Friomaramide, a Highly Modified Linear Hexapeptide from an Antarctic Sponge, Inhibits Plasmodium falciparum Liver-Stage Development

Article abstract of DOI:10.1021/acs.jnatprod.9b00362

The cold waters of Antarctica are known to harbor a rich biodiversity. Our continuing interest in the chemical analysis of Antarctic invertebrates has resulted in the isolation of friomaramide (1), a new, highly modified hexapeptide, from the Antarctic sponge Inflatella coelosphaeroides. The structure of friomaramide was determined using spectroscopic methods and its configuration established by Marfey's method. Friomaramide, which bears the unusual permethylation of the amino acid backbone and is the longest polypeptide bearing a tryptenamine C-terminus, blocks >90% of Plasmodium falciparum liver-stage parasite development at 6.1 μM.

Full text of DOI:10.1021/acs.jnatprod.9b00362

Note
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Friomaramide, a Highly Modified Linear Hexapeptide from an
Antarctic Sponge, Inhibits Plasmodium falciparum Liver-Stage
Development
Matthew A. Knestrick,† Nerida G. Wilson,‡ Alison Roth,§ John H. Adams,§ and Bill J. Baker*,†,§
†Department of Chemistry, University of South Florida, 4202 E. Fowler Avenue, CHE205, Tampa, Florida 33620, United States
‡Western Australia Museum and University of Western Australia Perth, Fremantle, Western Australia 6106, Australia
§Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida
33620, United States
S
* Supporting Information
ABSTRACT: The cold waters of Antarctica are known to harbor a rich
biodiversity. Our continuing interest in the chemical analysis of Antarctic
invertebrates has resulted in the isolation of friomaramide (1), a new, highly
modified hexapeptide, from the Antarctic sponge Inflatella coelosphaeroides. The
structure of friomaramide was determined using spectroscopic methods and its
configuration established by Marfey’s method. Friomaramide, which bears the
unusual permethylation of the amino acid backbone and is the longest
polypeptide bearing a tryptenamine C-terminus, blocks >90% of Plasmodium
falciparum liver-stage parasite development at 6.1 μM.
alaria remains one of the most deadly and widespread
ucts therefore remain a promising source of new and unique
Minfectious diseases, with almost half of the world’s chemical diversity.
This study of the Antarctic sponge Inflatella coelosphaeroides
population at risk and an estimated 219 million cases reported
in 2017.1 Plasmodium falciparum is the most prevalent and
lethal cause of malaria, with the highest concentrations in Sub-
Saharan Africa.1 Major successes in global malaria control and
elimination have been possible by treating the disease-causing
blood stage, but now drug resistance is emerging in the blood-
stage infection. Prevention of liver-stage forms of infection,
where the parasite numbers are few, represents a vulnerable
bottleneck for therapeutic intervention.2,3 Unfortunately, drug
discovery targeting these stages of Plasmodium has been
limited by the lack of efficient laboratory liver models capable
of supporting an affordable medium- to high-throughput drug
screening.4,5 With treatment options scarce, there is a dire need
for new lead compounds capable of targeting liver-stage forms
of Plasmodium.
reports the isolation and characterization of a new, highly
modified linear peptide, friomaramide (1), and its bioactivity
in a recently developed in vitro liver model of P. falciparum
infection in metabolically active primary human hepatocytes.6
Friomaramide is both structurally distinctive and potently
bioactive, blocking >90% of P. falciparum liver-stage develop-
ment at 6.1 μM.
In search of such malaria treatments, we have screened a
collection of natural products from Southern Ocean inverte-
brates in an in vitro liver-stage model.6 The cold water oceans
surrounding Antarctica are geographically and biologically
isolated from northern oceans7 and host a unique, highly
productive marine ecosystem.8 Sponges from these oceans are
morphologically and taxonomically diverse and play important
ecological roles in benthic ecosystems.9 Sponges are sessile and
soft-bodied and often rely on a high diversity of chemical
defenses to deter predation, prevent fouling, and cultivate their
extensive symbiotic microorganisms.10 Sponge natural prod-
I. coelosphaeroides was collected via trawling in the northern
reaches of the Scotia Arc in the Southern Ocean between April
and May 2013 (Table S1) at depths of approximately 100−400
Received: April 19, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00362
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Table 1. NMR Data for Friomaramide in MeOH-d4
b
δH, mult. (J in
a
b
δH, mult. (J in
a
pos
δC, type
Hz)
gCOSY
gHMBC
pos
δC, type
Hz)
gCOSY
gHMBC
Ile1-CO
Acetyl
CO
173.9, C
β
35.7, CH 1.98, m
α, β-Me, γ
Me
23.4, CH3 1.92, s
Ac-CO
β-Me 19.6, CH3 0.76, d (6.5)
β
δ
γ
α
N-Me-Phenylalanine
CO
γ
26.7, CH2 1.29, m
α
175.1, C
δ
16.7, CH3 0.60, d (6.5)
β
α
53.3, CH 5.14, br dd (8.5,
6.7)
40.2, CH2 2.90, m
3.05, m
β
α
N-Me, Ac-CO, Phe-
N-Me 34.3, CH3 3.01, s
N-Me-Isoleucine2
α, Ile1-CO
CO
β
γ, δ/δ′, Phe-CO
CO
173.8, C
α
61.2, CH 5.34, m
β
β-Me, γ, N-Me, Ile1-
CO,
γ
141.6, C
δ/δ′
ε/ε′
ζ
129.5, CH 7.28, br m
138.5, CH 7.25, br m
139.2, CH 7.23, br m
ε/ε′, ζ
δ/δ′, ζ
ε/ε′, δ/δ′
ζ
γ
Ile2-CO
δ
β
35.8, CH 2.10, m
α, β-Me, γ
γ
β-Me 16.9, CH3 0.81, d (6.5)
γ
β
δ
γ
α
N-Me 32.6, CH3 2.86, s
N-Me-Valine
α
26.7, CH2 1.16, m
21.2, CH3 0.78, d (6.5)
α
δ
β
CO
172.7, C
N-Me 31.7, CH3 3.09, s
α, Ile2-CO
α
62.9, CH 5.04, d (11.00)
β
γ, γ′, N-Me,
Tryptenamine
Phe-CO, Val-CO
1
2
129.3, CH 6.59, d (8.2)
133.5, CH 6.31, d (8.2)
138.0, CH 7.22, s
109.9, C
2
1
2, 2′, 3a′
1, 3′
3′, 3a′, 7a′
β
γ
29.8, CH 2.21, m
12.2, CH3 0.80, d (7.3)
16.4, CH3 0.83, d (7.3)
α, γ, γ′
β
α, γ′
α, γ
Val-CO
2′
3′
3a′
4′
5′
6′
7′
7a′
γ′
β
N-Me 34.6, CH3 2.78, s
N-Me-Alanine
129.5, C
128.7, CH 7.60, d (7.8)
130.9, CH 7.08, t (7.2)
132.3, CH 7.15, t (7.2)
121.9, CH 7.36, d (7.8)
138.5, C
5′
3′, 6′, 7a′
3a′, 7′
4′, 7′a
CO
175.8, C
4′, 6′
5′, 7′
6′
α
59.4, CH 5.17, m
β
α
N-Me, Val-CO, Ala-
CO
α, Ala-CO
α, Ala-CO
3a′, 5′
β
15.7, CH3 1.20, d (7.0)
N-Me 32.8, CH3 2.95, s
N-Me-Isoleucine1
CO
α
172.8, C
59.4, CH 5.09, d (11.0)
a
b
13C NMR spectrum recorded at 200 MHz. 1H NMR spectrum
β
β-Me, γ, N-Me, Ala-
CO,
recorded at 800 MHz, reported in ppm.
m. Sponges were freeze-dried and extracted with CH2Cl2/
MeOH (1:1). The extract was mounted on silica gel and
subjected to normal-phase MPLC. Further NMR-guided
purification of the EtOAc/MeOH (3:1) eluting fractions was
accomplished with reversed-phase HPLC eluted with MeCN,
yielding 1.5 mg of friomaramide (1) as an off-white,
amorphous solid.
Figure 1). Alanine, phenylalanine, valine, and two isoleucine
residues, all methylated, were identified.
Friomaramide was determined to have a molecular formula
1
of C46H67N7O6 based on HRESIMS, corroborated by H and
13C NMR data (Table 1). Evaluation of the NMR data, as well
as the correlations of protons to carbons in the gHSQC
spectrum, indicated the presence of five notable nitrogen-
bearing methyl groups, multiple aromatic signals, and six ester/
amide-type carbonyls, as well as two olefinic, eight methine,
three methylene, and eight aliphatic/acyl methyl carbons.
These initial observations were suggestive of a highly
methylated peptide.
The planar structure of 1 was determined spectroscopically.
Among the evident intact amino acid residues, gHMBC data
correlated each α-methine proton (δH 5.09−5.34) to two
amide-type carbonyls (δC 172.7−175.8), as well as a singlet N-
methyl (δH 2.78−3.01). These correlations suggested that the
molecule was an N-methylated peptide. Interestingly, all five of
the intact amino acids displayed nitrogen methylation. With
this knowledge, the gHMBC and gCOSY spectra were used to
construct each amino acid within the molecule (Table 1,
Figure 1. Important gCOSY (bold) and gHMBC (→) correlations
establishing the planar structure of friomaramide.
The sequence of the five methylated amino acids was
established using gHMBC correlations. In particular, the α-
methine protons, which displayed correlations to the adjacent
amide carbonyls, were critical to establishing the peptide
sequence. The α-methine of N-Me-Phe (δH 5.14) displayed an
HMBC correlation to the N-Me-Phe amide carbonyl (δC
175.1), but no other amino acid carbonyl, indicating it was
the terminal amino acid. The α-methine proton of N-Me-Val
(δH 5.04) displayed correlations to both the N-Me-Val (δC
172.7) and N-Me-Phe carbonyls; the N-Me-Ala α-methine (δH
5.17) displayed correlations to the N-Me-Ala (δC 175.8) and
N-Me-Val carbonyls; the N-Me-Ile1 α-methine (δH 5.09)
displayed correlations to the N-Me-Ile1 (δC 172.8) and N-Me-
B
DOI: 10.1021/acs.jnatprod.9b00362
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Figure 2. Friomaramide evaluated against P. falciparum liver-stage parasites and assessment of hepatocyte toxicity. (a) Blood-stage active
antimalarial dihydroartemisinin (DHA) shows no inhibition of P. falciparum liver-stage development at 5 μg/mL (17.6 μM), while friomaramide
and primaquine (PQ) show >90% inhibition at 5 μg/mL (19.3 and 6.1 μM, respectively). Graph bars represent means with SD for biological
replicates (n = 2) and experimental replicates (n = 3). (b) Friomaramide is nontoxic toward primary human hepatocytes at a 5 μg/mL
concentration (6.1 μM), where the dotted red line indicates mean hepatocyte nuclei counts for 0.1% DMSO control, comparable to DHA and PQ
at the same concentration.
Ala carbonyls; and finally the α-methine of N-Me-Ile2 (δH
5.34) displayed correlations to the N-Me-Ile2 (δC 173.8) and
N-Me-Ile1 carbonyls. The sequence of amino acids was thus
established as N-Me-Phe-N-Me-Ala-N-Me-Val-N-Me-Ile1-N-
Me-Ile2.
The N-terminus of the peptide was determined to be
acetylated, indicated by gHMBC correlation between the α-
methine proton of N-Me-Phe (δH 5.14) and an acetyl carbonyl
at δC 173.9.
subsequent drug treatment at 5 μg/mL (17.6, 19.3, and 6.1 μM
concentration of dihydroartemisinin, primaquine, and 1,
respectively). Media was changed daily with fresh addition of
drug for 3 days until chemical fixation on day 6. Friomaramide
showed 92% ( 14% standard deviation) inhibition of P.
falciparum liver-stage infection and development, which is
highly comparable to primaquine, one of the few known liver-
stage acting antimalarial drugs (Figure 2a). Further, treatment
with friomaramide elicited no toxic effects toward primary
human hepatocytes determined by quantification of fluores-
cently labeled hepatocyte nuclei, indicating viable nuclei
(Figure 2b). These results identify friomaramide as an
important new chemotype that can block infection and
development in human liver cells of P. falciparum, the most
prevalent and lethal cause of malaria.
Friomaramide brings a number of new structural features to
light. Tryptenamine as a C-terminus to a small peptide is found
only rarely in natural products. The terpeptins, terrestrial
fungal natural products, are prenylated tripeptides terminating
in tryptenamine.14 From the marine environment, a marine
Aspergillus produces terpeptin-like aspergillamides,15 while the
only compounds bearing tryptenamine from macroorganisms
are from red algae11,16 and a tunicate,17 though in those two
examples, the tryptenamine was not part of a peptide chain.
Friomaramide introduces a sponge source of this unusual
amino acid derivative and, taken with the peptidic nature of the
molecule, suggests that host-associated fungi may be the actual
producer. Few fungal tryptenamine C-terminating peptides are
fully N-methylated, although some small peptides lacking
tryptenamine are as highly N-methylated as friomaramide.18,19
Those highly methylated peptides that do not bear trypten-
amine can be up to eight amino acids in length, while those
bearing tryptenamine are never larger than three amino acids.
The six amino acid friomaramide appears to be a hybrid
between the terpeptin14,20/miyakamide21 type tripeptides and
larger peptides such as pterulamide22 or RHM-type18 products.
Acetylation on the N-terminus amino acid as found in
friomaramide is rare among fungal products. Peptide
methylation is attractive in drug discovery, as non-methylated
peptides have poor pharmacokinetic properties (short in vivo
half-life, poor oral availability).23
The C-terminus of friomaramide was found to bear an
eliminative decarboxylation product of tryptophan, i.e.,
tryptenamine. The tryptenamine residue could be constructed
by observation of gCOSY-coupled aromatic protons that
indicated the presence of an aromatic ring within the residue
(δH 7.08−7.60). The chemical shift of C-3a′ (δC 129.5) and C-
7a′ (δC 138.5), as well as gHMBC correlations of H-4′ (δH
7.60) and H-2′ (δH 7.22) with C-3′ (δC 109.9) and C-7a′,
respectively, verified the fused aromatic ring system of an
indole. The olefin comprising positions 1 (δC 129.3) and 2 (δC
133.5) were identified by gHMBC correlations between C-1
and C-2 with the H-2 and H-1, respectively, as well as the four-
bond correlation between H-2′ and C-2. The cis-orientation of
the olefin was assigned on the basis of J1,2 = 8.2 Hz as opposed
to the much higher J-coupling seen in trans-orientation
tryptenamine olefins.11,12 Finally, correlation between the
tryptenamine H-2 and the carbonyl of N-Me-Ile2 secured its
placement on the C-terminus of friomaramide.
Stereochemical analysis of friomaramide was performed by
Marfey’s method.13 Briefly, the peptide was hydrolyzed then
derivatized with Marfey’s reagent, FDAA (Nα-(2,4-dinitro-5-
fluorophenyl-L-alaninamide), which allowed the amino acid
isomers to be distinguishable from one another chromato-
graphically. These reaction products were evaluated using LC-
MS, by comparison to similarly derivatized amino acid
standards (Table S2). The absolute configuration of
friomaramide was determined to be D-N-Me-Phe-L-N-Me-
Val-L-N-Me-Ala-L-N-Me-Ile-L-N-Me-Ile.
Bioactivity of friomaramide was evaluated for inhibition of P.
falciparum liver cell infection and development in an in vitro
liver model,6 while being simultaneously measured for toxicity
against the primary human hepatocytes. Cryopreserved
primary human hepatocytes were seeded at 1.8 × 104 cells
per well in a 384-well microplate. Freshly isolated P. falciparum
sporozoites were inoculated at 2.0 × 104 per well with
In summary, friomaramide is a new, highly modified peptide
with potent activity against the liver stage of malaria. In the
face of rising antimalarial drug resistance emerging to
C
DOI: 10.1021/acs.jnatprod.9b00362
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
elution gradient of 3:1 MeCN/H2O to yield the purified
artemisinin combination therapies, the frontline antimalarial
drugs of choice, it is important to find new chemodiversity
sources. Friomaramide was found to block P. falciparum
sporozoite infection and subsequent liver-stage parasite
development, showing similar inhibitory activity to the
known liver-stage antimalarial drug primaquine. Structurally,
friomaramide contains a number of unique and uncommon
modifications for a small peptide. This chemical novelty and
biological specificity present a new framework on which a hit
to lead developmental strategy could be expected to bring
forward new candidates for malaria treatment.
friomaramide (1.5 mg).
Friomaramide (1): white, amorphous solid; [α]25 −150 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 230 (2.81); IR (thin film) 3450,
3050, 2980, 2960, 1700, 1680, 1635, 1500, 1240 cm−1; 1H NMR (800
MHz, MeOH-d4), Table 1; 13C NMR (200 MHz, MeOH-d4), Table
1; HRESIMS m/z 814.5226 [M + H]+ (calcd for C46H68N7O6,
814.5226), m/z 836.5051 [M + Na]+ (calcd for C46H67N7O6Na,
836.5045).
Marfey’s Analysis. A portion of 1 (0.3 mg) was hydrolyzed at 120
°C with 6 M HCl for 24 h. A 0.1 M NaHCO3 solution (200 μL) was
added to the dried hydrolysate of 1, as well as to L- and D-amino acids
standards of N-Me valine, N-Me alanine, N-Me phenylalanine, N-Me
isoleucine, and N-Me allo-isoleucine. A solution of Marfey’s reagent
and FDAA in acetone (25 mg in 50 μL) was added to each
hydrolysate and standard, and each was incubated at 90 °C for 10
min. To quench each reaction, 100 μL of 2 M HCl was added and
then diluted with 300 μL of MeCN. The Marfey’s derivatives of the
hydrolysate and standards were analyzed using LC-MS-ToF with a
Phemonenex Kinetex C-18 column (2.6 μm, 100 Å, 150 × 3 mm),
with an elution gradient of 3:1 H2O/MeCN acidified with 0.1%
formic acid to 1:3 H2O/MeCN acidified with 0.1% formic acid over
10 min at a 0.4 mL/min flow rate. Comparison of retention time and
mass of each Marfey’s derivative of the hydrolysate and standard was
used to determine the absolute stereochemistry of 1 (Table S2).
Biological Assay. Primary human hepatocytes (PHHs) were
plated at 18 000 per well in 40 μL of fresh media on day 0 and were
incubated overnight at 37 °C, 5% CO2. Of the original PHHs plated,
roughly 10 000 adhere to the plate. P. falciparum sporozoites were
added to each well on day 3 postseed of hepatocytes and allowed to
incubate for 24 h at 37 °C, 5% CO2. On days 0, 1, 2, and 3
postsporozoite infection, media was replaced with 40 μL of fresh
media, with simultaneous drug administration at a 40 nL volume of a
5 mg/mL drug using a pin tool (V & P Scientific). The pin tool
performs a 1000-fold dilution, thus making the final drug
concentration 5 μg/mL. The drug-treated media administered on
day 3 was removed on day 4 and replaced with fresh media with no
further drug administration. On day 6, the media was removed, and
the PHHs were chemically fixed using 4% paraformaldehyde, then
immunofluorescent staining was performed for quantification and
analysis of parasite infection as previously described.6 This mode is
termed “prophylactic”, where it is measuring the drugs ability to block
sporozoite invasion and subsequent liver-stage parasite development.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using an AutoPol IV polarimeter at 589 nm. UV
absorptions were measured by an Agilent Cary 60 UV−vis
spectrophotometer; IR spectra were recorded with a PerkinElmer
Spectrum Two equipped with a UATR (single reflection diamond)
sample introduction system. NMR spectra were recorded at 298 K on
Varian Direct Drive 800 MHz NMR spectrometers. Chemical shifts
are reported with the use of the residual MeOH-d4 signals (δH 3.31
ppm; δC 49.0 ppm) as internal standards for 1H and 13C NMR
spectra, respectively. The 1H and 13C NMR assignments were
supported by gCOSY, gHSQC, and gHMBC experiments. The high-
resolution electrospray ionization mass spectra were performed on an
Agilent 6230 TOF LC/MS. Medium-pressure liquid chromatography
(MPLC) was performed using a Combiflash Rf 200i MPLC, using
ELSD and UV detection with a RediSepRf 80 g silica column.
Reversed-phase HPLC was completed with a gradient of H2O to
MeOH on a semipreparative Phenominex C18 column (10 μm, 100
Å, 250 × 10 mm) or with a gradient of H2O to either ACN or MeOH
on an analytical Phenominex C18 column (5 μm, 100 Å, 250 × 4.6
mm) using ELSD and UV detection.
Biological Material. Sponge specimens were collected aboard the
R/V Nathaniel B. Palmer research vessel by trawl and immediately
frozen, with tissue samples taken for DNA analyses. Specimens of the
sponge Inflatella coelosphaeroides Koltun, 1964, include Scripps
Institution of Oceanography Benthic Invertebrate Collection
accession numbers BIC-SIO S20232, S20400, S20399, S20411,
S20437, S20454, and S20375 (see Table S1). Where possible,
individual sponges had their DNA extracted using a Qiagen DNeasy
kit using the manufacturer’s protocol. The DNA was used in a PCR
reaction using the forward primer SpongeCOI-F1 but without the
M13 tail24 and the reverse primer dgHCO25 with an annealing
temperature of 40 °C for the first 7 cycles and 50 °C for the
subsequent 35 cycles. Amplicons were sequenced at the Australian
Genome Research Facility and assembled and edited in Geneious
v9.1.8 (Biomatters Ltd.). These sequences were then blasted to the
NCBI database (megablast algorithm), with all sequences showing
high percentage matches (>99%) to a specimen of I. coelosphaeroides
collected from the Ross Sea.24 Two specimens, S20232 and S20399,
externally look similar to I. coelosphaeroides but have some minor
morphological details that differ. Since their LC/MS spectrometric
profiles matched with the remaining I. coelosphaeroides samples, all
were combined for mass extraction. Sequences were deposited in the
NCBI database with accession numbers MH892414-MH892417.
Extraction and Isolation. The collection of sponges were
extracted in 1:1 CH2Cl2/MeOH for two consecutive 24 h periods,
immediately followed by extraction in 1:1 MeOH/H2O for another
24 h. The 1:1 CH2Cl2/MeOH extract was dried, and the 6.4 g extract
was mounted on silica gel and subjected to NP MPLC with an elution
gradient of hexanes to EtOAc, followed by a 1:3 MeOH/EtOAc wash
on an 8 g silica column. Fraction E eluted in 1:3 MeOH/EtOAc,
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00362.
1
1D H and 13C, 2D gCOSY, gHSQC, and gHMBC
NMR spectra and HRESIMS for friomaramide; data
table of sponge accession numbers and photodocumen-
tation of specimens; data table of Marfey’s analysis
(PDF)
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +1 (813) 974-1967. Fax: +1 (813) 974-2876. E-mail:
bjbaker@usf.edu.
ORCID
Alison Roth: 0000-0001-6917-3485
John H. Adams: 0000-0003-3707-7979
Bill J. Baker: 0000-0003-3033-5779
1
displayed H NMR signals indicative of the methylated peptide, and
was selected for further purification. Semipreparative RP HPLC was
performed on fraction E with an elution gradient of 1:3 MeOH/H2O
to MeOH over 40 min, yielding four fractions. One of these fractions,
eluting with MeOH, was subjected to analytical RP HPLC with an
Notes
The authors declare no competing financial interest.
D
DOI: 10.1021/acs.jnatprod.9b00362
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
(22) Lang, G.; Mitova, M. I.; Cole, A. L.; Din, L. B.; Vikineswary, S.;
Abdullah, N.; Blunt, J. W.; Munro, M. H. J. Nat. Prod. 2006, 69,
1389−1393.
ACKNOWLEDGMENTS
■
We would like to thank the team of the research vessel of the
Nathanial B. Palmer and G. Rouse, for providing support
during many field seasons of collection, in particular the 2013
cruise, and A. Hickling for sequencing support at WAM. This
project was supported by the National Science Foundation
through grant ANT-1043749 (to N.G.W.), grants ANT-
0838776 and PLR-1341339 (to B.J.B.), and University of
South Florida (to J.H.A.).
(23) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem.
Res. 2008, 41, 1331−1342.
(24) Vargas, S.; Kelly, M.; Schnabel, K.; Mills, S.; Bowden, D.;
̈
Worheide, G. PLoS One 2015, 10, 0127573.
(25) Meyer, C.; Geller, J.; Paulay, G. Evolution 2005, 59, 113−125.
REFERENCES
■
(1) World Health Organization. World Malaria Report. https://
www.who.int/malaria/publications/world-malaria-report-2018/
report/en/.
(2) Lindner, S. E.; Miller, J. L.; Kappe, S. H. I. Cell. Microbiol. 2012,
14, 316−324.
(3) Rabinovich, R. N.; Drakeley, C.; Djimde, A.; Hall, B. F.; Hay, S.
I.; Hemingway, J.; Kaslow, D. C.; Noor, A.; Okumu, F.; Steketee, R.;
Tanner, M.; Wells, T. N. C.; Whittaker, M. A.; Winzeler, E. A.; Wirth,
D. F.; Whitfield, K.; Alonso, P. L. PLOS Med. 2017, 14, e1002456.
(4) Campo, B.; Vandal, O.; Wesche, D. L.; Burrows, J. N. Pathog.
Global Health 2015, 109, 107−122.
(5) Baird, K. Pathog. Global Health 2015, 109, 93−106.
(6) Roth, A.; Maher, S. P.; Conway, A. J.; Ubalee, R.; Chaumeau, V.;
Andolina, C.; Kaba, S. A.; Vantaux, A.; Bakowski, M. A.; Luque, R. T.;
Adapa, S. R.; Singh, N.; Barnes, S. J.; Cooper, C. A.; Rouillier, M.;
McNamara, C. W.; Mikolajczak, S. A.; Sather, N.; Witkowski, B.;
Campo, B.; Kappe, S. H. I.; Lanar, D. E.; Nosten, F.; Davidson, S.;
Jiang, R. H. Y.; Kyle, D. E.; Adams, J. H. Nat. Commun. 2018, 9, 1837.
(7) McClintock, J. B.; Amsler, C. D.; Baker, B. J. Integr. Comp. Biol.
2010, 50, 967−980.
(8) Clarke, A.; Murphy, E. J.; Meredith, M. P.; King, J. C.; Peck, L.
S.; Barnes, D. K. A.; Smith, R. C. Philos. Trans. R. Soc., B 2007, 367,
149−166.
(9) Hooper, J. N. A.; Van Soest, R. W. M. Systema Porifera; Springer:
Boston, MA, 2002.
(10) von Salm, J. L.; Schoenrock, K. M.; McClintock, J. B.; Amsler,
C. D.; Baker, B. J. The status of marine chemical ecology in
Antarctica: form and function of unique high-latitude chemistry. In
Marine Chemical Ecology; Puglisi, M. P.; Becerro, M. A., Eds.; CRC
Press: Boca Raton, FL, 2019; pp 27−60.
(11) Palermo, J. A.; Flower, P. B.; Seldes, A. M. Tetrahedron Lett.
1992, 33, 3097−3100.
(12) Ondeyka, J. G.; Dombrowski, A. W.; Polishook, J. P.; Felcetto,
T.; Shoop, W. L.; Guan, Z.; Singh, S. B. J. Ind. Microbiol. Biotechnol.
2003, 30, 220−224.
(13) Bhushan, R.; Bruckner, H. J. Chromatogr. B: Anal. Technol.
̈
Biomed. Life Sci. 2011, 879, 3148−3161.
(14) Kagamizono, T.; Sakai, N.; Arai, K.; Kobinata, K.; Osada, H.
Tetrahedron Lett. 1997, 38, 1223−1226.
(15) Toske, S.; Jensen, P. R.; Kauffman, C. A.; Fenical, W.
Tetrahedron 1998, 54, 13459−13466.
(16) Kirkup, M. P.; Moore, R. E. Tetrahedron Lett. 1983, 24, 2087−
2090.
(17) Appleton, D. R.; Page, M. J.; Lambert, G.; Berridge, M. V.;
Copp, B. R. J. Org. Chem. 2002, 67, 5402−5404.
(18) Boot, C. M.; Tenney, K.; Valeriote, F. A.; Crews, P. J. Nat. Prod.
2006, 69, 83−92.
(19) Boot, C. M.; Amagata, T.; Tenney, K.; Compton, J. E.;
Pietraszkiewicz, H.; Valeriote, F. A.; Crews, P. Tetrahedron 2007, 63,
9903−9914.
(20) Izumikawa, M.; Hashimoto, J.; Takagi, M.; Shin-Ya, K. J.
Antibiot. 2010, 63, 389−391.
(21) Shiomi, K.; Hatae, K.; Yamaguchi, Y.; Masuma, R.; Tomoda,
H.; Kobayashi, S.; Omura, S. J. Antibiot. 2002, 55, 952−961.
E
DOI: 10.1021/acs.jnatprod.9b00362
J. Nat. Prod. XXXX, XXX, XXX−XXX