Antischistosomal Properties of Sclareol and Its Heck-Coupled Derivatives: Design, Synthesis, Biological Evaluation, and Untargeted Metabolomics

Article abstract of DOI:10.1021/acsinfecdis.9b00034

Sclareol, a plant-derived diterpenoid widely used as a fragrance and flavoring substance, is well-known for its promising antimicrobial and anticancer properties. However, its activity on helminth parasites has not been previously reported. Here, we show that sclareol is active against larval (IC₅₀ ≈ 13 μM), juvenile (IC₅₀ = 5.0 μM), and adult (IC₅₀ = 19.3 μM) stages of Schistosoma mansoni, a parasitic trematode responsible for the neglected tropical disease schistosomiasis. Microwave-assisted synthesis of Heck-coupled derivatives improved activity, with the substituents choice guided by the Matsy decision tree. The most active derivative 12 showed improved potency and selectivity on larval (IC₅₀ ≈ 2.2 μM, selectivity index (SI) ≈ 22 in comparison to HepG2 cells), juvenile (IC₅₀ = 1.7 μM, SI = 28.8), and adult schistosomes (IC₅₀ = 9.4 μM, SI = 5.2). Scanning electron microscopy studies revealed that compound 12 induced blebbing of the adult worm surface at sublethal concentration (12.5 μM); moreover, the compound inhibited egg production at the lowest concentration tested (3.13 μM). The observed phenotype and data obtained by untargeted metabolomics suggested that compound 12 affects membrane lipid homeostasis by interfering with arachidonic acid metabolism. The same methodology applied to praziquantel (PZQ)-treated worms revealed sugar metabolism alterations that could be ascribed to the previously reported action of PZQ on serotonin signaling and/or effects on glycolysis. Importantly, our data suggest that compound 12 and PZQ exert different antischistosomal activities. More studies will be necessary to confirm the generated hypothesis and to progress the development of more potent antischistosomal sclareol derivatives.

Full text of DOI:10.1021/acsinfecdis.9b00034

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Article
Anti-schistosomal properties of Sclareol and its Heck-coupled derivatives:
design, synthesis, biological evaluation and untargeted metabolomics
Alessandra Crusco, Helen Whiteland, Rafael Baptista, Josephine E. Forde-Thomas, Manfred
Beckmann, Luis A.J. Mur, Robert J Nash, Andrew D. Westwell, and Karl F. Hoffmann
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00034 • Publication Date (Web): 13 May 2019
Downloaded from http://pubs.acs.org on May 17, 2019
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Anti-schistosomal properties of Sclareol and its Heck-coupled derivatives: design,
synthesis, biological evaluation and untargeted metabolomics
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Alessandra Crusco a,b, Helen Whiteland , Rafael Baptista , Josephine E. Forde-Thomas ,
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Manfred Beckmann , Luis A. J. Mur , Robert J. Nash , Andrew D. Westwell * and Karl F.
Hoffmann a*
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Institute of Biological, Environmental and Rural Sciences (IBERS), Penglais Campus,
Aberystwyth University, Aberystwyth SY23 3DA, United Kingdom
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School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3NB,
United Kingdom
c PhytoQuest Limited, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB Wales, UK
* Authors for correspondence:
KFH, e-mail: krh@aber.ac.uk
ADW, e-mail: WestwellA@cf.ac.uk,
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Sclareol, a plant-derived diterpenoid widely used as a fragrance and flavouring substance, is
well known for its promising antimicrobial and anticancer properties. However, its activity on
helminth parasites has not been previously reported. Here, we show that sclareol is active
against larval (IC50 ≈ 13 µM), juvenile (IC50 = 5.0 µM) and adult (IC50 = 19.3 µM) stages of
Schistosoma mansoni, a parasitic trematode responsible for the neglected tropical disease
schistosomiasis. Microwave-assisted synthesis of Heck-coupled derivatives improved activity,
with the substituents choice guided by the Matsy decision tree. The most active derivative 12
showed improved potency and selectivity on larval (IC50 ≈ 2.2 µM, SI ≈ 22 in comparison to
HepG2 cells), juvenile (IC50 = 1.7 µM, SI = 28.8) and adult schistosomes (IC50 = 9.4 µM, SI =
5.2). Scanning electron microscopy studies revealed that compound 12 induced blebbing of the
adult worm surface at sub-lethal concentration (12.5 µM); moreover, the compound inhibited
egg production at the lowest concentration tested (3.13 µM). The observed phenotype and data
obtained by untargeted metabolomics suggested that compound 12 affects membrane lipid
homeostasis by interfering with arachidonic acid metabolism. The same methodology applied to
praziquantel (PZQ)-treated worms revealed sugar metabolism alterations that could be ascribed
to previously reported action of PZQ on serotonin signalling and/or effects on glycolysis.
Importantly, our data suggest that compound 12 and PZQ exert different anti-schistosomal
activities. More studies will be necessary to confirm the generated hypothesis and to progress
the development of more potent anti-schistosomal sclareol derivatives.
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Keywords: diterpenoids, schistosomiasis, anthelmintic, sclareol, microwave synthesis,
untargeted metabolomics
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Sclareol is a labdane diterpenoid widely used as a fragrance1 as well as a food flavouring
substance2 and is generally considered safe for topical and oral administration.3 Originally
isolated from Salvia sclarea, sclareol is also found in essential oils derived from other Salvia spp
which are thought to be produced in response to infection by bacteria and fungi.4 These plant-
defence properties have led to further mechanistic investigations, which have demonstrated
sclareol’s potency as an antibacterial,5 antifungal6 and anticancer agent.7–10 However, to the
best of our knowledge, the activity of sclareol against parasitic helminths has not been
previously investigated. As part of our ongoing efforts in identifying and characterising
anthelmintic phytochemicals, we now report the activity of sclareol and new synthetic analogues
against the causative helminth responsible for schistosomiasis.
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Schistosomiasis is a chronic, debilitating helminthiasis caused by parasitic trematodes within the
genus Schistosoma and currently affects approximately 600 million people.11 This disease is
predominantly found in tropical areas of the world and, with up to 300,000 deaths per year, is
considered the most deadly neglected tropical disease (NTD).12 In the absence of a prophylactic
vaccine, schistosomiasis is primarily controlled by a single drug, praziquantel (PZQ). This
pyrazino-isoquinolone is active against the adult stages of all human-infective Schistosoma spp,
but not on immature forms and, therefore, requires repeat administrations to maximise
efficacy.13 Furthermore, the fear of selecting for PZQ insensitive or resistant parasites drives the
need to identify new anti-schistosomal drugs with a different mechanism of action to PZQ. The
identification of such compounds will contribute to the sustainable control of schistosomiasis into
the future.14
Towards this end, several diterpenoids with anti-schistosomal activity have been previously
reported,15–17 including several from our group.18,19 The collective results of these investigations
demonstrate that this molecule class could be a promising source of next generation
anthelmintic. In this study, the anti-schistosomal activity of sclareol was demonstrated on larval,
juvenile and adult stages of Schistosoma mansoni and this activity was considerably improved
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by the creation and testing of semi-synthetic derivatives. Based on initial structural-activity
relationships (SARs) derived from a first group of analogues, different Heck-coupled derivatives
were synthesised based on a previously reported synthetic route20 and significantly improved by
microwave-assisted synthesis. The choice of substituents was guided by the Matsy decision
tree,21 an updated version of the more classical Topliss scheme.22 This process led to the
synthesis of the most active compound 12 (IC50 ≈ 2.2 µM for schistosome larvae, SI > 20 in
comparison to HepG2 cells; IC50 = 1.7 µM for juveniles, SI = 28.8; IC50 = 9.4 µM for schistosome
adults, SI = 5.2). The effects of compound 12 on schistosome phenotype were investigated by
Scanning Electron Microscopy (SEM), revealing tegumental alterations at a sub-lethal dose,
characterised by swelling and bubble-like protrusions. Untargeted metabolomics of treated adult
worms revealed significant alterations in lipid balance and, in particular, arachidonic acid (ARA)
metabolism. These results are consistent with the previously reported effects of ARA on
schistosome surface membranes.23 Furthermore, the effect of compound 12 on schistosome
metabolism is quite distinct to that induced by PZQ where alteration of sugar metabolism was
predominantly observed. This metabolomics profile adds mechanistic support to the activity of
PZQ in modulating serotonin signalling24,25 and/or glycolysis25,26 in schistosomes. The overall
results of sclareol derivative synthesis, bioactivities and mechanism of action investigations
(distinct from PZQ) are presented and discussed here.
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RESULTS AND DISCUSSION
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Anthelmintic activity of (-)-sclareol.
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The activity of a small collection of plant-derived diterpenoids (see Supplementary Information,
S1) on S. mansoni schistosomula (larvae), using the high throughput screening platform
Roboworm,18,27 identified anthelmintic properties among labdane-type diterpenoids. Based on
this finding, we decided to ascertain the anthelmintic activity of enantiopure (-)-sclareol (Figure
1), the commercially available labdane diterpenoid (Figure 1A) more structurally-related to the
labdane hits in S1 (same condensed rings, open chain and allylic alcohol group) and widely
used in the cosmetic1 and food2 industries due to its excellent safety profile. Indeed, in vivo
toxicity studies in mice have demonstrated a LD50 > 5000 mg/kg for both topical and oral
delivery routes and a LD50 = 1000 mg/kg for the parental route.3 Moreover, as sclareol is also
well known for its promising antimicrobial and antifungal activities,5,6 we postulated that this
labdane diterpenoid possessed biological properties (low toxicity and anti-infective nature) to
warrant anthelmintic investigations. Therefore, sclareol’s ability to affect motility and phenotype
of S. mansoni schistosomula was first assessed (final concentrations of 50 - 1 µM) by the
Roboworm platform18,27 (Figure 1B). Here, this diterpenoid demonstrated a dose-dependent
anti-schistosomal activity with an IC50 of 14.2 µM for phenotype and 12.3 µM for motility (Figure
1B, 1F). Sclareol was then screened against juvenile (final concentrations of 50 - 0.8 µM) and
adult worms (final concentrations of 50 - 3.13 µM) where it negatively affected worm motility
(IC50 of 5.0 and 19.3 µM respectively) (Figure 1C, 1D, 1F) and inhibited production of the
pathogenic eggs at the lowest concentration tested (Figure 1E). We additionally confirmed the
low toxicity of sclareol3 on human HepG2 cells (CC50 = 74.1 µM) (Figure 1F). Considering the
relative low toxicity of sclareol, we decided to pursue further medicinal chemistry studies on this
diterpenoid to improve its anthelmintic characteristics.
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Figure 1. Structure and anti-schistosomal activity of (-)-sclareol. A) Structure and scaffold
numbering system of the diterpenoid (-)-sclareol. B) Sclareol affects the phenotype and motility
of S. mansoni schistosomula in a dose dependent manner (50 - 1 µM). Schistosomula co-
cultivated with negative (0.625 % DMSO) and positive (Auranofin 10 µM, 0.625% DMSO)
controls are also indicated. The screening was performed by the high throughput screening
platform Roboworm18,27 with each point being the average of two replicates (each replicate
contains 120 parasites/well). Hit compounds (within the hit threshold) affect ≥ 70% of the larvae.
Z’-scores28: 0.69 for phenotype and 0.48 for motility. C) Sclareol affects the motility of mixed-sex
S. mansoni juvenile worms in a dose dependent manner (50 – 0.8 µM) and of D) mixed-sex S.
mansoni adult worms (50 - 3.13 µM). Scores were calculated according to the Methods. Each
point represents a single worm (10 juveniles x 2 independent experiments, 3 worm pairs x 2
independent experiments) with mean +/- SD illustrated. E) Sclareol inhibits egg production of S.
mansoni adult worm couples when compared to control treatment (0.00). Values are mean +/-
SD of egg counts from 3 worm pairs x 2 independent experiments. F) Estimated IC50s (µM)
calculated from dose response curves on schistosomula phenotype and motility, on juvenile and
adult worm motility, estimated CC50 on HepG2 human liver cell and Selectivity Index (SI).
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Synthesis of sclareol derivatives
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To quantify structural activity relationships and improve upon the moderate anthelmintic activity
of sclareol, a small group of analogues was first synthesised (Figure 2). Oxidation of sclareol
with KMnO4 and MgSO4 in acetone29 led to the synthesis of compound 1, with the allylic alcohol
substituted by a keto group. Dehydration of compound 1 with I2 in toluene29 led to compound 2.
A further derivative of sclareol was obtained by acetylation of both alcoholic functions by acetyl
chloride in the presence of N,N-dimethylaniline in dichloromethane,30 giving compound 3, and
subsequent selective hydrolysis by KOH in ethanol30 leading to compound 4. The latter, was
subjected to an E1 elimination by using NaHCO3 in DMSO, as described by Rogachev et al.,30
with some modifications using microwave conditions (200 °C, 100 W, 11 min); this procedure
led to the synthesis of compound 5 as a geometric isomeric mixture (2:1 exo:endo isomer
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mixture, as determined by H-NMR integration, see Supplementary information, S2). The
obtained dehydrated derivative was subjected to the Heck-reaction,20 with yields and reaction
times improved by microwave conditions (130°C, 300 W, 12 min), leading to compound 6. The
same Heck-reaction was performed directly on sclareol, obtaining the direct derivative
compound 7.
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R
X
4
5
O
O
HO
6
7
OH
OH
A2
8
9
H
H
H
7
8
R = H
R = 4-Cl
y: 92%
y: 90%
y: 87%
y: 90%
y: 89%
y: 86%
y: 86%
A1
2
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C1
HO
y: 61%
y: 84%
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R = 3,4-diCl
R = 4-OCH3
R = 3-Cl
R = 4-F
R = 4-CF3
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OH
B1
H
R = 4-F; X = 4-F-C6H4 y: 10%*
(-)-Sclareol
HO
HO
HO
AcO
B2
B4
OAc
OAc
B3
H
H
H
H
6
3
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5
y: 91%
y:92%
y:52%
y: 68%
A1: KMnO4 / MgSO4 / acetone / rt / overnight A2: I2 / toluene / reflux / 3 h B1: AcCl / N,N-DMA / DCM / rt / 24 h B2: KOH / H2O /
EtOH / rt / 24 h B3: NaHCO3 / DMSO / 100 W / 200 °C / 11 min (microwave-assisted) B4 = C1: Ar-B(OH)2 / Pd(OAc)2 / Cu(OAc)2 /
NaOAc / DMF / 130 °C / 300 W / 12 min (microwave-assisted; *classic thermal for 14 at 80°C, 6 h). y = yield.
Figure 2. Derivatives of sclareol. Structures and synthetic scheme of the sclareol derivatives.
When not specified, X = H.
To investigate the impact of these changes on anthelmintic activity, this first small set of
compounds (1 - 7) was screened against the schistosomula stage of S. mansoni. Compounds 1
and 2 lost their potency (Table 1), showing that the allylic portion is important for anthelmintic
activity. The bis-acetylated compound 3 was inactive too, while the mono-acetylated compound
4 recovered activity, comparable to sclareol, showing again that the free allylic alcohol is
essential. The importance of the allylic alcohol for activity was also confirmed by comparison
with the previous diterpenoid collection screening results (see Supplementary information, S1),
where the two hit compounds S6 and S8 share this feature, while similar compounds presenting
an inverted allylic alcohol (S2, S10) or replacement of the function (S3, S9) were not active.
Compound 5 showed comparable activity to compound 4 (and to its plant-derived reference S6),
while the addition of a phenyl ring in position 15 led to loss of activity in compound 6.
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Interestingly, this did not happen when the addition was made directly on sclareol to obtain
compound 7, which showed an almost 3-fold improved activity (IC50 ≈ 5.5 µM) over sclareol.
Thus, we decided to pursue the synthesis of new Heck-coupled derivatives (compounds 7 - 13)
with different phenyl substituents. The previous described synthesis route,20 using Pd(OAc)2,
Cu(OAc)2, NaOAc in DMF at 80 °C, was followed and improved by microwave heating at 130
°C, leading to improved yields (from ~ 60% to ~ 90%) and improved reaction times (from 3 - 6 h
to 11 min). Under microwave conditions, mono-coupled products (7 - 13) with trans
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configuration, as shown by the H-NMR coupling constants of the alkene (J ≈ 16 Hz), were
obtained; when using classic thermal synthesis, Heck-coupling led to the bis-arylated products
(14) in addition to mono-coupled product (12).
To maximise the chance of finding more potent analogues against schistosomula while reducing
the number of synthesis steps, the Matsy decision tree,21 an updated version of the classical
Topliss tree,22 was followed (Figure 3). The first phenyl ring substitution with chlorine in the 4-
position (compound 8) improved anti-schistosomula activity of compound 7 to an IC50 ≈ 3.5 µM.
The 3,4-dichloro-substituted compound 9 was then synthesised as suggested by the Matsy tree,
but this led to decreased activity. Subsequently, the far right branch of the tree was followed,
leading to the synthesis of 4-OMe (compound 10) and 3-Cl (compound 11) substituted
analogues (the first with a solubility problem, the second with lower activity) as well as a 4-F
analogue (compound 12), which showed the most potent activity (IC50 ≈ 2.2 µM, 6-fold more
potent than sclareol). Taking into account the increased activity brought by the fluorine group, a
4-CF3 derivative was also synthesised (compound 13); the double addition side product
generated from the classical reaction of compound 12 (compound 14) was also tested. While
compound 13 was less potent, the activity of the side product (compound 14) was more potent
(IC50 ≈ 1.3 µM). The greater anti-schistosomula activity associated with the double addition side
product 14 could be correlated with increased lipophilia compared to the correspondent single
addition product 12, which in turn is more lipophilic and potent when compared to sclareol
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lacking a phenyl ring (calculated logP 7.9 > 6.4 > 4.7, respectively). Indeed, increased lipophilia
could be responsible for a greater passage of the compound through the schistosomula
membrane and, therefore, for better activity. However, changes in lipophilia brought about by
phenyl substituents did not always follow this correlation (e.g. 8 is more potent than 9, although
calculated logP are 6.8 and 7.4, respectively) and suggest that other factors likely contribute.
However, when compounds were tested on juvenile and adult worms, as fully described in the
next paragraph, compound 12 was the most potent.
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Table 1. Anti-schistosomal and HepG2 cytotoxicity activities
IC50 on
SI on
schistosomula
phenotype /
motility
IC50 on
juvenile
worms (µM)
CC50 on
HepG2 cells
(µM)
SI
SI
on adult
worms
schistosomula
phenotype /
motility (µM)
IC50 on adult
worms (µM)
Compounds
on juvenile
worms
Sclareol
14.2 / 12.3
>25
5.5
NA
NA
NA
NA
NA
NA
2.0
6.5
NA
NA
11.2
1.7
NA
6.3
19.3
NA
74.1
>100
>100
>100
>100
85.1
74.1
43.7
42.7
93.3
>100
53.7
49.0
63.1
42.7
5.2 / 5.9
NA
13.5
NA
NA
NA
NA
NA
NA
21.9
6.7
NA
NA
4.8
28.8
NA
6.8
3.8
NA
NA
NA
NA
NA
NA
2.3
3.4
NA
NA
1.9
5.2
NA
2.6
1
2
10
11
12
13
14
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>25
NA
NA
3
>25
NA
NA
4
11.6 / 12.1
12.9 / 10.4
>25
NA
NA
5
NA
6.6 / 8.2
NA
6
NA
7
5.7 / 5.3
3.5 / 3.6
27.6 / 10.6
>25
18.6
12.7
NA
7.7 / 8.2
12.2 / 11.9
3.4 / 8.8
NA
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9
10 *
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12
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14
NA
8.2 / 5.4
2.1 / 2.3
26.0 / 19.8
1.4 / 1.3
27.7
9.4
6.5 / 9.9
23.3 / 21.3
2.4 / 3.2
30.5 / 32.8
NA
16.4
Estimated IC50s (µM) calculated from dose response curves for S. mansoni schistosomula
phenotype/motility and S. mansoni adult and juvenile worm motility. Estimated CC50s (µM)
calculated from dose response curves for HepG2 cells and Selectivity Indices (SI) of the
anthelmintic activity when compared to the cell line. The values are mean results of experiments
in duplicate for helminths or triplicate for cells (IC50s and CC50s with 95% confidence intervals in
Supplementary Information, S3). * Solubility problem.
Figure 3. Matsy decision tree and pathway followed to generate more active anti-
schistosomal compounds. The figure shows the Matsy decision tree by Boyle et al.,21 an
updated version of the classic Topliss decision tree. According to the increased or decreased
activity brought by the different aromatic substituents, a pathway is followed leading to the
choice of new substituents. The pathway followed in this study is highlighted in green.
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Anthelmintic and cytotoxic screening of derivatives
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As described above, to help inform the progression of hit compound identification, compounds
were first titrated (50 to 1 µM) on S. mansoni schistosomula by using the automated platform
Roboworm, which scores parasites based on their phenotype and motility.18 A follow-on
secondary screen of all synthesised compounds was subsequently completed to validate these
results and to compare anti-schistosomal potency at 10 µM (Figure 4). In addition to this, all
compounds were assessed for overt cytotoxicity by screening against the human HepG2 liver
cell line (collective results shown in Table 1).
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Figure 4. Screening of sclareol derivatives on S. mansoni schistosomula at 10 µM. A) The
synthesised derivatives (at 10 µM) were assessed for their ability to affect phenotype and
motility of S. mansoni schistosomula when compared to the original sclareol (Scl, 10 µM) as well
as to the negative (0.625% DMSO) and positive (Auranofin 10 µM, 0.625% DMSO) controls.
The screening was performed by the high throughput platform Roboworm as previously
described.18 Hit compounds (within the hit threshold) affect ≥ 70% of the larvae. Each point
represents the average score of two replicates. Z’-scores28: 0.41 for phenotype and 0.23 for
motility. B) Images of the parasites after treatment with the controls and the five hits at 10 µM
are also shown. Control schistosomula presented a normal phenotype and high motility
(indicated by change in red outlines), while affected schistosomula presented an altered
phenotype (change in shape and darkening) and low or absent motility (no change in red
outlines).
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Compounds with IC50 values on schistosomula below 10 µM (7, 8, 11, 12 and 14) were then
selected for follow-on experiments against juvenile and adult schistosomes (Table 1;
Supplementary information, S4 and S5). Similar to the anti-schistosomula screens, compound
12 showed the greatest activity (IC50 of 1.7 µM on juveniles, 9.4 µM on adults; Table 1) and
inhibited egg production (despite worm recovery) at all concentrations tested (Figure 5).
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Figure 5. Screening of compound 12 on S. mansoni juvenile and adult worms. Compound
12 affects the motility of A) S. mansoni juvenile worms (50 – 0.8 µM) and B) S. mansoni adult
worms (50 – 3.13 µM). Scores were calculated according to the Methods. Each point represents
a single worm (10 juveniles x 2 independent experiments, 3 worm pairs x 2 independent
experiments) with mean +/- SD shown. C) Compound 12 inhibits egg production (3 worm
pairs/well x 2 replicate experiments) until the lowest concentration tested (3.13 µM) when
compared to the DMSO control (0).
Phenotypes of adult worms treated with a sub-lethal concentration (12.5 µM) of compound 12
were analysed by SEM (Figure 6). The surface of treated parasites was altered when compared
to the DMSO control and, in particular, blebbing, swelling of the tegument and bubble-like
protrusions were observed on both male and female worms (Figure 6A and 6C compared to 6B
and 6D). This suggested that the anti-schistosomal activity of compound 12 could be related to
membrane or tegumental disruptions, as previously reported for other terpenoids.16,17,31
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Figure 6. Scanning electron microscopy (SEM) images of S. mansoni worms co-
cultivated with compound 12. SEM images of untreated adult male (A) and female (C) worms
compared to male (B) and female (D) worms treated with compound 12 at 12.5 µM. The surface
of the treated parasite appears damaged when compared to the control and the presence of
bubble-like protrusions and swelling is evident.
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Untargeted metabolomics
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To further investigate the mode of action of compound 12, analysis of metabolomic changes
induced in adult worms co-cultured at its IC50 value was performed as described in the Methods,
adapting the procedure applied on bacteria by Baptista et al.32 and using MetaboAnalyst 4.033
for metabolite comparative quantification and annotation. Flow infusion electrospray ionisation
high resolution mass spectrometry (FIE-HRMS) was used to profile extracted metabolites
derived from treated (compound 12 vs PZQ) and control S. mansoni adult worms. Unsupervised
principal component analysis (PCA) revealed that the metabolomics profile of worms treated
with compound 12 is quite distinct from the metabolic profile of PZQ-treated and untreated
worms, suggesting a different mode of action (Figure 7).
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Figure 7. Principal component analysis (PCA) of S. mansoni adult worm metabolome.
PCA score plots (positive and negative ionisation mode together, coloured regions display 95%
confidence interval) of normalised m/z intensities of metabolites extracted from S. mansoni
adult worms after 24 h treatment with compound 12 (12, in red) at IC50 concentration (9.4 µM)
compared to worms treated with DMSO (0.625%) (D, in green) and to worms treated with PZQ
(P, in blue) at IC50 concentration (30 nM; Supplementary Information, S6).
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Metabolites responsible for separation between compound 12 treated and control worms
(DMSO) in PCA were identified using MetaboAnalyst 4.0.33 A total of 2123 m/z FIE-HRMS
features were found to be significantly different (p < 0.01) between the two samples considering
positive and negative ionisation mode together (1210 and 913, respectively). MetaboAnalyst 4.0
- MS peaks to pathway was used to identify these variables. The software used the mummichog
algorithm, which allows avoidance of the a priori identification of metabolites and the biased
manual assignment of spectral features to metabolites34 by looking for local enrichment after
plotting all possible matches in the metabolic network. This method provides accurate
reproduction of true activity, as the false matches distribute randomly.34 The run analysis
revealed that arachidonic acid (ARA), pentose phosphate, pyrimidine, amino sugar and fructose
and mannose metabolism were all significantly affected in compound 12 treated worms (Table
2A for significant pathways, Supplementary Data SD1-2 for all identified metabolites SD3-4 for
all identified pathways).
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Table 2. Significantly affected pathways in adult S. mansoni worms after 24h treatment with
compound 12 and PZQ (compared to DMSO control worms)
A. Compound 12 vs control worms
Pathway Hits tot.
total
Hits sig.
EASE
FET
Gamma
Arachidonic acid metabolisma
Pentose phosphate pathwayb
Pyrimidine metabolismb
Amino sugar and nucleotide sugarb
Fructose and mannose metabolismb
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0.10996 0.01598 0.00013
0.03670 0.00711 0.00008
0.05077 0.01550 0.00009
0.05509 0.01460 0.00009
0.09049 0.02083 0.00012
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B. PZQ vs control worms
Pathway
Hits tot.
Hits sig.
EASE
FET
Gamma
total
25
Glycolysis or Gluconeogenesisb
Citrate cycle (TCA cycle)b
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0.00830 0.00008
0.00890 0.00010
0.03936
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Fructose and mannose metabolismb
Pentose and glucuronate interconversionsb
Pentose phosphate pathwayb
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0.00890 0.00010
0.06189
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0.09122 0.01435 0.00012
0.11081 0.02864 0.00013
0.11081 0.02864 0.00013
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Alanine, aspartate and glutamate metabolismb
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a From metabolites identified in positive ionisation mode
b From metabolites identified in negative ionisation mode
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Pathways identifications were performed by using MetaboAnalyst 4.0,33 as described in the
Methods section, after analysis and identification (tolerance = 3ppm) of m/z features obtained
by high resolution mass spectrometry. The tables includes ranked enriched pathways, total
number of hits, significant hits (p < 0.05), their EASE (Expression Analysis Systematic Explorer)
score,35 their raw p-values (Fisher’s exact test, FET) and the p-values using a Gamma
distribution.
Within the positive ionisation mode, the only pathway found to be significantly affected by
compound 12 was ARA metabolism. Specifically, ARA and its downstream derivatives
prostaglandin (PG) E2 and/or D2 were found in higher abundance within the metabolite pool of
compound 12 treated worms when compared to controls (Figure 8A). Interestingly, exogenously
administered or elevated levels of host derived ARA have previously been shown to kill
schistosomes in vitro as well as in vivo.23,36 Because of this and an excellent safety profile, ARA
has recently undergone clinical trials in school children within highly endemic regions.37,38 While
ARA led to worm reductions similar to PZQ in both lightly- or heavily-infected children, it
potentiated the effect of this anthelmintic when co-administrated to both study populations. The
proposed mechanism of ARA action on schistosome viability has been reported to function
through activation of the tegument-associated neutral sphingomyelinase, nSMase; this enzyme
is responsible for sphingomyelin hydrolysis and a consequent increase in membrane
permeability and alteration of lipid balance, as well as exposure of surface antigens to antibody
binding.23,38,39 Interestingly, the reported phenotype for ARA-treated worms includes tegumental
disruption and the formation of “bubble-like lesions”,23 very similar to what was observed in
compound 12 treated worms. Indeed, sphingomyelin hydrolysis is associated with internal
release of ceramide,40 signalling molecules that have been previously associated with
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membrane blebbing and apoptosis.41 Collectively, these observations seem to suggest that
compound 12 affects schistosome survival by interfering with either ARA metabolism and/or
PGE2/D2 homeostasis. Although PG-producing cyclooxygenases have not yet been identified in
schistosomes,42 prostaglandins are a major component of the schistosome lipidome43 and are
thought to be involved in host immune system evasion44 as well as in cercarial penetration.45
Whether these altered PGE2/PGD2 levels, induced by compound 12, would also affect
schistosome survival in vivo is currently unknown. However, this hypothesis seems plausible
due to the importance of these downstream ARA products in modulating host interactions.
Finally, and similar to the phenomenon observed for other diterpenoids on human cells,46,47 the
lipophilic structure of compound 12 may directly affect schistosome survival by altering PG and
lipid balances essential for normal maintenance of the worm’s heptalaminate barrier.
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Figure 8. Significant affected key-metabolites found in treated schistosomes. Some key
metabolites with significantly (*p < 0.01, **p < 0.001) different concentrations found between
control and treatment. A) Compound 12 treatment induces increased production of arachidonic
acid and prostaglandins (e.g. PGE2 or PGD2 level; as the two prostaglandins are structural
isomers with the same exact mass and belonging to the same enriched pathway, the algorithm
is not able to distinguish between them). B) PZQ-treatment induces increased production of
glucose and lactate, while levels of glucose 6-phosphate were decreased.
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When examining the compound 12 induced pathways significantly enriched in the negative
ionisation mode, the involvement of sugar metabolism was evident. In particular, pentose
phosphate pathway (PPP) metabolites were less abundant. As one of the main functions of the
PPP is to generate metabolites essential for nucleic acid synthesis, it was not surprising that
nucleotides and nucleotide sugars were also less abundant in compound 12 treated
schistosomes. The other main function of the PPP is in the synthesis of NADPH that, in
mammals, is necessary for fatty acid synthesis; therefore, the PPP would be downregulated
when fatty acids are in abundance. Despite the lack of a proper de novo lipogenesis,
schistosomes can synthesise new fatty acids by modification of host-derived ones using
NADPH-dependent enzymes;48 therefore, similar to mammals, we speculate that the PPP was
likely downregulated in compound 12 treated worms due to the increase in abundance of fatty
acids (here ARA).
To determine if these metabolomics changes are specific to compound 12 or are generally
induced in schistosomes upon all anthelmintic stresses, interrogation of the metabolomics
signatures between PZQ-treated and untreated worms was subsequently performed. To our
knowledge, this has not been done before and may also represent a novel way of identifying
PZQ’s mechanism of action. A total of 2756 m/z HRMS features were found to be significantly
different (p < 0.01) between the two samples considering positive and negative ionisation
modes together (1725 and 1031, respectively). Affected pathways mainly belonged to sugar
metabolism (i.e. glycolysis or gluconeogenesis), citrate cycle, fructose and mannose
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metabolism, pentose and glucuronate interconversions, PPP and the metabolism of the
glucogenic amino acids alanine, aspartate and glutamate (see Table 2B for significant
pathways, Supplementary Data SD1-2 for all identified metabolites and SD5-6 for all identified
pathways). In particular, the main product of schistosome carbohydrate metabolism, lactate,
was found to be more abundant than in the control; other glucose pool metabolites (e.g. glucose
6-P) were found in decreased abundances, while glucose itself was found to be in much higher
concentration (Figure 8B). Considering the recent report describing PZQ as a human serotonin
5-HT2b receptor agonist,24 we assessed how our metabolomics data paralleled serotonin
pathway activation. In schistosomes, serotonin increases carbohydrate catabolism by increasing
glycogen utilization, stimulating glycolysis and, therefore, increasing production of lactate.49
Moreover, serotonin also causes an increase in glucose uptake.49 Disturbance of carbohydrate
metabolism, with an increase in glycogen breakdown, and an impairment of serotonin-
stimulation of carbohydrate metabolism have also been described in PZQ-treated
schistosomes.25 All these previously reported effects are compatible with the observed data,
supporting the idea that PZQ could alter serotonin signalling in schistosomes, too. A different
hypothesis that could justify an increased glucose concentration, associated with a decreased
concentration of glucose-6-phosphate, is a direct interference of PZQ in the glycolytic pathway.
In schistosomes, the enzyme responsible for glucose phosphorylation is a hexokinase with
intermediate characteristic between mammalian hexokinase and glucokinase and is a strong
point of glycolysis regulation.50 An antagonist effect of PZQ on hexokinase was recently
demonstrated on the trematode Clonorchis sinensis,26 whose hexokinase share 69% identity
with S. mansoni. A similar antagonist action of PZQ on S. mansoni hexokinase would justify a
decreased glucose phosphorylation. However, we cannot exclude that the observed effects on
glycolysis could also be a consequence of generalised cellular stress.
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CONCLUSIONS
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In summary, we demonstrated that sclareol, a nontoxic diterpenoid widely used in the cosmetic
and food industries, has anti-schistosomal properties. By pursuing the medicinal chemistry
development of 14 derivatives, we have created more active compounds. The most potent
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derivative, compound 12, showed a selective improved potency on larval schistosomes (IC50
≈
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2.2 µM, SI ≈ 22 when compared to HepG2 cells), juveniles (IC50 = 1.1 µM, SI = 44.5) and adult
worms (IC50 = 9.4 µM, SI = 5.2). Moreover, phenotypic analyses by SEM and metabolomics
investigation revealed that this diterpenoid likely acts by disrupting surface membranes through
alterations of ARA metabolism. This mechanism of action is quite distinct from that ascertained
by metabolomics-based investigation of PZQ-treated worms, which revealed carbohydrate
metabolism alterations similar to that observed upon serotonin signalling. Further mechanistic
analyses of sclareol derivatives could provide important information for the development of
novel anti-schistosomal molecules.
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METHODS
Chemistry
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Enantiomerically pure (-)-sclareol was purchased from Sigma Aldrich (357995-1G, CAS 515-03-
7) and used without further purification. The diterpenoid small collection was obtained by
PhytoQuest Ltd and all other reagents or solvents were obtained from Sigma Aldrich or Fisher
Scientific and used without purification. The synthesised sclareol derivatives were characterized
1
by high resolution mass spectrometry (HRMS), H, 13C and two-dimensional Nuclear Magnetic
Resonance (NMR) spectroscopy (2D COSY, HSQC). NMR spectra were recorded on a Bruker
Avance 500 MHz NMR spectrometer, with CDCl3 used as solvent and NMR spectra referenced
to the CDCl3 residual peak. All the reactions were checked by Thin Layer Chromatography
(TLC) on pre-coated TLC aluminium sheets of silica gel. All the synthesised derivatives were
purified by column chromatography on silica gel (35-70 mesh) using the eluents indicated. Mass
spectrometry was performed on an Orbitrap Fusion Thermo Scientific with a Dionex UltiMate
3000 UHPLC system. Microwave-assisted reactions were performed in a CEM discover
microwave synthesiser 2005 (908010, serial number DU9667).
The general methods for synthesis and the characterization (1H and 13C NMR, HRMS) of all the
1
synthesised compounds, as well as H and 13C NMR spectra of key compounds (7, 8, 11, 12
and 14) can be found in the Supplementary Information.
Compound handling and storage
(-)-Sclareol and all the synthesised derivatives were solubilised in DMSO (Fisher Scientific,
Loughborough, UK) and stored at -20°C at a stock concentration of 16 mM.
Schistosoma mansoni schistosomula culture and compound screening
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S. mansoni (Puerto Rican Strain, Naval Medical Research Institute - NMRI) schistosomula were
obtained by mechanical transformation51 of S. mansoni cercariae collected after exposure of
infected Biomphalaria glabrata (NMRI) snails to 2 h of light at 26 °C. Newly transformed
schistosomula were deposited in 384-well black-sided microtiter plates (Perkin Elmer, MA, USA)
and screened by the high throughput screening platform Roboworm as previously described,18,27
with a final DMSO concentration of 0.625%. The effect of compounds on phenotype and motility
of schistosomula was analyzed after 72 h using the image analysis model described by Paveley
et al.52 Phenotype and motility scores were used to calculate IC50 values derived from dose
response titrations (50, 25, 10 and 1 µM) by using GraphPad Prism 7.02.
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Schistosoma mansoni juvenile worm culture and compound screening
S. mansoni juvenile worms were recovered from hepatic portal veins by perfusion (Smithers and
Terry, 1965) three weeks after percutaneous exposure of TO mice (Harlan, UK) to ~ 4000 S.
mansoni cercariae and collected as previously described (Whiteland et al., 2018). Briefly,
collected worms were transferred into 50 mL falcon tubes and subjected to three series of
centrifugation (300 x g for 2 min) and washing (in phenol-red free DMEM) steps, with the final
washed parasites pelleted by gravity. Between ten and fifteen juvenile worms were cultured per
well in a 96-well tissue culture plate (Fisher Scientific, Loughborough, UK). Each well contained
200 µL of modified DMEM (Gibco, Paisley, UK) supplemented with 10% v/v Hepes (Sigma-
Aldrich, Gillingham, UK), 10% v/v Foetal Calf Serum (Gibco, Paisley, UK), 0.7% v/v 200 mM L-
Glutamine (Gibco, Paisley, UK) and 1% v/v Antibiotic/antimycotic (Gibco, Paisley, UK). After
incubation for 2 h at 37 °C in a humidified atmosphere containing 5% CO2, test compounds
were added to obtain the final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.65 and 0.8 µM (0.3%
DMSO final concentration). After 72 h, worms were scored manually using microscopic methods
previously described (Whiteland et al., 2018). Briefly, a score of 0 equaled no detectable
movement, 1 included movement of the suckers and/or slight body contraction, 2 represented
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slow movement of anterior and posterior regions, 3 equated to sluggish movement of the full
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body and 4 represented normal movement. IC50 values were determined using GraphPad Prism
7.02.
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Schistosoma mansoni adult worm culture and compound screening
Mature adult parasites were recovered from hepatic portal veins by perfusion53 seven weeks
after percutaneous exposure of TO mice (Harlan, UK) to 180 S. mansoni cercariae. Three adult
worm pairs were cultured per well in a 48-well tissue culture plate (Fisher Scientific,
Loughborough, UK). Each well included 1 mL of modified DMEM (Gibco, Paisley, UK) media
containing 10% v/v Hepes (Sigma-Aldrich, Gillingham, UK), 10% v/v Foetal Calf Serum (Gibco,
Paisley, UK), 0.7% v/v 200 mM L-Glutamine (Gibco, Paisley, UK), 1% v/v Antibiotic/antimycotic
(Gibco, Paisley, UK). After incubation for 2 h at 37 °C in a humidified atmosphere containing 5%
CO2, test compounds were added to obtain the final concentrations of 50, 25, 12.5, 6.25 and
3.13 µM (0.3% DMSO final concentration). After 72 h, worms were scored manually using
microscopic methods described in the literature54 and eggs collected and counted from each
well. IC50 values were determined using GraphPad Prism 7.02.
HepG2 cell culture and MTT assay
HepG2 human liver cancer cells were grown to ~ 80% confluency in a modified BME culture
media (containing 10% v/v Foetal Bovine Serum, 1% v/v MEM non-essential amino acid
solution, 1% v/v 200 mM L-Glutamine, 1% v/v antibiotic/antimycotic). Confluent cells were
subjected to cytotoxicity assays as previously described.18,27 Briefly, 2.5 x104 cells per well were
cultured in a black walled 96-well microtiter plate (Fisher Scientific, Loughborough, UK) and
incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. Test compounds were
then titrated from 100 to 3.13 µM (1.25 final % DMSO) and negative (DMSO; 1.25%) and
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positive (1% v/v Triton X-100)55 controls included. After 24 h incubation, the MTT assay was
performed as described.18,27 CC50 values were determined using GraphPad Prism 7.02.
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Adult worms, cultured for 72 h with sub-lethal concentrations of test compounds and negative
(DMSO) controls, were collected and prepared for SEM analysis. Several steps from Collins et
al.56 were adapted as previously described.18 Briefly, collected schistosomes were relaxed in
1mL of anaesthetic (1% ethyl 3-aminobenzoate methane (Sigma-Aldrich, Gillingham, UK)
dissolved in DMEM) and then killed in 1mL of 0.6mM MgCl2 (Fisher Scientific, Loughborough,
UK). After washing in PBS, worms were placed in SEM fixative (0.1M sodium cacodylate, 2.5%
v/v glutaraldehyde (Agar Scientific, Stansted, UK) in ultra-pure water) and stored at 4°C until
ready for SEM analysis. In preparation for SEM analysis, the stored samples were exposed to a
number of wash, staining and dehydration steps as described18 and finally mounted for the
imaging. SEM analysis was conducted using a Hitachi S-4700 FESEM microscope (Ultra High
Resolution, an accelerating voltage of 5.0kV with a working distance of 5.0 mm). Images were
captured at 2560x1920 resolution.
Metabolomics sample preparation and metabolite extraction
The procedure previously described by Baptista et al.32 was adapted to schistosome materials.
Adult male worms were collected and cultured in media as described in previous paragraphs. In
brief, 24-well plates were set up with 10 male worms per well (one biological replicate), for a
total of 18 wells (6 replicates per 3 time points) per treatment (compound 12 and PZQ, at their
respective IC50, 9.4 µM and 30 nM, and control). At each time point (0, 12, 24 h), worms were
removed from culture, immersed in liquid nitrogen to quench metabolism and stored at - 80 °C.
In preparation for the extraction, samples were thawed, centrifuged (10 °C, 2000 rpm) and
washed with phosphate buffer saline. Worms were disrupted by mill bead homogenizer and
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extraction performed in chloroform/methanol/water 2:5:2 solution. After a final centrifugation,
100 µL of solution were transferred in mass vials for FIE-HRMS analysis.
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Metabolomics analysis
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Extracted metabolites were analysed by flow infusion electrospray ionisation high-resolution
mass spectrometry (FIE-HRMS) in the High Resolution Metabolomics Laboratory (HRML),
Aberystwyth University. FIE-HRMS was performed by a Q-Exactive Plus mass analyser
equipped with an UltiMate 3000 UHPLC system, which includes Thermo-Scientific binary pump,
column compartment (not used) and auto sampler. Metabolite fingerprints were created in both
positive and negative polarity switching mode. Ion intensities were acquired between m/z 55
and 1200 for 3.5 min in profiling mode at a resolution setting of 280,000. 20 µl of each extracts
were injected by auto sampler into a flow of 100 µl*min-1 methanol:water (70:30, v/v).
Electrospray ionisation (ESI) source parameters were set in agreement with manufacturer’s
recommendations. An in-house data aligning routine in Matlab (R2013b, The MathWorks) was
used to join mass spectra around the apex of the infusion maximum into a single mean intensity
matrix (runs x m/z) for each ionisation mode. Data from the matrix were log10-transformed and
used for statistical analysis performed by MetaboAnalyst 4.0. - Statistical analysis.33
Metabolites and pathway identification were performed by MetaboAnalyst 4.0. - MS peaks to
pathway33 (tolerance = 3ppm, model organism = S. mansoni; time point = 24h, as this had the
highest number of significantly different features). MetaboAnalyst identified pathways based on
the mummichog algorithm, which predicts metabolome changes directly from mass
spectrometry data, without the a priori identification of metabolites and avoiding the biased
manual assignment of spectral features to metabolites 34. Mummichog looks for local
enrichment after plotting all possible matches in the metabolic network, providing accurate
reproduction of true activity, as the false matches will distribute randomly.34 Examples of key-
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metabolites were analysed for significant difference (t-test) between control and treatment on
Microsoft Excel and by GraphPad Prism 7.02.
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Ethics statement
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All procedures performed on mice adhered to the United Kingdom Home Office Animals
(Scientific Procedures) Act of 1986 (project license PPL 40/3700) as well as the European
Union Animals Directive 2010/63/EU and were approved by Aberystwyth University’s (AU)
Animal Welfare and Ethical Review Body (AWERB).
Author contribution
Conceived and designed the experiments: AC, ADW, KFH. Performed the experiments: AC
(compound synthesis and characterization), AC (S. mansoni and HepG2 cell screening), RJN
(providing plant-derived diterpenoids), HW (plant-derived diterpenoid screening), AC, RB, JEF,
MB, LM (metabolomics). Manuscript preparation: AC (original draft), ADW, KFH (editing and
revision).
Acknowledgment
We thank all of Prof. Karl F. Hoffmann’s laboratory for help in maintaining the S. mansoni life
cycle. We thank Prof. Andrea Brancale for the use of the microwave synthesiser.
Funding
We thank the Welsh Government, Life Sciences Research Network Wales scheme for financial
support to AC and RB. IBERS receives strategic funding from the BBSRC.
The authors declare no competing financial interest.
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Supplementary Information
Supplementary information include general methods for synthesis and characterization (1H-
NMR and 13C-NMR peak lists, HRMS) of all the synthesized compounds, 1H-NMR and 13C-NMR
spectra of key-compounds, screening of diterpenoid small collection on S. mansoni
schistosomula, anti-schistosomal and cytotoxicity data summary (IC50 and CC50 with 95%
confidence interval), scatter graphs for S. mansoni juvenile, adult worms and eggs count after
treatments with key-compounds, PZQ titration on adult worms.
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Supplementary Data Files
SD1: Identification (matched compounds as KEGG code) of metabolites found by positive
ionisation mode.
SD2: Identification (matched compounds as KEGG code) of metabolites found by negative
ionisation mode.
SD3: Ranked enriched pathways with total number of hits found by positive ionisation mode
after compound 12 treatment, significant hits (p < 0.05), their EASE (Expression Analysis
Systematic Explorer) score,35 their raw p-values (Fisher’s Exact Test, FET) and the p-values
using a Gamma distribution.
SD4: Ranked enriched pathways with total number of hits found by negative ionisation mode
after compound 12 treatment, significant hits (p < 0.05), their EASE (Expression Analysis
Systematic Explorer) score,35 their raw p-values (Fisher’s Exact Test, FET) and the p-values
using a Gamma distribution.
SD5: Ranked enriched pathways with total number of hits found by positive ionisation mode
after PZQ-treatment, significant hits (p < 0.05), their EASE (Expression Analysis Systematic
Explorer) score,35 their raw p-values (Fisher’s Exact Test, FET) and the p-values using a
Gamma distribution.
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SD6: Ranked enriched pathways with total number of hits found by negative ionisation mode
after PZQ-treatment, significant hits (p < 0.05), their EASE (Expression Analysis Systematic
Explorer) score,35 their raw p-values (Fisher’s Exact Test, FET) and the p-values using a
Gamma distribution.
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References
1.
2.
3.
Caniard A., Zerbe P., Legrand S., Cohade A., Valot N. Discovery and functional
characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (
L.) and their relevance for perfume manufacture. BMC Plant Biol. 2012;12(1):1. Doi:
10.1186/1471-2229-12-119.
Smith RL., Waddell WJ., Cohen SM., Feron VJ., Marnett LJ., Portoghese PS., Rietjens
IMCM., Adams TB., Gavin CL., Mcgowen MM., Taylor S V., Williams MC. GRAS 24: the
24th publication by the FEMA Expert Panel presents safety and usage data on 236 new
generally recognized as safe flavoring ingredients. Food Technol. 2009;63(6):46–105.
Rifm T., Panel E., Belsito D., Bickers D., Bruze M., Calow P., Greim H., Hanifin JM.,
Rogers AE., Saurat JH., Sipes IG., Tagami H. A toxicologic and dermatologic
assessment of cyclic and non-cyclic terpene alcohols when used as fragrance
ingredients. Food Chem Toxicol. 2008;46. Doi: 10.1016/j.fct.2008.06.085.
Sirikantaramas S., Yamazaki ÆM. Mechanisms of resistance to self-produced toxic
secondary metabolites in plants. Phytochem Rev. 2008;7:467–77. Doi: 10.1007/s11101-
007-9080-2.
4.
5.
6.
Tapia L., Torres J., Mendoza L., Urz A., Ferreira J., Pavani M., Wilkens M. Effect of 13-
epi -sclareol on the bacterial respiratory chain. Planta Med. 2004;70:1058–63. Doi:
10.1055/s-2004-832647.
Ma M., Feng J., Li R., Chen S., Xu H. Synthesis and antifungal activity of ethers,
alcohols, and iodohydrin derivatives of sclareol against phytopathogenic fungi in vitro.
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