Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays

Article abstract of DOI:10.1021/acschembio.6b00945

The identification of small molecules that bind to and perturb the function of microRNAs is an attractive approach for the treatment for microRNA-associated pathologies. However, there are only a few small molecules known to interact directly with microRN

Full text of DOI:10.1021/acschembio.6b00945

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Article
Discovery of Inhibitors of MicroRNA-21
Processing Using Small Molecule Microarrays
Colleen M. Connelly, Robert E. Boer, Michelle H. Moon, Peter Gareiss, and John S Schneekloth, Jr.
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00945 • Publication Date (Web): 13 Dec 2016
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Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule
Microarrays
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Colleen M. Connelly1, Robert E. Boer1, and Michelle H. Moon1, Peter Gareiss2, and John
S. Schneekloth, Jr.1*
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1Chemical Biology Laboratory, National Cancer Institute, Frederick MD 21702, USA
2Yale Center for Molecular Discovery, West Haven, Connecticut, United States
Corresponding Author:
*Email: schneeklothjs@mail.nih.gov
ABSTRACT
The identification of small molecules that bind to and perturb the function of
microRNAs is an attractive approach for the treatment for microRNAꢀassociated
pathologies. However, there are only a few small molecules known to interact directly
with microRNAs. Here, we report the use of a small molecule microarray (SMM)
screening approach to identify low molecular weight compounds that directly bind to a
preꢀmiRꢀ21 hairpin. Compounds identified using this approach exhibit good affinity for
the RNA (ranging from 0.8 – 2.0 ꢁM) and are not composed of a polycationic scaffold.
Several of the highest affinity compounds inhibit Dicerꢀmediated processing, while inꢀline
probing experiments indicate that the compounds bind to the apical loop of the hairpin,
proximal to the Dicer site. This work provides evidence that small molecules can be
developed to bind directly to and inhibit miRꢀ21.
INTRODUCTION
The goal of developing RNAꢀbinding small molecule therapeutics is an area of
increasing interest.1-4 However, the discovery of molecules that bind to RNA with good
affinity and specificity is a major challenge. RNA is a highly polar molecule with a
flexible, dynamic structure, and largely solventꢀexposed binding pockets that are
structurally distinct from small molecule binding sites on proteins. While historically
molecules like aminoglycosides have been studied as RNA binders, these compounds
suffer from poor specificity and systemic toxicity. Therefore, approaches that identify
small molecules with new RNAꢀbinding chemotypes are needed. Furthermore, recent
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discoveries of novel classes of nonꢀcoding, regulatory RNAs5 have provided substantial
evidence for a variety of roles for RNA in cancer.6-8
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One such class of nonꢀcoding RNAs is the family of microRNAs (miRNAs), ~22
nucleotide sequences produced by Drosha and Dicer processing of longer RNA hairpins.
MiRNAs suppress gene expression by binding to partially complementary sequences in
target mRNAs leading to either cleavage of the mRNA by the RNAꢀinduced Silencing
Complex (RISC) or inhibition of translation.9 Aberrant expression of these regulatory
miRNAs has been implicated in the development, metastasis, and progression of a wide
range of human cancers as well as other diseases including cardiovascular disease and
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immune disorders.
11
A powerful example of a diseaseꢀassociated miRNA is miRꢀ21,10,
the
overexpression of which has been observed in many human cancers including
glioblastomas, lymphomas, breast, pancreatic, cervical, colorectal, ovarian, liver, and
lung cancers. The overexpression of miRꢀ21 in various tumors leads to reduced levels
of miRꢀ21 targets in vivo, many of which are tumor suppressors.12 Knockdown of miRꢀ21
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with antisense agents induces apoptosis in glioblastoma,13,
breast cancer,15 and
hepatocellular carcinoma cell lines.16, 17 Further, a direct relationship between miRꢀ21
overexpression and preꢀBꢀcell lymphoma development has been demonstrated in a
mouse model, providing evidence for miRꢀ21 as a potential therapeutic target.12
The identification of small molecules that bind to “oncomiRs” such as miRꢀ21
could provide a viable approach for the inhibition of miRNAs involved in cancer
development and could lead to mechanistically new cancer therapies. Small molecules
that inhibit the cellular function of miRꢀ21 have been discovered; however, the
mechanism of action of these inhibitors has not been elucidated, and they do not appear
to bind directly to the RNA.18, 19 Similarly, aminoglycoside,20 peptide/protein,21 and
peptoidꢀbased22, 23 inhibitors of miRꢀ21 have been studied. A longꢀstanding goal has
been to develop small molecules that directly bind to miRꢀ21 in order to inhibit its
biogenesis and function, and to date, this has proven challenging, with only a small
number of compounds reported.20, 22, 23
Here, we employ a small molecule microarray (SMM) screen of a large library of
druglike molecules to identify new motifs that bind to the preꢀmiRꢀ21 hairpin. Hit
compounds that showed selective binding to miRꢀ21 over other oligonucleotides were
validated through a series of biochemical and biophysical experiments. Through
synthesis and analysis of a focused library, we demonstrate that this series of
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compounds exhibits
a
clear structureꢀactivity relationship.
Multiple affinity
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measurements show that several compounds display low micromolar to high nanomolar
binding to the RNA hairpin. Dicer processing and inꢀline probing experiments further
demonstrate that the compounds bind near the apical loop of the hairpin and inhibit
formation of the mature miRNA. The compounds described here provide evidence that
small molecules that bind directly to miRꢀ21 can be used to block miRNA biogenesis and
serve as miRꢀ21 inhibitors.
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RESULTS AND DISCUSSION
SMMs are a powerful tool for the high throughput screening of compound
libraries against biological targets of interest,24-29 and have successfully been used to
identify druglike small molecules that bind to nucleic acid targets including both DNA30
and RNA.31 Briefly, in this technique, small molecules are spatially arrayed and
covalently linked to a functionalized glass surface using a robotic microarrayer. Slides
are then hybridized with a fluorescently labeled oligonucleotide and imaged. Array
features that increase in fluorescence indicate binding to the oligonucleotide (Figure 1A).
To identify small molecules that bind to miRꢀ21, we performed a SMM screen with a
fluorescently labeled preꢀmiRꢀ21 hairpin. A 29ꢀmer RNA hairpin was designed
containing the preꢀmiRꢀ21 Dicer cleavage site and was labeled with a Cy5 fluorophore at
the 5’ end (Figure 1B). SMM slides were incubated with the hairpin at a concentration of
500 nM for 1 h, after which slides were washed and imaged using a previously reported
31
protocol.30,
In parallel, an identical screen was performed using the HIV
Transactivaiton Response (TAR) RNA hairpin to control for selectivity in binding. Next,
data analysis was performed to generate a composite Zꢀscore for each compound in the
library, and compounds with a Zꢀscore > 3 were considered hits. In total, a library of
20,000 compounds was screened, and 19 compounds were identified as binding to the
miRꢀ21 hairpin but not the TAR hairpin for a hit rate of 0.01 %.
From the 19 hit compounds that were identified in the SMM screen, 11 were
selected for further analysis and purchased from commercial vendors (Figure S1). Of
note, in the set of 19 total hits, seven of these compounds contained a common
chemotype. Thus, only the highest scoring members of this group of compounds were
purchased for initial followꢀup analysis. To evaluate the binding of hit compounds to
miRꢀ21, a series of biochemical assays and affinity measurements were performed.
Initially, each hit compound was analyzed in a differential scanning fluorimetry (DSF)
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assay, where the melting temperature (Tm) of the RNA was measured in the presence
and absence of each compound. Changes in the Tm of the hairpin indicate compound
interaction with the RNA. Of the 11 compounds, 1 and 2 (Figure 1C) showed
reproducible ꢀTm values of –5.1 ± 1.9 and –2.3 ± 0.3 °C, respectively, indicating binding
to the hairpin. These compounds were among the highest scoring hits from the SMM
screen, showed selectivity for miRꢀ21 over the TAR hairpin, and their chemotype was
represented in multiple screening hits. Therefore, these two compounds were selected
for further analysis.
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The DSF analysis provides reliable qualitative evidence for compound binding,
but is not used to measure equilibrium dissociation constant (Kd) values. To establish Kd
values for each compound, we utilized two orthogonal fluorescence titration assays.
First, each compound was titrated into a solution containing a 2ꢀaminopurineꢀlabeled (2ꢀ
AP) RNA hairpin, where A15 was replaced with 2ꢀAP.23 Compoundꢀinduced changes in
2ꢀAP fluorescence are used to derive a Kd value. In parallel, a fluorescence intensity
assay (FIA) was performed. Compounds were titrated into a solution containing the Cy5ꢀ
labeled hairpin used in the SMM screen, and changes in fluorescence intensity were
measured by fluorescence spectroscopy and fit to a binding curve. In the 2ꢀAP assay,
compounds 1 and 2 were found to have Kd values of 2.3 ± 0.5 ꢁM and 0.8 ± 0.2 ꢁM,
respectively. Similarly, when measured by FIA, 1 and 2 had Kd values of 3.2 ± 0.7 ꢁM
and 0.7 ± 0.1 ꢁM, respectively. Affinity measurements in the presence of excess yeast
tRNA were unaffected, indicating a specific binding interaction (Supplementary Figure
S2). The Kd values were in good agreement between the two assays, despite different
fluorophores and conditions, suggesting that both assays are faithfully reporting on
compound affinity measurements and would be valid for evaluating compound affinity.
To evaluate the structureꢀactivity relationship (SAR) of compounds 1 and 2, we
assembled a library of synthetic and commercially available analogs. We used an
efficient synthetic route that would enable rapid access to a variety of compounds
differing in the substitution patterns throughout the core scaffold.32, 33 Beginning with
commercially available 3,6ꢀdibromocarbazole, alkylation with epichlorohydrin produced
an epoxide,34 which could be readily opened with substituted anilines or other nitrogen
heterocycles in a BiCl3ꢀcatalyzed reaction. This approach was used to generate a series
of analogs (3–9) differing in aromatic ring substitution patterns (Table 1). Nine
commercially available analogs in this series were purchased as well (10–16, Table 1),
several of which were also identified as hits in the SMM screen. In addition to changing
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the anilineꢀcontaining functionality, we evaluated perturbations to the carbazole moiety
(Table 2) as well as the linker structure and length (Table S1). To generate analogs
satisfying these criteria, we modified the synthetic route to accommodate differentially
substituted carbazoles (17–23), bromoindoles (24), diarylamines (25–27), and different
linkers (SI10–12).35 In total, we assembled a library of 30 analogs, including
substitutions and modifications on almost all sites of the active structure.
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Once the library was complete, each compound was evaluated in the DSF, FIA,
and 2ꢀAP assays to compare binding affinity to the miRꢀ21 hairpin (Tables 1, 2, S1). In
general, the Kd values from both FIA and 2ꢀAP assays showed good agreement.
Additionally, most compounds that showed good affinity exhibited larger ꢀTm in the DSF
assay. Alteration of the aniline substituent (3–16) was permissive, with most analogs
displaying comparable binding affinity. Here, the best performing compounds contained
a simple aromatic ring with a single alkoxy, halo, or alkyl substituent. Presumably, this
class of compounds is conjugated to the SMM surface via reaction with the aniline
nitrogen (or the neighboring secondary alcohol). These groups would thus be near the
surface of the array, and are not likely to be involved in direct contact with the RNA. The
largest changes occurred with perturbation of the carbazole core, where removal of the
bromide substituents (17), removal of one of the arenes (24), or replacement of the
carbazole with a diarylamine (25–27), resulted in compounds with dramatically reduced
binding. Changing the substitution pattern of the halides on the carbazole (18–22) had a
more modest effect, as did changing bromoꢀ to chloroꢀ substitution (23). Alterations to
the linker between the carbazole and aniline substituents also had modest effects, with
longer linker lengths or modification of the secondary alcohol being associated with
slightly weaker binding (Table S1, SI10–12). The most active compounds in the series
had Kd values of ~1ꢀ3 ꢁM with large effects in the DSF assay (ꢀTm = –5 to –9 °C).
Taken together, these data indicate an SAR whereby the dibromocarbazole functionality
is important for interaction with the RNA, while linker length and aniline substitution
effects are less impactful.
Based on their relatively high affinities for the miRꢀ21 hairpin, six compounds
were assessed for their ability to repress the levels of mature miRꢀ21 through inhibition
of Dicerꢀmediated maturation of the preꢀmiRNA.22 As validated by the DSF and
fluorescence intensity assays, the small molecules bind to the truncated miRꢀ21 hairpin,
which contains many but not all of the secondary structural elements found in the wild
type preꢀmiRꢀ21 RNA. Binding of small molecules to the preꢀmiRꢀ21 hairpin could inhibit
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the ability of Dicer to recognize and efficiently process preꢀmiRꢀ21, thus inhibiting mature
miRꢀ21 biogenesis. To test this hypothesis, a Dicer cleavage assay was performed on a
full length 60 nt 5’ꢀAlexaFluor 647ꢀlabeled preꢀmiRꢀ21 hairpin (Figure 1B). This hairpin
was completely cleaved by Dicer to produce a 22ꢀnt fragment consistent with mature
miRꢀ21 formation. The consumption of preꢀmiRꢀ21 substrate was observed by inꢀgel
fluorescence. A doseꢀdependent inhibition of Dicer cleavage was observed upon
treatment of preꢀmiRꢀ21 with increasing concentrations of the initial SMM hit compound
1 (Figure 3A). Here, inhibition of Dicer processing occurred at concentrations near the Kd
values observed in affinity titration experiments. Preꢀincubation of the RNA with each of
six related carbazole derivatives at a concentration of 1 ꢁM led to a significant inhibition
of Dicerꢀmediated cleavage and an increase in the quantity of full length preꢀmiRꢀ21
after ribonuclease treatment (Figure 3B). Compounds 1 and 2, initially identified in the
SMM screen, displayed 47% and 59% inhibition, respectively. Notably, treatment with
analogs 6, 7, 11, or 13 produced levels of full length preꢀmiRꢀ21 that were equal to that
of control reactions performed in the absence of Dicer. Thus, Dicer cleavage was
abrogated upon compound binding. These results suggest that compound binding to the
RNA may either physically block Dicer access to the preꢀmiRNA or induce structural
changes that impair the efficiency of Dicer processing of the preꢀmiRNA.
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To gain insight into the region of binding of the small molecules to the miRꢀ21
hairpin, a Mg2+ induced inꢀline probing assay36 was performed with the pꢀethoxy 13 and
the 5’ꢀCy5ꢀlabeled miRꢀ21ꢀhp used in the SMM screen.22, 23 The 5’ꢀCy5ꢀlabeled miRꢀ21
hairpin (3 ꢁM) was incubated with or without 13 at increasing concentration (3−300 ꢁM)
in buffer containing 1 mM MgCl2 for 4 days. A previously reported miRꢀ21ꢀbinding
aminoglycoside, streptomycin, was used as a positive control for binding.20 The resulting
cleavage products were separated by PAGE, visualized by in gel fluorescence, and
quantified to assess the cleavage efficiency across the various conditions (Figure 4A). In
the absence of small molecule, Mg2+ induced inꢀline cleavage predominately in the apical
loop, with lower efficiency in the baseꢀpaired region. When the miRꢀ21 hairpin was
analyzed in the presence of 13, the resulting cleavage pattern was markedly different
from that generated in the absence of ligand, suggesting that compound binding affects
the conformation or flexibility of the RNA within those regions. Notably, cleavage after
residues C17, U18, C19 in and near the apical loop was significantly reduced with
increasing concentration of 13 (Figure 4A and B). Similarly, reductions in reactivity after
U11 and U12, located in and near a single nucleotide bulge proximal to the apical loop,
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were also observed. Changes were observed in multiple experiments, and a
representative experiment is shown in Figure 4A. Consistent with the literature reported
binding site determined by S1 nuclease probing, treatment with a known miRꢀ21ꢀbinding
aminoglycoside, streptomycin, also led to a reduction in cleavage after positions C17,
U18, and C19. In order to quantitate differences in cleavage patterns, replicate
experiments were analyzed in triplicate at three concentrations. Here, the most marked
and consistent decreases in reactivity were also observed after C17, U18 and C19
(Figure S3). These compoundꢀinduced changes in cleavage patterns indicate binding of
13 to the miRꢀ21 hairpin proximal to or within the apical loop region (Figure 4C).
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The identification of small molecules that bind directly to miRꢀ21 has proven
challenging to date. In this work, we demonstrate that a SMM screening approach can
be used to identify molecules that bind to miRꢀ21 and inhibit the processing of the preꢀ
miRꢀ21 hairpin by Dicer. Molecules within this series display a clear structureꢀactivity
relationship. More specifically, the dibromocarbazole functionality of the molecule
appears to play a key role in promoting the interaction of the compounds with the
hairpin. Modification of linker length or composition provided modest changes, as did
alteration of the aniline substituents. The best compounds in this series, such as 2 and
13, displayed 0.8 – 2.0 ꢁM equilibrium dissociation constants as measured by both 2ꢀAP
and FIA affinity measurements. When evaluated by DSF, these compounds uniformly
caused a decrease in the melting temperature of the 29ꢀmer miRꢀ21ꢀhp RNA. This is in
contrast to most binding interactions, which increase Tm, and indicates that the
compounds destabilize the hairpin structure upon binding. Several compounds within
this series also displayed inhibitory activity in a Dicerꢀmediated nuclease cleavage
assay, indicating that they interfere with mature miRꢀ21 biogenesis. Inꢀline probing
analysis of 13 demonstrates that C17, U18, C19, U11, and U12 are protected from
cleavage upon compound incubation, providing evidence that the compounds interact
with the hairpin at or near the apical loop. Thus, the compounds may be blocking Dicerꢀ
mediated hairpin processing by binding near the apical loop proximal to the Dicer site.
Taken together, the data presented here suggest that the longstanding goal of
inhibiting miRꢀ21 biogenesis using a small molecule inhibitor may in fact be possible.
The chemical structure of the inhibitors described in this manuscript is distinct from the
aminoglycoside, peptoid, or peptideꢀbased miRꢀ21ꢀbinding inhibitors previously reported.
The reported structures stand as relatively rare examples of nonꢀaminoglycoside small
molecules that bind to miRꢀ21. Molecules similar to 1 and 2 have previously been
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studied in other contexts. For example, P7C3 (10) and derivatives have been studied
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extensively as proneurogenic compounds in vivo, and have been shown to have good
bioavailability32,
P7C3 appears to exert neuroprotective effects by binding
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nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in NAD
salvage.38 Thus, molecules in this series may interact with RNA molecules as well as
proteins. However, the interaction between P7C3 derivatives and RNA has not been
explored previously. Notably, a highly neuroprotective fluoro derivative of P7C3 (SI12) is
less active than other derivatives in miRꢀ21 binding assays, highlighting differences in
SAR between RNA binding and proꢀneurogenic activity. Given that the compounds
reported here have established SAR, selectivity, and in vitro potency against a molecular
target, this work can be considered a first step toward developing a chemical probe for
miRꢀ21.39
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In summary, we have reported the discovery of a small molecule that binds to
miRꢀ21 and inhibits the maturation of the microRNA hairpin by binding near the apical
loop and preventing Dicer processing. An SMM screening approach was key to the
rapid identification of lead structures, which display good binding affinity for the RNA in
multiple biophysical assays. Future efforts will focus on the development of more potent
and selective analogs capable of blocking miRꢀ21 biogenesis in cellular models, which
would demonstrate a fundamentally novel approach for the treatment of a broad variety
of cancers. Finally, this work highlights the potential of SMM screening approaches for
future efforts toward identifying druglike small molecule inhibitors of other diseaseꢀ
associated RNAs.
MATERIALS AND METHODS
General RNA Information. To avoid RNase contamination, all buffers were prepared
with DEPCꢀtreated water and all surfaces and equipment were decontaminated with
RNaseZap (Ambion) prior to RNA handling.
Deprotected and HPLC purified
oligonucleotides were purchased from Dharmacon (ThermoFisher) or IDT DNA with the
following sequences:
Cy5ꢀlabeled miRꢀ21ꢀhp RNA: 5’ꢀCy5ꢀGGGUUGACUGUUGAAUCUCAUGGCAACCCꢀ3’
Cy5ꢀlabeled TAR RNA: 5’ꢀCy5ꢀGCAGAUCUGAGCCUGGGAGCUCUCUGCCꢀ3’
miRꢀ21ꢀhp RNA: 5’ꢀGGGUUGACUGUUGAAUCUCAUGGCAACCCꢀ3’
2ꢀAPꢀlabeled miRꢀ21ꢀhp RNA: 5’ꢀGGGUUGACUGUUGA(2AP)UCUCAUGGCAACCCꢀ3’
AlexaFluor
647 (NHS
Ester)ꢀlabeled preꢀmiRꢀ21ꢀhp RNA: 5’ꢀAlexa647Nꢀ
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UAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGG
GCUGUCꢀ3’
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Small Molecule Microarray (SMM) Screening. SMM slides were prepared according
to previously reported procedures.27,
Briefly, γꢀaminopropyl silane (GAPS)
30, 31
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microscope slides were functionalized with an Fmocꢀprotected amino polyethylene glycol
spacer (Fmocꢀ8ꢀaminoꢀ3,6ꢀdioxaoctanoic acid) in N,Nꢀdimethylformamide (DMF).
Following piperidine deprotection, 1,6ꢀdiisocyanatohexane was coupled to the surface to
provide isocyanateꢀfunctionalized microarray slides for immobilization of small molecule
library members containing either primary or secondary alcohols or amines (aliphatic or
aromatic). A total of 20,000 unique small molecules (10 mM in DMSO) containing at
least one primary or secondary alcohol or amine were purchased from commercial
vendors including ChemBridge and ChemDiv. The libraries were printed on four array
slides containing ~5000 distinct molecules printed in duplicate, in addition to dyes and
controls used for quality control validation. The arrays were exposed to pyridine vapor to
facilitate covalent attachment to the isocyanateꢀfunctionalized slide surface. Slides were
then incubated with a polyethylene glycol solution to quench unreacted isocyanate
surface.
The deprotected 5’ꢀCy5ꢀlabeled miRꢀ21 hairpin (Cy5ꢀmiRꢀ21ꢀhp) from
Dharmacon was dissolved in RNase free 1X PBS (12 mM NaH2PO4, 137 mM NaCl, 3
mM KCl, pH 7.4), and was annealed by heating to 95 °C for 3 min, followed by slowly
cooling to room temperature for 1 h. The hairpin was then diluted to 500 nM in PBST
(0.01% Tweenꢀ20) for screening. Printed microarray slides were incubated with the
RNA at a concentration of 500 nM for 1 h. Following incubation, slides were washed
three times with PBST and once with water to remove unbound RNA, and the slides
were dried by centrifugation for 2 min at 4000×g. Slides were imaged for fluorescence
(650 nm excitation, 670 nm emission) on a GenePix 4000a Microarray Scanner with a
resolution of 5 or 10 ꢂm. The scanned image was aligned with the corresponding
GenePix Array List (GAL) file to identify individual features. Hits were determined using
the following criteria: (a) coefficient of variance (CV) of duplicate spots <100, (b) Average
Z score for a compound >3, (c) [(ZꢀscoreRNA incubated) – (ZꢀscoreControl Array)]/ZꢀscoreControl
Array) > 3, (d) no activity with any other nucleic acid structures screened in parallel. Other
Cy5ꢀlabeled nucleic acids screened in parallel included HIV TAR RNA, the FOXO3 DNA
transcription factor binding domain, MYC G4 forming DNA, and CAG DNA repeat, all of
which were screened using the same method described above for the miRꢀ21 hairpin.
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Hits were further validated by visual inspection of array images and compounds for
further study were purchased from original suppliers.
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Differential Scanning Fluorimetry. miRꢀ21ꢀhp RNA from Dharmacon was dissolved in
PBS (pH 7.4), annealed by briefly heating to 95 °C for 3 min and cooling to room
temperature for 1 h, and then diluted to 10 ꢂM in PBS. Stock solutions of each small
molecule were prepared at a concentration of 1 mM in DMSO. In a white 96ꢀwell PCR
plate, small molecules were diluted to a final concentration of 30 ꢂM in PBS (50 ꢂL total
volume) in triplicate and were incubated with the miRꢀ21ꢀhp RNA at a final concentration
of 2 ꢂM for 10 min at room temperature. SYBR Green II was then added to each well at
a final concentration of 2X (5% final DMSO concentration), the solutions were mixed,
and the plate was centrifuged at 4000 rpm for 2 min. After a 10 min incubation, the
fluorescence was measured on a LightCycler 480 (Roche) as the samples were heated
from 20 to 80 °C with a ramp rate of 0.04 °C/s and an acquisition of 10 reads/1 °C using
an excitation wavelength of 465 nm and an emission wavelength of 510 nm. The
melting temperature (Tm) was determined by the maximum of the negative of the first
derivative of the fluorescence vs. temperature melting curves. The mean Tm and
standard deviations were determined from replicate wells.
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2-Aminopurine (2-AP) Fluorescence Titration.
Fluorescence titrations were
performed according previously reported protocols.23 Deprotected miRꢀ21 hairpin RNA
containing a 2ꢀAP label substituted for A15 in the apical loop was purchased from
Dharmacon and was dissolved in PBS (pH 7.4) to a concentration of 100 ꢂM. The RNA
was annealed by briefly heating to 95 °C for 3 min, followed by cooling to room
temperature for 1 h, and was subsequently diluted to 10 ꢂM in PBS. Small molecule
solutions were prepared as serial dilutions in DMSO. In a black 96ꢀwell plate, small
molecules were diluted to final concentrations ranging from 0.05 – 500 ꢂM in triplicate in
PBS (10% final DMSO concentration), and the miRꢀ21ꢀ2AP RNA was added at a final
concentration of 2 ꢂM. Background fluorescence of the small molecules was assessed
in wells containing compound at each final concentration in PBS in the absence of RNA.
After delivery of the RNA, the plate was centrifuged (1000 rpm, 2 min) and allowed to
incubate for 30 min at room temperature with shaking. Fluorescence was then
measured on a Synergy Mx microplate reader (BioTek) with an excitation wavelength of
310 nm and an emission wavelength of 365 nm. Background fluorescence of the small
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molecules in the absence of RNA was subtracted from wells containing RNA, and the
apparent dissociation constants were determined by fitting a sigmoidal doseꢀresponse
curve to the mean fluorescence of background subtracted triplicate measurements.
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Fluorescence Intensity Assay. Fluorescence titrations were performed using a 5’ꢀCy5ꢀ
labeled miRꢀ21 hairpin purchased from Dharmacon. The RNA was prepared in 1X PBS
at a concentration of 100 uM and was annealed by heating to 95 °C for 3 minutes,
followed by cooling to room temperature over 1 h. Small molecule solutions were
prepared as serial dilutions in DMSO. In a black 96ꢀwell plate, small molecules were
diluted to final concentrations ranging from 0 – 250 ꢂM in triplicate in PBS with a 5% final
DMSO concentration. After shaking for 5 minutes, 5’Cy5ꢀmiRꢀ21ꢀhp RNA was added to
a final concentration of 100 nM. The samples were allowed to equilibrate for 30 minutes
with shaking. The fluorescence intensity was then measured on a Synergy Mx
microplate reader (BioTek) at an excitation wavelength of 649 nm and an emission
wavelength of 670 nm. The fluorescence intensity was then normalized to the values
obtained for RNA incubated with a DMSO control and was plotted against small
molecule concentration. The apparent dissociation constants were determined using a
single site model to fit the curve.
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Inhibition of Dicer Cleavage. Dicer cleavage assays were performed on 5’ꢀAlexaFluor
647ꢀlabeled preꢀmiRꢀ21ꢀhp RNA and were monitored by the presence of full length
RNA.40 5’ꢀAlexaFlour 647ꢀlabeled preꢀmiRꢀ21 was dissolved in PBS (pH 7.4) to a
concentration of 100 ꢂM and was annealed by briefly heating to 95 °C for 3 min,
followed by slowly cooling to room temperature over 1 h. For assessing each compound
at a single concentration, the annealed RNA was preꢀincubated at a final concentration
of 10 nM in 20 mM Tris, pH 7.4, 12 mM NaCl, 2.5 mM MgCl2, 40 U/mL RNaseOUT
(Invitrogen), 1.0 mM fresh DTT with either a DMSO control (5% final concentration) or
each compound at a concentration of 1 ꢂM at room temperature for 30 min.
Recombinant Dicer (0.05 U, Genlantis) was then added, and digestion was performed at
37 °C for 2 h. Digestions were quenched by adding equal volume of 2X TBE/urea
sample buffer (BioRad) and heating to 95 °C for 5 min. Cleavage was analyzed using a
15% TBE/Urea polyacrylamide gel and was visualized by fluorescence of the 5’ꢀAlexa
Fluor 647 label (630 nm excitation, 670 emission) with an ImageQuant LAS 4000 gel
imager (GE Healthcare Life Sciences), and the backgroundꢀcorrected fluorescence
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intensity of the full length RNA was analyzed using ImageQuant software. The
fluorescence intensity for full length RNA in the small molecule treated samples was
normalized to DMSO controls with and without Dicer within the same experiment.
Digestions were performed in triplicate on separate days, and the normalized
fluorescence for each compound was averaged and standard deviations were
determined. For the doseꢀresponse experiment, the annealed RNA was preꢀincubated
at a final concentration of 500 nM in 20 mM Tris, pH 7.4, 12 mM NaCl, 2.5 mM MgCl2, 40
U/mL RNaseOUT (Invitrogen), 1.0 mM fresh DTT with either a DMSO control (5% final
concentration) or small molecule at concentrations ranging from 0.1–100 ꢂM at room
temperature for 30 min. Recombinant Dicer (0.5 U) was then added, and digestion was
performed at 37 °C for 30 min. Digestions were analyzed using a 15% TBE/Urea
polyacrylamide gel and visualized as described previously.
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Analysis of Mg2+-Induced In-line Cleavage. 5’ꢀCy5ꢀlabeled miRꢀ21ꢀhp RNA was
dissolved in 0.1X PBS (pH 7.4) to a concentration of 100 ꢂM and was annealed by
briefly heating to 95 °C for 3 min, followed by slowly cooling to room temperature for 1
h.22, 23 The annealed RNA was incubated at a final concentration of 3 ꢂM in 50 mM Tris,
pH 8.5, 10 mM KCl buffer with either a DMSO control (5% final concentration) or
compound at a concentration of 3, 10, 30, 100, or 300 ꢂM. MgCl2 was added at a final
concentration of 1 mM, and the reactions were incubated at room temperature in
darkness for 4 days. Alkaline hydrolysis was performed in 10 mM NaHCO3, pH 9.0 at 95
°C for 5 min. Ribonuclease T1 digestion was carried out with 0.1 U of ribonuclease T1
(Ambion) in 20 mM Tris, pH 7.5, 50 mM NaCl, 0.1 mM MgCl2 at room temperature for 20
min. The RNase T1 reaction was stopped by adding 0.2 volumes of 5 mM EDTA. Equal
volumes of loading buffer containing 7 M urea, 1X TBE, and 0.01% direct red dye was
added to each reaction, and the samples were heated to 95 °C for 5 min prior to analysis
by electrophoresis on a denaturing polyacrylamide sequencing gel (20%, 19:1
crosslinking, 7 M urea). The gel was visualized by fluorescence of the 5’ꢀCy5 label (630
nm excitation, 670 emission) with an Typhoon FLA9500 gel imager (GE Healthcare Life
Sciences) and was analyzed with ImageQuant software.
Supporting Information
The supporting information is available free of charge on the ACS publications
website at DOI:
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Available are Supplementary Figures 1ꢀ3, Supplementary Table 1, and synthetic
procedures and characterization data for compounds 3ꢀ9, 18ꢀ27, as well as for relevant
synthetic intermediates.
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ACKNOWLEDGMENTS
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This work was supported by the Intramural Research Program of the National
Institutes of Health, Center for Cancer Research, and the National Cancer Institute
(NCI), National Institutes of Health (1 ZIA BC011585 02), and Yale University. We thank
Dr. Stuart Le Grice for use of the Typhoon gel imager. We thank Drs. Jim Kelley and
Christopher Lai for assistance in collecting high resolution mass spectral data.
FIGURE CAPTIONS
Figure 1. Small molecule microarray screening. (A) Cartoon depicting a small molecule
microarray screen to identify compounds that bind to miRꢀ21ꢀhp RNA. (B) Sequence of
the truncated Cy5ꢀlabeled miRꢀ21 hairpin (miRꢀ21ꢀhp) used in the SMM screening and
subsequent inꢀline probing (top) and full length AlexaFluor 647ꢀlabeled precursor miRꢀ21
(preꢀmiRꢀ21) used in the Dicer cleavage assays (bottom). The sequence of mature miRꢀ
21 is indicated in blue. (C) Raw SMM images for hit structures (compounds are printed
in duplicate). The behavior of each compound in the negative control (buffer) incubated,
Cy5ꢀlabeled miRꢀ21ꢀhp RNA incubated, and Cy5ꢀlabeled HIV TAR hairpin RNA
incubated SMMs are shown for comparison.
Figure 2. Validation of hit compounds identified by SMM screening. (A) Differential
scanning fluorimetry (DSF) experiments demonstrating changes in the melting
temperature (Tm) of the unlabeled miRꢀ21ꢀhp RNA upon addition of 1 or 2 at 30 ꢂM in
triplicate compared to a DMSO control. (B) 2ꢀAminopurine fluorescence titration of 2ꢀ
APꢀlabeled miRꢀ21ꢀhp RNA in the presence of 1 or 2 at increasing concentrations. (C)
Fluorescence titration assay of 5’ꢀCy5ꢀlabeled miRꢀ21ꢀhp RNA in the presence of
increasing concentration of 1 or 2. Apparent Kd values for each compound were
determined from triplicate experiments using both the 2ꢀAP and fluorescence titration
assays. The error bars indicate the standard deviation determined from three
independent measurements.
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Table 1. Synthesis and evaluation of aniline modified analogs of 1 and 2. The ∆Tm and
apparent Kd values were determined by differential scanning fluorimetry, 2ꢀaminopurine
and fluorescence intensity assays.
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Table 2. Synthesis and evaluation of carbazole modified analogs of 1 and 2. The ∆Tm
and apparent Kd values were determined by differential scanning fluorimetry, 2ꢀ
aminopurine and fluorescence intensity assays.
Figure 3. Dicer cleavage assays of full length 5’ꢀAlexaFluor 647ꢀlabeled preꢀmiRꢀ21. (A)
Inꢀgel fluorescence of 5’ꢀAlexaFluor 647ꢀlabeled preꢀmiRꢀ21 cleaved with Dicer in the
presence of increasing concentration of 1. Arrow indicates the band corresponding to the
unprocessed preꢀmiRꢀ21, which was used for quantitation. (B) Quantification of inꢀgel
fluorescence of 5’ꢀAlexaFluor647ꢀlabeled preꢀmiRꢀ21 in the presence of Dicer and the
miRꢀ21 binding compounds at a concentration of 1 ꢁM. Treatments were performed in
triplicate, and compound treatment was normalized to DMSO controls with and without
Dicer within the same experiment. The error bars indicate the standard deviation
determined from the three independent experiments.
Figure 4. Analysis of small molecule binding to miRꢀ21ꢀhp RNA by Mg2+ induced inꢀline
cleavage. (A) Inꢀgel fluorescence of 5’ꢀCy5ꢀlabeled miRꢀ21ꢀhp RNA after treatment with
13 at concentrations of 3, 10, 30, 100, or 300 ꢁM or a DMSO control in the absence (–)
or presence (+) of 1 mM MgCl2. OH and T1 are a partial alkaline hydrolysis ladder and
ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the
cleavage efficiency was significantly altered by compound treatment. (B) Quantification
of fluorescent band intensity from above gel, where band intensity from 13 or
streptomycin treated samples was normalized to the average band intensity of DMSO
control treated samples. (C) Structure of the 5’ꢀCy5ꢀlabeled miRꢀ21ꢀhp RNA used for the
inꢀline probing. Significant changes to the cleavage pattern in the presence of small
molecule are indicated in blue. The red line indicates the putative Dicer cleavage site on
preꢀmiRꢀ21.
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ACS Chemical Biology
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ACS Chemical Biology
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Table 1
176x480mm (300 x 300 DPI)
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Table 2
247x369mm (300 x 300 DPI)
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