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6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 125
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miRNAs function, thus increasing miRNAs eects and restoring the expression of
tumor suppressor miRNAs.
Maiti and co-workers also reported the screening of dierent classes of small
molecules for their miRNome modulation potential. Noteworthy, three quinazoline
compounds, such as compound 2 (Figure 6.3), were shown to non-specically
upregulate tumor suppressor miRNAs in MCF-7 cancer cells and to inhibit cancer
cells proliferation with an EC50of 20 μM [30]. The authors demonstrated that these
compounds act by inducing apoptosis or cell cycle arrest. As for enoxacin, the eect
is non-specic and not related to a direct RNA binding.
As illustrated by these examples, activation of miRNAs expression by small
molecules cannot represent a specic approach, and it is proposed as a general
method to increase miRNAs levels without discriminating which miRNAs are
aected. After these few examples, there were no further developments, and the
research in the eld focused on the specic inhibition of oncogenic miRNAs as will
be described in the following sections.
6.4.2 Inhibition of miRNAs Production: Pre- and Pri-miRNA Binders
The strategy of inhibiting overexpressed miRNAs has been focused on inhibiting
their biogenesis. For example, interaction with the mature miRNA to prevent
interactions with mRNA (Step 6 in Figure 6.2), or inhibiting enzymes involved in
the dierent steps of miRNA biogenesis and maturation (Steps 2–5 in Figure 6.2).
This approach is thus based on the hypothesis that a molecule that is able to bind
eciently and specically to a miRNA precursor could inhibit its biogenesis [23].
MiRNA precursors (pre-miRNAs and pri-miRNAs) bear a particular secondary
and tertiary structure that associates single-stranded and double-stranded regions
and induces the formation of three-dimensional structures favorable to specic
interaction with small molecules. The interaction of a small molecule with one of
the oncogenic miRNA precursors can thus inhibit the corresponding biogenesis
step, such as the cleavage of the pri-miRNA by Drosha or of the pre-miRNA by
Dicer and thus block the synthesis of the overexpressed oncogenic miRNA [31].
It is also possible to interfere with RNA–protein interactions such as miRNA-Ago
or miRNA-Lin28, the latter being an essential protein for miRNA function.
The examples reported in the literature so far concern molecules identied by
high-throughput screening of large collections of compounds (>500 molecules), by
screening focused libraries of compounds known to interact with RNA or by designing selective ligands based on the structure of the targeted RNA. In the following
section we will discuss key examples of miRNA-inhibiting agents and the methodologies used to discover them.
6.4.2.1 Discovery of miRNAs Inhibitors by Intracellular Assays
High-throughput screening (HTS) allows the identication of molecules able to bind
to miRNAs or their precursors in order to inhibit their functions. Various screening
methods have been developed over time based on the use of uorescence and/or
luminescence, with cellular, in vitro or in silico assays. Intracellular assays have
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Figure 6.4 (a) Inhibitors of miRNAs biogenesis 3–8 discovered by intracellular assays.
(b) Primary and secondary structure of pre-miR-21.
been widely employed to screen compound libraries because they allow for the fast
evaluation of the phenotype as well as for the quantication of the targeted miRNA.
The rst library screen for the identication of oncogenic miRNA inhibitors
was performed by Deiters’ team in 2008 with an intracellular assay based on a
luciferase reporter system [32]. Screening of more than 1000 compounds against
miR-21, a widely studied oncogenic miRNA in a large number of cancers, followed
by structure–activity relationship studies led to the identication of the derivative
diazobenzene 3 (Figure 6.4a), capable of inhibiting miR-21 production. The study
of the mode of action of this analogue suggests that this compound inhibits the
transcription of the miR-21 gene into pri-miR-21. It should be noted that this
compound does not aect the expression of other miRNAs at the intracellular level,
suggesting specicity of action. Deiters’ team continued to exploit library screening
to nd small molecules that inhibit the production and/or action of oncogenic
miRNAs. Using the same type of intracellular assay, they screened more than
300,000 compounds, and structure–activity relationship studies led in 2018 to the
identication of compound 4 (Figure 6.4a) and of ether-amide 5 (Figure 6.4a), both
highly eective and selective inhibitors of miR-21 production [23, 33]. However,
studies regarding the mechanism of action show that 4 does not aect either
gene transcription or pri-miR-21 formation in cells, whereas the ether-amide
compound family to which 5 belongs inhibits miR-21 biogenesis at the level of
the transcription step with a similar action to the azobenzene molecule 3. Cellular
assays have also demonstrated the ability of compound 4 to restore the sensitivity
of kidney carcinoma cells to chemotherapy and the inhibition by treatment with
molecule 5 of proliferation and microtumor formation in cervical cancer cells.
These cellular proof-of-concept studies make these two anti-cancer molecules
6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 127
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particularly promising. Using a similar reporter system, the same authors have also
screened compounds that could inhibit miR-122 that is directly involved in the
proliferation of hepatitis C virus (HCV) [34]. A primary screen of more than 300,000
compounds was followed by various steps of selection led to the identication of
compound 6 as the most promising for further development. Medicinal chemistry
optimization of this compound led to improved analogs such as compound 7, which
shows similar potency to that of 6 (IC50= 12.5 μM in an intracellular assay for
the measurement of miR-122 inhibition) but also a high specicity to inhibit the
production of miR-122 without aecting the expression of other miRNAs. The study
of the mechanism of action showed that these compounds are likely to interact with
HNF4α transcription factor directly instead of with a nucleic acid involved in the
biogenesis pathway. These compounds are thus not acting as specic RNA binders.
An alternative strategy to HTS is to screen focused libraries that are enriched with
compounds known to have the ability to bind RNA. In this context, an intracellular
screening approach was used to test 15 aminoglycosides using a luciferase assay to
identify inhibitors of miR-21 activity [35]. Streptomycin (compound 8, Figure 6.4a)
was identied as the most eective inhibitor with a level of inhibition comparable
to that of a specic antisense oligonucleotide. Also tested on various other miRNAs, 8 showed only partial selectivity, proving that the molecule does not inhibit
Dicer which would result in non-selective and therefore probably toxic inhibitors.
Docking and footprinting studies have shown that streptomycin binds eciently to
pre-miR-21 (Figure 6.4b), near the apical loop, which blocks the access, and therefore the cleavage, of Dicer. To nalize the study, cell-based assays in Jurkat lines
were conducted. The level of miR-21 is well repressed and an increase in apoptosis is observed, the latter being linked to an increase in the level of PDCD4, an
apoptosis-inducing protein and one of the main targets of miR-21.
Intracellular assays proved to be eective in the discovery of specic miRNA
inhibitors, but it is clear that the mechanism of action of hits derived from screens
needs to be veried. As noted above, in some cases the mechanism did not involve
direct binding to RNA but rather involved inhibition of transcription. In vitro assays
that will be described in the following sections are more suitable to discover RNA
binders able to inhibit miRNA processing in a specic manner.
6.4.2.2 Target-Based In Vitro Assays
A large number of RNA binders have been discovered thanks to target-based assays
mimicking the intracellular context. Some of these ligands have subsequently been
tested in cells to verify their biological activity with very successful results, and
target-based in vitro assays proved to be particularly ecient for the discovery of
specic ligands. Fluorescence-based biochemical assays are the most common
approaches that have been employed for screening. In this context, Nakatani’s
team has developed an HTS screen via a uorescent indicator displacement
(FID) assay where a uorescent intercalator can be displaced by a ligand, thus
inducing a decrease of uorescence upon binding [36]. While this assay format is
particularly suited for identifying compounds that bind RNA via a non-selective
intercalation mode, some promising compounds have been identied. Indeed,
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these studies have allowed for the screening of several thousand compounds on
dierent pre-miRNAs for the identication of compounds inhibiting Dicer cleavage.
As an example, after an initial screen and structure–activity relationship studies,
compound 9 (Figure 6.5) could be identied as an inhibitor of pre-miR-29a cleavage
by Dicer [37]. This tricyclic compound containing a central isoxazole linked to
a phenyl and an oxadiazole shows binding to pre-miR-29a in SPR studies. Even
though the binding anity was not precisely quantied, this kind of chemical
structure could be a starting point for the search for new pre-miRNA ligands.
Using a similar approach, Herdewijin and co-workers screened 22 molecules as
potential inhibitors of Dicer-mediated cleavage of pre-miR-155, which is known
to be involved in normal and oncogenic immune functions [38]. Among these
molecules, intercalating agents such as ethidium bromide 10 and Hoechst 33258 11
as well as the aminoglycosides kanamycin B 12b and neomycin B 13 (Figure 6.5)
showed the best anity for the target. Aminoglycosides are well-known RNA
ligands that bind to the RNA of the prokaryotic ribosome and inhibit protein
synthesis in bacteria [39]. They are therefore used clinically as antibiotics [8].
However, only the intercalating agents were able to inhibit Dicer cleavage. Another
screen of 14 commercial aminoglycosides for their ability to inhibit Dicer cleavage
on pre-miR-27a, an oncogenic miRNA overexpressed in various cancers led to
the identication of streptomycin 8, neomycin 13, and tobramycin as ecient
inhibitors [40]. These compounds were then studied in MCF-7 cells that overexpress miR-27a with a luciferase assay to conrm the inhibitory activities in cells. All
three molecules decreased miR-27a expression by 35–50%.
In order to identify inhibitors of pre-miR-21 cleavage by Dicer, a uorescence
resonance energy transfer (FRET) assay was developed by Davies and Arenz in 2006
against miR-21 [41]. A rst study thus led to the identication of an aminoglycoside, kanamycin A 12a (Figure 6.5), as an inhibitor of pre-miRNA cleavage by Dicer.
Arenz’s team also worked on the synthesis of aminoglycoside mimetics and identied compound 14 (Figure 6.5) as an inhibitor of Dicer cleavage of pre-let-7 [42].
Aminoglycosides have thus been extensively studied for their ability to interfere with
oncogenic miRNA biogenesis.
A screen of several classes of antibiotics (aminoglycosides, tetracyclines,
macrolides, lincosamides, linezolid, chloramphenicol, and puromycin) to identify
inhibitors of Dicer cleavage was performed on four pre-miRNAs involved in various
cancers (−372, −373, −17, and −21) [43]. Neomycin B 13 was identied as the
most active molecule for the dierent pre-miRNAs as well as the best ligand for
the precursors of these miRNAs. Minocycline 15, which belongs to the tetracycline
family, also showed interesting activity. Tetracyclines, like aminoglycosides, bind to
the prokaryotic ribosome and are used clinically as antibiotics. Compounds 13 and
15 bind to pre-miRNAs at the Dicer binding site which prevents Dicer from binding
to its target and inhibits cleavage.
Another screen based on the FRET technique was conducted using 640 compounds to identify inhibitors of miR-372 biogenesis. Specically, the goal was to nd
molecules capable of binding to pre-miR-372 and, in turn, inhibit the biogenesis of
this oncogenic miRNA [44]. The miR-372 is oncogenic in several cancers such as
Figure 6.5 Chemical structures of compounds 9–18 discovered with in vitro target-based assays able to detect inhibition of the biogenesis of miRNAs.
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gastric adenocarcinoma [45], esophageal cancer [46], and thyroid cancer [47]. This
screen identied three active compounds, among which the spermine-amidine
compound 16 (Figure 6.5) was the most promising. Indeed, compound 16 inhibits
the biogenesis of miR-372 in vitro as well as in cancer cells. This compound showed
excellent specicity in the presence of other nucleic acids such as transfer RNA
or DNA. Moreover, the antiproliferative activity on gastric adenocarcinoma cells
overexpressing miR-372 is highly specic since no activity is observed on other
gastric epithelial cell lines. The miRnome study revealed that this compound acts
only on a small number of miRNAs with a similar interaction site. The set of
inhibited miRNAs also share a protein target: the tumor suppressor protein LATS2.
The latter is the target of miR-372, and its expression is restored at the cellular level
in the presence of the miR-372 inhibitor.
Disney’s team has also been heavily involved in the identication of oncogenic miRNA inhibitors using a very ecient screening methodology based
on two-dimensional combinatorial screening (2DCS) [48]. In a rst study, two
guanidinylated aminoglycosides (G-NeoB 17 and G-KanA 18, Figure 6.5) were
identied by screening a library of compounds bound to an agarose surface against
a large library of labeled RNAs containing 6-nucleotide internal loops [49]. Amplication and sequencing of compound-bound RNAs allowed the identication
of specic interactions between guanidinylated compounds and one (or more)
loop(s). The miRNA precursors containing these loops were then identied, and
indeed G-NeoB 17 (Figure 6.5) selectively recognizes an internal loop located on
the pri-miR-10a which corresponds to the catalytic site of Drosha. Noteworthy,
miR-10a is involved in many cancers and represents an interesting biological
target [50]. The study of the cellular activity on HeLa cells conrmed the inhibition
activity of compound 17; the biogenesis of miR-10a is specically inhibited at the
level of pri-miRNA cleavage, as proven by the accumulation of pri-miR-10a in the
studied cells.
In the search for new assays suitable for the discovery of compounds inhibiting the
production of miRNAs, a technique known as catalytic enzyme-linked click chemistry assay (cat-ELCCA) was developed. This assay is based on the combination of
click chemistry-based assays with catalytic signal amplication [51]. When performing cat-ELCCA, a biotinylated biomolecule is initially immobilized in the wells of a
microtiter plate coated with streptavidin. A click chemistry handle may already be
present in this substrate or may be added by an enzymatic or biomolecular interaction. An initial click reaction with labeled horseradish peroxidase (HRP) is used
to start the detection process. This is followed by the addition of a pro-absorbent,
-uorescent, or -chemiluminescent HRP substrate. The assay was created to check
the presence of the terminal loop of the immobilized pre-miRNA in the case of Dicer
cat-ELCCA. In the presence of Dicer, the loop is cleaved, yielding no signal; while in
the presence of an inhibitor, the loop remains intact resulting in signal production.
This provides a turn-on assay for inhibition detection [52]. Pre-miR-21 was chosen
as an initial RNA target for screening ∼50,000 small molecules and ∼33,000 natural product extracts (NPEs) leading to the identication of known but non-specic
RNA binders, tetracyclines being the strongest inhibitors. Despite the diculties in
6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 131
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Figure 6.6 Compounds 19, 20a,and20b able to bind precursors of miRNAs and discovered
upon in silico screenings. The compounds have been reported as racemic structures.
the identication of specic binders and inhibitors, this assay proved to be extremely
ecient for screening large HTS of compound libraries.
Beside intracellular and in vitro assays, in silico methodologies also showed to be
promising for the discovery of compounds able to interfere with miRNA biogenesis. Kang et al. screened 1990 molecules in silico based on the three-dimensional
structure of pre-miR-21 [53]. Five molecules were studied in more detail on epithelial cancer cells (glioblastoma, breast cancer, and gastric cancer) and compound
19 (AC1MMYR2, Figure 6.6) showed the best activity on dierent cell lines.
However, this compound also indirectly targets other miRNAs such as miR-181
and miR 200a/b. Despite this lack of specicity, AC1MMYR2 showed very good antitumor results in preclinical in vivo studies of glioblastoma and breast cancer [54].
Recently, 20,000 compounds were screened by the small-molecule microarray
(SMM) method for pre-miR-21 ligands [55]. After screening and optimization, two
molecules 20a and 20b (Figure 6.6) were identied as promising. The study of
their mechanism of action showed that these molecules bind to the apical loop of
pre-miR-21 and prevent the proper function of Dicer.
These examples demonstrate that despite the implicit theoretical results obtained
by in silico studies, experimental applications of the compounds discovered by
means of docking can be successful. Altogether, the target-based in vitro assays
described above have proven to be particularly promising for the discovery of
inhibitors of miRNAs biogenesis with the discovery of strong inhibitors in vitro and
in some cases in cells.
6.4.2.3 Design of Specific Ligands of Pre- and Pri-miRNAs
Although screening assays have identied very promising inhibitors, the rational
design of ligands specic for a particular miRNA would be the ideal methodology
to obtain ecient inhibition and also be able to modulate the activity of the
compounds and avoid toxic eects. To date, it remains extremely dicult to design
RNA ligands specic for a certain sequence/structure, but some examples have been
reported in the literature. Lu et al. were inspired by the rst structure identied by
HTS (compound 3, Figure 6.4) and synthesized several analogues considering that
the diphenylazene structure could be replaced by the more synthetically accessible
N-phenylbenzamide structure [56]. These analogues were rst tested for their ability to inhibit miR-21 biogenesis and function in HeLa and U-87 cells. Compound 21
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(a)
(c)
(d)
Figure 6.7 Chemical structure of RNA ligands designed to bind to pre-miRNAs.
(a) Compound 21. (b) Primary and secondary structures of pre-miR-372. Colored squares
indicate the binding sites of compounds 22–24 with the colors corresponding to the moiety
interacting at the indicated site. (c) Compounds 22–24 designed to target pre-miR-372.
Colors indicate the different moieties and their site of interaction on the target.
(d) Naphthyridine 25.
(b)
(Figure 6.7a) was identied as the most eective, showing inhibition of miR-21
production at 10 μM. In order to evaluate the specicity of this compound, 12 other
miRNAs were tested and showed that the inhibition was specic for miR-21. This
study conrmed that chemical optimization of a known ligand can lead to better
inhibitors.
Multifunctional ligands have also been designed as conjugates between dierent
RNA binding domains bringing both anity and selectivity for the target. In this
context, multifunctional compounds in which several motifs known to interact with
RNA in an ane and/or specic manner were conjugated on the same molecule.
The aim of our studies was initially to target the production of the oncogenic
miR-372 upon binding to pre-miR-372 (Figure 6.7b). As mentioned above, this
miRNA has a protein target called LATS2 which is a tumor suppressor protein
whose expression is inhibited by miR-372. The rst ligands studied consisted of two
interaction motifs: an aminoglycoside (neomycin) and an articial nucleobase [57].
A screen that had been performed previously identied neomycin as the most
favorable compound of this family for inhibiting miR-372 production [43]. It was
therefore chosen for the preparation of a rst set of ligands and was conjugated to
several natural and articial nucleobases. The latter have been previously described
in the context of the triple-helix strategy for interaction with DNA base pairs [58].
6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 133
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Indeed, these compounds form specic hydrogen bonds, known as Hoogsteen
bonds, as opposed to Watson–Crick bonds with DNA base pairs. These nucleobases
have therefore been diverted to form specic bonds with the base pairs of miRNA
precursors and in particular with pre-miR-372 (Figure 6.7b). This type of conjugate
is thus designed to interact at the single-strand/double-strand junctions of the target.
Among the compounds synthesized and tested as inhibitors of miR-372 production, compound 22 (Figure 6.7c) showed very promising results [57]. Indeed, this
compound inhibits pre-miR-372 cleavage in vitro, and the proliferation of gastric
adenocarcinoma (AGS) cells that specically overexpress miR-372 as the same compound has no eect on the proliferation of other gastric epithelium cells that do
not express the targeted miRNA. Quantication of miRNAs at the intracellular level
showed that miR-372 production is inhibited in a dose-dependent manner but also
that other miRNAs are aected. The study of the target protein LATS2 showed that
its translation is restored in the presence of ligand 22.
Medicinal chemistry optimization of compound 22 was performed in order to
better describe the pharmacophore essential for activity and to design more active
compounds [59]. By varying both the nucleobase, the aminoglycoside, and the
spacer used to link these two parts, it was possible to identify the analogues 23a and
23b in which the nucleobase moiety was extended by addition of another aromatic
ring [59]. These two molecules retain the same intracellular specicity and have
better inhibitory activities against AGS cell proliferation. The study of the binding
site on the pre-miR-372 target has claried the formed interactions and enabled
design of more eective ligands. In this context, new ligands containing neomycin,
nucleobase, and also an amino acid were designed [60]. Being known to interact
eciently with RNA as they are the main constituents of natural RNA ligands,
i.e., peptides, three basic amino acids (lysine, histidine, and arginine) were chosen.
This study resulted in the discovery of compound 24 that was selective for miR-372
and -373 (Figure 6.7c). In addition to the previously obtained interactions with the
neomycin and articial nucleobase moiety, the histidine side chain allows selective
interaction with the stem–loop junction, which enhances anity and increases
selectivity toward pre-miR-372 (Figure 6.7b). At the intracellular level, compound
24 maintains its specic antiproliferative activity on gastric adenocarcinoma cells
overexpressing miR-372.
The team of Nakatani and coworkers recently developed a chemical tool to specifically inhibit Dicer processing of pre-miRNAs upon stabilization of pre-miRNAs
dimers. Tothis aim, they employed a cytosine-binding compound, naphthyridine 25
(Figure 6.7d), to interfere with Dicer cleavage of pre-miR-29a. The latter contains a
C-bulge close to the cleavage site of Dicer, and the authors demonstrated that binding to this bulge eectively inhibited Dicer cleavage with a specic mechanism of
action [61]. Indeed, 24 binds to the loop residues of pre-miR-29a and induces the
dimerization of these RNA structures. The dimerization is responsible for the eventual inhibition of Dicer processing.
In order to improve the design of ligands specic to a particular sequence/
structure, the team of Disney and co-workers also developed an ecient and
original methodology called Inforna [62]. This approach is based on the 2-DCS
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methodology previously described above, and it is applied to a small-molecule
library in the presence of a large collection of RNA motifs (internal loops, bulges,
apical loops). Second, RNA sequences capable of binding to one or more ligands are
analyzed with structure–activity relationships through sequencing (StARTS) [63].
The latter is a statistical tool that compiles the characteristics of RNA motifs that are
responsible for interaction with ligands identied by 2-DCS and thus predicts the
anities and selectivities of a ligand/RNA interaction. This analysis indicates the
probability that a ligand binds and is specic for a particular RNA structure. Finally,
the combination of the results obtained by 2-DCS and StARTS with structural
information of the potential RNA targets leads to the identication of particularly
ecient and highly specic ligands for the RNA containing the targeted structure.
Inforna was initially applied to the precursors of 1024 miRNAs, and particularly
active ligands were identied for 22 oncogenic pre-miRNAs. Compounds 26, 27,
and 28 (Figure 6.8a) were identied as ligands for pri-miR-182, pri-miR-96, and
pre-miR-210, all of which are oncogenic and overexpressed in dierent cancers [64].
While compound 26 is a derivative of kanamycin A, the other two molecules contain
benzimidazole and methylpiperazine moieties that therefore appear to be preferred
motifs for interactions with the internal loops of miRNA precursors. Additional
studies were performed on compound 27, identifying it as more specic for miR-96
than a complementary LNA oligonucleotide sequence. This compound induced
cell apoptosis at micromolar concentrations in breast cancer cells by binding to
the pri-miR-96 at the level of a U-U internal loop (green region in Figure 6.8b).
Following development of the Inforna approach, it was applied to a large number
of small molecules. Similar to 27, compound 28 was identied as a specic binder
of the G-G internal loop of pri-miR-96 (blue region in Figure 6.8b). The two
molecules were thus conjugated to form a dimer (compound 29, TargaprimiR-96)
in Figure 6.8a) optimally designed to have each monomer at its binding site on
the pri-miRNA [65]. This molecule binds with greater anity to pri-miR-96 and
inhibits pri-miR-96 biogenesis 400-fold over 26 in breast cancer cells. In addition,
studies in in vivo models of triple negative breast cancer (TNBC) show that the
dimer specically inhibits tumor growth and induces apoptosis. Pharmacokinetic
studies evaluating the concentration of the dimer in the plasma of mice after 48h
show a concentration much higher than that required to obtain a biological eect,
proving the bioactive potential of TargaprimiR-96 in vivo.
A few years later, compound 28, called TargapremiR-210, was studied as a ligand for pre-miR-210 (Figure 6.8b) and an inhibitor of Dicer-mediated cleavage [66].
Although cellular target engagement was not established, the study identied the
C–C internal loop (blue circle, Figure 6.8b) as the primary target of TargapremiR-210
with an anity constant of 165 nM compared to more than 2 μM for any other loop.
This internal loop is located close to the catalytic site of Dicer in pre-miR-210 which
makes this molecule active against pre-miR-210 cleavage at a nanomolar level. This
study established a correlation between activity and anity when a ligand binds to a
functional site of a miRNA precursor. MiR-210 is of therapeutic interest because it is
highly expressed in cancer cells under hypoxic conditions, such as in MDA-MB-231
triple-negative breast cancer cells. TargapremiR-210was therefore tested in vitro and
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