Characterization of a structural leoligin analog as farnesoid X receptor agonist and modulator of cholesterol transport

Article abstract of DOI:10.1055/a-1171-8357

The ligand-activated farnesoid X receptor is an emerging therapeutic target for the development of drugs against metabolic syndrome-related diseases. In this context, selective bile acid receptor modulators represent a novel concept for drug development. Selective bile acid receptor modulators act in a target gene- or tissue-specific way and are therefore considered less likely to elicit unwanted side effects. Based on leoligin, a lignan-type secondary plant metabolite from the alpine plant Leontopodium nivale ssp. alpinum, 168 synthesized structural analogs were screened in a farnesoid X receptor in silico pharmacophore-model. Fifty-six virtual hits were generated. These hits were tested in a cell-based farnesoid X receptor transactivation assay and yielded 7 farnesoid X receptor-activating compounds. The most active one being LT-141A, with an EC₅₀ of 6 μM and an E_max of 4.1-fold. This analog did not activate the G protein-coupled bile acid receptor, TGR5, and the metabolic nuclear receptors retinoid X receptor α, liver X receptors α/β, and peroxisome proliferator-activated receptors β/γ. Investigation of different farnesoid X receptor target genes characterized LT-141A as selective bile acid receptor modulators. Functional studies revealed that LT-141A increased cholesterol efflux from THP-1-derived macrophages via enhanced ATP-binding cassette transporter 1 expression. Moreover, cholesterol uptake in differentiated Caco-2 cells was significantly decreased upon LT-141A treatment. In conclusion, the leoligin analog LT-141A selectively activates the nuclear receptor farnesoid X receptor and has an influence on cholesterol transport in 2 model systems.

Full text of DOI:10.1055/a-1171-8357

Published online: 2020-06-02
Original Papers
Characterization of a Structural Leoligin Analog as Farnesoid X
Receptor Agonist and Modulator of Cholesterol Transport#
Authors
Angela Ladurner , Thomas Linder , Limei Wang , Verena Hiebl , Daniela Schuster3, 4, Michael Schnürch2,
Marko D. Mihovilovic , Atanas G. Atanasov1, 5, 6, 7, Verena M. Dirsch1
1
2
1
1
2
Affiliations
Supporting information available online at
1
2
3
Department of Pharmacognosy, Faculty of Life Sciences,
http://www.thieme-connect.de/products
University of Vienna, Vienna, Austria
Institute of Applied Synthetic Chemistry, TU Wien, Vienna,
Austria
ABSTRACT
The ligand-activated farnesoid X receptor is an emerging ther-
apeutic target for the development of drugs against metabol-
ic syndrome-related diseases. In this context, selective bile
acid receptor modulators represent a novel concept for drug
development. Selective bile acid receptor modulators act in a
target gene- or tissue-specific way and are therefore consid-
ered less likely to elicit unwanted side effects. Based on leoli-
gin, a lignan-type secondary plant metabolite from the alpine
plant Leontopodium nivale ssp. alpinum, 168 synthesized struc-
tural analogs were screened in a farnesoid X receptor in silico
pharmacophore-model. Fifty-six virtual hits were generated.
These hits were tested in a cell-based farnesoid X receptor
transactivation assay and yielded 7 farnesoid X receptor-acti-
vating compounds. The most active one being LT-141A, with
an EC50 of 6 µM and an Emax of 4.1-fold. This analog did not
activate the G protein-coupled bile acid receptor, TGR5, and
the metabolic nuclear receptors retinoid X receptor α, liver X
receptors α/β, and peroxisome proliferator-activated recep-
tors β/γ. Investigation of different farnesoid X receptor target
genes characterized LT-141A as selective bile acid receptor
modulators. Functional studies revealed that LT-141A in-
creased cholesterol efflux from THP-1-derived macrophages
via enhanced ATP-binding cassette transporter 1 expression.
Moreover, cholesterol uptake in differentiated Caco-2 cells
was significantly decreased upon LT-141A treatment. In con-
clusion, the leoligin analog LT-141A selectively activates the
nuclear receptor farnesoid X receptor and has an influence
on cholesterol transport in 2 model systems.
Department of Pharmacy/Pharmaceutical Chemistry and
Center for Molecular Biosciences Innsbruck (CMBI), Uni-
versity of Innsbruck, Innsbruck, Austria
4
Department of Pharmaceutical and Medicinal Chemistry,
Institute of Pharmacy, Paracelsus Medical University,
Salzburg, Austria
5
6
7
Institute of Genetics and Animal Breeding of the Polish
Academy of Sciences, Jastrzebiec, Poland
Institute of Neurobiology, Bulgarian Academy of Sciences,
Sofia, Bulgaria
Ludwig Boltzmann Institute for Digital Health and Patient
Safety, Medical University of Vienna, Vienna, Austria
Key words
farnesoid X receptor, leoligin, structural analog, lignan,
cholesterol efflux, cholesterol uptake
received January 14, 2020
revised
April 24, 2020
accepted May 5, 2020
Bibliography
DOI https://doi.org/10.1055/a-1171-8357
published online | Planta Med © Georg Thieme Verlag KG
Stuttgart · New York | ISSN 0032‑0943
Correspondence
Dr. Angela Ladurner
Department of Pharmacognosy, Faculty of Life Sciences,
University of Vienna
Althanstraße 14, A-1090 Vienna, Austria
Phone: + 431427755239, Fax: + 431427755969
angela.ladurner@univie.ac.at
#
Dedicated to Professor Dr. Wolfgang Kubelka on the occasion of his 85th
birthday.
Ladurner A et al. Characterization of a… Planta Med

Original Papers
been proposed, including differential cofactor or DNA binding,
transactivation versus transrepression, and binding of ligands to
allosteric pockets (S2 pocket) [8].
ABBREVIATIONS
ABCA1
BSEP
CDCA
CETP
FXR
ATP-binding cassette transporter 1
bile salt export pump
In the basal state, FXR resides in the nucleus and is bound to-
gether with its heterodimer partner, the retinoid X receptor
(RXR), to respective response elements in promoter regions of
target genes together with co-repressors. Upon the binding of full
agonists, like obeticholic acid and the endogenous agonist cheno-
deoxycholic acid (CDCA), conformational changes lead to the re-
lease of these co-repressors and to the binding of co-activators,
resulting in target gene expression. In contrast to full FXR ago-
nists, SBARMs are partial agonists. The molecular basis of differen-
tial modes of FXR modulation has been investigated recently and
shows that the partial agonists DM175 and ivermectin can recruit
co-activator and co-repressor peptides at the same time, thereby
eliciting a fluid FXR modulation profile [11].
chenodeoxycholic acid
cholesteryl ester transfer protein
farnesoid X receptor
HMGCR
LXR
3-hydroxy-3-methyl-glutaryl-CoA reductase
liver X receptor
PBC
primary biliary cholangitis
phorbol 12-myristate 13-acetate
peroxisome-proliferator-activated receptor
retinoid X receptor
PMA
PPAR
RXR
SBARM
SHP
selective bile acid receptor modulator
small heterodimer partner
FXR can be activated by different compounds from diverse
sources, including a multitude of natural products. The most ob-
vious FXR modulators are compounds with a steroidal structure,
like the triterpene oleanolic acid, as this structure closely resem-
bles the endogenous agonists. However, there are also other
structural classes among FXR agonists like the sesquiterpene alco-
hol farnesol that gave FXR its name or the polyphenols xanthohu-
mol and epigallocatechin gallate [5,12].
Leoligin, the major lignan from the roots of L. nivale ssp. alpi-
num, is reported to influence cholesterol metabolism via choles-
teryl ester transfer protein (CETP), 3-hydroxy-3-methyl-glutaryl-
CoA reductase (HMGCR), and ATP-binding cassette transporters
A1 (ABCA1) and G1 (ABCG1) [13–15], and is thus an interesting
candidate for FXR modulation. In context of an interdisciplinary
research program [16] exploiting natural compound isolates to-
wards new potential drug leads [17], we established a stereoselec-
tive synthetic route for the generation of a compound library
based on the leoligin core structure [18]. In order to identify po-
tential and novel FXR agonists, we utilized FXR specific pharmaco-
phore models [19] to screen a database of leoligin structural ana-
logs. Selected virtual hits were tested in a cell-based FXR transac-
tivation assay and yielded 7 FXR activating compounds, the most
promising termed LT-141A. Furthermore, we characterized this
novel FXR agonist for its effects on cholesterol metabolism in hu-
man cell models for macrophage cholesterol efflux and intestinal
cholesterol uptake.
Introduction
In the last decades, metabolic syndrome-related diseases have be-
come an increasing public health issue [1]. Many metabolic path-
ways are regulated by nuclear receptors. These receptors act as
ligand-activated transcription factors, thereby regulating the ex-
pression of target genes involved in processes like lipid, glucose,
and cholesterol metabolism. One of these nuclear receptors is
the farnesoid X receptor (FXR). Endogenously, FXR is activated by
bile acids, which are synthesized from cholesterol in the liver [2,
3
]. FXR-dependent gene regulation results in downregulation of
lipogenesis in the liver as well as bile acid synthesis from choles-
terol, hepatic bile acid uptake, and intestinal absorption. Bile acid
modification and secretion, on the other hand, are upregulated by
FXR [4]. Altogether, this depicts the importance of FXR in meta-
bolic processes, and cholesterol metabolism in particular. FXR is
mainly expressed in metabolic tissues such as the liver and intes-
tine but also in the kidney and adrenal glands and to a lesser
amount in macrophages [5, 6]. The therapeutic potential of FXR
lies in the treatment of cholestatic liver diseases, hypercholester-
olemia, hypertriglyceridemia, nonalcoholic steatohepatitis, and
type 2 diabetes mellitus [7]. Of note, 1 FXR agonist, obeticholic
acid (Ocaliva), has already been approved in the U.S. and Europe
in a fast track procedure for the treatment of primary biliary cho-
langitis (PBC) [8]. Despite its beneficial impact in the therapy of
PBC, obeticholic acid has several adverse effects, such as pruritus,
fatigue, and gastrointestinal symptoms. Moreover, disadvanta-
geous serum lipid and cholesterol profiles may occur with FXR
agonist treatment [9, 10]. Thus, it is of great interest to develop
novel FXR modulators with a minimal side effect profile and with
a wide area of treatment indications.
Results
All pharmacophore models reported in reference [19] were used
to screen 168 synthetic leoligin analogs (▶ Fig. 1a). Nearly all
compounds fitted into the FXR ligand models 3fli-1 and 3fli-1-s
(▶ Fig. 2), which pointed towards this compound class as interest-
ing test substances. Fifty-nine compounds were predicted by at
least 1 additional model.
To validate the in silico predictions, 65 virtual hits, preferably
those found by 3fli- and additional models, were tested in a cell-
based FXR transactivation assay. This yielded 7 FXR activating
compounds, with the most active one being LT-141A (▶ Fig. 1b
and Table 1). LT-141A concentration-dependently transactivated
FXR with an EC50 of 6.36 µM and an Emax of 4.1-fold compared to
High affinity full agonists of FXR activate the whole plethora of
FXR target genes and are therefore more likely to elicit undesir-
able effects. Therefore, increasing focus is directed at the identifi-
cation and development of selective bile acid receptor modulators
(
SBARM) that act in a tissue- or gene-specific manner with the ul-
timate goal to design FXR agonists for specific therapeutic pur-
poses (reviewed in [8]). This aim is very challenging as the mech-
anisms underlying gene- and tissue-selectivity are still being eluci-
dated. Several hypotheses about how to achieve selectivity have
Ladurner A et al. Characterization of a… Planta Med

the vehicle control in a transactivation assay using the bile salt ex-
port pump (BSEP) promoter coupled to a luciferase gene. Interest-
ingly, LT-141A showed a similar response as the endogenous FXR
agonist CDCA (▶ Fig. 3a). We also compared LT-141A to its par-
ent compound leoligin in the same assay. Leoligin only slightly in-
creased FXR transactivation with an Emax of 1.5-fold compared to
the vehicle control (▶ Fig. 3b). Taken together, this strong in-
crease in FXR transactivation was achieved by the exchange of
the angelic acid ester of leoligin with an in o-position methylated
benzoic acid ester (▶ Fig. 1).
▶
Fig. 1 Chemical structures of leoligin and LT-141A.
In order to investigate if, in addition to its agonistic properties,
LT-141A can act as an antagonist, we determined the reduction in
CDCA-induced FXR activity in a full-length receptor transactiva-
tion assay. In this assay, LT-141A was able to reduce CDCA-in-
duced FXR activity to 59.45%, thereby showing clear antagonistic
properties (▶ Fig. 3c).
2-fold. The weaker endogenous FXR agonist CDCA and LT-141A
had no effect on the mRNA of this target gene (▶ Fig. 6c).
In order to evaluate a putative effect of LT-141A on cholesterol
homeostasis, we investigated its influence on cholesterol uptake
in intestinal Caco-2 cells. A decreased uptake of cholesterol in
these cells implies a lower systemic cholesterol burden and a high-
er fecal cholesterol excretion, making it beneficial especially for
metabolic diseases. As a positive control the LXR agonist
GW3965 was used, which significantly decreased cholesterol up-
take. LT-141A showed a comparable effect to GW3965, whereas
the 2 FXR agonists CDCA and GW4064 did not elicit a significant
decrease (▶ Fig. 7a). Next, we investigated LT-141A’s influence on
ApoAI-mediated cholesterol efflux in THP1-derived macrophages.
Cholesterol efflux from macrophages is an important mechanism
in the prevention of atherosclerosis via inhibition of foam cell for-
mation. LT-141A at 10 µM increased cholesterol efflux approxi-
mately 2.5-fold, being even stronger than the positive control pio-
glitazone (10 µM), a PPARγ agonist (▶ Fig. 7b). Investigation of
the responsible mechanism behind the increased cholesterol
efflux from macrophages revealed that not only the mRNA
(▶ Fig. 7c) but also the protein level (▶ Fig. 7d) of the cholesterol
transporter ABCA1 is dose-dependently elevated upon treatment
with LT-141A, suggesting a direct link. Interestingly, CDCA but not
GW4064 significantly increased ABCA1 mRNA levels (▶ Fig. 7c).
As ABCA1 is not only an important cholesterol transporter in mac-
rophages but also in intestinal and liver cells, we measured ABCA1
protein levels in these cell types. The positive controls GW3965
and T0901317, 2 LXR agonists, significantly increased ABCA1 pro-
tein in both cell types, as expected. However, LT-141A as well as
the FXR agonists GW4064 and CDCA had no effect on ABCA1 pro-
tein expression (▶ Fig. 7e and f). These results suggest a cell type-
specific effect of LT-141A that seems to be only partially depen-
dent on FXR activation.
To better understand if LT-141A can either directly or indirectly
activate FXR, we performed an FXR-Gal4 assay. In this assay, LT-
1
41A showed an EC50 of 5 µM and Emax of 14.2-fold compared to
the vehicle control (▶ Fig. 4a), whereas leoligin had a marginal
effect with an Emax of 1.4-fold compared to the vehicle control
(
▶ Fig. 4b). In addition, LT-141A reduced CDCA-induced FXR-
Gal4 activity to 44.11% (▶ Fig. 4c). Furthermore, we performed
FXR binding pose predictions with CDCA and LT-141A, showing
that the residues Arg331 and Ser332 are particularly important
for LT-141A binding (▶ Fig. 5).
To further strengthen the assumption that LT-141A binds to
the FXR ligand binding domain (LBD), we performed an FXR co-
activator assay. LT-141A successfully recruited the co-activator
peptide SRC2-2 to the FXR LBD with an EC50 of 2.5 µM (▶ Fig. 4d).
The endogenous full FXR ligand CDCA recruited SRC2-2 with an
EC50 of 12.7 µM but with much higher affinity, further illustrating
the partial agonistic nature of LT-141A. Next, we evaluated the se-
lectivity of LT-141A toward the nuclear receptors retinoid X recep-
tor α (RXRα), the liver X receptors α and β (LXRα and β), the per-
oxisome proliferator-activated receptor β/δ, and γ (PPAR β/δ, and
γ) and the G-protein coupled bile acid receptor TGR5 (GPBAR1),
all of which are involved in the regulation of metabolic processes.
We used cell-based transactivation assays to determine activities
on the mentioned receptors. Interestingly, LT-141A was not able
to activate any of the tested receptors (Supplementary Fig. 1S,
Supporting Information).
To study the consequences of FXR activation, we investigated a
direct and indirect FXR target gene in the liver cell line HepG2. In-
duction of small heterodimer partner (SHP), a bona-fide FXR target
gene, is a major mechanism to suppress gene expression [20]. SHP
mRNA expression was significantly increased by the FXR agonist
GW4064 as expected, and also LT-141A increased its expression
significantly between 30 and 1 µM, although not concentration-
dependently (▶ Fig. 6a). CYP7A1 is an indirect FXR target gene,
downregulated via SHP, and a key enzyme in bile acid synthesis.
Upon FXR activation, this gene is downregulated as observed with
GW4064. LT-141A, however, had no effect on the expression of the
CYP7A1 gene when compared to vehicle control (▶ Fig. 6b). Weal-
so investigated the mRNA expression levels of the FXR target gene
fibroblast growth factor 19 (FGF19) in intestinal Caco-2 cells.
GW4064 significantly upregulated this gene, however, only around
Discussion
In the present study, we identified a novel SBARM, LT-141A, that
had no cross-activity on other metabolic nuclear receptors
(PPARs, LXRα/β, and RXR) and, more importantly, did not activate
TGR5 (GPBAR1). Off-target effects of FXR modulators often in-
clude the activation of this bile acid responsive G-protein coupled
receptor. TGR5 activation is a possible reason for the occurrence
of pruritus, besides other molecular causes, which is the most
common reason for treatment discontinuation of FXR agonists in
patients.
Ladurner A et al. Characterization of a… Planta Med

Original Papers
▶
Fig. 2 Pharmacophore-based virtual screening. A structure-based pharmacophore model for FXR agonists based on the PDB entry 3fli [31]
mapped the majority of leoligin analogs from a virtual library. a The co-crystallized FXR agonist XL335 (WAY-362450) with 4 hydrophobic contacts
yellow spheres) and a hydrogen bond acceptor (red arrow) anchoring the compound in the binding site. b Leoligin matching the 5 pharmacophore
features of the 3fli-based model.
(
▶
Table 1 Leoligin analogs identified as FXR modulators.
common structure
Compound ID
R1
R2
Fold activation at 10 µM
LT-141A
H
4.79 ± 0.92
LT-071A
H
3.75 ± 0.13
LT-095A
SOGE-21
H
2.44 ± 0.49
2.23 ± 0.37
OCH3
Leoligin
H
1.84 ± 0.28
LT-075A1
LT-104A
H
H
1.81 ± 0.23
1.56 ± 0.16
Leoligin, a lignan-type secondary metabolite, originally iso-
lated from the roots of the alpine plant L. nivale ssp. alpinum has
been reported to reduce cholesterol levels and inhibit HMGCR in
ApoE− mice, to activate CETP in vivo and in vitro, and to promote
cholesterol efflux from human THP1-derived macrophages via up-
regulation of the cholesterol transporters ABCA1 and ABCG1 [13–
15]. These overall favorable in vivo and in vitro properties on cho-
lesterol metabolism encouraged us to use leoligin as parent com-
/−
Ladurner A et al. Characterization of a… Planta Med

▶
Fig. 3 Concentration-dependent FXR transactivation by LT-141A and leoligin. HEK-293 cells were cotransfected with an hFXR expression plas-
mid, a BSEP luciferase reporter plasmid, and an EGFP plasmid as internal control. Cells were treated with 3 µM GW4064 and 50 µM CDCA as positive
control or the indicated concentrations of (a) LT-141A, (b) leoligin for 18 h, or (c) CDCA for 18.5 h and LT-141A for 18 h. The measured luciferase-
derived luminescence was normalized to the obtained EGFP-derived fluorescence and to the vehicle control. Each bar represents the mean ± SD of
at least 3 independent experiments performed in quadruplicate and evaluated by 1-way ANOVA with the Bonferroni post-test or Studentʼs t-test.
*
*** p < 0.0001, ***p < 0.001, **p < 0.01 compared with vehicle control (DMSO), ns not significant versus DMSO.
▶
Fig. 4 Concentration-dependent FXR-Gal4 transactivation by LT-141A and leoligin. HEK-293 cells were cotransfected with an FXR-Gal4 plasmid,
tk(MH1000)4xLuc luciferase reporter plasmid and an EGFP plasmid as internal control. Cells were treated with 3 µM GW4064 and 50 µM CDCA as
positive control or the indicated concentrations of (a) LT-141A, (b) leoligin for 18 h, or (c) CDCA for 18.5 h and LT-141A for 18 h. The measured
luciferase-derived luminescence was normalized to the obtained EGFP-derived fluorescence and to vehicle control. Each bar represents the mean ±
SD of at least 3 independent experiments performed in quadruplicate and evaluated by 1-way ANOVA with the Bonferroni post-test or Studentʼs
t-test. ***p < 0.001, *p < 0.05 compared with vehicle control (DMSO), ns not significant versus DMSO. d A LanthaScreen TR-FRET FXR Co-activator
assay was performed with LT-141A and CDCA in various concentrations. Results represent 4 experiments performed in quadruplicates.
pound to identify new FXR modulators. For this purpose, we used
a previously synthesized library of 168 leoligin analogs [18] that
were screened in an in silico structure-based pharmacophore
model. LT-141A was able to potently activate FXR in a transactiva-
tion assay and bind the FXR ligand-binding domain in a Gal4-
based assay with EC50 values of 6.36 µM and 5 µM, respectively.
Additionally, LT-141A was active in an FXR co-activator recruit-
ment assay with an EC50 of 2.5 µM. Compared to the endogenous
full FXR agonist CDCA, LT-141A showed a lower affinity to the co-
activator peptide SRC2-2, thereby indicating partial agonism and
binding to the FXR LBD. Leoligin, on the other hand, had no or
only marginal influence in the Gal4-based assay. This particular
Ladurner A et al. Characterization of a… Planta Med

Original Papers
▶
6
(
Fig. 5 FXR binding pose prediction for LT-141A. a Binding pose of the positive control CDCA in the human FXR co-crystal structure (PDB code
hl1). b Predicted binding pose of LT-141A in the same structure. Electrostatic anchoring via hydrogen bonds (arrows) and charged interactions
star) are depicted for comparison.
▶
Fig. 6 Influence of LT-141A on the expression of different FXR target genes. HepG2 cells were treated with the indicated test substances for 24 h.
The mRNA levels of (a) SHP and (b) CYP7A1 were detected by RT-qPCR. c Differentiated Caco-2 cells were treated with the indicated test sub-
stances for 24 h. The mRNA levels of FGF19 were detected by RT-qPCR. Bar graphs represent mean ± SD from at least 3 independent experiments,
evaluated by 1-way ANOVA with the Bonferroni post-test or Studentʼs t-test. ***p < 0.001, **p < 0.01, *p < 0.05 compared with vehicle control
(
DMSO), ns not significant versus DMSO.
change in pharmacological activity seems to be an immediate
consequence of the significant structural change from an olefinic
to an aromatic ester group within the leoligin scaffold. In addition,
LT-141A reduced CDCA-induced FXR activity in FXR transactiva-
tion and Gal4 assays. These results corroborate the partial ago-
nism of LT-141A and are in line with the observation that some
partial FXR agonists cause a structural conformation able to bind
co-activator and co-repressor peptides [11].
Target gene expression studies revealed that LT-141A does not
act as a classical agonist, but as a SBARM. CYP7A1, a key enzyme
in bile acid biosynthesis, is downregulated by FXR agonists like
GW4064. LT-141A, however, did not change the expression level
of this gene. Another direct target gene of FXR is FGF19. FGF19 is
expressed in intestinal cells and secreted to reach the liver, where
it regulates gene expression. FXR agonists upregulate this gene;
however, LT-141A had no effect on FGF19 mRNA levels in Caco-2
cells. The reason for this might be the general low response of FXR
agonists on this gene in our experimental setting. SHP, on the oth-
er hand, was upregulated with LT-141A as with the FXR agonist
GW4064.These results clearly show that LT-141A acts as a gene-
selective FXR agonist.
FXR is a key regulator of bile acid biosynthesis and therefore
also a major player in cholesterol metabolism. This prompted us
to investigate the effect of LT-141A in functional settings for cho-
lesterol homeostasis. Cholesterol uptake from dietary sources oc-
curs in the intestine. A reduced intestinal cholesterol uptake cor-
responds to a reduced systemic cholesterol load and is therefore
beneficial in the therapy of metabolic syndrome-related diseases.
Remarkably, LT-141A reduced the cholesterol uptake to a similar
extent as the LXR agonist GW3965, whereas the FXR agonists
CDCA and GW4064 had no effect. The FXR agonistic property of
LT-141A seems therefore not to be responsible for the decreased
cholesterol uptake in Caco-2 cells.
We additionally examined if LT-141A can increase cholesterol
efflux from macrophages. This would suggest an anti-atheroscler-
otic effect as accumulation of cholesterol in macrophages leads to
the formation of foam cells, which are a major indicator for the de-
velopment of atherosclerotic plaques. LT-141A increased ApoAI-
Ladurner A et al. Characterization of a… Planta Med

▶
Fig. 7 LT-141A regulates cholesterol transport. a Cholesterol uptake in intestinal Caco-2 cells was determined as described in the material and
methods section. b Cholesterol efflux from THP1-macrophages was determined as described in the material and methods section. c THP1-mac-
rophages were treated with the indicated test substances for 24 h. The mRNA levels of ABCA1 were detected by RT-qPCR. d THP1-macrophages
were treated with the indicated test substances for 24 h. The ABCA1 protein levels were detected by western blot. e Differentiated Caco-2 cells
were treated with the indicated test substances for 48 h. The ABCA1 protein levels were detected by western blot. f HepG2 cells were treated with
the indicated test substances for 24 h. The ABCA1 protein levels were detected by western blot. Bar graphs represent mean ± SD from at least
3
independent experiments, evaluated by 1-way ANOVA with the Bonferroni post-test or Studentʼs t-test. ***p < 0.001, **p < 0.01, *p < 0.05
compared with vehicle control (DMSO), ns not significant versus DMSO.
mediated macrophage cholesterol efflux even stronger than the
PPARγ agonist pioglitazone. PPARγ increases cholesterol efflux via
the induction of LXR expression [21,22]. Increased expression of
the well-studied cholesterol transporter ABCA1 seems to be re-
sponsible for the enhanced cholesterol efflux upon treatment with
LT-141A. Interestingly, GW4064 did not increase ABCA1 mRNA lev-
els, but CDCA did, pointing to a different mechanism underlying
cholesterol efflux than FXR agonism. In the liver cell line HepG2,
ABCA1 expression is, however, neither influenced by the FXR ago-
nists, GW4064 or CDCA, nor by LT-141A. A similar pattern was ob-
served in the intestinal cell line Caco-2, where neither the FXR ago-
nist GW4064 nor LT-141A had an effect on ABCA1 mRNA levels.
CDCA slightly upregulated ABCA1 mRNA in this setting, suggest-
ing again an alternative mechanism of ABCA1 regulation. The LXR
agonists GW3965 and T0901317 increased ABCA1 levels in all
tested cell types as is suggested by literature [23–26].
ABCA1 expression can be regulated at the transcriptional, post-
transcriptional, and post-translational level, a plethora of possible
mechanisms exists for the induction of both ABCA1 mRNA and
protein expression by LT-141A.
All the obtained results not only suggest target-gene but also
cell-type selectivity of LT-141A. Such tissue-selective FXR agonists
may provide a beneficial side effect profile and are therefore inter-
esting for the development of novel drugs. Tissue-specificity
could be achieved via differential availability of transporters or by
reducing the retention time of a substance in a specific tissue.
Moreover, differential expression of co-regulatory proteins in
these cell types might lead to tissue-specific target gene expres-
sion. Several intestinal-specific FXR agonists are already known
(fexaramine, TC-100, ivermectin, ECGC) and are suggested to be
safer for the treatment of metabolic syndrome-related diseases
than systemic FXR agonists [8].
It is kind of puzzling that LT-141A is not activating LXR as it
clearly upregulated the expression of the LXR target gene ABCA1
in macrophages. Also, the FXR agonist GW4064 did not mimic the
effects of LT-141A on ABCA1 mRNA expression, but CDCA did. As
Taken together, we could identify a novel FXR agonist, LT-
141A, with an EC50 of 6.36 µM in an FXR transactivation assay
and 5 µM in an FXR-Gal4 assay, without activating other metabolic
nuclear receptors and the G-protein coupled bile acid receptor
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Original Papers
▶
Fig. 8 Synthesis pathway for leoligin and LT-141A.
TGR5. LT-141A has been shown to enhance cholesterol efflux
from THP-1-derived macrophages via upregulation of the choles-
terol transporter ABCA1. Additionally, LT-141A also decreased
cholesterol uptake in intestinal cells. These results display poten-
tial beneficial effects of LT-141A on cholesterol metabolism and
characterize LT-141A as target gene and cell-type selective FXR
agonist.
droxy group with either angelic acid or 2-methylbenzoic acid de-
livered leoligin and LT-141A, respectively. By varying the aryl
halide coupling partner in the Suzuki-Miyaura step and the car-
boxylic acid in the esterification, a library of leoligin analogs can
be synthesized. Details for all reaction steps and analytical data
of all compounds can be found in the Supporting Information.
Pharmacophore-based virtual screening
Pharmacophore-based virtual screening was performed using
DiscoveryStudio version 2015 (BIOVIA, Dassault Systèmes
Discovery Studio Modeling Environment: Dassault Systèmes,
Materials and Methods
Synthesis of leoligin and leoligin analogs
2
015). From the 2D structures of 168 leoligin analogs, a 3D multi-
A synthetic route for the total synthesis of leoligin (▶ Fig. 8),
which can be applied as well for synthesizing leoligin analogs has
been established previously [18]. Standard addition of vinylma-
gensium bromide to 1 delivered the substrate for the first key step
of our synthesis: establishing chirality upon lipase-mediated ki-
netic resolution of rac-2 to (S)-2 employing Amano lipase PS in
high optical purity. The further synthesis consisted of etherifica-
tion of (S)-2 to 3, followed by standard m-CPBA epoxidation to 4.
Subsequent radical cyclization [27,28] delivered the tetrahydro-
furan scaffold 5. Noteworthy, the synthesis of 5 from (S)-2 could
be carried out in a single operation without isolation of intermedi-
ates 3 and 4. The hydroxymethyl group in intermediate 5 was pro-
tected with the sterically demanding TBDMS group since its steric
bulk ensures 3,4-cis diastereoselective hydroboration with com-
monly used 9-borabicyclo(3.3.1)nonane (9-BBN) as reagent. The
hydroboration product was again not isolated but subjected in
situ to a Suzuki-Miyaura coupling with 5-iodoveratrole to deliver
conformational database was calculated with a maximum of
500 conformers per molecule using BEST conformational analysis
settings. This database was screened against a previously estab-
lished FXR ligand pharmacophore model set [19] using BEST
FLEXIBLE fitting. This means that the screening ligand conforma-
tion is energetically minimized during the pharmacophore match-
ing so that it better fits into the model.
Cell culture reagents, chemicals, and plasmids
DMEM, containing 4.5 g/L glucose, L-glutamine, Eagleʼs minimum
essential medium (EMEM) without L-glutamine, RPMI-1640, non-
essential amino acids, benzylpenicillin, and streptomycin were ob-
tained from Lonza. FBS and trypsin was supplied by Gibco via
Invitrogen. Phorbol 12-myristate 13-acetate (PMA), water-soluble
cholesterol, ApoAI, sodium taurocholate, lysophosphatidylcho-
line, oleic acid, monooleoylglycerol, GW4064 (purity ≥ 97%),
CDCA (purity ≥ 96%), GW3965 (purity ≥ 98%), pioglitazone (pu-
rity ≥ 98%), lithocholic acid (purity ≥ 95%), and T0901317 (purity
6
after concomitant deprotection. A final esterification of the hy-
Ladurner A et al. Characterization of a… Planta Med

≥
98%) were purchased from Sigma-Aldrich. Fatty acid-free bo-
Cells were then lysed and the luminescence of the firefly luciferase
and the fluorescence of EGFP were quantified with a Tecan Spark
plate reader. The luciferase-derived luminescence was normalized
to the EGFP-derived fluorescence from each well to account for
differences in transfection efficiency or cell number. For the FXR
transfections, 5 µg hFXR or 1 µg FXR-Gal4 plasmid, 5 µg reporter
plasmid (BSEP‑Luc plasmid or tk(MH1000)4xLuc plasmid, respec-
tively) were used. For the RXRα, LXRα/β, PPARγ transfections, 6 µg
expression plasmid and 6 µg reporter plasmid (RXRE‑Luc, ABCA1-
Luc, and PPRE‑Luc, respectively) were used. For the PPARβ/δ
transfection, 1 µg expression plasmid and 6 µg reporter plasmid
were used. For the TGR5 transfection, 5 µg expression plasmid
and 5 µg CRE-reporter plasmid were used. As internal control,
3 µg EGFP plasmid was transfected in all assays.
vine serum albumin (BSA) was obtained from Carl Roth. [3H]-cho-
lesterol was provided by PerkinElmer. Human plasma was ob-
tained from young, healthy volunteers.
The hFXR expression plasmid, the bile salt export pump (BSEP)-
Luc plasmid, and the FXR‑Gal4 plasmid were kindly provided by
Prof. Dr. Schubert-Zsilavecz (Goethe University Frankfurt). The
PPAR luciferase reporter construct (tk-PPREx3-luc) and the report-
er plasmid for the Gal4 assay (tk(MH1000)4xLuc) was a kind gift
from Prof. Ronald M. Evans (Howard Hughes Medical Institute).
The expression plasmids for human PPAR subtypes (pSG5-hPPAR-
β, pSG5-PL-hPPAR-γ1) were a kind gift from Prof. Walter Wahli and
Prof. Beatrice Desvergne (Center for Integrative Genomics, Univer-
sity of Lausanne). The ABCA1-Luc Plasmid was kindly provided by
Prof. Ira G. Schulman (University of Virginia). The human RXRα,
LXRα, and β expression plasmids were bought from Missouri S&T
cDNA Resource Center and the RXRE‑Luc plasmid from Panomics.
The TGR5 expression plasmid, GPBAR1, transcript variant 3,
NM_170699.1 (OG-SC123315) was obtained from Origene via
Biomedica. The CRE‑Luc plasmid, pGL4.29[luc2P/CRE/Hygro], was
obtained from Promega. The plasmid encoding enhanced green
fluorescence protein (pEGFP‑N1) was obtained from Clontech.
The primary antibody against ABCA1 (catalog no. NB400-105)
was obtained from Novus Biologicals. The anti-actin antibody
LanthaScreen TR-FRET FXR co-activator assay
The LanthaScreen TR-FRET FXR Co-activator Assay Kit (Invitrogen)
was used according to the manufacturerʼs instructions. In short,
GST-tagged FXR LBD is added to varying concentrations of LT-
141A and CDCA (300 µM–3 nM). Then the fluorescein-labeled
SRC2-2 co-activator peptide and terbium-labeled anti-GST anti-
body is added in a 384-well plate to a total volume of 20 µl. After
1 h of incubation at room temperature, fluorescence was mea-
sured at 520 nm and normalized to the fluorescence measured at
495 nm (Tecan Spark). The 520 nm/495 nm ratios were used to
determine the binding of the test compounds to the FXR LBD.
(
catalog no. 8691002) was acquired from MP Biologicals. Goat
antimouse secondary antibody, HRP conjugate (catalog no. 12-
49), was purchased from Millipore, and antirabbit IgG, HRP-
3
THP-1, HepG2, and Caco-2 cell culture
linked secondary antibody (catalog no. 7074S), was obtained
from Cell Signaling via New England Biolabs. The peqGOLD Total
RNA Kit was purchased from PeqLab, and the High Capacity cDNA
Reverse Transcription Kit was from Applied Biosystems. The
LightCycler 480 SYBR Green I Master was purchased from Roche.
ABCA1 (Hs_ABCA1_1_SG QuantiTect Primer Assay, catalog no.
QT00064869), CYP7A1 (Hs_CYP7A1_1_SG QuantiTect Primer
Assay, catalog no. QT00001085), SHP (Hs_NR0B2_1_SG Quanti-
Tect Primer Assay, catalog no. QT00061460), GAPDH
THP-1 cells were obtained from ATCC and maintained in RPMI-
1640 medium supplemented with 10% heat-inactivated FBS,
100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-gluta-
mine at 37°C in an incubator with 5% CO . THP-1 macrophages
2
were acquired upon stimulation with 200 nM PMA for 72 h.
HepG2 cells (ATCC) were maintained in DMEM medium supple-
mented with 10% heat-inactivated FBS, 100 U/mL penicillin,
100 µg/mL streptomycin, and 2 mM L-glutamine at 37°C in an in-
(
Hs_GAPDH_1_SG QuantiTect Primer Assay, catalog no.
cubator with 5% CO . Caco-2 cells were obtained from DSMZ and
2
QT00079247), FGF19 (Hs_FGF19_2_SG QuantiTect Primer Assay,
catalog no. QT02452289), and BSEP (Hs_ABCB11_1_SG
QuantiTect Primer Assay, catalog no. QT00035049) oligonucleo-
tide primers were purchased from Qiagen.
maintained in EMEM medium supplemented with 20% heat-inac-
tivated FBS, 2 mM L-glutamine, and 1 × nonessential amino acids
at 37°C in an incubator with 5% CO . Test substances were dis-
2
solved in dimethyl sulfoxide (DMSO). The final DMSO concentra-
tion in all experiments was equal to 0.1%.
Luciferase reporter gene assays
RNA extraction and reverse transcription-quantitative
polymerase chain reaction (RT-qPCR)
For the luciferase reporter gene assays, HEK-293 cells (ATCC) were
maintained in DMEM containing 4.5 g/L glucose supplemented
with 2 mM L-glutamine, 10% FBS, 100 U/mL benzylpenicillin, and
6
THP-1 cells were seeded at a density of 0.2 × 10 /mL in a volume
1
00 µg/mL streptomycin. Cells were grown in 15 cm dishes at a
of 4 mL per well in 6-well plates and differentiated for 72 h, then
loaded with cholesterol (10 µg/ml) and treated with the indicated
test substances for 24 h, as described previously [15]. After treat-
ment, total RNA was extracted from cells using the peqGOLD Total
RNA Kit. Concentration of total RNA was measured with
NanoDrop 2000c. cDNA was synthesized from 1 µg total RNA
based on the protocol from the High Capacity cDNA Reverse Tran-
scription Kit. In combination with the LightCycler 480 System
from Roche, LightCycler 480 SYBR Green I Master was used for
quantification of mRNA expression. Relative mRNA levels were
quantified with the ΔCT method, using human GAPDH as an en-
6
density of 6 × 10 cells per dish for 19 h and then transfected via
the calcium phosphate precipitation method. After 6 h, the cells
were reseeded to 96-well plates (5 × 10 cells/well) in DMEM con-
taining 4.5 g/L glucose supplemented with 2 mM L-glutamine,
4
1
00 U/mL benzylpenicillin, 100 µg/mL streptomycin, and 5% char-
coal-stripped FBS. Cells were treated with indicated concentra-
tions of test compounds, positive control (50 µM CDCA and 3 µM
GW4064 for FXR, 1 µM GW3965 for LXRα/β, 10 nM GW0742 for
PPARβ/δ, 10 µM pioglitazone for PPARγ, and 10 µM lithocholic acid
for TGR5) or solvent vehicle (0.1% DMSO) and incubated for 18 h.
Ladurner A et al. Characterization of a… Planta Med

Original Papers
dogenous control. HepG2 cells were seeded at a density of
(200 rpm). Cells were then lysed with NP40 lysis buffer (150 mM
NaCl; 50 mM HEPES (pH 7.4); 1% NP40). The radioactivity in these
lysates was then measured by liquid scintillation counting. Choles-
terol uptake was calculated by normalization to protein content as
determined by the Bradford method.
6
1
× 10 /mL in 6-well plates. After 24 h, cells were treated in DMEM
supplemented with 5% stripped FBS, 2 mM L-glutamine, 100 U/
mL penicillin, and 100 µg/mL streptomycin with test substances
for another 24 h. Total RNA extraction and RT-qPCR were
performed like for THP-1 cells. Caco-2 cells were seeded at
6
2
Cholesterol efflux from THP-1-derived macrophages
0
.06 × 10 cells/cm on PET transwell inserts (Sarstedt) and culti-
vated for 19 days under asymmetric conditions (EMEM with L-glu-
tamine, streptomycin, penicillin, and non-essential amino acids on
the apical side and the same medium with additional 20% heat-in-
activated FBS on the basolateral side). Transepithelial electrical re-
sistance measurements were routinely performed to evaluate the
integrity of the cell monolayer. After 19 days, the medium was
changed to serum-free medium in the apical and medium supple-
mented with 0.5% BSA on the basolateral compartment for 24 h.
After that, cells were treated with test compounds for another
Cellular cholesterol efflux was measured as previously described
[29]. In short, THP-1 cells were seeded at a density of 0.2 × 106/
mL per well in 24-well plates and differentiated for 72 h. After
being washed twice with PBS, macrophages were treated with
test substances and labeled by incubation in RPMI-1640 medium
supplemented with 2.5% FBS, [3H]-cholesterol (0.5 µCi/mL), and
cholesterol (10 µg/ml). Cells were washed with PBS and then incu-
bated with fresh serum-free medium containing 10 µg/mL ApoA1
for 6 h to induce macrophage cholesterol efflux. Effluxed (super-
natant) and intracellular (cell lysate) [3H]-cholesterol was counted
by liquid scintillation. Cholesterol efflux (percentage of total cho-
lesterol) was determined by the ratio of radio-labeled cholesterol
in the supernatant to that of both supernatant and cells. The spe-
cific efflux is calculated as the difference between the efflux in the
presence and absence of the acceptor (blank): Specific efflux (%) =
Cholesterol efflux (%) – Blank efflux (%).
2
4 h. Total RNA extraction was performed with the TRIzol reagent
from Invitrogen. RT-qPCR was performed as described for THP-1
cells. GAPDH was used as housekeeping gene.
Western blot analysis
THP-1 macrophages, Caco-2 cells, or HepG2 cells were prepared
as described for RNA extraction. THP-1 macrophages and HepG2
cells were treated for 24 h with the indicated substances and
Caco-2 cells for 48 h. Cells were washed with cold PBS (4°C) and
lysed with NP40 buffer (THP-1, Caco-2; 150 mM NaCl; 50 mM
HEPES (pH 7.4); 1% NP40) or RIPA buffer (HepG2; 500 mM NaCl;
Statistical analysis
Statistical analyses were performed with the GraphPad prism
software version 4.03. Nonlinear regression (sigmoidal dose re-
sponse) was used to calculate the EC50 values and maximal fold
activation. The data are presented as mean ± SD. Statistical evalu-
ation was performed by 1-way analysis of variance (ANOVA) with
the Bonferroni post-test or Studentʼs t-test, whichever was appro-
priate. Differences between groups with a p-value < 0.05 were
considered statistically significant.
5
0 mM TrisHCl pH 6.8; 1% Nonidet P40; 0.5% deoxycholat; 0.1%
SDS; 0.05% sodium azide), respectively, containing a protease in-
hibitor mixture (1% Complete (Roche); 1% phenylmethylsulfonyl
fluoride; 0.5% Na VO ; 0.5% NaF). Cell lysates were harvested
3
4
and centrifuged at 16000 g for 20 min at 4°C to remove cell de-
bris. Protein concentration was determined by the Bradford meth-
od. An equal amount of protein samples was resolved via sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and then transferred onto a PVDF membrane (Bio-Rad). The
membranes were blocked with 5% low-fat milk and sequentially
incubated with the primary antibodies [ABCA1 (1:500); actin
Molecular docking
Docking studies were conducted using AutoDock Vina 1.1. [30] as
implemented in LigandScout 4.4. (LigandScout 4.4., Inte:Ligand
GmbH) The protein data bank (PDB) structure 6hl1 [11] describ-
ing the endogenous ligand CDCA bound to human FXR was se-
lected for the binding mode calculations. Pose prediction accura-
cy was assessed in a re-docking experiment with excellent results
(RMSD < 0.1). Default settings were used.
(
1:5000)] and appropriate secondary antibodies [goat antimouse
secondary antibody, HRP conjugate (1:5000) or antirabbit IgG,
HRP-linked secondary antibody (1:500)] according to the manu-
facturerʼs instructions. Luminescence was detected after incuba-
tion with ECL-reagent by a LAS-3000 (Fujifilm). Densitometric
analysis was performed using the Multi Gauge software (Fujifilm).
Actin protein levels were used as loading control.
Supporting Information
Supplementary Fig. 1S and details for all reaction steps and ana-
lytical data on the synthesis of leoligin and leoligin analogs are
available in the Supporting Information.
Cholesterol uptake in intestinal Caco-2 cells
Caco-2 cells were prepared as described for RNA extraction. They
were treated for 48 h with the indicated test substances. Cells
were then washed with PBS and 1.8 mL of serum-free DMEM sup-
plemented with 2 mM L-glutamine, 1× nonessential amino acids,
and 1% human plasma was added to the basolateral compart-
ment. A micellar solution containing 0.05 mM cholesterol, 1 µCi/
mL [3H]-cholesterol, 2 mM sodium taurocholate, 0.2 mM lyso-
phosphatidylcholine, 0.6 mM oleic acid, and 0.2 mM monooleoyl-
glycerol, together with test substances, was added to the apical
compartment and incubated for 2 h at 37°C on the orbital shaker
Acknowledgements
This project was supported by the Austrian Science Fund (FWF): S10710,
S10711, and S10704 (NFN-Drugs from Nature Targeting Inflammation)
and by the Vienna Anniversary Foundation for Higher Education
(H-268438/2018). We thank Daniel Schachner, Scarlet Hummelbrunner,
Alexandra Holzer, Lenka Kovářová, Nadja Kühne, and Ulrike Kirchweger
for their excellent technical assistance. We also thank Inte:Ligand GmbH
(Vienna) for an academic license for LigandScout 4.4.
Ladurner A et al. Characterization of a… Planta Med

[
16] Waltenberger B, Atanasov AG, Heiss EH, Bernhard D, Rollinger JM, Breuss
JM, Schuster D, Bauer R, Kopp B, Franz C, Bochkov V, Mihovilovic MD,
Dirsch VM, Stuppner H. Drugs from nature targeting inflammation
Conflict of Interest
The authors declare that they have no conflict of interest.
(DNTI): a successful Austrian interdisciplinary network project. Monatsh
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