described by a 6H singlet (δH-64/65 2.34) that was HSQC-correlated
to a prominent 13C resonance (δC-64/65 41.3) and 15N-gHMBC-
correlated to a shielded 15N resonance (δN 24.6). Fortunately, nine
of ten N atoms in 1 were observed in the latter 15N-gHMBC8
experiment which showed additional correlations from five N-
methyls to δN 105.6, 108.5, 113.4, 117.5, 120.5, two R-methyls
(Ala and N-Me-Ala) to δN 115.1, 122.4, and H2-7 of O-Me-Tyr to
δN 118.2. Hydroxyisovaleric acid (HIV) was assigned on the basis
of TOCSY correlations from deshielded CH-55 (δH 5.00, δC 74.7)
to isopropyl methine (δH-56 2.21) and methyl (δH3-57/58 1.06)
resonances. At this point, it remained to assign 114 mass units
(interpreted as C5H8O2N ) N-Me-Thr or N,O-diMe-Ser), to
determine the carboxyl terminus and to establish the sequence of
residues in the depsipeptide chain. COSY correlations were
observed between an unassigned methyl doublet at δ 1.07 (H3-40)
and an oxygenated methine multiplet at δ 5.50 (H-39). Strong
ROESY correlations, but no COSY or TOCSY correlations, were
observed between this methyl-oxymethine pair and a very broad,
partially obscured signal (δ 2.89, CDCl3; 3.11 ppm, C6D6). Variable
temperature experiments in CDCl3 (298-328K, 700 MHz, 1 mm
The marine filamentous cyanobacterium Leptolyngbya sp. was
collected by hand using SCUBA from the Coiba National Park,
Panama. A crude organic extract of this material was subjected to
bioassay-guided fractionation via normal phase vacuum liquid chro-
matography (NP-VLC) using a stepped gradient of hexanes to EtOAC
to MeOH. In preliminary biological activity screening, the 100%
EtOAc eluting fraction was cytotoxic (IC50 300 ng/mL) to NCI-H460
† Oregon State University.
‡ Southwest Foundation for Biomedical Research.
§ Wyeth Research.
| INDICASAT.
Scripps Institution of Oceanography and UCSD.
9
6324 J. AM. CHEM. SOC. 2008, 130, 6324–6325
10.1021/ja801383f CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
N-Me-L-Ala, and either N-Me-L-Thr or N-Me-L-allo-Thr. The presence
of N-Me-L-Thr is proposed from computational models9 of the two
possible coibamide structures, constrained by ROESY correlations
between N-CH3-4 and CH3-40, and between R-H-2 and N-CH3-48.
Coibamide A displayed potent cytotoxicity to NCI-H460 lung
cancer cells and mouse neuro-2a cells (LC50 < 23 nM), but did
not interfere with tubulin or actin in cytoskeletal assays. Flow
cytometric studies showed that 1 caused a significant dose-
dependent increase in the number of cells in the G1 phase of the
cell cycle with little change in G2/M and a loss of cells in S phase
(see Supporting Information). Coibamide A was evaluated against
the NCI’s in vitro panel of 60 cancer cell lines and produced mean
cytostatic (GI50 and TGI with range) and cytotoxic (LC50 and range)
parameters as follows: log GI50, -8.04 (2.96); log TGI, -5.85
(3.43); log LC50, -5.11 (2.66). These log mean values of < -4
with log range values of >2 indicate both potency and histological
selectivity. Coibamide A showed highest potency (GI50) to MDA-
MB-231 (2.8 nM), LOX IMVI (7.4 nM), HL-60(TB) (7.4 nM),
and SNB-75 (7.6 nM) and good histological selectivity for breast,
CNS, colon, and ovarian cancer cells (see Supporting Information).
Coibamide A was COMPARE negative,10 indicating that it likely
inhibits cancer cell proliferation through a novel mechanism.
In summary, coibamide A (1) is a promising lead agent in cancer
drug discovery, with a potentially new mechanism of action. Further
investigation of the molecule is being pursued via chemical
synthesis, since the producing organism has not yet been cultured
successfully in the laboratory.
Figure 1. Partial structures A and B assembled from key ROESY correlations
and mass fragments; structure C shows the key mass spectrometric fragments
supporting the position of N-Me-Thr and cyclization of 1.
cryoprobe) resolved this broad peak into a 3H singlet which was
HSQC-correlated to an N-methyl resonance (δC-41 29.7). Further-
more, careful examination of C6D6 HSQC data revealed an
additional heteroatom-substituted methine (δH-38 6.72, δC-38 56.6),
which showed weak TOCSY correlations to the above-described
methyl-oxymethine pair. Hence, the remaining residue was as-
signed as N-Me-Thr.
Two partial structures (Figure 1, A and B) could be assembled
based on a combination of mass spectrometric data and ROESY
correlations between each N-methyl and the R-proton of the adjacent
residue. Additionally, a ROESY correlation between N-CH3-36 and
the N-Me-Thr ꢀ- and γ-protons (H-39, H3-40) positioned this
residue at the N terminus of partial structure A.
A ROESY correlation between γ-H3-40 of the N-Me-Thr and R-H-
43 (N-Me Leu) in combination with MS fragments of m/z 535 and
567 (Figure 1, C) oriented N-Me-Thr as the fifth residue in the
depsipeptide backbone, thus linking partial structures A and B. This
sequence of residues was also consistent with MS2 fragments observed
by LC-MS of the base hydrolysate of 1, which comprised four major
linear products (2-5, pS30). Finally, an HMBC correlation from H-39
to carbonyl C-1 (δC 170.4) indicated an ester linkage from N-Me-Thr
to the C-terminal N-Me-Ala to complete the planar structure of
coibamide A (1).
Acid hydrolysis of 1 followed by various HPLC-MS and GC-MS
methodologies was used to determine the absolute configuration of
coibamide A. While some standards were commercially available (N-
Me-Leu, N-Me-Ile, N-Me-Ala, Ala, HIV and O-Me-Tyr), others
required laboratory synthesis by standard methods (N-Me-Thr, N,N-
diMe-Val, and N,O-diMe-Ser). Chiral HPLC (Phenomenex Chirex
phase 3126 (D), 4.6 × 250 mm) established the presence of O-Me-
L-Tyr, two N,O-diMe-L-Ser residues, N,N-diMe-L-Val, and L-Ala, while
chiral GC-MS (CyclosilB, 30.0 m × 250 µm × 0.25 µm) of meth-
ylated standards and the natural product hydrolysate identified L-HIV.
Treatment of the acid hydrolysate of 1 with Marfey’s reagent, followed
by C18 HPLC established the presence of N-Me-L-Ile, N-Me-L-Leu,
Acknowledgment. Financial support from the NIH Fogarty
International Center (ICBG Grant TW006634) and NCI (CA52955),
and from the William Randolph Hearst Foundation (to S.L.M.) is
gratefully acknowledged. We thank the Smithsonian Tropical Research
Institute, crew of R/V Urraca, T. Capson, C. Guevara, and R. Thacker
for collection of the cyanobacterium and Autoridad Nacional del
Ambiente (ANAM), Panama, for permission to make this collection,
OSU Biochemistry NMR Facility for 600 MHz spectrometer time, J.
Morre and the OSU EIHS Center for MS data acquisition (NIEHS
P30 ES00210), Prof. F. D. Horgen for N,O-diMeSer standards, and
C. Anklin (Bruker Biospin) for 700 MHz NMR data.
Supporting Information Available: Experimental section and
analytical data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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