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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5644_Библиотеки_им_академика_М_И_Перельмана
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Figure 6.8 (a) Chemical structures of ligands 26–28 identified with InfoRNA. (b) Primary and secondary structures of pre-miR-210 and part of pri-miR-96.
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in vivo to evaluate its therapeutic potential. This molecule selectively recognizes
pre-miR-210 in both cases and showed biological activity inducing cell apoptosis
in vitro and tumor growth reduction in vivo.
Using the same kind of approach, a compound specic for the inhibition of
the production of miR-18a, a miRNA belonging to the miR-17-92 cluster and
overexpressed in prostate cancer, was identied. This compound (30, Figure 6.9)
strongly and specically interacts with pre miR-17, -18a, and -20 in a U, G, or A
bulge regions, all three located in the catalytic site of Dicer [67]. Treatment of
prostate cancer cells with this ligand, called Targapremir-18a, showed inhibition of
miR-18a production, restoration of the expression of the tumor suppressor protein
serine/threonine kinase 4 (STK4), and induction of apoptosis. The study of the
cellular targets of Targapremir-18a was pursued using the Chem-CLIP (Chemical
Cross-Linking and Isolation by Pull Down) technique [68]. This approach is based
on the combination of a specic RNA ligand with a chemical scaold capable of
covalently binding to RNA (most common are chlorambucil or diazirines [highlighted in green in compound 31, Figure 6.9]). Furthermore, a chemical handle
allowing the subsequent isolation of the complex is added to the compound and is
generally represented by biotin (highlighted in orange in compound 31, Figure 6.9).
The resulting probe molecule will bind to the RNA target followed by covalent bond
formation whereupon the ligand/target complex can be isolated with magnetic
beads coated with streptavidin. The RNA target can subsequently be isolated and
identied by RT-qPCR. In the case of TargapremiR-18a 31, specic binding to the
miR-18a precursor could be conrmed as the primary target, with pre-miR-17 and
-20a as secondary targets.
Other oncogenic miRNAs have been targeted using the same approach. After
demonstrating both in vitro and in vivo that it is possible to inhibit miRNA biogenesis by using small molecules to target the Drosha or Dicer cleavage sites, the
Disney group studied additional miRNAs including miR-544, which is upregulated
in tumor cells in response to hypoxia [69]. Compound 32 bearing a naphthyridine
scaold substituted with amines, pyrrolidines, and a cyano group, was identied
by Inforna as the most eective against this target. This compound inhibits the
action of Dicer and has similar eects to ASOs at 25-fold lower concentrations.
This inhibitor also conrmed a biological role for miR-544 in apoptosis resistance,
tumor growth, and chemotherapy resistance, making it a prime target for cancer
therapy [69]. Compound 33, consisting of an azido-neomycin, decreases the level
of miR-525 in vivo through action on the pri-miR-525, and by binding to the
Drosha catalytic site [70]. This miRNA is particularly overexpressed in liver cancers
indicating its therapeutic importance as it inhibits the invasive properties of a
hepatocellular carcinoma cell line. Although this molecule can bind to a large
number of cellular RNAs, only binding to sites such as the Drosha catalytic site
for miR-525 allows a biological response. A relevant example of the potential of
the InfoRNA approach in the design of miRNAs inhibitors is the discovery of
compound 34 that specically interacts with a UA double base pair of pre-miR-200c
and of compound 35 that interacts with an internal UU loop adjacent to the UA
base pairs [71]. MiR-200 family associated with type 2 diabetes includes ve distinct
Figure 6.9 Chemical structure of ligands 30–36 discovered by InfoRNA and specific for a particular structure of pre-miRNA. The conjugation of 34 and
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35 led to new ligand 36, highly specific for pre-miR-200c.
138 6 MicroRNAs as Targets for Small-Molecule Binders
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members, but overexpression of miR-200c is sucient to induce β-cell apoptosis.
The combination of 34 and 35 resulted in ligand 36 that is highly specic for
miR-200c compared to the other members of the same family.
Collectively, these examples illustrate that while pure rational design remains
a challenge, tools are available to facilitate design of specic compounds. Furthermore, these tools are continually evolving to further enable design of ecient
binders. Compounds exhibiting high specicity for a chosen miRNA precursor
induced the desired biological eect. Furthermore, such compounds also represent
important chemical tools to elucidate the role of miRNAs in cancer and other related
pathologies. Based on these considerations, other classical medicinal chemistry
strategies have been developed for the targeting of miRNAs with small molecules
as it will be described in the following sections.
6.4.2.4 Fragment-Based Drug Design
The same medicinal chemistry approaches commonly used for the discovery of
compounds targeting proteins can be employed for the identication of RNA
binders specic for a particular structure. In this context, fragment-based drug
design (FBDD) has recently been utilized to nd RNA binders that target miRNA
precursors such as those illustrated in the previous sections [72]. FBDD involves
screening of libraries of fragments, which are compounds with low molecular
weight (generally <300 Da) and with low number of functionalities. The aim is to
identify small chemical scaolds that can bind to a target of interest, often with low
anity and/or activity. Following validation of target binding through orthogonal
biophysical techniques, these fragment hits are subsequently optimized by addition
of substituents (fragment growing) or linking two fragments with a close binding
site on the target (fragment linking) leading compounds with high anities and
activities. NMR is a frequently employed technique for fragment-based screening
since it allows for assessment of anity and identication of the binding sites.
NMR was recently applied to the search for fragments that inhibit the biogenesis
of miR-21 upon binding to pre-miR-21 [73]. A screen of 420 compounds led to the
identication of 18 hits. After rening the screen and validating the identied hits,
thiadiazole fragment 37 (Figure 6.10) was demonstrated to bind in close proximity
Figure 6.10 Compounds 37, 38 discovered using a fragment-based drug design approach,
and compounds 39, 40 discovered using InfoRNA as probes for the development of
fragment screening.
6.4 Targeting MicroRNAs with Small-Molecule RNA Binders 139
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to the Dicer cleavage site on pre-miR-21. Further assays would be needed to assess
the actual biological activity of this kind of compound, but it represents a starting
point for the development of new ligands targeting this oncogenic miRNA.
Advanced FBDD was then performed combining this approach with InfoRNA.
This latter approach, described in the previous section, has been applied to
the screening of fragment libraries [74]. The largest collection of RNA-focused
small-molecule fragments to date (n = 2500) was created by examining features in
all published compounds that bind RNA. The most relevant interactions between
fragments and RNA were identied. Approximately 12.8 million interactions were
found by examining the RNA-binding landscape for each fragment following a
library-versus-library selection using an RNA library displaying a discrete structural
feature. Mining of this dataset across the human transcriptome led to the identication of a drug-like fragment (compound 38 in Figure 6.10) as a potent and specic
inhibitor of miR-372 biogenesis, thus alleviating invasive and proliferative oncogenic phenotypes in gastric cancer cells. Noteworthy, 38 has favorable properties,
including favorable anity for the RNA target of 300 ± 130 nM, a molecular weight
of 273 Da, and quantitative estimate of drug-likeness (QED) score of 0.8. Thus,
these studies demonstrate that a low-molecular-weight, fragment-like compound
can specically and potently modulate RNA targets.
InfoRNA allowed for the identication of compound 39 that binds in the Dicer
cleavage site of pre-miR-21 [75]. To develop a methodology for fragment screening,
compound 40 was prepared by addition of a diazirine group for the photoactivated
capture of bound RNA targets and a terminal alkyne handle that can be bioorthogonally coupled to an azide-containing purication tag through click chemistry. Evaluation of pre-miR-21 binding by 40 was performed using the ChemCLIP approach
conrming that the binding site was similar to 39. A fragment library of compounds
containing the diazirine and the alkyne moieties were then generated, and the application of the same strategy allowed for the identication of several fragments able to
bind pre-miR-21. The combination of these fragments produced a particular active
compound with increased potency to inhibit pre-miR-21 processing and to decrease
the levels of mature miR-21. In triple-negative breast cancer cells, the substance had
selective eects on the transcriptome and reduced an invasive phenotype linked to
miR-21.
6.4.2.5 DNA-Encoded Libraries (DELs)
The use of DNA-encoded libraries (DELs) in drug discovery has already led to
successful results and led to the discovery of new drug candidates currently in
clinical trials. However, only recently has this technology been applied to the search
for RNA binders. In a relevant example, a screen of a DNA-encoded library against
a library of RNA structures enabled the evaluation of 300 million interactions in
total and resulted in identication of numerous ligand/target pairs [76]. Among
them, ligands specic for the 5′-GAG/3′-CCC internal loop present in an oncogenic
primary miRNA (pri-miR-27a) were identied. Compound 41 (Figure 6.11) has
nanomolar binding anity for the target, decreased miR-27a expression in four
dierent cancer cell lines at nanomolar concentrations, and exhibits high selectivity
across miRnome in triple-negative breast cancer cells.
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Figure 6.11 Chemical structure of ligand 41 identified using DELs.
Similar to FBDD, DEL screening approaches have high potential for the identication of novel RNA binders that exhibit the desired specicity. While only few
examples have been reported to date, these techniques are surely extremely promising and will probably lead to bioactive compounds in the near future.
6.5 Inhibition of RNA–Protein Interactions in miRNAs
Pathways
Beside direct binding of RNA binders to one of the miRNAs precursors, it is also
possible to target the interactions formed between miRNAs and miRNA precursors with the partner proteins essential for miRNA functions. Abell et al. designed
oligonucleotide-small-molecule conjugates to inhibit the interaction between the
miRNA–Ago2 complex and mRNA [77]. The goal of such conjugates is the specic
recognition of the miRNA target by the oligonucleotide moiety, which will guide
the small-molecule inhibitor of Ago2 close to the targeted protein. The designed
compounds contain a short oligonucleotide sequence (tetramer) complementary to
miR-122 linked to an Ago2 inhibitor (compound 42, Figure 6.12) that was identied
by an in silico screen of 627,000 compounds. Optimization of these conjugates led
to the synthesis of compounds 43 and 4 4 containing a miR-122-specic 5′-TCAC-3
peptide nucleic acid (PNA) tetramer. Fluorescence assays to evaluate the ability of
′
Figure 6.12 Compounds inhibiting the interaction between Ago2 and miRNA (42–46) and
between Lin28 and pre-Let-7 (47, 48).
6.5 Inhibition of RNA–Protein Interactions in miRNAs Pathways 141
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the conjugates to inhibit miRNA–Ago2 interaction with mRNA as well as its cleavage
were performed. These conjugates thus revealed a ten-fold higher IC50than the PNA
tetramer sequence alone, demonstrating the potential of such a strategy to inhibit
miRNA functions. Liang et al. employed a conjugation strategy to inhibit pre-miR-21
cleavage by Dicer [78, 79]. In this study, the compounds were obtained by conjugation of two types of oligonucleotides (morpholino or PNA) of dierent lengths and a
2-hydroxy-isoiquinoline-1,3-dionemotif (molecule 45, Figure 6.12), previously identied as a weak inhibitor of Dicer [79]. Such molecules can be used because oligonucleotides allow vectorization and provide an increasein local eective concentration.
Biological evaluation of the dierent conjugates was performed by electrophoresis
and led to the identication of the bifunctional molecule 46 containing an 11-mer
PNA with a GAGATTCAACA sequence specic to the apical loop of pre-miR-21.
This conjugate inhibits pre-miR-21 cleavage by Dicer with an IC50of 0.5 μM compared to 100 μM for the Dicer inhibitor alone. Although the use of shorter ASOs
compared to oligonucleotides may provide benets in terms of specicity and cellular distribution, the bifunctional molecules in this study are not cell permeable and
require further optimization.
Another important interaction that has been considered in miRNA-targeting
studies is the one between the miRNA-binding proteins LIN28 and let-7 [80].
Let-7 miRNAs function as a tumor suppressor by downregulating the expression
of oncogenes including RAS, c-MYC, HMGA2 [81]. LIN28 is a post-transcriptional
regulator protein that binds to pri- and pre-let7, thus blocking let-7 maturation and
inducing its degradation. Many primary human tumor cells overexpress LIN28, and
this has been connected to poor clinical prognosis. Inhibition of LIN28/pre-let-7
interaction is thus considered a potential anticancer approach, and various studies
reported small-molecule inhibitors of this interaction [82]. Recently, Wu and
coworkers identied tetrahydroquinoline (THQ) as a weak inhibitor of LIN28 and
decided to perform the medicinal chemistry optimization of this scaold [83]. This
led to compounds 45, 4 6 having low micromolar IC50for LIN28 inhibition but
devoid of intracellular activity. Despite this drawback, only few examples of LIN28
inhibitors have been reported so far and these remain promising scaolds for future
medicinal chemistry improvements.
Recently, new specic assays are being developed to identify novel compounds
that inhibit RNA/protein interactions. The eld includes RNA interaction with
protein-mediated complementation assay, or RiPCA [84]. In this assay, cells are
engineered to express the small subunit of the split luciferase, NanoLuc, fused to HT,
an engineered dehalogenase that covalently binds to chloroalkane-containing ligands leading to a fusion protein SmHT. The cells are then transiently co-transfected
with a plasmid encoding the RBP-of-interest fused to the large subunit of NanoLuc
and a chloroalkane-modied RNA probe, which allows covalent conjugation to
SmHT. Subsequent interaction between the RBP and RNA drives reconstitution
of NanoLuc, generating chemiluminescence upon treatment of cells with the
NanoLuc luciferase substrate. This assay was used to prove the interaction of
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a pre-miRNA, pre-let-7, with Lin28 and was particularly useful to detect this
interaction intracellularly. This methodology could thus be employed in the future
to screen for inhibitors of this interaction.
6.6 Adding Cleavage Properties to miRNAs Interfering
Agents
As illustrated by all the examples above, small-molecule binders of miRNA precursors proved to be ecient tools to inhibit the intracellular expression of pathological
miRNAs and sometimes also to induce the desired eect in vivo. A step further in the
search for ecient miRNA inhibitors is to introduce additional properties to ligands
that not only bind to the target, but also induce its degradation and cleavage in cells
and eventually in vivo. In a rst attempt to nd compounds able to bind an RNA
target and induce its cleavage, bleomycin A5 (compound 49, Figure 6.13), a natural
product known to induce DNA and RNA strand breaks and used as an anticancer
agent, was studied as a binder and cleaving agent on dierent RNA motifs and structures [85]. It was shown that bleomycin A5 preferentially cleaves motifs containing
A-U base pairs as well as purine-rich sequences. In vitro and in vivo assays were carried out on pre-miR-10b, which contains these motifs and is an oncogenic miRNA
that is overexpressed in many cancers and involved in invasion and metastasis. All
assays conrmed the action of the compound on the intended target. Bleomycin
was then employed to introduce cleavage properties to other specic pre-miRNA ligands. As a typical example, targaprimir-96 (conjugate 29, Figure 6.8) was coupled to
bleomycin A5 (conjugate 50, Figure 6.13) by introducing an azido group on the spermidine side chain and coupling using 1,3-dipolar cycloaddition reaction. This led to
very ecient inhibition of Drosha processing and to the cleavage of pri-miR-96 in
intracellular assays [86].
A major advance in the eld of RNA ligands in general and miRNA inhibitors in
particular has been made recently with the design of chimeric compounds called
RIBOTACs capable of targeting a miRNA precursor and inducing its degradation
by recruitment of a ribonuclease in a manner similar to that performed on protein
targets by PROTACs (described in further detail in Chapter 9) [87]. As an example,
compound 29 was conjugated to a 2′-5′-poly(A) oligonucleotide capable of recruiting an endogenous RNase L inducing degradation of pri-miR-96 at the intracellular
level and in sub-stoichiometric amounts (conjugate 51, Figure 6.13). This strategy
was applied successfully to other pre-miRNAs ligands. In a relevant example, a
specic ligand of pre-miR-21 (compound 52, Figure 6.13) identied with Inforna
was optimized by the preparation of dimer 53 and then coupled to bleomycin or to
a small-molecule compound able to recruit RNase L, leading to conjugate 54 [88].
The latter induces the degradation of pre-miR-21 at the intracellular level and in
sub-stoichiometric amounts. It has 20-fold higher activity than the corresponding
dimer in reducing the intracellular level of miR-21 and 10-fold higher than the
bleomycin conjugate. Finally, the in vivo study of this molecule showed that cleavage
of pre-miR 21 leads to inhibition of breast cancer metastasis to the lung.
Figure 6.13 Examples of conjugates able to induce the cleavage of the targeted RNA thanks to the presence of bleomycin (compounds 49, 50)ortothe
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presence of a RNase L recruiter (compounds 51–54).
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The addition of cleavage properties to specic RNA binders thus showed to be
particularly promising to induce the desired biological eect thanks to the degradation of the RNA target. The synthesized conjugates may still have to be optimized
for therapeutic application, but in vivo studies clearly demonstrated the potential of
this strategy.
6.7 Conclusions
In conclusion, the examples of RNA ligands described in this chapter illustrate the
feasibility of the approach and the possibility of obtaining specicity of action in vitro
as well as at the cellular level and in vivo. miRNAs represent particularly promising
targets not only for anticancer therapies, which are currently the most studied, but
also for other pathologies in which these short non-coding RNAs are involved as
well as for antiviral approaches. However, the miRNA network is extremely rich and
complex since thousands of miRNAs have been identied, and each one controls
the expression of hundreds of proteins. Modulation of this network may have
important eects on the biology of the cell, and toxicity could be a major limitation
of the approach based on the targeting of these RNAs. Furthermore, the miRNA
precursors that usually represent the target of small-molecule miRNA inhibitors
have very similar three-dimensional structures, thus limiting the possibilities for
selective binding. Despite this limitation, very specic ligands have been identied
showing biological activity in cells and in vivo, which encourages the scientic
community to pursue this strategy. Finally, the eld of RNA ligands for therapeutic
applications is broad and rapidly expanding for targeting a large number of targets
such as viral, bacterial, and other eukaryotic non-coding RNAs. Altogether, the
gathered results will play a major role in dening the main features for RNA
binders and in opening the possibility for the rational design of ecient and specic
inhibitors.
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