Design and Implementation of Synthetic RNA Binders for the Inhibition of miR-21 Biogenesis

Article abstract of DOI:10.1021/acsmedchemlett.0c00682

Targeting RNAs using small molecules is an emerging field of medicinal chemistry and holds promise for the discovery of efficient tools for chemical biology. MicroRNAs are particularly interesting targets since they are involved in a number of pathologies

Full text of DOI:10.1021/acsmedchemlett.0c00682

pubs.acs.org/acsmedchemlett
Letter
Design and Implementation of Synthetic RNA Binders for the
Inhibition of miR-21 Biogenesis
Chloé Maucort, Duc Duy Vo, Samy Aouad, Coralie Charrat, Stéphane Azoulay, Audrey Di Giorgio,
and Maria Duca*
Cite This: ACS Med. Chem. Lett. 2021, 12, 899−906
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ABSTRACT: Targeting RNAs using small molecules is an emerging field of medicinal chemistry and holds promise for the
discovery of efficient tools for chemical biology. MicroRNAs are particularly interesting targets since they are involved in a number
of pathologies such as cancers. Indeed, overexpressed microRNAs in cancer are oncogenic and various series of inhibitors of
microRNAs biogenesis have been developed in recent years. Here, we describe the structure-based design of new efficient inhibitors
of microRNA-21. Starting from a previously identified hit, we performed biochemical studies and molecular docking to design a new
series of optimized conjugates of neomycin aminoglycoside with artificial nucleobases and amino acids. Investigation about the mode
of action and the site of the interaction of the newly synthesized compounds allowed for the description of structure−activity
relationships and the identification of the most important parameters for miR-21 inhibition.
KEYWORDS: RNA ligand, microRNA, cancer, inhibitor, RNA targeting
argeting the biogenesis of oncogenic microRNAs using
Tsynthetic small molecules has recently been raised as a
promising strategy to affect cancer cell growth.1,2 MicroRNAs
(miRNAs or miRs) are short noncoding RNAs that control
gene expression upon binding to mRNAs and inhibition of
related protein synthesis.3 Each miRNA is responsible for the
expression of hundreds of proteins, and the resulting regulation
process is widespread in eukaryotic cells.4,5 Modifications in
miRNA expression have been also recognized as major causes
in various pathologies such as cancer.6 The miRNAs that are
overexpressed in cancer are called oncogenic and inhibit the
translation of tumor suppressor proteins, while miRNAs that
are underexpressed are tumor suppressors and inhibit the
translation of oncogenic proteins. Various approaches have
thus been developed to tackle these deregulation processes,
such as the use of oligonucleotides that replace a lacking tumor
suppressor miRNA or directly inhibit the function of an
oncogenic one.7 Small molecules have also been developed as
inhibitors of the biogenesis of oncogenic miRNAs, and they
have led to great successes in terms of activity and
specificity.1,8,9 More complex compounds, such as nucleo-
base-peptide libraries, also led to promising results in the
context of miRNA inhibition.10 Among the most important
examples, compounds identified using InfoRNA showed high
specificity toward the miRNA target, inhibiting its production
upon binding to the corresponding pri-miRNA or pre-
miRNA.11,12 Other recently developed methods to predict
RNA−ligand interactions, such as RLDOCK, together with
target-directed screenings and structure-based probing offer
promising perspectives for the identification of RNA bind-
ers.13−16
Complementary to these works, we recently developed
strong pre-miRNAs ligands directed against oncogenic miR-
372 biogenesis and able to bind to its precursor pre-miR-
Special Issue: RNA: Opening New Doors in Medicinal
Chemistry
Received: December 29, 2020
Accepted: May 3, 2021
Published: May 14, 2021
https://doi.org/10.1021/acsmedchemlett.0c00682
© 2021 American Chemical Society
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Figure 1. (A) Chemical structure of the conjugate 1. (B) Dissociation constant (KD) evaluation for compound 1 and its affinity for the pre-miR-21
sequence. The reported KD value is expressed in nM and calculated over three independent experiments performed in duplicate. (C) IC50
◆
evaluation for compound 1 inhibition of pre-miR-21 processing in the presence of E. coli RNase III purified enzyme ( ) and in the presence of
○
MCF-7 lysates ( ). Human Recombinant Dicer has also been used and led to very similar values. The reported IC50 value is expressed in μM and
calculated over three independent experiments performed in duplicate. (D) Structure of pre-miR-21 as reported in the miRbase (www.mirbase.org)
with the binding site of 1 highlighted in yellow. (E) Docking of 1 with the pre-miR-21 hairpin loop performed using AutoDock 4, in which the grid
boxes were fixed on the entire RNA sequence. (F) Compound 1 and direct interacting residues of pre-miR-21 sequence together with the formed
interaction in the complex.
372.17−19 miR-372 is overexpressed in various cancers, such as
gastric cancers, being directly linked to the proliferation of
these cancer cells.20 To identify new inhibitors of this
oncogenic miRNA, we designed conjugates between the
aminoglycoside neomycin, artificial nucleobases, and amino
acids. Aminoglycosides are known RNA ligands employed in
the clinic as antimicrobials since they bind to prokaryotic
rRNA, thus impairing protein synthesis in bacteria.21 These
antibiotics have known for a long time, but interest in their
conjugation for the improvement of biological activity and/or
pharmacological profile is still great.22 Neomycin belongs to
this class and represents a strong yet nonspecific RNA ligand.23
Artificial nucleobases are heterocyclic compounds able to form
specific hydrogen bonds with DNA and RNA base pairs.24
Finally, amino acids, especially basic ones, are also known to
bind to RNA as the main constituent of natural RNA ligands,
i.e., peptides. The resulting conjugates bear nucleobase S
reported to interact with an A·U base pair25 as well as an
amino acid (Lys, Arg, or His) and the aminoglycoside
neomycin. The strongest inhibitor of this series was conjugate
1 containing histidine amino acid (Figure 1A).17 Compound 1
showed excellent binding and selectivity for pre-miR-372 in
vitro. Also, it inhibited the proliferation of gastric adenocarci-
noma (AGS) cells highly overexpressing miR-372, and this
inhibition was specific since other cells that do not overexpress
miR-372 were not affected. These studies demonstrated that
the antiproliferative effect could be linked to the inhibition of
miR-372 expression in AGS cells and that this kind of
conjugate inhibited in a dose-dependent manner another small
set of miRNAs inside cells. Among them, miR-21 was
particularly inhibited. miR-21 is a general oncogenic miRNA
in various cancers and is responsible for the repression of
several important tumor suppressor proteins, thereby repre-
senting an interesting anticancer target.26 Small molecules able
to target this oncogenic miRNA have been reported to show
the efficacy of this strategy for the development of
antiproliferative compounds.27−31
To assess how compound 1 affects miR-21 biogenesis, we
characterized its binding to pre-miR-21 in vitro. First, the
dissociation constant (KD) was evaluated using previously
reported fluorescence-based assays.18 This kind of assay is
based on the fact that binding to RNA induces a conforma-
tional change that influences the environment of the probe.32
The measurement of fluorescence variation upon binding of
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Figure 2. (A) General structure of the new series of conjugates synthesized in this work. (B) Docking of the new conjugate containing neomycin,
histidine, and nucleobase S (6c) with the pre-miR-21 hairpin loop performed using autodock 4. (C) Compound 6c and direct interacting residues
of pre-miR-21 sequence together with the interactions in the complex.
increasing concentrations of ligands to a 5′-labeled pre-miR-21
thus leads to KD measurements. As illustrated in Figure 1B, the
KD of compound 1 was 128 12 nM, 10 times lower than the
that for neomycin. Selectivity of 1 for pre-miR-21 in the
presence of other nucleic acids competitors was then studied,
since it is an important parameter for the design of pre-
miRNAs inhibitors. We thus measured the KD in the presence
of a large excess (100 equiv) of competitors, such as tRNA and
DNA, two intracellular abundant nucleic acid structures. These
competition experiments allow for a better understanding of
the selectivity against intracellularly highly expressed nucleic
acids but also about nonspecific backbone and intercalation
interactions of the studied ligands. Compound 1 showed
selectivity for pre-miR-21 since KD values were maintained in
the presence of tRNA or DNA (Table 1, entries 1 and 2).
Following these preliminary data, the inhibition of Dicer
processing of pre-miR-21 was measured using a FRET-based
assay where a 5′-fluorescein-3′-dabcyl-labeled pre-miR-21 was
employed.18 Normal cleavage by Dicer enzyme induces an
increase in fluorescence, while binding of an appropriate ligand
that inhibits cleavage blocks the appearance of fluorescence.
Performing this kind of assay over a range of ligand
concentrations allows for the measurement of IC50 values.
The obtained results for compound 1 showed an IC50 of 1.26
0.6 μM (Figure 1C and Table S1). This promising result
prompted us to evaluate the selectivity of the inhibition
activity. We thus measured IC50 using the same cell-free assay
but in the presence of MCF7 cell lysates instead of
recombinant Dicer enzyme, since a large number of protein
and nucleic acid competitors are present in the mixture and
could impair the activity of 1 on pre-miR-21 if the compound
acts nonselectively. Interestingly, promising selectivity of
inhibition could be observed when lysates were employed,
since the IC50 value was 3.10 0.9 μM that represents a 2.5
ratio between the two values. Altogether, these results are
promising for the development of compounds of that type
against miR-21 production. We thus decided to explore the site
of interaction of compound 1 on the pre-miR-21 structure
using molecular docking. While Figure 1D shows the primary
and secondary structure of pre-miR-21 as reported in the
miRbase (www.mirbase.org), Figure 1E and F illustrates the
docking of compound 1 performed using AutoDock obtained
using the pre-miR-21 hairpin loop after applying the MC-Fold/
MC-Sym pipeline to construct a 3D model.33 The docking
studies, that previously showed their valuable results in the
study of the interactions between various RNA ligands and
pre-miR-21,19,34 suggested that conjugate 1 interacted with
residues U27 to G28 and A50 to C52 that are located close to
the cleavage site of the Dicer enzyme (A29/C46) but in the
stem part of the pre-miR-21 structure.35 It has been
demonstrated that the stem region of each pre-miRNA (the
so-called ruler) is important for the correct positioning of
Dicer and that ligands interacting with this region can be
efficient inhibitors of pre-miRNAs processing.36,37 In the
context of this possible binding complex, the interaction of 1
with pre-miR-21 could involve the formation of hydrogen
bond interactions between the imidazole of histidine and U27
and G28 as well as electrostatic interactions between the
neomycin moiety and residues A50 to C52. The triazole seems
to also be important as it interacts with residue C49. Finally, it
can be noted that the nucleobase does not seem to be involved,
illustrating that the spatial distribution of the three binding
domains could be suboptimal for efficient binding. We thus
decided to explore the possibility to improve the inhibition of
miR-21 biogenesis upon chemical modifications that could
increase and optimize the interaction network formed. Based
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Scheme 1. Solid-Phase Synthetic Pathway for the Preparation of New Conjugates 6a/a′−6c-c′ Containing Neomycin, Artificial
a
Nucleobase S, and Amino Acids Alanine, Lysine, and Histidine
a
Final compounds were obtained as TFA salts even if the neutral structure was written for clarity. Reagents: (a) piperidine, DMF, rt, 30 min; (b)
Fmoc-Ala-OH, Fmoc-Lys-OH, or Fmoc-His-OH, HBTU, DIPEA, DMF, rt, 2 h; (c) Ac2O, pyr, DMF, rt, 10 min; (d) piperidine, DMF, rt, 30 min;
(e) Fmoc-propargyl-Gly, HBTU, DIPEA, DMF, rt, 2 h; (f) Ac2O, pyr, DMF, rt, 10 min; (g) piperidine, DMF, rt, 30 min; (h) succinic anhydride,
DMF, rt, 1 h; (i) Ac2O, pyr, DMF, rt, 10 min; (j) S or SAr,18 iodomethylpyridinium iodide, DIPEA, DMF, rt, 1.5 h; (k) Neo(Boc)6N3,17 CuI,
DIPEA, DMF, rt, overnight; (l) TFA, TIS, rt, 2 h.
on the intracellular inhibition of miR-21 expression,17 on the
promising values of KD and IC50 obtained during biochemical
assays, and on the molecular docking studies described above,
we designed compounds bearing a more flexible linker between
the neomycin moiety and the nucleobase as well as a different
distribution of the RNA binding domains. To this aim, we
envisioned modification of the chemical structure of 1 by
addition of an aliphatic linker between the triazole and the
nucleobase and by placing the amino acid residue on this linker
as illustrated in the general structure of Figure 2A. Molecular
docking helped us to understand if such compounds could be
better pre-miR-21 binders than compound 1. The docking of a
compound of this new series containing histidine as the amino
acid (6c) is illustrated in Figure 2B and C, and it showed that
this compound could indeed interact with pre-miR-21. The
docking suggests that the three moieties histidine, S, and
neomycin could participate to the interaction, opening the
possibility for better selectivity and activity compared to 1.
Furthermore, the model suggests that compound 6c could
interact with residues U15 to A17 as well as A50 to C51 via an
increased number of hydrogen bonds and electrostatic
interactions compared to compound 1.
Based on these results, we decided to synthesize a new series
of compounds bearing the general structure illustrated in
Figure 2A. To this aim, we conjugated neomycin to nucleobase
S that is the one used in the docking study, but also to SAr, a
similar artificial nucleobase containing a supplementary aryl
substituent that was previously shown to be very promising for
better interaction with pre-miRNAs.19 Furthermore, we
decided to compare histidine amino acid with alanine to
measure the influence of a hydrophobic side chain and with
lysine that contain a basic but aliphatic side chain to compare
with the histidine imidazole. A control compound containing
uracil instead of nucleobase S was also prepared to study the
influence of the nucleobase on the affinity and biological
activity (compound 15, Scheme S2).
The synthesis of conjugated aminoglycosides for better RNA
binding is a challenging field. Many analogues have been
reported in the literature, and even if a number of synthetic
routes have been explored to conjugate various moieties at
different positions on the aminoglycoside, new synthetic
approaches are always needed.38−40 Here, we decided to use
a solid-phase synthetic strategy as illustrated in Scheme 1 to
avoid purification steps. Rink amide MBHA resin was first
deprotected in the presence of piperidine in DMF and the
resulting free amine was charged at 50% with the amino acid
Na-Fmoc-L-alanine (as in 2a), Na-Fmoc-Nw-Boc-L-lysine (as
in 2b) or Na-Fmoc-NIm-Boc-L-histidine (as in 2c) in the
presence of HBTU, DIPEA in DMF. After capping the
unreacted amines with acetic anhydride in the presence of
pyridine in DMF, the added amino acid was deprotected by
piperidine in DMF and Na-Fmoc-L-propargyl-glycine was
coupled to the free amine by HBTU in the presence of
DIPEA in DMF. Capping by acetic anhydride in pyridine and
DMF led to desired intermediates 3a−c. The Fmoc group was
removed again, and the resulting amine was reacted with
succinic anhydride in the presence of DIPEA in DMF. Final
capping led to desired compounds 4a−c. This scaffold now
contains a free carboxyl group for the introduction of the
nucleobase and an alkyne group for the introduction of an
azido-substituted neomycin derivative. First, nucleobases S and
SAr17,19 were coupled to the carboxyl group in the presence of
iodomethylpyridinium chloride, DIPEA, and DMF, leading to
compounds 5a/a′−5c/c′. Then, 1,3-dipolar cycloaddition was
performed with Boc-protected 5″-azido-neomycin (Neo-
(Boc)6N3)18 in the presence of CuI, DIPEA, and DMF. This
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Table 1. Dissociation Constants (KD, nM) Measured for Pre-miR-21 Binding Alone or in the Presence of 100 equiv Excess of
tRNA (KD′, nM) or 100 equiv Excess of DNA Duplex (KD″, nM)
a
entry
ID
KD [nM] pre-miR-21
KD′ [nM] (tRNA competition)
KD′/KD
KD″ [nM] (DNA competition)
KD″/KD
1
2
3
4
5
6
7
8
9
neomycin
1
1390 126
128 12
133 20
128 7.0
50.7 7.4
348 34
50.3 11
162 40
641 165
1415 110
134 14
151 13
170 48
51.0 8.7
405 138
50.4 7.0
168 10
807 270
1.0
1.0
1.1
1.3
1.0
1.2
1.0
1.0
1.3
1950 20.2
171 16
187 24
261 60
95.7 22
877 79
80.9 3.2
274 30
1459 70
1.4
1.3
1.4
2.0
1.9
2.5
1.6
1.7
2.3
6a
6a′
6b
6b′
6c
6c′
15
a
Binding studies were performed on 5′-FAM-pre-miR-21 in buffer A (20 mm Tris−HCl (pH 7.4), 12 mm NaCl, 2.5 mm MgCl2, and 1 mm DTT).
Figure 3. IC50 values of compounds 6a/a′−6c/c′ against pre-miR-21 72-mer fragment labeled at the 5′-end with fluorescein and at the 3′-end with
dabcyl measured upon processing with recombinant enzyme (black circle). Selectivity of compounds 6a/a′−6c/c′ for pre-miR-21 in the presence
of MCF7 cell lysates (blue circle). IC50 values were determined from duplicates performed over three independent experiments.
step needed 4 equiv of CuI (2 × 2 equiv ) as Cu adsorbs on
the solid support. Then, removal of Boc groups by a mixture of
TFA/TIS led simultaneously to resin cleavage as well as the
release of large quantities of copper with desired conjugates
6a/a′−6c/c′ which had to be removed by successive
treatments with Chelex resin to capture Cu, leading to low
overall yields < 20%. To circumvent this constraining
purification step, we performed the deprotection/cleavage
step before the 1,3-dipolar cycloaddition in order to carry out
the click reaction in liquid-phase and to avoid copper
accumulation on the solid support. This strategy allowed us
to obtain compounds 6a/a′ and 6b′ with increased global
yields (between 35% and 45%). For comparison, the other
compounds (6b and 6c/c′) were prepared through a complete
liquid-phase synthesis (Supporting Information Scheme S1)
and led to final compounds with overall yields similar to the
solid phase methodology but with the need for more
purification steps. The synthesis of the desired conjugates
was thus successful even if their preparation remains complex.
Compounds 6a/a′−6c/c′ were designed in order to
improve affinity and inhibition activity against pre-miR-21
and its biogenesis as previously described for 1. We thus
decided to first study the binding affinity toward pre-miR-21
and compare the obtained values with nonconjugated
neomycin and parent compound 1. As mentioned before, we
also prepared compound 15 (Scheme S2) containing natural
nucleobase uracil instead of artificial nucleobase S. As
illustrated in Table 1 (and in Supporting Information Figure
S1), all conjugated compounds (entries 3−8) bear a KD in the
low nanomolar range, thus showing great improvement in the
binding affinity compared to unconjugated neomycin.
Compounds containing nucleobase S (1 and 6a−c) bear
better affinity than the ones containing SAr nucleobase (6a′−
6c′), suggesting that the addition of a phenyl ring on the
nucleobase probably hinders the interaction with the target.
Indeed, compounds 6a′, 6b′, and 6c′ show KD values of 128,
348, and 162 nM, respectively, thus being weaker binders than
the corresponding S derivatives 6a, 6b, and 6c. While
compound 6a showed a KD value of 133 nM, compounds 6b
and 6c showed KD values of 50.7 and 50.3 nM, respectively,
representing a 2.5-fold improvement in comparison with
compound 1. This also illustrates that a positively charged
and hydrophilic amino acid side chain is important for
interaction since 6b and 6c containing lysine and histidine,
respectively, have better affinity for the target than 6a
containing alanine. Interestingly, compound 15 containing
uracil instead of S bears a KD of 641 nM that is a 13-fold higher
value compared to that of S conjugates, illustrating that the
artificial nucleobase also plays an important role in pre-miR-21
binding. This suggests that the spatial distribution of these new
compounds could be more favorable for the involvement of the
nucleobase in the interaction compared to what observed for
compound 1. Selectivity studies in the presence of tRNA or
DNA showed that the ratios between KD′ (dissociation
constant in the presence of tRNA) or KD″ (dissociation
constant in the presence of DNA) and KD are close to 1 (Table
1, entries 3−9). Only compounds 6a′, 6b′, and 15 showed
slightly higher values.
Based on these binding data, we wondered whether these
compounds could be good inhibitors of its processing. We thus
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Figure 4. (A) Thermodynamic profiles for the high-affinity binding of heterocycle-spermine conjugates 1 and 6a/a′−6c/c′ to the pre-miR-21 at 20
°C: ΔG° (black bars), ΔH° (dark gray bars), and −TΔS° (light gray bars). (B) Molecular docking of 6b in interaction with pre-miR-21 hairpin
loop performed using AutoDock 4 wherein the grid boxes were fixed on the entire RNA sequence. (C) Compound 6b and direct interacting
residues of pre-miR-21 sequence together with the interactions in the complex.
performed the FRET-based assay described above for the study
of the inhibition activity of 1. The obtained results (Figures 3
and S2; Table S1) indicate that all conjugates inhibit
processing of pre-miR-21 in the low micromolar range with
compounds 1, 6a′, 6b, 6c, and 6c′ being the best inhibitors
with IC50’s values below 5 μM. Selectivity studies performed
using the same assays in the presence of MCF7 lysates showed
that compounds 6b and 6c bear the best selectivity in the
tested conditions (Table S1). It must be noted that even if the
affinity of 6b and 6c for pre-miR-21 was improved compared
to that of compound 1, the inhibition activity remained in the
same range as that for 1.
To gain a better understanding about the molecular
mechanism of interaction, we performed molecular docking
studies for compounds 6b and 6c. Docking of 6c with pre-
miR-21 was previously illustrated in Figure 2B and C, while
docking of compound 6b is illustrated in Figure 4B and C.
These studies suggested that both compounds seem to interact
with the same site. More specifically, 6b could interact with
residues C16−U21 and C49-A53 while 6c with U15-A17 and
A50-C51 upon formation of hydrogen bonds, π−π interactions
as well as electrostatic interactions. This hypothetical binding
site seems to be similar to the one of compound 1 and
experimental results about binding and selectivity illustrate that
both histidine and lysine are favorable amino acids for the
preparation of this type of ligands. Noteworthy, nucleobases S
and SAr seem to be closely involved in the interaction and the
spatial distribution of the three binding domains is more
accurate than the one of compound 1, as demonstrated also
experimentally by KD values. While 6b and 6c are the best pre-
miR-21 ligands and inhibitors, in terms of both affinity and
selectivity, 6c′ bears a three-time higher KD value than 6b and
6c but very similar inhibition activity. Comparison with 6c′
shows that the latter should interact with a different binding
site located close to the cleavage site of Dicer, i.e., U27 to G28
and C41 to A42 (Supporting Information Figure S3) but with
a lower number of interactions, thus being a weaker binder.
The interactions proposed by the model are mainly hydrogen
bonds and electrostatic interactions, but further intramolecular
interactions are involved, probably hindering the correct
interaction with the target and causing the observed increase
in the KD value.
To complete these studies, we decided to measure the
thermodynamic parameters of the complex formed with pre-
miR-21 for all the synthesized compounds in comparison with
1. Free Gibbs energies (ΔG°) were first calculated from the
dissociation constants (ΔG° = −RT ln KD) and were found to
be very similar (from −36.6 to −41.5 kJ/mol), with 6b and 6c
forming the most favorable complexes (Figure 4C). To
complete the thermodynamic binding profiles, enthalpic
(ΔH°) and entropic (−TΔS°) energy contributions were
determined after the determination of ΔG°T at several
temperatures (278−308 K). The obtained values allow for a
comparison of ligands binding to the target. In all cases, the
formation of ligand/pre-miR-21 complexes is driven by
entropy, indicating a strong desolvation effect during
interaction. The ΔG° value can be divided into the ΔG°nel
value, which reflects the contribution of nonelectrostatic
interactions to the total free energy, such as nonionic
hydrophobic effects driven by entropy, and specific inter-
actions, including H-bonds, van der Waals interactions, and π-
stacking, and the pure electrostatic (polyelectrolyte) contribu-
tion, ΔG°el, which reflects the ionic interactions occurring
between two groups of opposite charge and is highly
dependent on the salt concentration. We found that the
interactions mainly involve nonelectrostatic interactions
because the ΔG°nel component represents 87−99% of the
overall free energy (Table S2). Although this is an important
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̂
parameter that should contribute to the observed selectivity,
binding could be probably improved by noncovalent
interactions such as H-bonds, π-stacking, and π-ions for an
increase in the enthalpy component.
Stéphane Azoulay − Université Cote d’Azur, CNRS, Institute
of Chemistry of Nice (ICN), 06100 Nice, France
̂
Audrey Di Giorgio − Université Cote d’Azur, CNRS, Institute
of Chemistry of Nice (ICN), 06100 Nice, France
In this work, we took advantage of an original binder that
proved to inhibit miR-21 biogenesis in cancer cells to perform
the design of a new series of pre-miR-21 ligands, based on the
potential configuration of the binder/RNA complex as
proposed by initial docking. These ligands were synthesized
upon conjugation of three RNA binding domains, and the in
vitro study of their affinity, selectivity, and inhibition activity
allowed for the identification of two compounds, 6b and 6c,
that are strong and selective binders. The study of their
binding to pre-miR-21 confirmed that the three binding
domains, i.e., artificial nucleobase S, neomycin, and histidine or
lysine, act cooperatively since each separate moiety is not able
to bind the target. Docking studies suggested that the binding
site is located in the stem part of pre-miR-21 where two bulges
induce the distortion of the double helix. Altogether, these
results support the idea that the inhibition efficacy of an RNA
binder depends upon both the affinity and the binding site on
each pre-miRNA. Future works will be devoted to the
optimization of these compounds and reducing the size of
the aminoglycoside moiety with the aim to increase selectivity
and enthalpic contribution to binding and to confirm their
activity in cell-based studies.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00682
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Thanks are due to Institut de Recherche Servier for fellowship
to C.M., to Agence Nationale de la Recherche for support and
fellowship to D.D.V. (ANRJS07-011-1, ) and to IDEX-JEDI of
̂
Université Cote d’Azur for fellowship to S.A.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00682.
■
sı
Binding curves and inhibition curves of compounds 1
and 6a/a′−6c/c′; docking of the new conjugate
containing neomycin, histidine, and nucleobase SAr
(6c') with the pre-miR-21 hairpin loop; salt effect for
compounds 1 and 6a/a'−6c/c'; IC50 values for the
inhibition of pre-miR-21 cleavage; thermodynamic
parameters for ligand−pre-miR-21 interactions; dissoci-
ation constants for pre-miR-372 binding; liquid-phase
synthetic pathway for the preparation of new conjugates
6b−6c/c′; solid-phase synthetic pathway for the
preparation of uracil-containing conjugate 15 containing
neomycin, nucleobase uracil, and amino acid alanine;
experimental procedures; NMR spectra of reported
compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
■
(13) Sun, L. Z.; Jiang, Y.; Zhou, Y.; Chen, S. J. RLDOCK: A New
Method for Predicting RNA-Ligand Interactions. J. Chem. Theory
Comput. 2020, 16, 7173−83.
̂
Maria Duca − Université Cote d’Azur, CNRS, Institute of
Chemistry of Nice (ICN), 06100 Nice, France; orcid.org/
0000-0002-2666-6180; Email: maria.duca@univ-
cotedazur.fr
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Correlated Chemical Probing of RNA. J. Am. Chem. Soc. 2020, 142,
18735−40.
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Huynh, K.; Hou, Y.; Cullen, B. R.; Al-Hashimi, H. M. Understanding
the characteristics of nonspecific binding of drug-like compounds to
canonical stem-loop RNAs and their implications for functional
cellular assays. RNA 2021, 27, 12−26.
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Alkyne Cycloaddition for Assembling HIV-1 TAR RNA Binding
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Darfeuille, F.; Staedel, C.; Duca, M. Building of neomycin-nucleobase-
Authors
̂
Chloé Maucort − Université Cote d’Azur, CNRS, Institute of
Chemistry of Nice (ICN), 06100 Nice, France
Duc Duy Vo − Université Cote d’Azur, CNRS, Institute of
Chemistry of Nice (ICN), 06100 Nice, France
Samy Aouad − Université Cote d’Azur, CNRS, Institute of
Chemistry of Nice (ICN), 06100 Nice, France
Coralie Charrat − Université Cote d’Azur, CNRS, Institute of
Chemistry of Nice (ICN), 06100 Nice, France
̂
̂
̂
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https://doi.org/10.1021/acsmedchemlett.0c00682
ACS Med. Chem. Lett. 2021, 12, 899−906

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