Biology Reference
In-Depth Information
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1
Fig.
1
Small molecule inhibitor of miR-122 discovered in a pilot screen of the NCI Diversity Set II
setup HTS
assay for
miR-122
increased
luciferase signal
in miR-122
assay?
yes
activity in
psiCHECK-
control
assay?
no
activity in
miRNA control
luciferase
assay?
miR-122 silencing
by RT-PCR and no
effect on other miRNAs
no
screen small
molecule
library
no
yes
yes
yes
compound is
not a miR-122
inhibitor
compound activates
luc in a non-miRNA
specific fashion
general miRNA
pathway inhibitor
validated miR-122
inhibitor that can be
further investigated
Fig.
2
Steps for the identifi cation of specifi c miR-122 inhibitors using a high-throughput screen and a set of
counter screens
line was generated (
see
Chapter
11
for a detailed discussion of the
assay development). In this cell line, the mature miR-122 binds its
target sequence and causes a decrease in the
Renilla
luciferase
expression. In the presence of a small molecule miR-122 inhibitor,
luciferase expression is restored, enabling the identifi cation of small
molecule inhibitors of miR-122 function. The developed reporter
assay was applied in a pilot screen of small molecule inhibitors of
miR-122, followed by several counter screens to: (1) eliminate
molecules that increase or decrease luciferase expression in a non-
miRNA controlled way (
see
Subheading
3.4
), (2) eliminate com-
pounds that act on the miRNA pathway in a general fashion and
are not specifi c for miR-122 (
see
Subheading
3.5
), and (3) validate
the effect of the small molecules on mature miR-122 levels (
see
Subheading
3.6
). The Diversity Set II from the NCI Developmental
Therapeutics Program was screened at a 10
M concentration in a
96-well format and several initial hit compounds were identifi ed as
potential miR-122 inhibitors that induced a
μ
≥
fi vefold increase in
the relative luciferase signal.
After further validation following the decision tree outlined in
Fig.
2
, several of the initial hits were dismissed due to toxicity or
non-miRNA-specifi c interactions with the luciferase reporter.
Importantly, the small molecule
1
(Fig.
1
) was verifi ed as a potent,
low-micromolar miR-122 inhibitor. When Huh7 cells transfected
with the psiCHECK-control plasmid were treated with
1
, no
change in luciferase signal was observed, indicating that the inhibi-
tor does not increase the luciferase expression in a non-miRNA-
specifi c fashion. Further, the compound showed no effect in a
