Regulating miRNA-21 Biogenesis by Bifunctional Small Molecules

Article abstract of DOI:10.1021/jacs.7b00610

We report a new strategy to regulate microRNAs (miRNAs) biogenesis by using bifunctional small molecules that consist of a pre-miRNA binding unit connected by a linker to a Dicer inhibiting unit. In this effort, fluorescence polarization-based screening was used to identify neomycin as a pre-miR-21 binding ligand. Although neomycin cannot inhibit miR-21 maturation, linking it to the RNase inhibitor 1 forms the bifunctional conjugate 7A, which inhibits the production of miR-21. We expect that this strategy will be applicable to design other molecules for miRNA regulation.

Full text of DOI:10.1021/jacs.7b00610

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Communication
Regulating miRNA-21 Biogenesis by Bi-functional Small Molecules
Hao Yan, Umesh Bhattarai, Zhi-Fo Guo, and Fu-Sen Liang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00610 • Publication Date (Web): 13 Mar 2017
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Regulating miRNA-21 Biogenesis By Bi-functional Small Molecules
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Hao Yan, Umesh Bhattarai, Zhi-Fo Guo, Fu-Sen Liang*
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Department of Chemistry and Chemical Biology, University of New Mexico, 300 Terrace Street NE, Albuquerque, NM
87131, United States
Supporting Information Placeholder
It is not uncommon that small molecules, which are selected
using binding-based screening against miRNA precursors, are
unable to block miRNA maturation.10 This phenomenon might be
a consequence of promiscuous targeting and/or poor cell permea-
bility of these small molecules. In addition, these small molecules
might possess binding affinities that are insufficiently low to
compete with the extensive macromolecule interactions occurring
between RNAs and other binding partners. To enhance binding
between small molecules and targeted miRNA precursors, ration-
ally designed bi-functional molecules have been developed in
which additional binding moieties are conjugated to original
RNA-recognizing molecules. For example, Duca and co-workers
designed neomycin-nucleobase conjugates that have additional
base pairing driven affinities against pre-miRNAs to give new
inhibitors that block maturation of miR-372 and -373.11 Maiti and
co-workers developed dual binding neomycin-bisbenzimidazole
conjugates, which contain an intercalating unit to enhance binding
of neomycin, and showed that the conjugates serve as inhibitors
for oncogenic miR-27a.12
ABSTRACT: We report a new strategy to regulate microRNAs
(miRNAs) biogenesis by using bi-functional small molecules that
consist of a pre-miRNA binding unit connected by a linker to a
Dicer inhibiting unit. In this effort, fluorescence polarization-
based screening was used to identify neomycin as a pre-miR-21
binding ligand. Although neomycin cannot inhibit miR-21 matu-
ration, linking it to the RNase inhibitor 1 forms the bi-functional
conjugate 7A, which inhibits the production of miR-21. We ex-
pect that this strategy will be applicable to design other molecules
for miRNA regulation.
MicroRNAs (miRNAs) are small non-coding RNAs, which
regulate gene expression by targeting mRNAs for translational
repression or destabilization. Various biological processes are
controlled by miRNAs,1 and dysregulation of miRNAs is associ-
ated with many human diseases.2 As a result, methods to regulate
the expression or function of miRNAs should be useful in studies
aimed at probing their function as well as the development of
novel therapies for human diseases.
Chemically modified antisense oligonucleotides (ASO) have
been used for blocking the function of miRNA.3 However, appli-
cations of ASO are limited by their poor cell permeability, unpro-
ductive accumulation in endosomes, inherent off-target effects,
and poor pharmacokinetic and bio-distribution profiles.4 These
limitations demonstrate that alternative methods are needed to
regulate miRNAs. Consequently, the development of small mole-
cules capable of modulating miRNA production has emerged as a
promising approach to overcome these limitations.5
The biogenesis of miRNAs is a multistep process,6 that starts
with transcription of the miRNA gene in the nucleus to generate
the primary transcript (pri-miRNA). Pri-miRNA is processed by
the RNase Drosha to form precursor miRNA (pre-miRNA), which
is then exported to the cytoplasm and further processed by the
RNase Dicer to produce the miRNA duplex. One strand of the
duplex, the mature miRNA, is incorporated into the RNA-induced
silencing complex (RISC) to guide the translational regulation of
targeted mRNAs.
A small molecule that targets any step in this sequence can po-
tentially serve as a miRNA regulator.5 For example, Deiters and
co-workers showed that miRNAs can be regulated by using small
molecules that repress their transcription.7 In addition, Disney and
co-workers developed a computational approach to identify small
molecules that bind to Drosha or Dicer processing sites within pri-
miRNA or pre-miRNA, which thereby inhibit the biosynthesis of
the mature miRNA.8 Other inhibitors interfering with the miRNA
maturation process have been discovered through screening of
chemical libraries.5,9
Figure 1. A schematic illustration of the new approach to regulate
miRNA biogenesis by using bi-functional small molecule that
target pre-miRNA.
In this work, we devised and tested a new strategy to regulate
miRNA biogenesis that relies on the use of newly designed bi-
functional small molecules (Figure 1). The purpose of previously
developed bi-functional molecules is to target two closely located
binding sites and utilize bi-valent recognition to enhance binding
affinity. In contrast, our bi-functional molecules are designed to
deliver a specific Dicer inhibitor to the Dicer-catalyzed cleavage
site in the target pre-miRNA/Dicer complex. The new conjugates
consist of a high affinity pre-miRNA binding unit linked to a low
affinity Dicer inhibition unit. The Dicer inhibitor in the conjugate
is selected so that it alone does not inhibit Dicer to a significant
extent. As a result, the pan-cellular function of Dicer will not be
disrupted by the bi-funtional molecule. However, recognition of a
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target pre-miRNA by the RNA binding unit in the conjugate
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brings the Dicer inhibitor unit into close proximity with the pre-
miRNA cleavage site of bound Dicer, thus causing blockage of
the activity of Dicer. Importantly, by functing in this manner, the
inhibition unit will promote inhibition of the production of the
target miRNA.
Oncogenic miR-21 is upregulated in many cancers.13 As a
result, small molecules that inhibit the production of miR-21 can
serve as useful tools to study the roles played by miR-21 in
cancers and as potential cancer therapeutic agents. To demostrate
the feasibility of the new strategy described above, we designed,
prepared and tested bi-functional inhibitors that block the
biogenesis of human miR-21.
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The first stage of assembling the newly designed bi-functional
Figure 2. Fluorescence polarization of KOF in the absence or
presence of pre-miR-21 and different aminoglycosides. The error
bars represent the standard error of mean (N = 3).
miR-21 inhibitors involves identification of
a pre-miR-21
targeting small molecule. Fluorescence polarization (FP), a
powerful tool for studying molecular interactions, was utilized for
this purpose.14 In this assay, the degree of FP from a fluorophore-
labeled small molecule increases when it forms a complex with a
macromolecule owing to a reduction in the rate of fluorophore
tumbling. To identify small molecules that bind pre-miR-21, we
used the FP-based competitive binding assay to screen ligands
that can compete with a moderate affinity ligand for binding to
pre-miR-21. Because kanamycin binds to several RNAs,15 we
expected that it would bind pre-miR-21 and, as a result, serve as
the standard ligand in the FP-based screening protocol. To verify
miR-21 is reflected in a decrease of the FP value. Among the
tested substances, neomycin was found to reduce the FP of
KOF/pre-miR-21 to a value that matches that of free KOF, indi-
cating a complete displacement (Figure 2). To determine the
binding affinity of neomycin to pre-miR-21, the hydroxyl
fluorophore-tagged derivative of neomycin, NF, was prepared
(Scheme S2). Using the FP assay, we determined that the Kd of
NF to pre-miR-21 is 38 ± 2 nM (Figure S2), which is 10 fold
smaller than that of KOF. This result confirms that neomycin
strongly binds to pre-miR-21 and led to the selection of neomycin
as the pre-miR-21 binding unit in the new bi-functional mole-
cules. Importantly, the above result indicates that modification of
the primary hydroxyl group on neomycin does not disrupt
binding, therefore, this hydroxyl group can be a suitable position
to conjugate the Dicer inhibition unit.
The active site of Dicer is located within the RNase III domain,
which cleaves pre-miRNAs through a mechanism in which two
metal ions (Mg2+) catalyze the phosphoryl transfer reaction.17 The
distance between the two Mg2+ is around 4 Å, similar to those in
active sites of other mechanistically similar RNases. Many RNase
inhibitors with metal chelating properties have been developed,18
including the N-hydroxyimide 1 (Scheme 1a) that inhibits
influenza endo-nuclease at micomolar concentrations.18b As a
result, 1 was used as the Dicer inhibiting unit in the new bi-
functional molecules.
this assumption, kanamycin derivatives, containing
a 1,8-
naphthalimide fluorophore at either the primary hydroxyl (KOF)
or the primary amine group (KNF) (Scheme S1, ESI) were
prepared and their binding to pre-miR-21 was determined using
the FP assay (Figure S1, ESI). Based on the saturation binding
curve of KOF versus pre-miR-21 from the FP assay, a binding
affinity (Kd) of 419 ± 83 nM was obtained (Figure S1b). On the
other hand, the binding affinity of KNF to pre-miR-21 was found
to be too small to be determined (Figure S1c).
Based on these results, KOF was employed as the standard pre-
miR-21 binding ligand in the FP-based screening protocol.
Screening was carried out by incubating the pre-formed KOF/pre-
miR-21
complex
with
13
commercially
available
aminoglycosides, which are one of the best studied classes of
molecules that target RNAs.14-16 Displacement of KOF from pre-
miR-21 by an aminoglycoside with higher affinity against pre-
Scheme 1. (a) Structure of an influenza endo-nuclease inhibitor. (b) Synthesis of inhibitor building blocks. (c) Synthesis of
bi-functional small molecules.
(b)
(a)
O
O
O
O
CO2H
CO2H
b
a
N
O
N
PMB
N
O
OH
nO
nO
O
OH
58-65%
n = 0
81%
O
O
O
2, 3 7 A
Compound
&
2
n = 0
4
1
3
B
C
D
1
2
3
NH2
NHR'
O
(c)
O
HO
HO
HO
HO
R = R' =
NH2
NHR'
O
H2N
R'HN
O
5
O
N
N
N
O
NH2
OH
H
Neomycin
5
O
NHR'
S
NH2
O
R
N
O
c
48%
43%
OH
N
H
OH
O
HO
OH
n
O
H2N
Boc
O
S
O
NHR'
d
OH
H
OH
OH
N
Boc
6
e
O
O
N3
S
5
O
OH
O
OH
Compound 3
H2N
O
R'HN
7
18-40%
Reagents and conditions: (a) para-methoxybenzyloxyamine, toluene, reflux; (b) trifloroacetic acid, DCM; (c) i) (Boc)2O, Et3N, DMF,
o
H2O, 60 C; ii) TPS-Cl, pyridine, r.t.; iii) Cysteamine hyrochloride, Cs2CO3, DMF; (d) 6-azido-hexanoic acid, EDCI, TEA, DCM; (e) i)
CuSO4, sodium ascorbate, DMSO/H2O; ii) trifloroacetic acid, DCM.
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The route to synthesize bi-functional inhibitors began with
Because 7A is the most potent Dicer inhibitor in the in vitro as-
say, it was used for in cell investigations. To study the in cell
inhibitory activity of 7A, mature miR-21 expression levels were
determined by utilizing the RT-qPCR assay. HEK293T cells were
transfected with pCMV-miR21 plasmid expressing pre-miR-21
and treated at the time of transfection with neomycin, a
commericial phosphorothioate-based anti-miR-21 ASO (anti-
miR-21 B), or various concentrations of 7A. As shown in Figure
4a, 7A inhibits the expression of miR-21 in a dose-dependent
manner (76% reduction at 5 µM when compared to non-treated
cells). The activity of 7A is similar to that of anti-miR-21 B. In
contrast, neomycin does not cause a significant inhibition of miR-
21 expression at 100 µM. When 7A was added 4 h after
transfection, no inhibititory activtiy was observed, which suggests
the transfection procedure may assist cellular delivery of 7A.
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preparation of a series of homophthalic acids (2A-D) possessing
alkynyl polyethylene glycol tails of different lengths (Scheme 1b
& S3). Reactions of 2A-D with para-methoxybenzyl (PMB)
oxyamine produced O-PMB-oxyimides 3A-D that were later
coupled to neomycin. The free imide 4, used as a control in
activity assays, was produced by removing the PMB group in 3A
with trifloroacetic acid. Employing the reported method,14 the
amino groups in neomycin were protected and the primary
hydroxyl group was tranformed to an amino group to give 5
(Scheme 1c). Reaction of 5 with 6-azido-hexanoic acid gave the
azide 6, which was then coupled with 3A-D using click chemistry.
Subsequent removal of the Boc and PMB protecting groups from
the click products by using trifloroacetic acid generated bi-
functional molecules 7A-D containing tethers of different length
linking the two functional units.
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Figure 3. (a) The representative image of the electrophoresis
analysis of Dicer-mediated pre-miR-21 cleavage in the presence
of tested compounds. (b) Densitometric quantitative analysis of
miR-21 levels from three independent assays as in (a). The error
bars represent the standard error of mean (N = 3).
Figure 4. (a) RT-qPCR analysis of mature miR-21 expression
levels. (b) The representative image of western blotting analysis
of PDCD4 levels in pre-miR-21 expressing HEK293T cells with
or without 7A treatment. (c) Densitometric quantitative analysis
of PDCD4 levels from three independent assays as in (b). The
error bars represent the standard error of mean (N = 3).
The ability of 7A-D to inhibit Dicer-mediated pre-miR-21
cleavage in vitro was determined. For this purpose, 32P-labeled
pre-miR-21 was prepared by in vitro transcription,19 and then
incubated (37 °C, 2.5 h) with recombinant human Dicer in the
absence or presence of 7A-D, or either the Dicer inhibition unit
alone (i.e. compound 4), the pre-miR-21 binding unit alone (i.e.,
neomycin), or a commercial morpholino-based anti-miR-21 ASO
designed to block miR-21 maturation (anti-miR-21 A). The extent
of cleavage of pre-miR-21 (72 nt) into mature miR-21 (22 nt) was
then determined utilizing a 15% denaturing polyacrylamide gel
and phosphor-imaging (Figure S3). As shown in Figure 3, Dicer
cleaves pre-miR-21 completely to form mature miR-21. In addi-
tion, anti-miR-21 A, as a positive control, inhibits cleavage by
70% at 5 µM, indicating that the assay performs correctly. Im-
portantly, bi-functional molecule 7A inhibits Dicer promoted
cleavage in a dose dependent manner, reducing the level of miR-
21 formation by 90% at 5 µM (Figure 3 & S4). A comparison of
the results coming from studies with 7A-D shows that Dicer cata-
lyzed miR-21 formation increases as the length of the linker be-
tween the two functional units increases, demonstrating that inhib-
itory activity is critically altered by the distance between the two
functional units. In contrast to the bi-functional molecules, un-
modified neomycin, at concentrations up to 1 mM, does not inhib-
it Dicer activity (Figure 3 & S5). Furthermore, the inhibitor unit
alone (i.e., 4) displays no inhibition at concentrations up to 50 µM
(Figure 3 & S6). The combined results clearly show that conjuga-
tion of neomycin, the pre-miR-21 recognition unit, to the Dicer
inhibitory unit 1 significantly enhances the Dicer inhibition po-
tency (>200 fold comparing 7A to neomycin) and that the pres-
ence of a linker of proper length between the two units is essential
for the activity.
Programmed cell death protein 4 (PDCD4) is a tumor
suppressor protein, whose mRNA is a direct target of miR-21.20
Expression of miR-21 represses PDCD4 expression in HEK293T
cells and repression of miR-21 should de-repress PDCD4
production. As expected, the expression level of PDCD4 protein,
determined by using western blotting and compared to non-treated
cells, increases by 90% when cells are treated with 7A (Figure 4b
& 4c). The cellular assay results demonstrate that 7A is highly
active in repressing miR-21 formation in cells.
In the design of bi-functional molecules employed in this study,
we utilized aminoglycosides as the pre-miRNA binding unit
because they are well-known RNA-binders. However,
aminoglycosides, including neomycin, are known to target a
number of RNA sequences and, as a result, the new bifunctional
substances could suffer from selectivity issues.11-12,14-16
To investigate the miRNA regulation selectivity of the
neomycin-based bi-functional molecule 7A, its ability to inhibit
another randomly chosen miRNA, miR-34a, in HEK293T cells
was determined using RT-qPCR. The results show that 7A indeed
reduces the expression of miR-34a (40% reduction at 5 µM when
compared to non-treated cells), but not at the same level of
potency as it does against miR-21 at the same concentration
(Figure S7). Thus, although 7A displays some degree of
selectivity, the incorporation of more selective pre-miRNA
recognition moieties other than aminoglycosides in the bi-
functional design may lead to improved RNA selectivity.
Because neomycin is known to target ribosomal RNA and
interfere translation, effects of 7A on translation were tested by
transfecting HEK293T and CHO cells with an EGFP reporter
followed by 7A treatment.8b No significant change on EGFP
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(14) (a) Kirk, S. R.; Luedtke, N. W.; Tor, Y. J. Am. Chem. Soc. 2000,
122, 980; (b) Michael, K.; Wang, H.; Tor, Y. Bioorg. Med. Chem. 1999,
7, 1361.
expression was observed at the concentration (5 µM) that inhibits
miR-21 maturation (Figure S8). Furthermore, no cytotoxicity was
observed (analyzed by the MTT assay) when cells were treated
with 7A at the same concentration (Figure S9). These results show
that under the condition of miR-21 inhibition, 7A does not affect
translation or cause toxicity.
In summary, a new approach to regulate miRNA maturation
was developed that relies on the use of a Dicer-inhibiting bi-
functional small molecule. The study showed that 7A, which con-
tains neomycin as a pre-miR-21 binding unit and the N-
hydroxyimide 1 as the Dicer inhibitory unit, inhibits miR-21 mat-
uration in vitro and in cells. Together, these observations demon-
strate that the new bi-functional strategy is applicable to the de-
sign of active miR-21 inhibitors based on an otherwise non-active
pre-miR-21 binder. We expect that the strategy will be affective in
generating new bi-functional regulators for miRNAs.
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(19) Huang, C.; Yu, Y.-T. In Curr. Protoc. Mol. Biol.; John Wiley &
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H.; Colburn, N. H.; Post, S.; Allgayer, H. Oncogene 2008, 27, 2128.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS
Publications
website.
Figure S1-S9, Scheme S1-S3 and experimental details (PDF)
AUTHOR INFORMATION
Corresponding Author
fsliang@unm.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This research was supported by an Institutional Development
Award (IDeA) from the National Institute of General Medical
Sciences of the National Institutes of Health (P20GM103451).
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