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6
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MicroRNAs as Targets for Small-Molecule Binders
Maria Duca
Université Côte d’Azur, CNRS, Institute of Chemistry of Nice, 28 avenue Valrose, 06100 Nice, France
6.1 Introduction
Among the various therapeutic targets exploited by currently marketed drugs,
proteins represent the large majority and are also the most studied for the development of new therapies. However, it is known that a large majority of the human
genome is transcribed into RNA and that only 1.5% of this RNA is then translated
into proteins [1]. Moreover, among these proteins only a very small percentage
is correlated with a pathology and many of them are qualied as “undruggable,”
because these proteins do not contain suitable binding sites in which small
molecules can bind with good anity and specicity. The remaining part of the
genome produces non-coding RNAs (ncRNAs), which represent major players for
the regulation of many cellular processes, such as transcription, translation, or the
regulation of gene expression. The discovery, characterization, and in-depth study
of these RNAs have led to the discovery that these structures may represent valid
therapeutic targets [2]. This allows for a much broader spectrum of therapies than
is currently available and oers hope for a number of diseases that have no existing
or eective treatment to date.
Over the last 20 years, several strategies have been developed to target RNA [3].
Among these approaches, the use of oligonucleotides is the most common method
and involves short nucleotide sequences capable of specically recognizing the target RNA. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs),
for example, are two classes of oligonucleotides that inhibit a particular RNA by
complementary recognition of RNA sequences [4]. To date, many oligonucleotides
are in clinical trials and several have been marketed. It should be noted, however,
that there are a number of limitations to the use of oligonucleotides as therapeutics, such as cost, biodistribution to the site of action, and metabolic stability. The
use of peptides represents a second strategy to target RNA [5]. These compounds of
intermediate molecular weight can provide a large surface area for interaction with
RNA and have good potential to form complexes with high anity and specicity.
More easily modied and optimized than oligonucleotides, peptides still present
119
RNA as a Drug Target: The Next Frontier for Medicinal Chemistry, First Edition.
Edited by John Schneekloth and Martin Pettersson.
© 2024 WILEY-VCH GmbH. Published 2024 by WILEY-VCH GmbH.
120 6 MicroRNAs as Targets for Small-Molecule Binders
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limitations for in vivo applications due to stability and biodistribution. The third
approach for targeting RNA is the use of small molecules that are able to interact with the target RNA through the recognition of RNA tertiary structural features rather than the primary sequence [6]. Indeed, the association of single- and
double-stranded regions of most biologically relevant RNAs induces the formation
of three-dimensional structures that are more similar to the structures of a protein than that of a DNA double helix [7]. These structures can thus be targeted by
small molecules with the advantage that they can in principle overcome the constraints of oligonucleotides and peptides thanks to much more favorable pharmacological properties. The use of small molecules to target RNA for chemical biology
and medicinal chemistry studies is thus a particularly promising approach although
many challenges still need to be addressed. Among the most studied targets, one
can cite ribosomal RNAs (rRNAs) and riboswitches in bacteria; genomic RNAs in
viruses; extended RNA repeats, splicing of precursor messenger RNAs (pre-mRNAs)
and microRNAs (miRNAs) in eukaryotes.
The main issue in all these studies is the identication of ligands able to bind
specically to the targeted RNA with high anity. Nevertheless, several successful
examples are already in the literature and, even more importantly, on the market
(Figure 6.1). Indeed antibiotics, such as aminoglycosides, chloramphenicols,
lincosamides, macrolides, oxazolidinones, or tetracyclines are known to bind to
specic sites on the RNA of the prokaryotic ribosome and inhibit protein synthesis
in bacteria [8]. These compounds were the rst to demonstrate the feasibility of
using small molecules to target RNA. More recently, risdiplam has been approved
by the FDA for the treatment of spinal muscular atrophy (SMA) [9]. This drug
is able to modify splicing upon binding to a pre-mRNA and thereby correct the
expression of SMN protein that is altered in this pathology. These eorts have also
led the way to the discovery of other splicing modulators [10].
Among the most exploited RNA targets in the literature, miRNAs, belonging to
the class of ncRNAs, have been widely studied. Beside ribosomal RNA (rRNA) and
transfer RNA (tRNA), both of which are linked to the translation of mRNAs into
proteins, many classes of ncRNAs exist and are generally divided into two families:
long non-coding RNAs (lncRNAs) and small non-coding RNAs including miRNAs.
Figure 6.1 Examples of marketed drugs acting as RNA binders as the main molecular
mechanism of action.
6.2 MicroRNAs 121
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The discovery and identication of miRNAs and of their functions in the
regulation of gene expression is one of the most important discoveries of the
last decades. In particular, the deregulation of miRNAs has been associated with
the initiation and development of a large number of diseases including cancers.
As a consequence, miRNAs represent a new class of therapeutic targets for the
development of anti-cancer therapies. Given the recent success of small molecules
targeting RNA, targeting miRNAs is currently of high interest to the scientic
community and could represent a particularly eective strategy in the treatment of
cancers or other miRNA-associated pathologies. Furthermore, chemical probes able
to interfere with the miRNA network are important tools to elucidate intracellular
pathways that have not yet been completely understood. In this chapter, we will
illustrate in detail how miRNAs have been targeted by small molecules and which
strategies can be employed to eciently and specically inhibit therapeutically
relevant miRNAs.
6.2 MicroRNAs
As mentioned above, miRNAsare small ncRNAs consisting of short oligonucleotides
of 18–25 nucleotides. MiRNAs are involved in the regulation of gene expression
through selective interaction with one or more mRNAs to induce their degradation
and inhibit translation. The rst miRNA, Lin-4, was discovered in 1993 in the worm
species Caenorhabditis elegans [11]. Lin 4 regulates lin-14 messenger RNA leading
to a decrease in production of its associated proteins. This research led to characterization of a new mechanism of regulation of gene expression and revolutionized
the understanding of molecular biology. Seven years separate this discovery from
that of a second miRNA, let-7, again in C. elegans, but also found in animals and
humans [12]. Since then, many miRNAs have been identied in animals, plants,
and viruses, some of which are present in all of these species [13]. Numerous studies
have been undertaken to precisely link each miRNA to the genes it regulates. Some
miRNAs have high levels of expression, others very low, and all vary from species to
species. Aided by increasingly sophisticated biotechnologies, the discovery of these
new players in living organisms has grown exponentially since the early 2000s.
In each species, miRNAs can be grouped into families. Members of these families
are transcribed by adjacent genes and have the same core sequence (called the “seed
sequence”), i.e. the sequence responsiblefor the recognition of targeted mRNAs [14].
The number of miRNAs varies within these families and also varies from species to
species. Regarding nomenclature, several suxes can be found in miRNA names.
When two miRNAs have an identical sequence but come from a dierent precursor,
the suxes added are numbers (e.g. miR-1-1 and miR-1-2). When two miRNAs come
from the same precursor but not from the same strand, they are annotated with a
sux “-3p” or “-5p” depending on the strand where they are located if they are found
in similar proportions in the cell (e.g. miR-146b-3p and miR-146b-5p). If one of these
two miRNAs is much more abundant than the other form, an asterisk will follow the
name of the less abundant one (e.g. miR 146*). Finally, the sux will be a letter if the
122 6 MicroRNAs as Targets for Small-Molecule Binders
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miRNAs have identical sequences except for one or two nucleotides (e.g. miR-146a
and miR146b). In order to understand the strategies that are being used to target
miRNAs, it is important to understand how they are formed and how they function,
and this will be the subject of the following section.
6.3 MicroRNAs Biogenesis
MiRNA biogenesis is a multistep process involving key enzymes that begins in
the nucleus with the transcription of a miRNA gene into primary miRNAs (primiRNAs) consisting of 500 to 3000 nucleotides (Figure 6.2, Step 1) [15]. Once
produced and structured in the nucleus, the pri-miRNA is cleaved by the microprocessor, a protein complex formed by Drosha and DCGR8 [16]. Drosha is a nuclear
ribonuclease III (RNAse III) that interacts specically with double-stranded
RNAs (Figure 6.2, Step 2) [17]. This cleavage induces the formation of a sequence
consisting of 70 nucleotides called pre-miRNA. Pri-miRNA and pre-miRNA have a
stem–loop structure, characteristic of many biologically relevant non-coding RNAs,
inducing the formation of particular binding pockets and oering the possibility
for small-molecule RNA ligands to bind. The pre-miRNA is then recognized by the
Exportin 5 (Exp5)–RanGTP complex, which is a transporter that transfers molecules
and macromolecules from the nucleus to the cytoplasm (Figure 6.2, Step 3) [18].
Once in the cytoplasm, the pre-miRNA is cleaved by another RNAse III called
Dicer (Figure 6.2, Step 4) that begins recognition by binding to the 3′-end of
Transcription
Step 1
NUCLEUS
CYTOPLASM
Figure 6.2 Biogenesis of miRNAs inside human cells. The most important proteins and
enzymes involved in the process are Drosha (PDB:6LXE), Dicer (PDB:7XW3), TRBP
(PDB:4WYQ), and Argonaute2 (PDB:4OLA).
Primary miRNA
(pri-miRNA)
Step 2
Drosha
cleavage
Precursor miRNA
(pre-miRNA)
Step 4
Dicer
Exportin 5
Step 3
miRISC complex formation
and interaction with mRNAs
mRNA degradation and
translation inhibition
cleavage
Step 6
miRNA duplex
Unwinding
Step 5
miRNA
6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 123
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the substrate [19]. The catalytic domain of the enzyme will then cleave the 5
and 3′strands of the double-stranded RNA (dsRNA) within about 20–25 base
pairs of their ends. Unlike Drosha, Dicer is able to cleave pre-miRNA alone.
However, its activity has been shown to be modulated by associated proteins,
particularly the TAR RNA-binding protein (TRBP). The resulting sequence is a
double-stranded RNA, 18–25 nucleotides long, called miRNA duplex, or miRNA/
miRNA*, these two strands being guide and passenger, respectively. After cleavage,
the Dicer–TRBP–miRNA duplex complex is able to interact with a protein of
the Argonaut family (Ago). The guide strand is then loaded onto Ago, while the
passenger strand is unwound and detached from the duplex before being degraded
(Figure 6.2, Step 5). Thus, the RNA-induced silencing complex (RISC) [20] ready to
interact with the target mRNA(s) is formed, containing Dicer, TRBP, and Ago which
carries the mature single-stranded miRNA. Once formed, the RISC complex guides
the miRNA to its target mRNA to bind to the 3′-untranslated regions (3′-UTR)
and regulate the expression of one or more proteins by inhibiting translation or
inducing degradation of the corresponding mRNA (Figure 6.2, Step 6).
It has been shown that each miRNA can regulate hundreds of mRNAs and that the
production of the majority of protein-coding genes is under the control of miRNAs
and therefore many, if not all, biological processes.Given their degreeof involvement
in the regulation of gene expression, it is not surprising that a small deregulation of
miRNA expression levels can lead to a destabilization of the cellular machinery [21].
Thus, the complex intracellular miRNA network is essential for cellular homeostasis, but it also represents a very delicate equilibrium that can be deregulated by the
overexpression or the underexpression of one or more miRNAs. Several pathologies
such as cancers, cardiovascular or neurodegenerative diseases have been correlated
with a deregulation of certain miRNAs [22].
′
6.4 Targeting MicroRNAs with Small-Molecule RNA
Binders
Given the pivotal role of miRNAs in various pathologies, many studies have
been devoted to the search for small molecules able to interfere with the miRNA
network by either inducing or inhibiting the production and function of deregulated miRNAs [23]. The role of miRNAs in cancer has been particularly studied
since miRNAs represent important biomarkers but also promising anticancer
targets [24]. As mentioned above, in cancers some miRNAs are overexpressed,
inhibit the synthesis of tumor suppressor proteins, and are called oncogenes. Other
miRNAs are underexpressed, inhibit the synthesis of oncogenic proteins, and are
called tumor suppressors [24, 25]. These deregulation processes tend to promote
oncogenesis and tumor maintenance. However, the classication of miRNAs as
oncogenes or tumor suppressors cannot be generalized since some of them can be
oncogenic in one type of cancer and tumor suppressors in others. It is therefore
important to identify the biological functions and targets of miRNAs involved in
the cancer under study before considering these miRNAs as potential drug targets.
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Several strategies have been proposed both for the activation of tumor suppressor
miRNAs and for the inhibition of oncogenic miRNAs. Oligonucleotides have largely
been applied to this aim [26]. Indeed, these high-molecular-weight molecules can
increase expression of tumor suppressor miRNA or directly inhibit the function
of an oncogenic one. This approach is very eective and specic, but limitations
remain in its clinical application. Small molecules are now considered a viable and
similarly specic approach based on structure recognition of one of the precursors
in the biogenesis process (indirect mechanism) instead of the miRNAs themselves
(direct mechanism). In the following sections, we will detail some of the main
examples of small molecules discovered to interfere with the miRNAs network with
a particular attention to their application in cancer.
6.4.1 Induction of miRNAs Expression: Tackling the Decrease of Tumor
Suppressor miRNAs
A few examples of small molecules have been reported as inducers of expression
of under-expressed tumor suppressor miRNAs in cancer cells. The basis for this
approach is the observation that a large number of miRNAs are under-expressed
in cancer cells and that a non-specic activation of their expression could have
a positive outcome in inhibiting cancer cell proliferation [27]. One of the rst
reported examples is enoxacin (compound 1, Figure 6.3), a uoroquinolone
antibiotic, reported by Shan and co-workers after screening 2000 US FDA-approved
drugs using an intracellular reporter system based on enhanced green uorescent
protein (EGFP) [28]. Enoxacin was able to selectively increase the expression levels
of some miRNAs. The reported studies suggested that enoxacin could facilitate the
interaction between TAR RNA binding protein (TRBP) and RNAs inside cells and
promote the loading of miRNAs onto RISC. This enhancement is largely dependent
on the levels of expression of miRNAs inside cells rather than on specic RNA
sequences. Compound 1 is thus a non-specic activator of miRNAs function.
Further studies demonstrated that enoxacin has a powerful cancer-specic growth
inhibitory eect in human cell cultures and in in vivo mouse models [29]. This
biological activity is due to the binding of enoxacin to TRBP, a protein essential for
Figure 6.3 Chemical structure of compounds able to activate miRNAs biogenesis and
function: enoxacin (1) and quinoxalines (2).
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