Small Molecules in the Cone Snail Arsenal

Article abstract of DOI:10.1021/acs.orglett.5b02389

Cone snails are renowned for producing peptide-based venom, containing conopeptides and conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features, genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice, indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom.

Full text of DOI:10.1021/acs.orglett.5b02389

Letter
pubs.acs.org/OrgLett
Small Molecules in the Cone Snail Arsenal
Jorge L. B. Neves,†,∥ Zhenjian Lin,‡,∥ Julita S. Imperial,§ Agostinho Antunes,† Vitor Vasconcelos,†
Baldomero M. Olivera,*,§ and Eric W. Schmidt*,§,‡
†Interdisciplinary Centre of Marine and Environmental Research, CIIMAR/CIMAR, and Faculty of Sciences, University of Porto,
40150-123 Porto, Portugal
§
‡Departments of Medicinal Chemistry and Biology, University of Utah, Salt Lake City, Utah 84112, United States
S
* Supporting Information
ABSTRACT: Cone snails are renowned for producing peptide-based venom, containing conopeptides and
conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features,
genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice,
indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom.
arine mollusks of the genus Conus produce bioactive
peptides that are used in medicine.1 Each snail
NMR analysis. The 1H NMR spectrum showed only two
methylene triplets (∂ 3.00, 4.43 ppm, t, J = 7.5 Hz) and one
methyl singlet (∂ 2.74 ppm, s). An HSQC spectrum indicated
chemical shifts consistent with adjacency of the methylenes to
nitrogen and the methyl to an aromatic carbon. Indeed, a 15N-
HMBC spectrum revealed connectivity to two nitrogen units,
as well as the proximity of the methylene and methyl groups. A
13C-HMBC experiment indicated the substructure as shown in
Figure 1a.
M
synthesizes an array of different peptides, each of which targets
a specific receptor or ion channel subtype, often with exquisite
selectivity. The combination of these peptides, referred to as a
“cabal”, creates a unique response in prey animals, incapacitat-
ing prey and enabling consumption by the cone snail.2
Although cone snails eat fish, polychaete worms, or other
mollusks, because of the striking conservation of many ion
channels and receptors across higher animals, the peptides are
often highly selective to human proteins as well.
Because of these properties, cone snail research has focused
on venom peptides, with good reason. Recently, we found
potent neuroactivity in a venom fraction containing small
molecules. Here, we describe the discovery of a novel guanine
derivative, genuanine, which induces paralysis in mice. Given
the extensive previous chemical characterization in cones, even
in one of the species described below, it is remarkable that this
bioactive guanine derivative with novel modifications of the
purine ring was never characterized prior to the present study.
Natural small molecule derivatives of nucleic acids, such as
cytosine arabinoside, often have novel biological activity,
sometimes with important biomedical applications.
Figure 1. Structure of genuanine. (A) Key HMBC data used to
determine the right ring substructure. Red arrows, 15N-HMBC; black
arrows, 13C-HMBC. (B) Structure of genuanine.
Despite insufficient NMR data, the molecular mass (m/z =
238.0935) and a characteristic UV signature led us to speculate
that the genuanine was a derivative of guanine (Figure 1b).
Genuanine was synthesized from guanine in two steps. The
Conus genuanus was collected in Sao Vicente, Cape Verde. C.
̃
genuanus belongs to a small number of cones with a pigmented
venom duct, prompting us to search for small molecule
pigments, which have not yet been characterized in cones. The
red pigment is asymmetrically distributed when the venom is in
situ in the venom duct of the snail. Dry venom collected from
the red portion of the venom duct was extracted, providing a
mixture of small molecules that was neuroactive when injected
into mice. Careful examination of the venom components
indicated several compounds, including unstable pigments that
are under investigation. However, one of these, genuanine, was
stable and exhibited a very similar paralytic activity in mice to
what was seen in the crude small-molecule fraction.
1
synthetic material initially appeared on the basis of H NMR
spectroscopy to be different from the natural genuanine.
However, upon mixing the natural and synthetic material
together, it became apparent that the two are identical (Figure
S1, 3.11 and 3.12), but that chemical shifts are extremely
sensitive to slight changes in conditions (Figure S2).
Combination of synthetic and natural genuanine in 35% DCl
enabled measurement of a 13C spectrum, providing further
Genuanine was isolated as a colorless solid (1 mg). Solubility
issues and the small amount of available material limited the
Received: August 18, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b02389
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
evidence of the structure as shown. Thus, the structure of
genuanine was simultaneously elucidated and confirmed.
Compound 1 exhibited profound activity at doses as low as
40 nmol per mouse, in which the mouse was paralyzed in all
four limbs for >2 h. However, best activity was obtained with
fresh samples, and activity decreased with storage time (see
Supporting Information for a complete table of all conditions
and doses tested). This activity mimicked the potent paralytic
activity found in the venom extract. The reason for this loss of
activity over time has yet to be elucidated, but it was
reproducible with both natural and synthetic 1.
Initial attempts to improve synthetic yield also led to an array
of regioisomers of genuanine, compounds 2−4 (Figure 2), for
Figure 3. Out of the ∼800 known species of cone snails, C. imperialis
(left) and C. genuanus (center) have the rare property of venom ducts
containing red pigment at the distal end (right).
Figure 2. Synthesis of genuanine (top)3 and synthetic isomers used in
biological assays (bottom).
which no activity was detected (Table S1), suggesting a
possible structure−activity relationship underlying neuroactiv-
ity. Other nucleic acid analogues with neuroactivity have
previously been described. For example, a series of 8-
oxoisoguanines from sponges inhibit GABAergic transmission
in mice, while a sea anemone adenosine derivative caissarone is
an adenosine receptor antagonist.4 In the synthetic arena,
guanine derivatives have been synthesized as purinergic
receptor agonists and antagonists that act centrally,5 among
many other activities. It is highly speculative to connect any of
these previous results to our observed activity here, but they
provide further confidence that nucleic acids can affect paralysis
and the CNS. It is noteworthy that genuanine is present in
sufficient quantity to exert the same effect in the crude venom.
Thus, we propose that 1 is an active constituent in the cone
snail prey-capturing arsenal and not just a passive ingredient in
the venom duct.
Figure 4. Venom components from C. genuanus. HPLC-UV traces
show that the distal and proximal venom ducts contain quite different
components (top). Enumeration of major peaks in HPLC-MS spectra
show that, in contrast to the proximal duct, the distal duct is
dominated by small molecules (bottom).
low-molecular weight materials, of which genuanine 1 is the
major stable metabolite.
Genuanine belongs to a rare family of C-8 methylated nucleic
acid derivatives. Nucleosides are methylated at C-8 by radical
alkylators, such as procarbazine.7 In nature, a C-8 methylated
adenosine derivative is known as a component of bacteria
rRNA;8 its biosynthesis utilizes radical SAM enzymes.9 To the
best of our knowledge, these compounds were not known from
eukaryotes nor was the methylated guanine or guanosine
known in nature. Genuanine is also modified by propionate on
N-9, which is a previously unknown modification of nucleic
acids. Speculatively, this group could possibly also be added
from methionine via a radical SAM enzyme, among other
possible routes, possibly based upon tRNA metabolism.
Alternatively, in other mollusks pigmented organs are
associated with oxidative/radical metabolism10 consistent with
C-8 modification. A large family of nucleic acid analogues from
marine animals. Most recently, several nucleosides from a
sponge were produced by bacteria cultivated from the same
sponge.11 Both cone snails and their associated bacteria are
known to produce compounds,12 but determination of the
ultimate source of genuanine awaits experiment.
Venom peptides vary greatly between cone snail species,1b so
we sought to determine whether genuanine was widespread or
restricted to C. genuanus. At random, we selected nine
additional cone snails collected from various locations in the
Pacific Ocean for chemical analysis. We also selected C.
imperialis from Oahu, Hawaii. While C. imperialis is not closely
related to C. genuanus, it shares in common a pigmented venom
duct (Figure 3) and, to the best of our knowledge, is the only
other cone with a similar venom duct, having two regions of
different colors. Only C. genuanus and C. imperialis contained
genuanine. Other venom duct extracts were rich in nucleic
acids, but these were the primary metabolites guanine and
hypoxanthine.
Recently, it has been shown that Conus geographus venom
peptides vary in different regions of the venom duct.6 Similarly,
in C. genuanus, the red (distal) and yellow (proximal) venom
duct products are very different (Figures 4 and S6). While the
proximal venom duct contains largely peptidic, high-molecular
weight components, the distal venom duct is dominated by
B
DOI: 10.1021/acs.orglett.5b02389
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Organic Letters
Letter
(9) Grove, T. L.; Benner, J. S.; Radle, M. I.; Ahlum, J. H.; Landgraf, B.
J.; Krebs, C.; Booker, S. J. Science 2011, 332, 604.
(10) Derby, C. D. Biol. Bull. 2007, 213, 274.
(11) Bertin, M. J.; Schwartz, S. L.; Lee, J.; Korobeynikov, A.;
Dorrestein, P. C.; Gerwick, L.; Gerwick, W. H. J. Nat. Prod. 2015, 78,
493.
(12) (a) Hillyard, D. R.; Olivera, B. M.; Woodward, S.; Corpuz, G. P.;
Gray, W. R.; Ramilo, C. A.; Cruz, L. J. Biochemistry 1989, 38, 358.
(b) Lin, Z.; Torres, J. P.; Ammon, M. A.; Marett, L.; Teichert, R. W.;
Reilly, C. A.; Kwan, J. C.; Hughen, R. W.; Flores, M.; Tianero, M. D.;
Peraud, O.; Cox, J. E.; Light, A. R.; Villaraza, A. J.; Haygood, M. G.;
Concepcion, G. P.; Olivera, B. M.; Schmidt, E. W. Chem. Biol. 2013,
20, 73.
The finding of paralytic small molecules as venom
components in cone snails was unanticipated. To the best of
our knowledge, only one previous report documents a small
molecule: serotonin was found in small amounts in the venom
duct of C. imperialis.13 Thus, it appears that small molecules are
important and previously unrecognized contributors to the
toxicity of cone snail venom.
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.5b02389.
(13) McIntosh, J. M.; Foderaro, T. A.; Li, W.; Ireland, C. M.; Olivera,
B. M. Toxicon 1993, 31, 1561.
Materials and methods, NMR, and MS spectra (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: olivera@biology.utah.edu.
*E-mail: ews1@utah.edu.
Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was funded by NIH R01GM107557 to E.W.S.,
NIGMS P01GM048677 to B.M.O., NIH ICBG
U01TW008163 to E.W.S. and B.M.O., and UID/Multi/
04423/2013, MARBIOTECH, and NOVOMAR. We thank J.
Cox of the Utah Mass Spectrometry and Proteomics Core for
mass measurements reported in this study; and the Foundation
for Science and Technology (FCT-SFRH/BD/51477/2011),
Fundaca̧ o Calouste Gulbenkian, and University of Cape Verde
̃
(UniCV) for sample collection support.
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