Article

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX pubs.acs.org/jnp

Antiviral Compounds from Codiaeum peltatum Targeted by a Multi-

informative Molecular Networks Approach
Florent Olivon,† Simon Remy,† Gwendal Grelier,† Ceć ile Apel,† Ceć ilia Eydoux,‡
Jean-Claude Guillemot,‡ Johan Neyts,§ Leen Delang,§ David Touboul,† Fanny Roussi,†
and Marc Litaudon*,†
†
Institut de Chimie des Substances Naturelles, CNRS-ICSN, UPR 2301, Université Paris-Saclay, 91198, Gif-sur-Yvette, France
‡
Aix Marseille University, CNRS, AFMB, AD2P, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France
§
Laboratory for Virology and Experimental Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium
*
S Supporting Information
Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/25/19. For personal use only.
J. Nat. Prod.

ABSTRACT: From a set of 292 Euphorbiaceae extracts, the use of a molecular networking (MN)-based prioritization
approach highlighted three clusters (MN1−3) depicting ions from the bark extract of Codiaeum peltatum. Based on their
putative antiviral potential and structural novelty, the MS-guided purification of compounds present in MN1 and MN2 afforded
two new daphnane-type diterpenoid orthoesters (DDO), codiapeltines A (1) and B (2), the new actephilols B (3) and C (4),
and four known 1,4-dioxane-fused phenanthrene dimers (5−8). The structures of the new compounds were elucidated by NMR
spectroscopic data analysis, and the absolute configurations of compounds 1 and 2 were deduced by comparison of
experimental and calculated ECD spectra. Codiapeltine B (2) is the first daphnane bearing a 9,11,13-orthoester moiety,
establishing a new major structural class of DDO. Compounds 1−8 and four recently reported monoterpenyl quinolones (9−
12) detected in MN3 were investigated for their selective activities against chikungunya virus replication and their
antipolymerase activities against the NS5 proteins of dengue and zika viruses. Compounds 3−8 exhibited strong inhibitory
activities on both dengue and zika NS5 in primary assays, but extensive biological analyses indicated that only actephilol B (3)
displayed a specific interaction with the NS5 targets.

R ecent outbreaks of chikungunya (CHIKV), dengue

(DENV), and zika (ZIKV) massive epidemics highlighted
the worldwide public health threat that emerging and
reemerging viruses represent.1 These viruses, which belong
to the Alphavirus (CHIKV) and Flavivirus (ZIKV and DENV)
genera, are responsible for severe infectious diseases associated
with fever, rashes, and headache.2,3 ZIKV is also associated
with neurological disorders such as Guillain-Barre syndrome
and other congenital syndromes in newborns such as
microcephaly.4−6 In Flaviviruses, the nonstructural protein 5
(NS5) polymerase plays an essential role in viral replication.7−9
Since DENV and ZIKV NS5 proteins harbor highly similar
RdRp domains, these enzymes are considered as targets of
particular interest in the prospect of discovering dual
inhibitors.10 The discovery of such dual inhibitors could be
particularly relevant given that the similarity of dengue and zika
© XXXX American Chemical Society and
American Society of Pharmacognosy
symptoms renders their differential diagnosis difficult.11,12 In
order to identify novel CHIKV inhibitors, several Euphorbia-
ceae species have been investigated in the past few years using
bioassay-guided purification procedures. This has resulted in
the isolation of tigliane, jatrophane, flexibilane, daphnane, and
myrsinane diterpenoids showing micro- to nanomolar range
activities.13−19 However, bioassay-guided fractionation has
several limitations. As a noteworthy example, its workflow,
which relies on bioactivity concerns only, does not take full
advantage of the inherent structural diversity embedded in
crude extract collections. Thus, this approach frequently leads
to the reisolation of known bioactive compounds or analogues
Received: September 20, 2018
A DOI: 10.1021/acs.jnatprod.8b00800
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Figure 1. Extract and compound prioritization strategy using multi-informative molecular networking: (A) Generation of a molecular network from
LC-MS2 analyses of 292 EtOAc extracts of New Caledonian Euphorbiaceae. (B) Detection of bioactive clusters by mapping CHIKV, DENV, and
ZIKV activities over networks. (C) Selected MN1, MN2, and MN3 with a taxonomical mapping consisting in overlaying the 20 Euphorbiaceae
genera represented in the extract collection to detect genus- or extract-specific clusters. Node sizes are proportional to MS1 peak area values.

and slows down the natural products (NPs) drug discovery
process.
Recently, we developed a molecular networking (MN)-
based strategy consisting in deciphering the relationship
between spectral networks and biological activities and further
exploiting it to prioritize the isolation of bioactive NPs.20,21
The core concept of this approach is based on the cross-linking
of various information layers within a massive molecular
network to spot bioactive scaffolds. Assuming that spectral
B DOI: 10.1021/acs.jnatprod.8b00800
dissimilarity within a taxonomically homogeneous set of
samples could imply chemical uniqueness, the generation of
these multi-informative maps unifying structural data,
taxonomical information, and bioassay results allows bio-
activities to be associated with taxon-specific scaffolds. From a
network made of approximately 1200 clusters and constructed
from 292 LC-MS2 analyses of EtOAc Euphorbiaceae extracts,
this approach highlighted the original chemical composition of
Codiaeum peltatum, a species endemic from New Caledonia. A
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Chart 1
first chemical investigation of this extract afforded four unusual
chlorinated monoterpenyl quinolones (9−12).21
A careful analysis of the networks with CHIKV, DENV, and
ZIKV bioactivity overlays revealed two additional clusters of
interest (MN1 and MN2) associated with this C. peltatum bark
extract. This paper deals with the MS-guided purification of
these compounds that led to the isolation of new bioactive
daphnane-type diterpenoid orthoesters (DDO) (1 and 2) and
1,4-dioxane-fused phenanthrene dimers (3−8).
■ RESULTS AND DISCUSSION
A collection of 292 EtOAc extracts of New Caledonian
Euphorbiaceae was first filtered over polyamide to remove
polar compounds that might interact with biological assays and
induce false-positive responses. This set of samples was
prepared from 107 different species, of which 93% are
endemic. The extracts were prepared from leaves (47%),
bark or twigs (48%), whole plants (3%), or fruits (2%). The
Euphorbiaceae family was chosen for its large structural
diversity, characterized by unique classes of structurally diverse
diterpenoids endowed with a wide range of biological activities,
such as antiviral activity against human immunodeficiency
virus or modulation of P-glycoprotein and protein kinase
C.22−29
The 292 samples were profiled by LC-HRMS2 and MS2 data
were organized as molecular networks to map the spectral
space of the extract collection (Figure 1A). Concurrently, this
set was screened over three biological targets: (i) a CHIKV
cytopathogenic effect (CPE) inhibition assay affording EC50
values ranging from 0.02 to 100 μg.mL−1; (ii) a DENV NS5
inhibition assay affording a percentage of inhibition at 50 μg·
mL−1; and (iii) a ZIKV NS5 inhibition assay affording a
C DOI: 10.1021/acs.jnatprod.8b00800
Article
percentage of inhibition at 50 μg·mL−1. For each target, the
bioactivity results were first split up according to their activity
levels. The division into various bioactivity levels is much more
informative than a simple active/nonactive categorization, as
has been shown in our previous study.20 The bioactive clusters
were highlighted by mapping these bioactivity degrees over
networks using a color gradient applied to all nodes (Figure
1B). CHIKV EC50 values were layered into five activity ranges
(0 to 5; 5 to 10; 10 to 20; 20 to 100; and >100 μg·mL−1),
DENV NS5 percentages of inhibition were layered into five
groups (0 to 40; 40 to 50; 50 to 60; 60 to 80; and >80%),
whereas ZIKV NS5 percentages of inhibition were sliced into
five ranges (0 to 60; 60 to 70; 70 to 80; 80 to 90; and >90%).
The significance levels of 40% for DENV NS5 and 60% for
ZIKV NS5 roughly correspond to values above average plus
one standard deviation.
Starting from the whole Euphorbiaceae network showing
1231 constellations made of at least three nodes (Figure 1A),
this first filtering step allowed the number of constellations of
interest to be reduced to 27 for CHIKV, 28 for DENV, and 30
for ZIKV. The second step of the prioritization strategy
consisted in targeting spectral uniqueness to detect uncommon
secondary metabolites. Indeed, genus- or species-specific
compounds can be distinguished from other ubiquitous
metabolites in networks by using a taxonomical mapping.20,21
Each Euphorbiaceae genus was assigned a specific color tag
(see Figure 1C), and this layout was used to highlight clusters
of nodes that would be restricted to a given taxon and thus a
given color.
This methodology stressed the bioactivity potential and the
original chemical composition of the EtOAc bark extract of
Codiaeum peltatum (see Figure 1C). This extract was active
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Figure 2. Key COSY (bold), HMBC (blue arrows), and NOESY or ROESY (red arrows) correlations for compounds 1 and 3.
toward the three targets studied and was thus selected for an
in-depth investigation. Among the high chemical diversity of
this extract (Figure S1, Supporting Information) three
constellations were spotted either for bioactivity (MN1) or
bioactivity associated with a putative structural originality
(MN2 and MN3).
Besides sample prioritization, this multi-informative MN
approach allows a targeted isolation of compounds of interest.
After a first semipreparative HPLC fractionation step directed
toward the purification of MN1−3 associated compounds
traceable in the crude extract, all fractions obtained were
analyzed in LC-MS2. As the amount of starting extract was
relatively low (600 mg), only fractions showing ions detected
in high abundances and belonging to one of the targeted
clusters were selected for further isolation efforts.
The MN1 cluster displayed ions shared between bark
extracts of Codiaeum peltatum and C. oligogynum and bark and
wood extracts of Trigonostemon cherrieri. This information
together with the presence of chlorinated ions in the
Trigonostemon subunit of MN1 allowed this cluster to be
associated with DDO analogues.13,30,31
Compound 1 was obtained as a white, amorphous powder.
Its HRESIMS showed a protonated molecular ion peak at m/z
727.2752 (calcd 727.2749), which, in conjunction with the 13C
NMR data, was consistent with the molecular formula
C41H42O12. The 1H and 13C NMR data, along with the
HSQC spectrum, revealed three methyl groups, eight methine
groups, of which five are oxygenated, three aromatic rings, two
carbonyls, an exomethylene and an olefinic carbon, two
methylene groups (one oxygenated), five oxygenated tertiary
carbons, and one deshielded sp3 trioxygenated carbon at δC
118.2 typical of an orthobenzoate moiety.32 Additionally, two
protons showing no correlation in the HSQC spectrum were
attributable to the exchangeable protons of two hydroxy
groups.
The A−B−C rings of 1 can be constructed by comparison of
NMR literature data and analysis of COSY and HMBC
correlations (Figure 2).33−35 The spin system connecting H2-1
(δH 2.07, m; 2.27, ddd), H-2 (δH 1.93, m), H-3 (δH 5.09, d),
H-10 (δH 3.12, dd), and H3-19 (δH 1.08, d) together with
HMBC correlations from H3-19 to C-1 (δC 34.7), C-2 (δC
35.9), and C-3 (δC 81.9) and from 4-OH (δH 3.00, s) to C-4
(δC 80.6) and C-10 (δC 48.2) indicated the presence of a
saturated cyclopentane oxygenated at C-3 for ring A. HMBC
correlations from H-7 (δH 3.44, brs) to C-6 (δC 64.4), C-8 (δC
34.8), C-9 (δC 84.3), and C-20 (δC 66.6) and from H2-20 (δH
3.82, d; 3.87, d) to C-6 and C-7 (δC 64.4) confirmed the
presence of an epoxide moiety at C-6/C-7.34 The presence of
D DOI: 10.1021/acs.jnatprod.8b00800
Article
5-OH and 20-OH were supported by the downfield resonances
of the C-5 methine (δH 4.18, s; δC 74.5) and C-20 methylene
groups. Correlations from 11-OH (δH 3.48, s) to C-11 (δC
77.0) and C-18 (δC 24.6) revealed the presence of an unusual
oxidation at C-11. Two benzoyloxy groups were located at C-3
and C-12, as supported by HMBC correlations from H-3 to C-
1‴ (δC 168.1) and H-12 (δH 5.46, s) to C-1″ (δC 166.6). The
three remaining oxygen atoms were assigned to C-9 (δC 84.3),
C-13 (δC 86.1), and C-14 (δC 82.2). Eventually, the presence
of the 9,13,14-orthobenzoate moiety was confirmed by the
downfield shifts of the latter together with HMBC correlations
from H-14 (δH 4.73, d) and H-3′/H-7′ (δH 7.79, m) to C-1′
(δC 118.2).
The relative configuration of 1 was confirmed by 1H NMR
coupling constant values and key NOESY correlations as
depicted in Figure 2. The α-orientation of the 11-hydroxy
group was supported by the strong NOESY correlation
between H3-18 (δH 1.54, s), H-12, and H-8 (δH 3.08, d).
For establishing the absolute configurations, electronic circular
dichroism (ECD) calculations were carried out for compound
1 at the B3LYP/6-31G* level (Figure 3). Comparison of the
experimental and calculated ECD spectra supported the
absolute configuration of codiapeltine A (1)
(2S,3S,4S,5S,6R,7S,8S,9R,10R,11R,12S,13R,14R).
Compound 2 showed a protonated molecular ion at m/z
727.2749 (calcd 727.2749) in the HRESIMS, corresponding to
the molecular formula C41H42O12, the same as compound 1.
From this formula and similar spectroscopic data, it was
apparent that 1 and 2 shared the same DDO core, but with a
different C-ring substitution. Indeed, when compared with
compound 1, the upfield resonances of the oxygenated
secondary carbon C-12 (δC 68.8) and oxygenated tertiary
carbons C-13 (δC 81.8) and C-14 (δC 75.6) and the downfield
resonances of the 8-methine and oxygenated tertiary carbons
C-9 (δC 89.1) and C-11 (δC 84.1) of compound 2 suggested
that the orthobenzoate moiety is attached to different C-ring
positions. From the HMBC correlation of H-12 (δH 5.24, s) to
carbonyl C-1″ (δC 166.6) it was confirmed that a benzoyloxy
group is attached at C-12, as was the case in compound 1. In
addition, strong COSY correlations between H-14 (δH 4.20,
dd) and H-8 at δH 3.40 (d, J = 5.8 Hz) and a proton at δH 3.11
(d, J = 12.5 Hz) showing no correlation in the HSQC
spectrum, together with HMBC correlations from the latter
and C-8, C-13, and C-14, suggested the presence of a hydroxy
group at C-14 (δC 75.6). These results and HMBC correlations
from the 18-methyl protons to the deshielded carbons C-9 and
C-11 suggested that the orthobenzoate unit is attached at C-9,
C-11, and C-13. Coupling constants and NOESY correlations
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Table 1. NMR Data for Compounds 1 and 2 (500 and 125

MHz, CDCl3)
1 2
pos. δC δH (J in Hz) δC δH (J in Hz)
1α 34.7 2.07 m 34.0 1.97 ma
1β 2.27 ddd (14.6, 13.2, 11.5) 2.20 m
2 35.9 1.93 m 36.0 1.99 ma
3 81.9 5.09 d (4.7) 82.7 4.98 ma
4 80.6 80.2
4-OH 3.00 s 3.06 s
5 74.5 4.18 s 73.9 4.19 sa
5-OH 3.94 ma
6 61.4 61.5
7 64.4 3.44 brs 62.9 3.58 s
8 34.8 3.08 d (2.7) 40.8 3.40 d (5.8)
9 84.3 89.1
10 48.2 3.12 dd (13.2, 6.8) 46.8 3.39 ma
11 77.0 84.1
11-OH 3.48 s
12 75.4 5.46 s 68.8 5.24 s
13 86.1 81.8
14 82.2 4.73 d (2.7) 75.6 4.20 ma
14-OH 3.11 d (12.5)
15 141.6 142.8
16-a 114.6 4.98 brs 114.4 4.97 brs
Figure 3. Experimental (black) and calculated (blue) ECD spectra of
codiapeltines A (1) and B (2) in MeOH. Vertical bars represent the 16-b 5.24 brs 5.19 brs
experimental Δε in L·mol−1·cm−1 (left) and the B3LYP/6-31G*- 17 19.9 1.78 s 19.5 1.75 s
computed rotational strengths in 10−40 cgs units (right). 18 24.6 1.54 s 18.1 1.65 s
19 13.5 1.08 d (6.7) 13.6 1.12 d (6.4)
20-a 66.6 3.82 d (12.3) 67.0 3.77 d (12.2)
observed for compound 2 were comparable to 1, allowing its
20-b 3.87 d (12.3) 3.93 d (12.2)
relative configuration to be depicted as shown. TDDFT
1′ 118.2 116.0
calculation of the ECD spectrum of 2 was conducted at the
2′ 134.8 135.4
B3LYP/6-31G* level. The absolute configuration of codia-
3′−7′ 126.6 7.79 m 126.3 7.82 m
peltine B (2) was defined as (2S,3S,4S,5S,6R,-
4′−6′ 128.5b 7.42 m 128.5 7.45 m
7S,8R,9R,10R,11R,12R,13R,14R) by comparison of its exper-
5′ 130.2 7.42 m 130.1 7.45 m
imental and calculated ECD curves (Figure 3).
1″ 166.6 166.6
Unlike all reported diterpenoids sharing the 5−7−6 tricyclic
2″ 129.4 129.5c
ring system, i.e., daphnane- and tigliane-type diterpenoids,
3″−7′′ 130.4 8.09 d (8.1) 130.3 8.04 d (8.0)
codiapeltines A (1) and B (2) bear an unusual but unique C-
4″−6′′ 128.6b 7.40 m 128.6d 7.40 m
ring 9,11,12,13,14-oxygenation pattern. Furthermore, com-
5″ 133.5 7.55 m 133.6 7.55 m
pound 2 is the first daphnane possessing a 9,11,13-orthoester
1‴ 168.1 168.4
linkage, establishing a new major structural class of
2‴ 129.4 129.6c
DDO.23,32,36
3‴−7″′ 129.8 7.97 d (8.1) 129.8 7.96 d (8.1)
The cluster MN2 was composed of ions found exclusively in
4‴−6″′ 128.9b 7.40 m 128.9d 7.40 m
bark extracts of the endemic C. oligogynum and C. peltatum
5‴ 133.9 7.55 m 133.9 7.55 m
species. Based on the molecular formula deduced from the four a b,c,d
protonated ions at m/z 581.219 and 609.253 of MN2, the Overlapping signals. Values are interchangeable.
known actephilol A (5), epi-actephilol A (6), fimbricalyx C
(7), and fimbricalyx D (8), which shared the original dioxane comparable to those of actephilol A (5) but revealed the
phenanthrene dimeric scaffold, were dereplicated and sub- presence of only one methoxy group and two additional
sequently isolated.37−39 Interestingly, these phenanthrene carbonyls at δC 178.6 and 182.4. The structure of compound 3
dimers showed characteristic absorption bands at 224, 247, may be divided in two different monomeric units, A and B
and 290 nm in their UV spectra. This typical UV profile has (Figure 1). While the 1H and 13C NMR chemical shifts of
also been observed for a third analogue (compound 3) that subunit A were close to those of compound 5, those of subunit
was not included in MN2 but appeared in a separated cluster B showed marked differences. The ortho-quinone motif was
in the massive MN representation. As shown in Figure S2, deduced through the observation of the key HMBC
Supporting Information, some spectral differences indicated correlations from H-8′ (δH 7.71, s) to C-6′ and a carbonyl
that 3 was not part of MN2. at δC 178.6 on one hand and from H3-11′ (δH 2.65, s) to C-1′,
Compound 3 was isolated as a brownish-red resin and was C-2′, C-10a′, and a carbonyl at δC 182.4, which established the
assigned the molecular formula C34H28O9 by analysis of its 9′,10′-dione function. Other HMBC correlations fully
positive HRESIMS (m/z 581.1796 [M + H]+, calcd for supported the proposed structure depicted in Figure 2 for
C34H29O9, 581.1806). The spectroscopic data of 3 were closely subunit B.
E DOI: 10.1021/acs.jnatprod.8b00800
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Journal of Natural Products Article

Table 2. NMR Data for Compounds 3 (600 and 150 MHz, DMSO-d6) and 4 (600 and 150 MHz, Methanol-d4)
3 (A unit) 3 (B unit) 4 (A unit) 4 (B unit)
position δC δH position δC δH position δC δH position δC δH
1 47.2 1′ 132.1 1 48.8 1′ 120.3
2 204.2 2′ 140.6 2 207.0 2′ 141.6
3 96.7 3′ 146.3 3 98.5 3′ 141.4
3-OMe 52.1 3.42 s 4′ 110.1 7.23 s 3-OMe 53.0 3.49 s 4′ 108.2 7.74 s
4 72.5 5.71 s 4′a 121.7 4 74.5 5.80 s 4′a 120.7
5 112.1 4′b 135.9 5 116.2 4′b 132.6
6 142.8 5′ 109.7 7.20 s 6 144.6 5′ 107.2 7.64 s
7 103.3 6.74 s 6′ 164.1 7 105.8 6.79 s 6′ 156.8
8 155.2 7′ 126.3 8 156.3 7′ 126.5
9 118.1 8′ 132.1 7.71 s 9 120.5 8′ 125.7 7.93 s
10 123.8 7.93 s 8′a 131.2 10 125.5 8.01 s 8′a 122.2
11 126.0 9′ 178.6 11 128.2 9′ 154.3
12 156.4 9′-OMe 12 159.9 9′-OMe 56.2 4.02 s
12-OMe 10′ 182.4 12-OMe 56.5 3.84 s 10′ 96.8 6.83 s
13 104.0 7.38 s 10′a 124.8 13 101.4 7.35 s 10′a 129.5
14 133.6 11′ 13.4 2.65 s 14 135.4 11′ 11.8 2.61 s
15 28.7 1.35 s 12′ 15.5 2.13 s 15 30.0 1.46 s 12′ 16.9 2.34 s
16 27.9 1.58 s 16 28.9 1.65 s
17 16.7 2.32 s 17 17.1 2.34 s

Table 3. Antimetabolic and Antiviral Activities of Compounds 1−12 in Vero Cells against CHIKV and Their Inhibitory
Activities against RNA-Dependent RNA NS5 of Dengue and Zika Viruses
CHIKV ZIKV DENV
a
compound CC50 (Vero) EC50a SI b
IC50a IC50a
1 52 ± 12 10.0 ± 2.3 5 >69 >69
2 49 ± 17 4.4 ± 0.5 11 >69 >69
3 >172 >172 8.1 ± 0.9 12.1 ± 1.9
4 ndc nd 10.9 ± 2.5 14.5 ± 2.2
5 >172 >86 4.8 ± 0.9 4.5 ± 0.7
6 >129 >172 8.1 ± 1.0 9.1 ± 1.2
7 nd nd 13.6 ± 1.2 21.7 ± 2.0
8 nd nd 12.7 ± 1.3 17.8 ± 3.1
9 >208 >277 >138 >119
10 >288 >288 nd nd
11 >239 >239 >138 >119
12 >277 >277 nd nd
chloroquine 89 ± 28 11 ± 7 8
3′dATP 0.14 ± 0.01 0.07 ± 0.005
a
Data in μM. Values are the median ± absolute deviation calculated from at least three independent assays. bSI or window for antiviral selectivity is
calculated as CC50 Vero/EC50 CHIKV. cnd = not determined.

The relative configuration of compound 3 was established
from the comparison of NMR data with those of compound 5
and ROESY analysis (Figure 2). The cis-configuration of the C-
3−C-4 ring junction was confirmed by a strong ROESY
correlation between 3-OMe (δH 3.42, s) and H-4 (δH 5.71, s).
Actephilol B (3) is the seventh member of the 1,4-dioxane-
fused phenanthrene dimer family.37
Compound 4, displayed in MN2 (Figure 1C), was isolated
as a brownish-red resin, and its HRESIMS data showed a
protonated ion at m/z 595.2316 (calcd for C36H35O8,
595.2326). The spectroscopic data of compound 4 were
close to those of actephilol A (5) and epi-actephilol A (6).
However, a strong HMBC correlation between a methyl group,
MeO-12 (δH 3.84, s), and C-12 (δC 159.9) indicated the
presence of an additional methoxy in position 12. The relative
configuration of actephilol C (4) was supported by the strong
F DOI: 10.1021/acs.jnatprod.8b00800
ROESY correlation between 3-OMe (δH 3.49, s) and H-4 (δH
5.80, s).
As previously reported, the MS-guided purification of
compounds displayed in MN3 afforded the first naturally
occurring monoterpenyl quinolin-4-one isochloroaustralasine
A (12) along with three exceptionally rare chlorinated
monoterpenyl quinolin-2-ones, chloroaustralasines A−C (9−
11, respectively).21
Compounds 1−12 were evaluated for their antiviral activity
in a CHIKV cell-based assay (CPE inhibition) and against
DENV- and ZIKV-NS5 (Table 3). In the CHIKV assay,
compounds 1 and 2 showed anti-CHIKV activities compared
to the reference compound, chloroquine, with EC50 values of
10 ± 2.3 μM (SI = 5) and 4.4 ± 0.5 μM (SI = 11),
respectively. Although the anti-CHIKV activity of DDO
derivatives had already been reported,13,30,31 these results
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Journal of Natural Products
confirmed the predictions made from the multi-informative
MN approach used in this study.
In the DENV and ZIKV assays, results also met our
expectations, as compounds 3−8 exhibited strong inhibitory
activities, although ∼100 times less potent than the reference
compound, 3′-dATP, on both enzymes with IC50 values in the
μM range. To confirm these results, two series of experiments
were conducted. First, the IC50 values of compounds 3−8 were
determined in the presence of detergent, Triton X-100, at
0.01% final concentration. Using a detergent in the assay below
the critical micellar concentration indicates whether a
compound is a potential aggregator for one or more partners
of the assay. Thus, a nonspecific inhibition would induce an
increase of the IC50 value.40 Under these conditions
compounds 4−8 lost their inhibitory potential (IC50 > 100
μM) on both NS5 proteins, while compound 3 showed IC50
values of 36.0 ± 4.7 and 67.2 ± 20.0 μM on ZIKV-NS5 and
DENV-NS5, respectively.
Using an orthogonal assay based on fluorescence polar-
ization, the apparent Kd values of these compounds on ZIKV-
NS5 and DENV-NS5 were determined. This assay shows
whether a compound can have a specific interaction with the
enzyme by measuring its potential to shift the interaction
between the GTP bodipy (boron-dipyrromethene) and the
protein. Compounds 4−8 showed no fixation on the enzyme
(Kdapp > 100 μM). These results, correlated with the loss of
inhibition in the detergent assays, confirmed that these
compounds act as aggregators and not inhibitors.
However, compound 3 showed a Kdapp of 32.1 ± 4.5 and 9.3
± 3.6 μM on ZIKV-NS5 and DENV-NS5, respectively. It
agreed with the detergent assays and indicated a specific
interaction toward the NS5 targets. Taken together, these
results indicate that actephilol B (3) is the first natural dual
inhibitor of ZIKV and DENV NS5.
Time and money spent in tedious bioassay-guided
fractionation and isolation processes represent major bottle-
necks in NP drug discovery. Here, we reported an example of
the multi-informative MN methodology set up to reduce
workload and associated costs of the NP hit detection
workflow. This strategy facilitated the extract prioritization
step by highlighting the original chemical composition of C.
peltatum and also permitted a straightforward MS-guided
purification of targeted compounds. The study of the C.
peltatum bark extract led to the characterization of three
different chemical series. Among the compounds isolated,
codiapeltine B (2) is the first daphnane bearing a 9,11,13-
orthoester moiety, establishing a new major structural class of
daphnane-type diterpenoid orthoesters. Moreover, actephilol B
(3), a member of the rare 1,4-dioxane-fused phenanthrene
dimer family, has been found as the first natural dual inhibitor
of DENV and ZIKV NS5 yet characterized.
Without any prior chemotaxonomic knowledge or biblio-
graphic preconceived assumptions, the multi-informative MN
prioritization approach allows bioactive scaffolds to be spotted.
As a complement to bioactivity observations, the taxonomical
input pinpoints genus- or species-specific metabolites.
Combining biological and chemical profiling results in an
MN environment enables a clear view of the structural breadth
and distribution within taxonomically homogeneous extract
collections and drastically increases the chances of discovering
new lead compounds.
G DOI: 10.1021/acs.jnatprod.8b00800
■
Article
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured at 25 °C on an Anton Paar MCP 300 polarimeter. UV
spectra were recorded on a Varian Cary 100 UV−vis spectropho-
tometer. ECD spectra were measured at 25 °C on a JASCO J-810
spectropolarimeter. NMR spectra were recorded on a Bruker 500
MHz instrument (Avance 500) for compounds 1, 2, 7, and 8, on a
Bruker 600 MHz instrument (Avance 600) for compounds 3 and 4,
and on a Bruker 300 MHz instrument (Avance 300) for compounds 5
and 6. Chemical shifts are in ppm, and coupling constants are in Hz.
Kinetex analytical and semipreparative C18 columns (250 × 4.6 mm
and 250 × 10 mm; 5 μm Phenomenex) and a Nucleodur PFP
semipreparative column (250 × 10 mm; 5 μm Macherey-Nagel) were
used for HPLC separations using a Dionex system equipped with a
sample manager (Gilson 215 liquid handler), a column fluidics
organizer, a binary pump (Dionex HPG-3200BX), a UV−vis diode
array detector (190−600 nm, Dionex UVD340U), and a PL-ELS
1000 ELS Polymer Laboratory detector. HPLC separations were also
undertaken using a Waters Alliance system equipped with a binary
pump (Waters 2525), a UV−vis diode array detector (190−600 nm,
Waters 2996), and an ELS detector (Waters 2424). Disconnected
from ELSD, this system was also used in preparative mode to purify
small amounts of products. All solvents were purchased from Carlo
Erba (France) and SDS (Peypin, France).
Data-Dependent LC-ESI-HRMS2 Analysis. LC analyses were
performed with a Dionex Ultimate 3000 RSLC system equipped with
an Accucore C18 column (2.1 × 100 mm; 2.6 μm, ThermoScientific).
The mobile phase consisted of H2O−MeCN with 0.1% formic acid
(20:80) during 5 min, then a gradient from 20:80 to 100:0 in 20 min,
at a flow rate of 350 μL·min−1. The temperature of the column oven
was set at 30 °C and the injection volume at 5 μL.
LC-ESI-HRMS2 analyses were achieved by coupling the LC system
to an Agilent 6540 hybrid quadrupole TOF mass spectrometer
(Agilent Technologies, Massy, France) equipped with an ESI dual
source, operating in positive-ion mode. Source parameters were set as
follows: capillary temperature at 325 °C, source voltage at 3500 V,
sheath gas flow rate at 7 L.min−1, nebulizer pressure at 30 psi, drying-
gas flow rate at 10 L·min−1, drying-gas temperature at 350 °C, stealth-
gas temperature at 350 °C, skimmer voltage at 45 V, fragmentor
voltage at 150 V, and nozzle voltage at 500 V.
MS scans were operated in full-scan mode from m/z 100 to 1000
(0.1 s scan time) with a mass resolution of 20 000 at m/z 922. An
MS1 scan was followed by MS2 scans of the five most intense ions
above an absolute threshold of 3000 counts. Selected parent ions were
fragmented with collision energy fixed at 35 eV and an isolation
window of 1.3 amu.41
Calibration solution, containing two internal reference masses
(purine, C5H4N4, m/z 121.0509, and HP-921 [hexakis(1H,1H,3H-
tetrafluoropentoxy)phosphazene], C18H18O6N3P3F24, m/z 922.0098),
routinely led to mass accuracy below 2 ppm. LC-UV data were
analyzed with Chromeleon software (Dionex), and MS data
acquisition and processing were performed using MassHunter
Workstation software (Agilent Technologies, Massy, France).
MZmine 2 Data-Preprocessing Parameters. Networks were
generated using the feature-finding-based methodology previously
described.42 The 292 MS2 data files were converted from the .d
(Agilent) standard data format to .mzXML format using the
MSConvert software, part of the ProteoWizard package.43 All
.mzxml were processed using MZmine 2 v31.44 The mass detection
was realized keeping the noise level at 0. The ADAP chromatogram
builder was used using a minimum group size of scans of 4, a group
intensity threshold of 3000, a minimum highest intensity of 4000, and
m/z tolerance of 0.005 (or 20 ppm).45 The ADAP wavelets
deconvolution algorithm was used with the following standard
settings: S/N threshold = 8, minimum feature height = 4000,
coefficient/area threshold = 20, peak duration range 0.05−1 min, tR
wavelet range 0.01−0.07 min. However, these parameters needed to
be reoptimized for a few specific samples in this data set. MS2 scans
were paired using an m/z tolerance range of 0.02 Da and tR tolerance
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
range of 0.5 min. Isotopologues were grouped using the isotopic peak
grouper algorithm with an m/z tolerance of 0.005 (or 20 ppm) and a
tR tolerance of 0.2 min. Peak alignment was performed using the join
aligner module [m/z tolerance = 0.004 (or 10 ppm), weight for m/z =
2, weight for tR = 1, absolute tR tolerance 0.5 min]. The peak list was
gap-filled with the same tR and m/z range gap filler module [m/z
tolerance of 0.004 (or 10 ppm)].
Eventually, the .mgf preclustered spectral data file and its
corresponding .csv metadata file (for tR, areas, and formulas
integration) were exported using the dedicated “Export for GNPS”
and “Export to CSV file” built-in options. See further documentation
and tutorials at https://ccms-ucsd.github.io/GNPSDocumentation/
featurebasedmolecularnetworking/.
Molecular Network Analysis. The molecular networks were
created using the online workflow at Global Natural Products Social
molecular networking (http://gnps.ucsd.edu).46 MS2 spectra were
window-filtered by choosing only the top six peaks in the ±50 Da
window throughout the spectrum. The data were not clustered with
MS-Cluster. A network was created where edges were filtered to have
a cosine score above 0.7 and at least four matching peaks. Further
edges between two nodes were kept in the network if and only if each
of the nodes appeared in each other’s respective top 10 most similar
nodes. The library spectra were filtered in the same manner as the
input data. All matches kept between network spectra and library
spectra were required to have a score above 0.7 and at least 4 matched
peaks.
Plant Material. Stem and bark of Codiaeum peltatum were
collected at “Mont Aoupinié” (New Caledonia) at an altitude of 487
m in November 2001 and authenticated by V. Dumontet. A voucher
specimen (DUM-0109) has been deposited at the Herbier IRD de
Nouméa, New Caledonia.
Extraction and Isolation. The plant material (200 g, dry wt) was
extracted with EtOAc (3 × 0.5 L each). The EtOAc extract was
concentrated in vacuo at 40 °C to yield 1.5 g of residue. This extract
was dissolved in MeCN (0.25 L) and subjected to a liquid/liquid
partition with n-heptane (2 × 0.25 L) to afford 0.6 g of an MeCN-
soluble fraction. This fraction was subjected to a semipreparative
HPLC (Kinetex C18, H2O−MeCN 35:65 + 0.1% formic acid to 0:100
in 45 min at 4.5 mL·min−1) to afford 20 fractions of decreasing
polarity, F1−F20. Purification of fraction F12 (9.2 mg) by
semipreparative HPLC (Nucleodur PFP, H2O−MeCN 45:55 +
0.1% formic acid at 4.5 mL·min−1) afforded compounds 1 (2.5 mg)
and 2 (1.5 mg) (tR: 17.5 and 15.5 min, respectively). Fraction F7 (9.1
mg) was purified by semipreparative HPLC (Nucleodur PFP, H2O−
MeCN 50:50 + 0.1% formic acid at 4.5 mL·min−1) to afford
compound 3 (1.1 mg) (tR: 18.0 min). Fraction F5 (17.4 mg) was
purified by semipreparative HPLC (Nucleodur PFP, H2O−MeCN
30:70 + 0.1% formic acid at 4.5 mL.min−1) to afford compound 4 (1.4
mg) (tR: 13.4 min). Fraction F9 (20.9 mg) was subjected to a
semipreparative HPLC (Nucleodur PFP, H2O−MeCN 45:55 + 0.1%
formic acid at 4.5 mL.min−1) to afford compound 5 (2.9 mg) and
compound 6 (4.1 mg) (tR: 18.5 and 20.5 min, respectively). Fraction
F4 (27.8 mg) was purified by semipreparative HPLC (Nucleodur
PFP, H2O−MeCN 18:82 + 0.1% formic acid at 4.5 mL·min−1) to
afford compounds 7 (0.62 mg) and 8 (0.47 mg) (tR: 11.3 and 12.0
min, respectively).
Codiapeltine A (1): white, amorphous powder; [α]24D −53 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 230 (4.44) nm; 1H and 13C
NMR, see Table 1; HRESIMS m/z 727.2752 [M + H]+ (calcd for
C41H43O12, 727.2749); GNPS ID: CCMSLIB00004681455 ([M +
H]+).
Codiapeltine B (2): white, amorphous powder; [α]24D −43 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 230 (4.46) nm; 1H and 13C
NMR, see Table 1; HRESIMS m/z 727.2749 [M + H]+ (calcd for
C41H43O12, 727.2749); GNPS ID: CCMSLIB00004681456 ([M +
H]+).
Actephilol B (3): brownish-red resin; [α]24D −9 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 224 (4.45), 247 (4.40), 290 (4.31), 337
(3.86) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 581.1796
H DOI: 10.1021/acs.jnatprod.8b00800
Article
[M + H] + (calcd for C 34 H 29 O 9 , 581.1806); GNPS ID:
CCMSLIB00004681457 ([M + H]+).
Actephilol C (4): brownish-red resin; [α]24D +8 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 250 (5.13), 289 (4.86), 317 (4.49) nm; 1H
and 13C NMR, see Table 2; HRESIMS m/z 595.2316 [M + H]+
(calcd for C36H35O8, 595.2326); GNPS ID: CCMSLIB00004681458
([M + H]+).
Actephilol A (5): brownish-red resin; HRESIMS m/z 581.2179 [M
+ H ] + ( c a lc d f o r C 3 5 H 3 3 O 8 , 5 8 1 .2 1 70 ) ; G N P S I D :
CCMSLIB00004681459 ([M + H]+).
Epi-actephilol A (6): brownish-red resin; HRESIMS m/z 581.2176
[M + H] + (calcd for C 35 H 33 O 8 , 581.2170); GNPS ID:
CCMSLIB00004681460 ([M + H]+).
Fimbricalyx C (7): brownish-red resin; HRESIMS m/z 609.2467
[M + H] + (calcd for C 37 H 37 O 8 , 609.2483); GNPS ID:
CCMSLIB00004681461 ([M + H]+).
Fimbricalyx D (8): brownish-red resin; HRESIMS m/z 609.2466
[M + H] + (calcd for C 37 H 37 O 8 , 609.2483); GNPS ID:
CCMSLIB00004681462 ([M + H]+).
ECD Calculation. Computational calculations were performed
using the Gaussian ’09 software package, revision D01.47 Conforma-
tional analyses and geometry optimizations were performed at the
B3LYP/6-31G* level in MeOH. The ECD calculations were
performed using TDDFT at the B3LYP/6-31G* level in MeOH.
The ECD spectra were obtained by weighing the Boltzmann
distribution rate of each geometric conformation with a bandwidth
σ of 0.3 eV. Conformer structures of 1 and 2 and their optimized
coordinates at the B3LYP/6-31G* level in MeOH are available in
Figures S3 and S4, Supporting Information.
Virus-Cell-Based Anti-Chikungunya Assay. The antiviral
activity of the extracts and pure compounds against Chikungunya
virus was determined using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based
cytopathic effect reduction assay. Chikungunya virus (Indian Ocean
strain 899), kindly provided by Prof. C. Drosten (Institute of
Virology, University of Bonn, Germany), was grown on Vero (African
green monkey kidney) cells. Antiviral assays were performed in
DMEM medium [MEM Rega3 (cat. no. 19993013; Invitrogen), 2%
FCS (Integro), 5 mL of 200 mM L-glutamine, and 5 mL of 7.5%
NaHCO3]. Cells were seeded in a 96-well plate (Falcon, BD
Biosciences) and allowed to adhere overnight. Serial dilutions of the
test compounds were made in 100 μL of assay medium in a 96-well
microtiter. Subsequently, 50 μL of a 4× virus dilution in assay
medium was added (MOI 0.01), followed by 50 μL of a cell
suspension. This suspension, with a cell density of 25 000 cells/50 μL,
was prepared from a Vero A cell line subcultured in cell growth
medium (MEM Rega3, supplemented with 10% FCS, 5 mL of L-
glutamine, and 5 mL of NaHCO3) in a 1:4 ratio. The assay plates
were returned to the incubator (37 °C, 5% CO2, 95−99% relative
humidity) for 5 days, a time at which maximal virus-induced cell death
or CPE is observed in untreated, infected controls. Subsequently, the
assay medium was aspirated, replaced with 75 μL of a 5% MTS
(Promega) solution (MTS-phenazinemethosulfate (MTS/PMS)
stock solution [(2 g·L−1 MTS (Promega, Leiden, The Netherlands)
and 46 mg·L−1 PMS (Sigma−Aldrich, Bornem, Belgium) in PBS at
pH 6−6.5)], diluted 1/20 in MEM (Life Technologies, Gent,
Belgium) in phenol-red-free medium, and incubated for 1.5 h.
Absorbance was measured at a wavelength of 498 nm (Safire2,
Tecan), with the optical densities (OD values) reaching 0.6−0.8 for
the untreated, uninfected controls. The optical density values were
converted to control percentages, and logarithmic interpolation was
used to calculate the EC50 and CC50. The EC50 was defined as the
compound concentration that protected 50% of cells from virus-
induced CPE. Adverse effects of the drug on the host cell were also
assessed by means of the MTS method, by exposing uninfected cells
to the same concentrations of the compounds. The CC50 was defined
as the compound concentration that reduced the number of viable
cells by 50%. All assay conditions producing an antiviral effect that
exceeded 50% were checked microscopically for signs of a cytopathic
effect or adverse effects on the host cell (i.e., altered cell or monolayer
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
morphology). A sample was considered to elicit a selective antiviral
effect on virus replication only when, following microscopic quality
control, at least at one concentration no CPE or any adverse effect
was observed (image resembling untreated, uninfected cells). The
antiviral experiments have been performed in a biosafety screening
facility that has been validated for handling of Chikungunya virus as
well as the manipulation of molecules of unknown chemical safety
risk. All studies have been performed by trained staff.
DENV and ZIKV Assays. Products and Reagents. Homopoly-
meric uridine (Poly U) RNA template was obtained from GE
Healthcare. Natural extracts or pure compounds were resuspended at
10 or 1 mg·mL−1 in 100% DMSO and stored at −20 °C. The Quant-it
Picogreen dsDNA assay kit (ref P11496) was obtained from
Molecular Probes-Invitrogen.
Expression and Purification of Zika and Dengue 2 NS5
Recombinant Proteins. The viral NS5 proteins of Zika and Dengue
serotype 2 (DENV-2) virus coding sequences were cloned in fusion
with a N-terminus hexahistidine tag in gateway plasmids (pQE30).
The proteins were expressed in E. coli cells and purified following a
previously described protocol.48
Determination of the Inhibitory Potential of Compounds by a
Picogreen Assay on Zika and Dengue NS5. Principle of the Assay.
A picogreen kinetic assay was based on polymerase activity of NS5
protein, which catalyzes de novo reaction of a poly(U) template and
adenosine triphosphate (ATP). The reaction was carried out at 30 °C
with 50 mM Hepes pH 8, 10 mM DTT, 10 mM KCl, 2 mM MgCl2, 2
mM MnCl2, and the selected concentrations in substrate (Poly U and
ATP) and enzyme. Each reaction assay was robotized on a Biomek
4000 in 40 μL final volume for screening assay and IC50. Control
plates, using only DMSO at 5% final concentration instead of
compounds, were used to determine the Z′ factor, the background,
and the gain value of the assay. Quality of measurements was assessed
by calculating the Z′ factor for each plate: Z′ factor = 1 − ([3SD(neg)
+ 3SD(pos)]/[av(neg) − av(pos)]), where SD(neg) and SD(pos)
stand for the standard deviation obtained for negative and positive
controls, respectively, and where av(neg) and av(pos) are averages for
negative and positive controls, respectively. Assay plates contained
positive and negative controls distributed in the first and the last
columns, respectively. Positive and negative controls consisted of the
reaction mixture supplemented by ATP and 5% DMSO or 20 mM
EDTA, respectively. In the case of the screening assay, 3′dATP was
also used as an internal control in the last column at 10 μM final
concentration. Time incubation (from 10 to 30 min), substrate
concentrations (poly U and ATP), and enzyme concentration were
optimized, based on the previous experimental results to obtain the
best reproducibility (Z′ > 0.6; background <10%, gain value <120).
Screening Assay Determination Based on the Fluorescent
Picogreen Assay. The assay was organized in 96-well Nunc plates,
each containing 80 samples that are natural extracts or pure
compounds. Each of 80 samples was added to the assay using a
BioMek I5 workstation (Beckman) to a final concentration of 50, 10,
or 1 μg·mL−1 and/or 50, 10, and 1 μM in 5% DMSO.
Reactions were conducted in 40 μL final volume. For each assay, 20
μL of the enzyme mix was first distributed in plate wells using a
Biomek 4000 (Beckman), containing 2 μL of the samples. Reactions
were initiated by addition of 18 μL of the nucleotide mix (100 and 80
μM ATP for Zika NS5 and DENV-2 NS5, respectively) and incubated
at 30 °C for 20 and 30 min for Zika NS5 and DENV-2 NS5,
respectively. Assays were stopped by the addition of 20 μL of EDTA
(100 mM).
Reaction mixes were transferred to a Greiner plate using a Biomek
I5 Automate (Beckman). Picogreen fluorescent reagent was diluted to
1/800 in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5, with no
NaCl added) according to manufacturer’s data, and 60 μL of reagent
was distributed into each well of the Greiner plate. The plate was
incubated for 5 min in the dark at room temperature, and the
fluorescence signal was read at 480 nm (excitation) and 530 nm
(emission) using a TecanSafire2.
For each compound, the percentage of inhibition was calculated as
follows: Inhibition % = 100(raw_data_of_compound − av(pos)/
I DOI: 10.1021/acs.jnatprod.8b00800
Article
(av(neg) − av(pos)). Compounds leading to a 70% inhibition or
more at 50 μg·mL−1 and presenting a dose/response effect were
qualified as hits. Selected hits were then confirmed in triplicate, and
IC50 values determined only on pure compounds.
IC50 Determination Based on Fluorescent Picogreen Assay. The
concentrations of the compounds leading to 50% inhibition of NS5-
mediated RNA synthesis were determined in IC50 buffer (50 mM
HEPES pH 8.0, 10 mM KCl, 2 mM MnCl2, 2 mM MgCl2, 10 mM
DTT) containing 100 or 40 nM homopolymeric uridine RNA
template for Zika NS5 and DENV-2 NS5, respectively, seven various
concentrations of compound, and 40 nM of enzyme. For the IC50
determination in the presence of detergent, Triton X100 was added at
0.01% final concentration in the assay. Five ranges of inhibitor were
available (0.01 to 5 μM; 0.1 to 50 μM; 0.5 to 50 μM; 1 to 100 μM; 5
to 400 μM). According to the inhibitory potency of the compound
tested, a range was selected to obtain a more accurate IC50 based on
the best repartition of the points surrounding the range. Reactions
were conducted in 40 μL volume on a 96-well Nunc plate. All
experiments were robotized by using a BioMek 4000 Automate
(Beckman). A 2 μL amount of each diluted compound in 100%
DMSO was added to the wells to the chosen concentration (5%
DMSO final concentration). For each assay, the enzyme mix was
distributed in wells. Reactions were started by the addition of the
nucleotide mix (100 and 80 μM ATP for Zika NS5 and DENV-2
NS5, respectively) and were incubated at 30 °C for 10 min. Reaction
assays were stopped by the addition of 20 μL of EDTA (100 mM).
Positive and negative controls consisted respectively of a reaction mix
with 5% DMSO final concentration or EDTA (100 mM) instead of
compounds. Reaction mixes were then transferred to a Greiner plate
using a Biomek I5 Automate (Beckman). Picogreen fluorescent
reagent was diluted to 1/800 in TE buffer according to the
manufacturer’s data, and 60 μL of reagent was distributed into each
well of the Greiner plate. The plate was incubated for 5 min in the
dark at room temperature, and the fluorescence signal was read at 480
nm (excitation) and 530 nm (emission) using a TecanSafire2. The
IC50 value was determined using the following equation: % of active
enzyme = 100/(1 + I2/IC50), where I is the concentration of inhibitor
and 100% activity is the fluorescence intensity without inhibitor. IC50
was determined from curve-fitting using Prism software. For each
value, results were obtained using triplicates in a single experiment.
Apparent Kd Determination of Compounds by a Fluorescence
Polarization Assay. The concentration of compounds leading to a
shifting of the fixation of GTP bodipy to the D2 or Zika NS5 protein
was determined in the binding buffer (50 mM Tris pH 7.5, 50 mM
NaCl, 0.01% NP40, 1 mM DTT) containing 50 nM GTP bodipy, 300
nM D2 NS5, or 150 nM Zika NS5 and seven various concentrations
of compound. Reactions were conducted in 50 μL volume on a black
96-half-well Greiner plate. Each diluted compound (2.5 μL) in 100%
DMSO was added in plate wells to the chosen concentration (5%
DMSO final concentration). For each assay, 10 μL of a 5×
concentration of the enzyme was distributed in plate wells. Reactions
were started by the addition of the GTP bodipy (10 μL of a 5×
concentration) and were incubated at room temperature for 15 min.
Total fluorescence and fluorescence polarization were measured using
a microplate reader (Pherastar) with a fluorescence polarization
module (485 nm excitation and 520 emission wavelengths).
Instrument settings were fixed as follows: 100mP target mP, 200
flashes per well, 0.5 s settling time. Focus and gain values were
adjusted prior to measurement.
The positive control consisted of a reaction mix with 5% DMSO
final concentration in place of the compound. GTP bodipy alone (n =
8) was used to determine the minimal fluorescence polarization
intensity, and the average of this background value was subtracted
from each value of the assay. The fluorescence polarization of the
positive control is 100% fixation, and the residual fluorescence
polarization signal (mP %) is determined for each concentration of
the compound. The apparent Kd (Kdapp) of the compound was
determined from curve-fitting using Prism software. For each value,
results were obtained using triplicates in a single experiment.
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00800.
1
H, 13C, HSQC, HMBC, COSY, and ROESY NMR
spectra and HRESIMS data for compounds 1−8;
conformer structures of 1 and 2 and their optimized
coordinates at the B3LYP/6-31G* level in MeOH
(PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +33 1 69 82 30 85. E-mail: marc.litaudon@cnrs.fr.
ORCID
David Touboul: 0000-0003-2751-774X
Fanny Roussi: 0000-0002-5941-9901
Marc Litaudon: 0000-0002-0877-8234
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors are grateful to the North Province of New
Caledonia, which has facilitated our field investigation, and V.
Dumontet for his contribution to plant material identification
and species collection. We thank C. Collard and C.
Vanderheydt for their excellent technical assistance in the
acquisition of the antiviral data. This work has benefited from
an “Investissement d’Avenir” grant managed by Agence
Nationale de la Recherche (CEBA, ref ANR-10-LABX-25-
01). The tree and leaves icons in the graphical abstract were
created by Sayan Mukerjhee, Alice Noir, Iejank, Lance
Hancock, and LSE Designs from the Noun Project.
■
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