Discovery and structure-activity relationship studies of indole derivatives as liver X receptor (LXR) agonists

Article abstract of DOI:10.1016/j.bmcl.2007.03.076

A structurally novel liver X receptor (LXR) agonist (1) was identified from internal compound collection utilizing the combination of structure-based virtual screening and high-throughput gene profiling. Compound 1 increased ABCA1 gene expression by eightfold and SREBP1c by threefold in differentiated THP-1 macrophage cell lines. Confirmation of its agonistic activity against LXR was obtained in the co-factor recruitment and reporter transactivation assays. Structure-activity relationship studies on compound 1 are described.

Full text of DOI:10.1016/j.bmcl.2007.03.076

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3473–3479
Discovery and structure–activity relationship studies of
indole derivatives as liver X receptor (LXR) agonists
Farid Bakir,a Sunil Kher,a Madhavi Pannala,a Norma Wilson,a Trang Nguyen,a Ila Sircar,a
Kei Takedomi,d Chiaki Fukushima,d James Zapf,b Kui Xu,c Shao-Hui Zhang,c
Juping Liu,c Lisa Morera,c Lisa Schneider,c Naoki Sakurai,c
Rick Jackc and Jie-Fei Chenga,*
aDepartment of Chemistry, Tanabe Research Laboratories USA, Inc., 4540 Towne Centre Court, San Diego, CA 92121, USA
bDepartment of Computational Discovery, Tanabe Research Laboratories USA, Inc., 4540 Towne Centre Court,
San Diego, CA 92121, USA
cDepartment of Biology, Tanabe Research Laboratories USA, Inc., 4540 Towne Centre Court, San Diego, CA 92121, USA
dDepartment of Chemistry, Tanabe Seiyaku Company Ltd, Toda, Japan
Received 26 February 2007; revised 20 March 2007; accepted 23 March 2007
Available online 27 March 2007
Abstract—A structurally novel liver X receptor (LXR) agonist (1) was identified from internal compound collection utilizing the
combination of structure-based virtual screening and high-throughput gene profiling. Compound 1 increased ABCA1 gene expres-
sion by eightfold and SREBP1c by threefold in differentiated THP-1 macrophage cell lines. Confirmation of its agonistic activity
against LXR was obtained in the co-factor recruitment and reporter transactivation assays. Structure–activity relationship studies
on compound 1 are described.
Ó 2007 Elsevier Ltd. All rights reserved.
Liver X receptors (LXRa and LXRb) belong to the type
2 family of the nuclear hormone receptor superfamily
that function as transcription factors.1 LXRa is ex-
pressed at high levels in liver, adipose tissue, and macro-
phages, while LXRb is ubiquitously expressed. LXRs
function as heterodimers with the retinoid X receptors
(RXR) and regulate the expression of a number of genes
involved in cholesterol and fatty acid metabolism.2,3
Upon agonist binding, the DNA binding domain
(DBD) of LXR interacts with LXR response elements
on target genes to initiate transcription. One LXR target
gene is the ATP-binding cassette transporter ABCA1,
which is involved in reverse cholesterol transport
(RCT) from macrophages in the atherosclerotic plaques
to high-density lipoproteins (HDL) in the plasma.4,5 As
such, increasing RCT by LXR agonism is a potential
therapeutic approach for a number of pathophysiologi-
cal states including dyslipidemia, atherosclerosis, and
diabetes.6 To date, several distinct classes of agonists
have been described in the literature7–9 and patents10
which include natural ligands 24(S),25-epoxycholesterol
(EPC, I), N,N-dimethyl-3b-hydroxy-cholenamide (DMHCA,
II), and non-steroidal synthetic ligands: GW3965 (III)
and T0901317 (IV) (Fig. 1). Both natural and synthetic
N
O
O
HO
HO
DMHCA (II)
24(S), 25-Epoxycholesterol (I)
CF3
OH
CF3
O
O
S
HOOC
N
O
N
CF3
T0901317 (IV)
Keywords: Virtual screening; High-throughput gene profiling; LXR
agonist; Liver X receptor.
Cl
GW3965 (III)
CF3
*
Corresponding author. Tel.: +1 858 622 7029; fax: +1 858 558
9383; e-mail: jcheng@trlusa.com
Figure 1. Natural and synthetic LXR agonists.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.076

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F. Bakir et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3473–3479
LXR agonists have been shown to increase the expres-
sion of ABCA1. Synthetic LXR agonists raise plasma
HDL levels in mice and show anti-diabetic activity in
rodent models of type 2 diabetes.11,12 However, LXR
agonists also activate sterol response element binding
protein-1c (SREBP1c) expression, which controls the
entire fatty acid biosynthetic pathway and promotes
hyperlipidemia and hepatic steatosis. Thus, there is a
clear need for selective LXR modulators that are devoid
of lipogenic activity and could offer potential pharmaco-
logical benefits.13
ergy transfer (HTRF) assay.16 Herein, we describe the
structure–activity relationship (SAR) studies on this no-
vel chemotype, as LXR agonists. Strategically, com-
pound 1 was divided into three portions: N-Boc-indole
(left portion), indane (linker), and benzene sulfonamide
(right portion), and systematic SAR studies were con-
ducted in each portion (Fig. 2).
A general synthesis for initial hit 1 and its analogs is
shown in Scheme 1. The amine 2 was converted to the
requisite sulfonamides (3 and 3a–b) followed by Suzuki
coupling leading to the indole sulfonamides (1, 1a–l, 35,
and 36). Alternatively, 2 was protected as the trifluoro-
acetamide that was coupled with N-Boc-indole-boronic
acid followed by de-protection to yield the amine inter-
mediate 4. Compound 4 was derivatized to yield various
targets (5a–y and 37–41). The enantiomerically pure iso-
mers, 1m (R) and 1n (S), were synthesized from the cor-
responding chiral amines 2a (R)- and 2b (S), which in
turn were obtained in excellent optical purity by co-crys-
tallization of 2 with (S)- and (R)-camphorsulfonic acids,
respectively.17
In the search for novel LXR modulators with appropri-
ate gene regulation profiles, we adopted a high-through-
put gene profiling platform for our primary screening.
Fifteen hundred compounds were selected from an inter-
nal compound collection through a homology-based
virtual screening14 and subsequently evaluated by
high-throughput genomic technology (HTG, Inc., Tuc-
son, AZ)15 for gene expression profiling including 12
LXR target genes. Boc-indole compound (1) was identi-
fied as an initial hit that showed an excellent induction
ratio of ABCA1 over SREBP1c genes in the differenti-
ated THP-1 macrophages. It was further confirmed to
be an LXR agonist with a cofactor peptide recruitment
study using homogeneous time-resolved fluorescence en-
A convergent synthesis was also developed to explore
the N-1 substitution on the indole ring of compound 1
(Scheme 2).18 Thus, 2-nitrobenzaldehyde was converted
into dibromovinyl aniline 9 in two steps in high yields.
The aniline was functionalized (10) to yield the desired
N-alkyl (X = CH2), N-acyl (X = CO), and urea
(X = CONH) compounds which were subsequently re-
acted with the indane boronic ester 11 to provide the
corresponding N-substituted indole derivatives.
Left portion
Right portion
Linker
O
S
O
NH
Fold induction @ 10 μM:
ABCA1: 8.4
SREBP1c: 3.1
EC50 (μM): 0.21 (LXRβ)
N
O
Compounds wherein the linker attachment position was
moved from the 2-position on the N-Boc-indole moiety
(1) to the 4- and 6-positions (28 and 29, Fig. 3), respec-
tively, were synthesized from requisite boronic acids
according to Scheme 1. The N-Boc-pyrrole and N-Boc-
O
1.42 (LXRα)
1
Figure 2. Initial HTG hit and SAR strategy.
a
e
NSO2Ph
R2
NH2
NHCOCF3
Br
Br
Br
3: R2 = H
2
b
3a: R2 = CH3
d
d, f
3b: R2 = CH2Ph
i
NHSO2R
NH2
c or d
N
N
Boc
Boc
5a-y
NSO2Ph
R2
4
R1
g or h
N
R3
NHX
1:R1 = H; R2 = H; R3 = Boc
1a-l:R2 = H; R3 = Boc
R
N
Boc
j
35:R1 = H; R2 = CH3; R3 = Boc
36:R1 = H, R2 = CH2Ph; R3 = Boc
6: R1 = H; R2 = H; R3 = H
37-40: X = CO
41: X = CONH
Scheme 1. Reagents and conditions: (a) PhSO2Cl, DIEA, DCM, quantitative; (b) R2X, NaH, DMF; (c) N-Boc-indole-2-boronic acid (optionally
substituted), Pd[(Ph)3P]4, Na3PO4, DMF, 100 °C, 20–40%; (d) N-Boc-indole-2-boronic acid (optionally substituted), PdCl2(PPh3)2, K2CO3, toluene/
ethanol/water (10:1:1), microwave, 10 min; (e) TFAA, DIEA, DCM, 91%; (f) 10% Na2CO3, MeOH, rt, 83%; (g) RCOCl, DIEA, DCM, 70–80%; (h)
RNCO, DIEA, DCM, 50%; (i) similar to (a) with substituted PhSO2Cl; (j) TFA, DCM.

F. Bakir et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3473–3479
3475
Br
Br
Br
Br
Br
Br
CHO
NO2
a
b
c or d or e or f
NH
X
NH2
NO2
9
7
10
R
8
H
N
H
N
O
S
O
O
B B
O
h
S
O
O
O
O
3
N
X
B
O
g
O
R
11
12-16: X= -COO
17:
X = -SO2
18-21: X = -CO
22-27: X = -(CH2)n
Scheme 2. Reagents and conditions: (a) CBr4, PPh3, DCM; (b) SnCl2, ethanol, 100 °C, 2 h, 82%; (c) RCH2Br, K2CO3, THF; (d) RCOCl, Et3N,
DCM, 0 °C–rt, 18 h, 50–70%; (e) RNCO, Et3N, DCM; (f) RSO2Cl, Et3N, DMAP, DCM, 0 °C–rt, 18 h, 50–70%; (g) KOAc, Et3N, Pd(dppf)Cl2,
dioxane, 100 °C, 90%; (h) 10, Pd[(Ph3P)]4, Cs2CO3, toluene/ethanol/water (10:1:1), 10–20%.
O
O
S
NH
R =
;
;
;
R
N
NHBoc
N
Boc
N
N
Boc
Boc
Boc
29
28
30
31
Figure 3. Linker attachment position.
naphthalene compounds (30 and 31, Fig. 3) were pre-
pared in an analogous manner.
As indicated in Table 2, removal of the t-Boc group at
the indole N-1 position (6) resulted in the complete loss
of activity. Other carbamates such as ethyl, isobutyl,
and neopentyl (13–15) were 8- to 10-fold less potent
than the parent tert-butyl carbamate (t-Boc) derivative
1. Methyl carbamate (12) or more bulky alkyl carba-
mates such as 16 were inactive. No activity was seen
with the phenyl sulfonamide compound 17. While cyclo-
pentyl, n-pentyl or cyclopropyl methyl carboxamides
(18–20) retained micromolar potency, tert-butylmethyl
amide 21 wherein a methylene group substituted an oxy-
gen atom in 1 was completely inactive. The alkyl modi-
fications (22–27) were inactive indicating that either the
pocket size is limited around this area or the carbonyl
group is critical for activity.
Compounds 32–34 with three different 1-aminoindane
linkers (1,4;1,5, and 1,6) were prepared in two steps from
the requisite bromoindan-1-ylamine (Scheme 3), which in
turn were prepared from the corresponding 1-indanone
via a modified Leuckart reaction in excellent yields.19
SAR compounds prepared above were tested in the
cofactor recruitment assay and were rank-ordered based
on EC50 values. Those compounds tend to have slight
selectivity for LXRb over LXRa. Initial SAR studies
indicated that only small groups such as methyl or hy-
droxy at the 6-position of the indole ring were tolerated
(compare 1k and 1l with 1, Table 1). Replacement of the
6-hydroxy in 1l with 6-methoxy group (1j) reduced ꢀ2-
fold potency, whereas total loss of activity was observed
with the corresponding benzyloxy analog 1i. Any group
at the C-5 position, even a fluorine atom (1e), decreased
the binding activity, indicating that the interaction of 1
with the LXR binding site is tight around the indole
ring. Compound 1n (S-) isomer was fivefold more potent
than the (R-) isomer 1m.
The attachment point of the indole ring on the amin-
oindan linker seems to play a critical role. Switching
the aminoindan linker from the 2-position in 1 to
the 4- or 6-positions (28 and 29, Fig. 3), respectively,
was detrimental to the potency (EC50 > 30 lM).
Replacement of N-Boc-indole with N-Boc-pyrrole or
N-Boc-naphthalene resulted in a complete loss of
activity (30 and 31).
a, b
32: 1,4-
33: 1,5-
34: 1,6-
c, d
Br
Br
N
Boc
1
NHSO2Ph
O
NH2.HCl
Scheme 3. Reagents and conditions: (a) HCOONH4/NaCNBH3, MeOH, 65%; (b) 10% concd HCl in MeOH, quantitative; (c) PhSO2Cl, DIEA,
DCM, 90%; (d) N-Boc-indole-2-boronic acid, Pd[(Ph3P)]4, DMF, 100 °C or K2CO3, toluene/ethanol/water (10:1:1), microwave, 10 min, 20–40%.

3476
F. Bakir et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3473–3479
Table 1. Effect of substitution on the indole ring
Table 3. SAR of the sulfonamide moiety
O
O
R
O
S
O
S
NH
NH
N
N
Boc
Boc
R
a
a
Compound
R
EC50 (lM) LXRb
Compound
R
EC50 (lM) LXRb
5a
5b
5c
5d
5e
5f
4-Cl
4-F
1.16
0.46
0.65
0.43
0.73
1.81
1.33
0.96
0.46
0.6
1
1a
H
0.21
>30
>30
>30
>30
1.5
4-OH
4-OBn
5-OH
5-OBn
5-F
4-Et
1b
1c
4-Me
4-CF3
3-Cl
1d
1e
5g
5h
5i
3-Me
3-OMe
3-CN
2-CO2Me
2-CO2H
2-Me
2-OMe
2-CF3
2-CN
2-F
1f
1g
5-CN
5-OMe
5-Me
6-OBn
6-OMe
6-Me
6-OH
H
26
28
1h
1i
18
5j
>30
1.6
0.3
5k
5l
3.35
0.2
1j
1k
5m
5n
5o
5p
5q
5r
0.6
0.8
1l
0.67
0.66
0.13
1m(R)-isomer
1n(S)-isomer
0.06
0.10
0.09
0.76
H
a Results shown are mean values of duplicate samples in a single
experiment.
2,6-DiF
2-Cl
a Results shown are mean values of duplicate samples in a single
experiment.
Table 2. SAR of N-substitution of indole
O
Table 4. SAR of additional sulfonamides
O
S
NH
O
O
S
R
NH
N
R
N
a
Boc
Compound
R
EC50 (lM) LXRb
a
6
12
13
14
15
16
17
H
COOMe
>30
>30
1.6
Compound
R
EC50 (lM) LXRb
S
COOEt
COOCH2(i-Bu)
COOCH2(t-Bu)
5s
0.11
1.3
1.4
COOCH2CH(Me)(CH2)3Me >30
SO2Ph
>30
1.16
5t
0.67
0.13
2.1
S
O
N
18
19
CO
O
N
Me
5u
5v
5w
Me
0.9
2.1
CO
Ph–Ph(4)
20
21
22
23
24
25
26
27
CO(CH2)4Me
COCH2t-Bu
Me
>30
>30
>30
>30
>30
>30
>10
0.52
CH2COO t-Bu
(CH2)3OBn
CH2Ph
5x
5y
1
Me
Et
0.21
0.40
0.19
CH2Ph(4-OMe)
(CH2)2Ph
Ph
a Results shown are mean values of duplicate samples in a single
experiment.
a Results shown are mean values of duplicate samples in a single
experiment.
The importance of the relative position of the right por-
tion and left portion on the central indan ring (2,5 in
compound 1) was explored. Compound 33 with a 1,5
linker retained potency (EC50 = 0.22 lM), while com-
pounds with 1,4 (32) and 1,6 (34) linkers were not toler-

F. Bakir et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3473–3479
3477
Table 5. SAR of the carboxamide/urea analogs
Selected compounds were tested for their ability to acti-
vate LXRb in a reporter transactivation assay20 and the
regulation of LXR target genes such as ABCA1 and
SREBP1c in either THP-1 differentiated macrophages
or HepG2 cells with RT-qPCR assays (Table 6). In gen-
eral, those compounds maintained good potency in the
reporter assay. It is worth noting that those selected
compounds demonstrated somewhat better gene expres-
sion profiles than known LXR agonists such as GW3965
and T0901317. For instance, compound 5b caused 11-
fold increases in ABCA1 gene expression while the
SREBP1c gene induction was only twofold. Cell-based
functional assays, such as cholesterol efflux and lipogen-
esis assays,21,22 also indicated that these indole-based
compounds induced lipogenesis to a lesser extent than
did T0901317 or GW3965 (Table 6). It is interesting to
point out that compound 5t, which showed an activity
against LXRa, stimulated ABCA1 gene expression and
induced significant cholesterol efflux in the THP-1 cells,
indicating LXRb activity might be the determining fac-
tor for this cellular activity. However, correlation be-
tween LXRb activity and ABCA1 gene regulation is
not straightforward (5b vs 5q), partially due to com-
pound properties.
R
X
NH
N
Boc
a
Compound
X
R
EC50 (lM) LXRb
37
38
39
40
41
CO
CO
H
2-F
0.19
0.17
0.47
0.53
1.5
CO
CO
2-OMe
2-OH
H
CONH
a Results shown are mean values of duplicate samples in a single
experiment.
ated (EC50 > 30 lM) indicating the importance of over-
all shape of the compounds for binding.
Unlike the N-Boc-indole moiety, the sulfonamide region
could tolerate a wide range of structural variations
(Tables 3–5). In general, small substitutions (Me, F,
CN) on the phenyl sulfonamide were tolerated although
the ortho position was favored over the meta or para (5l,
5o, 5p, and 5r vs 5g, 5i, 5b, and 5f, respectively) posi-
tions. Addition of a second fluorine atom at the 6-posi-
tion of the phenyl ring in 5p did not improve the potency
any further (5q vs 5p). The 2-CN analog (5o) was the
most potent one with an EC50 value of 60 nM. The cor-
responding carboxylic acid analog (5k) lost ꢀ17-fold po-
tency. The heterocyclic analogs 5s (2-thienyl) and 5u
(isoxazolyl) increased potency slightly(Table 4). Intrigu-
ingly, even the methyl sulfonamide (5x) retained potency
similar to the phenyl analog 1. Substitution on the sul-
fonamide nitrogen in 1 was not tolerated. While the
Docking studies suggested that compound 1 fits nicely in
the LXRb ligand binding domain. The indole aromatic
ring system is close to Trp457 in the AF-2 (helix 12)
and forms a p–p interaction, while the Boc carbonyl
group forms a hydrogen bond with the conserved
His435 in helix 10/11, thereby locking it into an agonis-
tic conformation.23 The binding mode is very similar to
that of GW3965. The plausible binding mode is consis-
tent with the limited room for modification around the
Boc-indole moiety. The sulfonamide group may have
an interaction with Arg319 (Fig. 4).
N-Me
analog
35
retained
weak
activity
In summary, a structurally novel series of LXR ago-
nists was identified utilizing the combination of two
powerful technologies, virtual screening and high-
throughput gene profiling. Systematic SAR studies
were conducted and several potent and efficacious
LXRb agonists were identified. The docking study re-
vealed that the indole moiety in these compounds
may interact with conserved histidine 435 in helix 10/
10 and tryptophan 437 in the helix 12 (AF-2) to lock
(EC50 = 2.65 lM), the corresponding N-Bn analog 36
was inactive (EC50 > 30 lM) implying the requirement
of an H-bond donor at this site for binding.
While the replacement of the sulfonamide moiety with
the corresponding carboxamides (37–40) was tolerated,
the urea analogs (e.g., 41) were not favored (Table 5),
indicating the sulfonamide group may be able to interact
with the protein in the active site.
Table 6. In vitro and cell functional activities for selected LXR agonists
ABCA1 inductionb
(THP-1)
SREBP-1c
inductionb
(HepG2)
Efflux EC50
(THP-1)
Maximum
effluxc
Lipogenesis
Inductionc
(HepG2)
a
Compound
HTRF
a
EC50
LXRa
HTRF
a
EC50
LXRb
Reporter
a
EC50
LXRb
1
5b
1.5
4.3
0.21
0.065
0.12
8.4
11.4
7.5
3.1
2.0
2.1
2.9
3.0
5.2
6.7
0.680
0.570
0.590
0.570
0.21
1.6
1.7
1.7
1.5
1.7
1.8
1.6
3.1
2.0
1.2
1.7
3.2
2.7
3.6
0.464
0.666
0.108
0.093
0.035
0.015
5t
>30
0.336
0.363
0.052
0.015
0.011
5s
5q
0.54
0.57
0.367
0.04
7.3
7.5
GW3965
T0901317
10.7
8.4
0.010
0.033
a Values shown are in lM.
b Values shown are in ratio of compound (10 lM) versus control (vehicle only).
c Values shown are in ratio of compound (10 lM) versus control (vehicle only).

3478
F. Bakir et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3473–3479
11. Grefhorst, A.; van Dijk, T. H.; Hammer, A.; van der
Sluijs, F. H.; Havinga, R.; Havekes, L. M.; Romijn, J. A.;
Groot, P. H.; Reijngoud, D. J.; Kuipers, F. F. Am. J.
Physiol. Endocrinol. Metab. 2005, 289, 829.
12. Cao, G.; Liang, Y.; Broderick, C. L.; Oldham, B. A.;
Beyer, T. P.; Schmid, R. J.; Zhang, Y.; Stayrook, K. R.;
Suen, C.; Otto, K. A.; Miller, A. R.; Dai, J.; Foxworthy,
P.; Gao, H.; Ryan, T. P.; Jiang, X. C.; Burris, T. P.;
Eacho, P. I.; Etgen, G. J. J. Biol. Chem. 2003, 278, 1131.
13. Michael, L. F.; Schkeryantz, J. M.; Burris, T. P. Mini-Rev.
Med. Chem. 2005, 5, 729.
14. Virtual screening was performed with the program
GLIDE (Schro¨dinger). A set of 145 LXR agonists
reported in the literature were used to establish conditions
using the crystal structure of human LXRb (P8D.pdb). A
collection of 135,000 compounds was screened and the
1290 top scoring compounds were selected for assay.
15. High-throughput genomics screening was performed using
the ArrayPlate technology in a contract service provided
by HTG, Inc. The ArrayPlate technology is a quantitative
nuclease protection assay for multiplex gene profiling
(http://www.htgenomics.com/).
16. The HTRF cofactor peptide recruitment assay was modi-
fied from a previous report.24 Polyhistidine-tagged human
LXRa (2 nM) or b (1 nM) ligand-binding domain (Roche
Diagnostics, Indianapolis, IN) was mixed with the test
compound, 20 nM biotin-SRC1 peptide (Synpep, Dublin,
CA), 5 nM streptavidin–allophycocyanin, and europium-
labeled anti-polyhistidine antibody (1 and 0.5 nM for a and
b, respectively) in 50 mM Tris, pH 7.5, 50 mM KCl, 1 mM
EDTA, 0.1% BSA, and 1 mM DTT. The final volume of the
mixture was 40 lL in a 384-well assay plate. The mixture
was incubated at room temperature for 1 h with shaking.
Time-resolved fluorescence was measured at 615 and
665 nm on a LJL Analyst plate reader. The ratio of 665/
615 was used to calculate EC50 values of test compounds.
17. Prashad, M.; Hu, B.; Har, D.; Repic, O.; Blacklock, T. J.;
Acemoglu, M. Adv. Synth. Catal. 2001, 343, 461.
Figure 4. Plausible binding mode of compound 1 in LXRb LBD.
LXR into the agonistic conformation. Selected indole
compounds were shown to regulate LXR target genes
and they tended to show slightly improved profiles in
cell based functional assays as compared to GW3965
and T0901317.
Acknowledgment
We thank Dr. Fabio Tucci for his comments on the
manuscript.
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2 mg/ml BSA. Subsequently, 5 lg/ml of APO-AI was
added in fresh serum-free medium along with vehicle or
compound for additional 18 h incubation. Cholesterol
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22. To measure compound induction of lipogenesis, HepG2
cells in 48-well tissue culture plates were pre-treated with
vehicle or compound for 24 h. One microcurie of [14C]glyc-
erol was added and cells were cultured for another 48 h.
Cellular triglycerides were extracted, separated by TLC,
and quantified on a Storm 820 phosphorimager (GE
Healthcare, Giles, UK). Lipogenesis was presented as
fold-induction versus control (vehicle only). Results shown
are mean values of triplicate samples in a single experiment.
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