We also expressed RXR along with LXR to confirm the
ligand-binding specificity to LXR but not RXR in the LXR-RXR
heterodimer (Figure 7). Compounds 3–7, which have a tertiary
amide, showed stronger activity compared with compound 1 in
both the LXR and LXR assays. Once again, they showed
stronger activity in the LXR assay than the LXR assay. The
activity of compounds 5, 6, and 7 under conditions of RXR co-
expression was higher than that of LXR or LXR alone.
less potent compared with compound 1 (Table 3), compound 14
was more efficacious than compound 1 (Table 3). By contrast, -
lactam compound 17 and -lactam compound 18 showed lower
transactivation activity than compound 1. These results indicate
that among the compounds with restrained conformations, only
lactam compound 14 should have a conformation of the amide
oxygen suitable for binding the receptor. Comparison of those
amide (lactam) derivatives led us to determine the receptor-
bound conformation of cholenamide compound 1. This putative
receptor-bound conformation was consistent with that of the
chloenamide complexed in the ligand-binding site of the receptor,
as shown in Figure 3.
Next, we calculated EC50 by dose-curve using the similar
luciferase assay with cells expressing LXR. Presumably due to
the differences in the number of expressed receptors, the EC50
values deviated in each time of assay. Therefore, we included
compound 1 in each assay as a control to normalize the deviation
in each assay. T0901317 (T) was also included along with
compound 1 (Table 3).
In summary, we designed compounds exhibiting good affinity
for LXRs by modeling structures of LXRs in complex with their
ligands. In luciferase assays, the tertiary amide compounds 3 and
4 exhibited higher activity. In LXR-RXR assays, compounds
with hydrophobic moieties showed higher activity than
compound 1. The stereochemistry of the C23 of the lactams was
found to be important for determining the EC50 values and
transactivation of the genes, thus providing information
regarding the receptor-bound conformation of the side chain of
the cholenamide derivatives.
Table 3. Kinetic analyses of cholic acid derivatives for
LXR-dependent transcription stimulating activity
LXR
LXR
exp.
cpd
exp.
cpd
EC50
Efficacy/%
EC50
Efficacy/%
(M)
(M)
1
2
0.423
0.862
0.344
1.107
0.141
0.165
0.127
0.124
1.291
–
100
85
1
5
0.464
0.623
0.786
1.024
0.206
3.896
1.992
0.154
0.239
–
100
109
108
102
100
39
Acknowledgments
a
b
3
211
148
100
99
6
We thank Dr. Spyridon Theofilopoulos of Karokinska
Institute for providing LXR plasmids and LXRE reporter
plasmid.
4
7
1
1
c
e
g
8
d
f
10
11
1
Supplementary Material
9
134
100
66
65
1
100
117
0.69
100
98
Supplementary data associated with this article can be found,
12
13
1
14
15
1
0.54
100
62
References and notes
0.382
0.643
0.462
0.334
0.030
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