Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching

Article abstract of DOI:10.1021/ja210088v

Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a

Full text of DOI:10.1021/ja210088v

Article
pubs.acs.org/JACS
Design of a Bioactive Small Molecule That Targets the Myotonic
Dystrophy Type 1 RNA via an RNA Motif−Ligand Database and
Chemical Similarity Searching
Raman Parkesh,†,# Jessica L. Childs-Disney,†,# Masayuki Nakamori,‡ Amit Kumar,† Eric Wang,§
Thomas Wang,§ Jason Hoskins,‡ Tuan Tran,† David Housman,§ Charles A. Thornton,‡
and Matthew D. Disney*,†
†Department of Chemistry, Scripps Florida, 130 Scripps Way, Jupiter, Florida 33458, United States
‡Department of Neurology, University of Rochester, Rochester, New York 14642, United States
§Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, 68-132, Cambridge, Massachusetts 02139, United
States
S
* Supporting Information
ABSTRACT: Myotonic dystrophy type 1 (DM1) is a triplet repeating
disorder caused by expanded CTG repeats in the 3′-untranslated region of
the dystrophia myotonica protein kinase (DMPK) gene. The transcribed
repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC
motif that binds the RNA splicing regulator muscleblind-like 1 protein
(MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes
splicing defects in a subset of pre-mRNAs including the insulin receptor, the
muscle-specific chloride ion channel, sarco(endo)plasmic reticulum Ca2+
ATPase 1, and cardiac troponin T. Based on these observations, the
development of small-molecule ligands that target specifically expanded
DM1 repeats could be of use as therapeutics. In the present study, chemical
similarity searching was employed to improve the efficacy of pentamidine
and Hoechst 33258 ligands that have been shown previously to target the
DM1 triplet repeat. A series of in vitro inhibitors of the RNA−protein complex were identified with low micromolar IC50’s, which
are >20-fold more potent than the query compounds. Importantly, a bis-benzimidazole identified from the Hoechst query
improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/
kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif−ligand database, these
studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach
that combines analysis of known RNA−ligand interactions with chemical similarity searching.
the chloride ion channel and the IR pre-mRNAs explain the
myotonia and insulin insensitivity, respectively, associated with
myotonic dystrophy. The presence of expanded rCUG repeats
also causes reduction in the nucleocytoplasmic transport of the
DMPK mRNA and thus the expression level of DMPK.21,29
Reduced transport occurs because the expanded repeats bind to
various proteins and form nuclear foci.22,30−35
The disease model in which expanded rCUG repeats bind
and inactivate MBNL1 points to a therapeutic strategy in which
a small molecule or oligonucleotide binds the repeats and frees
MBNL1. This therapeutic avenue is supported by several
studies,36−39 including the correction of splicing defects by
overexpression of MBNL1.20 Several groups have identified
small molecules that inhibit the rCUG-MBNL1 interaction,
including a Hoechst derivative,40 an aminoglycoside deriva-
INTRODUCTION
■
RNA plays important roles in biological processes. For example,
RNA splicing patterns control the protein isoforms derived
from a single primary transcript, and microRNAs regulate
processes such as developmental timing by controlling the
lifetime of an mRNA.1−5 RNA-mediated diseases6−11 can be
caused by an RNA loss-of-function or a gain-of-function. RNA
gain-of-function causes or contributes to triplet and tetra-
repeating disorders such as the myotonic dystrophies,12−14
Huntington’s disease,15 spinocerebellar ataxia,16,17 and fragile X
syndrome.18 In myotonic dystrophy type 1 (DM1), an
expanded rCUG repeat in the 3′-untranslated region (UTR)
of the dystrophia myotonica protein kinase (DMPK) mRNA
binds and inactivates muscleblind-like 1 protein
(MBNL1).19−22 Sequestration of MBNL1 by the expanded
repeats results in the mis-splicing of a subset of pre-mRNAs
including the muscle-specific chloride ion channel23 and the
insulin receptor (IR)24−26 among others.27,28 Splicing defects in
Received: November 5, 2011
Published: February 2, 2012
© 2012 American Chemical Society
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Figure 1. Structures of the most potent lead compounds that inhibit formation of the r(CUG)12-MBNL1 complex. The compounds were identified
from chemical similarity searching in which pentamidine (P) and Hoechst 33258 (H) were used as query molecules. Left, the most potent
pentamidine-like compounds; right, the most potent Hoechst 33258-like compounds.
tive,41 an acridine−triazole conjugate,42 pentamidine,38 and
peptides and derivatives thereof.36,43 Pentamidine38 and
oligonucleotides37,39 improve splicing defects in cell culture
and animal models.
Chemical Similarity Searching Methodology. Hoechst
33258 (H) and pentamidine (P) (Figure 1) were used as query
molecules to identify compounds in the NCI and eMolecules
databases that are similar in shape and/or structure. The NCI
database was used because it is an easily accessible, medium-
sized database (250,000 compounds). It contains the most
complete collection of compounds that represents broad
chemical space, and the compounds are available free of charge
for academic purposes. All compounds used for virtual
screening in the NCI database have complete stereochemistry
specification, allowing reliable 3D coordinates of stereoisomers
to be calculated. The eMolecules database (8,000,000
compounds) was used as it contains the most comprehensive,
commercially available collection of small molecules for virtual
screening.
Pentamidine and Hoechst 33258 are attractive lead
compounds for virtual screening because of their relative
structural simplicity, which leads to computationally less
demanding 3D shape comparison. Three-dimensional compu-
tational shape comparison was completed using the 3D
alignment software Rapid Overlay of Chemical Structure
(ROCS) because of its fast and efficient implementation of a
3D shape algorithm.45 ROCS treats atoms as a Gaussian
function, and thus the overlaps between two (or more) atoms
are also Gaussian functions. This algorithm implemented in
ROCS speeds up the 3D shape-based calculations, as it involves
calculating the maximal intersection of the volumes of two
molecules.
For both compounds, 100 conformations were generated
with Omega 2.3.2 software from OpenEye Scientific Software
(www.eyesopen.com) and were compared to up to 100
conformations of each compound in the NCI and eMolecules
databases using ROCS (version 3.0.0).46 (The number of
conformers generated for some rigid molecules in the database
was less than 100.) Shape-based comparisons were quantified
using two different metrics: 3D similarities, as quantified by the
shape Tanimoto coefficient,47 and 3D chemistry alignment, as
quantified by the color score.48 Chemistry alignment is
specified by the color force field (implicit Mills−Dean48) and
accounts for the positioning of hydrogen bond donors,
hydrogen bond acceptors, anions, cations, hydrophobic groups,
It is likely that the small molecules that inhibit the rCUG-
MBNL1 interaction represent privileged scaffolds for binding
rCUG repeats. Therefore, we used Hoechst 33258 and
pentamidine as query molecules to identify potential lead
ligands with improved potencies and biological activity in the
National Cancer Institute (NCI) and eMolecules databases;
that is, we identified the small molecules that are similar in
shape and/or the positioning of functional groups, rings,
hydrophobic groups, charge, etc. Once identified, the potencies
of the compounds were tested in an in vitro assay. Many ligands
are more potent than the parent pentamidine and Hoechst
33258 compounds; the five most potent compounds showed a
>20- to >100-fold improvement for disruption of the rCUG-
MBNL1 complex. A bis-benzimidazole, which was identified by
the Hoechst query, improves DM1-associated pre-mRNA
splicing defects in cellular and animal models. Since Hoechst
33258 was identified to bind the repeating 5′CUG/3′GUC
motif in the DM1 RNA using an RNA motif−ligand database,
these studies suggest that the database approach can be useful
to identify lead ligands targeting RNA that can be further
improved by using chemical similarity searching.
RESULTS AND DISCUSSION
■
Previous reports have shown that small molecules40−43 and
oligonucleotides39 can disrupt formation of the toxic RNA−
protein interaction that causes myotonic dystrophy in vitro.
Two of these small molecules, Hoechst 33258 (H) and
pentamidine (P) (Figure 1), are interesting lead ligands that are
amendable to chemical similarity searching. Hoechst 33258 was
identified as a lead ligand by analysis of an RNA motif−ligand
database,40,44 while pentamidine was identified from a previous
report.38 The goal of chemical similarity searching is to identify
compounds with similar shapes or positioning of functional
groups that have improved biological activity and/or improved
pharmacodynamic or pharmacokinetic properties.
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Figure 2. Schematic of the qTR-FRET assay used to determine the potencies of compounds for disrupting the r(CUG)12-MBNL1 interaction. The
r(CUG)12 oligonucleotide is labeled with a 5′-biotin, while MBNL1 contains a His6 tag. (Left) In the absence of inhibitor, MBNL1 binds to
r(CUG)12. Binding is quantified by using two antibodies that form a FRET pairan anti-His6 antibody labeled with Tb that binds to MBNL1 and
streptavidin labeled with XL665 that binds to r(CUG)12. The two fluorophores are within close enough proximity to form a FRET pair. Tb is excited
at 345 nm; the resulting emission (∼545 nm) excites XL665, which emits at 665 nm. (Right) In the presence of inhibitor, the r(CUG)12-MBNL1
interaction is disrupted, and two fluorophores are not within close enough proximity to form a FRET pair. Therefore, emission is only observed at
545 nm (due to Tb). XL665 emission is not observed.
Table 1. IC50’s for Displacement of MBNL1 from rCUG Repeats and Degree of Similarity to the Query Molecule
a
(TanimotoCombo Score)
pentamidine-like
Hoechst-like
small molecule
TanimotoCombo score
IC50 (μM)
small molecule
TanimotoCombo score
IC50 (μM)
pentamidine
−
>1000
10
Hoechst 33258
−
>1000
50
P1
P2
P3
P4
P5
P6
P7
P8
1.45
1.47
1.30
1.13
1.36
1.33
1.07
1.55
H1
H2
H3
H4
H5
H6
H7
H8
H9
1.63
1.21
1.13
1.12
1.04
1.13
1.15
1.03
1.08
50
60
60
125
200
300
375
500
500
500
200
250
375
375
1000
a
The error in the IC50 measurements is 10%.
and rings. Complete overlap for either shape or color
(chemistry alignment) is assigned a score of “1”, while no
overlap is assigned a score of “0”. The molecules in the
databases were then ranked on the basis of the “Tanimoto-
Combo” score (sum of the shape Tanimoto coefficient and the
color score; range = 0−2).
Since the compounds are similar to either Hoechst 33258 or
pentamidine, it is likely that the compounds have similar
binding preferences for RNA internal loops.49 A subset of ∼40
compounds was chosen for small molecules identified to be
most similar to pentamidine; another subset of ∼40
compounds was chosen for small molecules identified to be
similar to Hoechst 33258 (Table S-3). These subsets were
selected by visual inspection of the top 500 compounds to
allow for maximal chemical diversity and their availability from
the NCI or eMolecules repositories.
XL665 (binds to the biotinylated RNA oligonucleotide) and
Tb-Anti-His6 (binds to MBNL1) are added. If the compound
does not disrupt the r(CUG)12-MBNL1 interaction, then
XL665 and Tb are within close enough proximity to form a
FRET pair, and the TR-FRET can be measured. TR-FRET is
not observed if the compound inhibits formation of the
r(CUG)12-MBNL1 complex. It should be noted that the RNA−
protein complex is preformed prior to addition of the
compound of interest. Therefore, in order to be scored as an
active compound, the small molecule must displace MBNL1
from the RNA.
We identified 17 compounds from the qTR-FRET screening
assay that inhibited ≥85% of r(CUG)12-MBNL1 complex
formation at 1 mM (Figure 1 and Table S-3). Interestingly,
pentamidine and Hoechst only inhibit ∼10% of r(CUG)12-
MBNL1 complex formation at 1 mM. The active compounds
were then purified by HPLC, and the IC50’s were measured
(Table 1). The IC50’s for the pentamidine-like compounds
range from 10 to 1000 μM, while the IC50’s for the Hoechst-
like compounds range from 50 to 500 μM. Note that in 2D
shape comparison the compounds are chemically quite distinct,
but they show excellent shape similarity and functional group
similarity as quantified by the combo score (Table 1). The best
hits (P1, P2, P3, H1, and H2), which have IC50’s ≤60 μM, were
Identification of Inhibitors Using Quantitative Time-
Resolved Fluorescence Resonance Energy Transfer
(qTR-FRET). In order to screen the ligands identified via
chemical similarity searching for disrupting the r(CUG)6-
MBNL1 complex, a qTR-FRET assay was used.50−52
A
schematic of the assay is shown in Figure 2. Briefly, 5′-
biotinylated r(CUG)12 and MBNL1-His6 are pre-equilibrated,
and then the compound of interest is added. Next, streptavidin-
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structurally analyzed to determine an active chemotype. Each
structure has two basic nitrogen atoms that are separated by
similar distances, 15.1−18.0 Å (Figure 3).
Figure 3. Structural analysis of the five most potent compounds that
inhibit formation of the r(CUG)12-MBNL1 complex using the TR-
FRET assay depicted in Figure 2. Each structure has a similar distance
between two carbon atoms that display a basic nitrogen.
Specificity of the Small Molecules for the DM1 Motif.
It is important that the small molecules that inhibit the rCUG-
MBNL1 interaction show some level of specificity for the DM1
motif, 5′CUG/3′GUC. To estimate specificity, competition
dialysis was employed. In these experiment, RNAs displaying
the DM1 motif, a fully paired RNA, RNAs displaying other 1×1
nucleotide internal loops, an oligonucleotide mimic of the
human aminoacyl tRNA site (A-site), and an oligonucleotide
mimic of the cardiac troponin T (cTNT) pre-mRNA were used
(Figure 4). The human A-site mimic was used because it is a
likely cellular bystander RNA in mammalian cells and because it
contains a 1×1 nucleotide UU internal loop motif.
Furthermore, the human A-site is accessible for small-molecule
binding in the context of the ribosome,53−55 and it is a highly
abundant cellular RNA.56 cTNT pre-mRNA is a natural
substrate for MBNL1.21 It was used in order to determine if
the small molecules might inhibit splicing of endogenous genes
due to promiscuous binding. Compounds P1, P3, H1, and H2
were investigated; the spectral properties of P2 prevented study
by competition dialysis, as the wavelengths at which it absorbs
overlap with those of RNA.
First, the specificities of the compounds for the DM1 motif
over a fully paired RNA and RNAs displaying other 1×1
nucleotide internal loops were investigated (Figure 5A). The
most selective compound is P1, as it binds the DM1 motif
preferentially over all other RNAs investigated. In contrast, P3
binds similarly to RNAs containing 5′CCG/3′GAC, 5′CGG/
3′GAC, 5′CCG/3′GUC, 5′CAG/3′GCC, and the DM1 motif.
Although the greatest amount of binding for H1 is observed to
the DM1 motif, significant binding is also observed to 5′CUG/
3′GCC, 5′CGG/3′GGC, and 5′CAG/3′GAC. H2 binding
preferences are 5′CGG/3′GGC > 5′CCG/3′GUC ≈ 5′CUG/
3′GUC ≈ 5′CGG/3′GAC.
Figure 4. Secondary structures of the RNAs used to determine the
specificities of P1, P3, H1, and H2, which are four of the most potent
inhibitors of the r(CUG)12-MBNL1 complex. RNAs containing only
one copy of a motif are referred to as 5′CXG/3′GXC.
of 5′CUG/3′GUC, an oligonucleotide mimic of the human A-
site, an oligonucleotide mimic of the cTNT pre-mRNA, and
MBNL1 (Figure 4). MBNL1 was included in competition
dialysis experiments since significant binding of the small
molecules to MBNL1 could inhibit its normal function. In both
cases, increased binding of the ligand is observed as the number
of 5′CUG/3′GUC motifs present in the RNA increases (Figure
5B). The binding of H1 appears to be approximately
stoichiometric, in good agreement with a previous report for
another Hoechst derivative.40 Moreover, H1 binds preferen-
tially to RNAs with multiple DM1 motifs over MBNL1 and
oligonucleotide mimics of the human A-site and cTNT pre-
RNA. In contrast, P1 does not bind stoichiometrically as the
number of DM1 motifs increases from one to six. P1 also binds
similarly to the human A-site and RNAs containing three or
more DM1 motifs, and significant binding is observed to the
mimic of the cTNT pre-mRNA (Figure 5B).
The affinities of H1 for an RNA containing one 5′CUG/
3′GUC (DM1) motif and a fully paired RNA (Figure 4) were
determined by fluorescence anisotropy. In good agreement
with our competition dialysis results (Figure 5), H1 binds
approximately 2-fold more tightly to the RNA with the DM1
motif (70 32 nM) than to the fully paired RNA (190 61
nM) (Supporting Information, Figure S-25).
A Bichromatic Reporter System and Flow Cytometry
Analysis To Assess Bioactivity. To investigate whether P1,
P2, P3, H1, and H2 are biologically active, we performed an
initial cell-based screen using a modified bichromatic reporter57
of alternative splicing (Figure 6A). The reporter contains
human muscleblind exon 5 and adjacent introns. The splicing
of this reporter is controlled by MBNL1 itself, through binding
Next, we investigated whether the amount of binding of P1
and H1 increased as a function of the number of DM1 motifs
present in an RNA (Figure 5B). Thus, competition dialysis
experiments were completed for RNAs containing 1−6 copies
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Figure 5. Results of competition dialysis experiments used to determine the specificities of a subset of the most potent compounds identified from
the qTR-FRET assay. Not all of the compounds could be further investigated due to their spectroscopic properties; that is, their absorbance spectra
overlapped with the absorbance from the RNAs or protein. (A) The specificities of four compounds were investigated by competition dialysis. Each
compound was incubated with the 11 RNAs shown in the top panel of Figure 4. The concentration of bound ligand was determined using UV
spectroscopy. P1 and H1 were the most promising ligands, as they bind an RNA containing 5′CUG/3′GUC more than the other RNAs. (B) The
specificities of P1 and H1 were further investigated by competition dialysis with RNAs containing one to six copies of the DM1 motif (5′CUG/
3′GUC), oligonucleotide mimics of the human A-site and the cTNT pre-mRNA, and MBNL1. The human A-site RNA and the cTNT pre-mRNA
are representative bystander RNAs. The binding of H1 and P1 to r(CUG)exp (shaded bars) was extrapolated on the basis of the presence of 479
copies of the 5′CUG/3′GUC motif in the DM1 cellular model system used in these studies.
and repression close to the 3′ splice site. The reporter was
designed such that exon 5 exclusion leads to translation of the
DsRed reading frame, and exon 5 inclusion leads to a read-
through of the +2 reading frame of DsRed, followed by
subsequent translation of the EGFP reading frame. Splicing
behavior was assessed by flow cytometry, where the number of
cells expressing DsRed and EGFP at various levels can be
divided into several quadrants (Figure 6B).
The behavior of the splicing reporter is consistent with that
of endogenous MBNL1 exon 5, as observed upon transfection
into HeLa cells. When transfected alone, ∼55% of cells express
DsRed or a combination of DsRed and EGFP. Upon co-
transfection with a plasmid encoding mouse MBNL1 coding
sequence, the percentage of cells that express DsRed or DsRed
and EGFP increases to ∼70% (Figure 6C,D). This is consistent
with increased levels of MBNL1 blocking inclusion of the test
exon, leading to increased DsRed production relative to EGFP.
Likewise, when the reporter is co-transfected with a mini-gene
containing 960 CUG repeats in the DMPK context (DT960),21
∼0% of cells express DsRed (Figure 6C,D). This is consistent
with sequestration of endogenous MBNL1 by CUG repeats,
leading to exon inclusion and increased EGFP production
relative to DsRed production. Taken together, these results
confirm that this bichromatic splicing reporter, which contains
MBNL1 exon 5 as a test exon, can be used as a sensitive
method for assessing cellular perturbations that affect MBNL1
activity.
To assess the biological activity of our compounds, we co-
transfected our bichromatic splicing reporter, along with
DT960, into three different cell lines. These lines include two
different C2C12 mouse myoblast lines; one is a subclone of the
original line (obtained from ATCC), and the other is the same
subclone containing a stably integrated control shRNA hairpin
that has been designed to not target any endogenous
transcripts. After co-transfection of 250 ng of the reporter
and 25 ng of DT960, the cells were treated with 20 μM P1, P2,
P3, H1, or H2. Two days later, cells were analyzed by flow
cytometry.
fraction of cells that express DsRed. In the presence of DT960,
compounds P1, P2, P3, and H2 led to a further reduction in the
fraction of cells that express DsRed as compared to untreated
cells containing DT960. In contrast, H1 led to a modest
increase in the fraction of cells expressing DsRed, suggesting it
can block or rescue the effects of CUG repeats on endogenous
MBNL1 activity.
As an additional control, the effect of H1 on the bichromatic
reporter in the absence of DT960 was also tested in C2C12 and
HeLa (Figure 6G,H). Addition of 20 μM H1 to cells in the
absence of DT960 led to a slight decrease in the fraction of cells
expressing DsRed. However, addition of 20 μM H1 in the
presence of DT960 led to a modest rescue relative to untreated
cells in the presence of DT960, as evidenced by increased
fractions of C2C12 and HeLa cells expressing DsRed (Figure
6G,H). These results suggest that of all the compounds, H1
exhibits biological activity in a manner favorable to counter-
acting the downstream effects of CUG repeats in the context of
DM1.
H1 Improves Splicing Defects in a DM1 Cell Culture
Model. Next, we determined if the five most potent
compounds could improve splicing defects in a DM1 cellular
model system38 using a cTNT mini-gene.21 cTNT pre-mRNA
is mis-spliced in myotonic dystrophy patients.21,58,59 In normal
cells, MBNL1 binds upstream of exon 5 in the cTNT pre-
mRNA and represses its inclusion.58,60 In the absence of rCUG
repeats, approximately 65% of exon 5 is included in cTNT
mRNA, while in the presence of rCUG repeats, approximately
90% of exon 5 is included (Figure 7A). HeLa cells were co-
transfected with plasmids containing a DM1 mini-gene that
encodes 960 CTG repeats in a 3′UTR21 and the cTNT pre-
mRNA mini-gene.21 The compound of interest was then added
at a final concentration of 500 μM in growth medium. No
improvement in splicing was observed for P1, P2, P3, or H2
(Supporting Information, Figure S-23), in good agreement with
a bichromatic splicing reporter (Figure 6).57
In contrast, modest improvement of splicing is observed
when the cells are treated with 500 μM H1 (Figure 7A). More
significant improvement is observed when cells are treated with
1 mM and 2 mM H1, improving splicing patterns almost to
Similar results were observed in both C2C12 lines (Figure
6E,F). As expected, co-transfection with DT960 reduces the
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Figure 6. (A) Schematic of a bichromatic splicing reporter. The test region is human muscleblind exon 5 and adjacent introns, and replaces test
exons in previous versions of the reporter.57 Inclusion of the exon leads to EGFP expression, and exclusion leads to DsRed expression. (B) A simple
method for quantitation of reporter behavior uses quadrants thresholded on EGFP and DsRed expression in cells. The fraction of cells in Q1, Q2,
and Q4 relative to transfected cells (Q1 + Q2 + Q4) can be used as a metric for reporter behavior. (C) Representative flow cytometry plot of the
reporter transfected alone, with MBNL1 coding sequence, or with 960 CUG repeats. The DsRed/EGFP ratio is responsive to MBNL1 activity. (D)
Representative quantitation of reporter behavior in response to MBNL1 coding sequence or 960 CUG repeats, of which the former leads to
increased DsRed expression, and the latter leads to increased EGFP expression. (E−H) Quantitation of reporter response to CUG repeats and
compounds in combination. As illustrated in panels E and F, compound H1 exhibits the highest DsRed/EGFP ratio, relative to all other compounds
in the presence of CUG repeats, in two different C2C12 cell subclones (C2C12L and C2C12E5). Panels G and H show instances in which
compound H1 can partially rescue the effect of CUG repeats on reporter splicing patterns in both C2C12 and HeLa cells. Note that compound H1
alone, in the absence of CUG repeats, leads to a slight increase in EGFP expression relative to DsRed.
wild-type (WT). (The difference in inclusion percentage for
WT cells (absence of r(CUG)exp) and r(CUG)exp-expressing
cells treated with 1 mM or 2 mM of H1 is not considered to be
significant as determined by statistical analysis.) The difference
in inclusion percentage for r(CUG)exp-expressing cells that are
untreated (Figure 7A, column 2) and treated with 1 mM or 2
mM of H1 is statistically significant. The two-tailed p-values are
0.0149 and 0.0192 for cells treated with 1 mM and 2 mM H1,
respectively. Interestingly, Hoechst 33258 does not improve
splicing defects in cTNT processing at these concentrations
(Supporting Information, Figure S-24).
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alone. Notably, treatment with P1 and P2 yields a large number
of observed events with low forward scatter area and height
values, indicating that most observed events represent cell
debris. Treatment of cells with P3 also leads to loss of cell
integrity but to a lesser extent. Following H1 treatment, over
half of the cells still remain within each gate, indicating that cell
integrity is largely preserved. In summary, H1 is by far the least
cytotoxic compound as compared to P1, P2, and P3
(Supporting Information, Figure S-26).
H1 Disrupts Formation of Nuclear Foci in a Cell
Culture Model of DM1. Another hallmark of myotonic
dystrophy is the formation of nuclear foci, which consist of
expanded rCUG repeats and proteins including
MBNL1.30,34,35,61 In order to determine if H1 is able to
disrupt or prevent formation of nuclear foci, a fluorescence in
situ hybridization (FISH) assay was used. HeLa cells were co-
transfected with a plasmid encoding the DM1 mini-gene. After
incubation with H1, the cells were fixed, permeabilized, and
incubated with a DY547-labeled, 2′OMe oligonucleotide
complementary to the rCUG repeats.38 H1 decreases the
percentage of cells with nuclear foci when the cells are dosed
with 2 mM and 1 mM compound (Figure 7B) but not with 500
μM.
H1 Improves Splicing Defects in a Mouse Model of
DM1. A mouse model of DM1 has been reported in which 250
rCUG repeats are expressed using an actin promoter (human
skeletal actin long repeat, HSALR).22 The presence of the
repeats causes the mis-splicing of the chloride ion channel
(chloride channel 1, skeletal muscle, Clcn1) and the
sarcoplasmic/endoplasmic reticulum calcium ATPase 1
(Serca1/Atp2a1) pre-mRNAs. Normal adult mice have a
Clcn1 exon 7a exclusion rate of 96 0.2%; DM1 mice have
an exclusion rate of 54 1.4%. When DM1 mice are dosed
with 80 mg/kg and 100 mg/kg, the exclusion rate is partially
rescued to 66 0.6% and 69 1.4%, respectively (Figure 8).
These improvements in splicing are statistically significant as
determined by a t test. (The two-tailed p-values are 0.0066 for
treatment with 80 mg/kg of H1 and 0.0016 for treatment with
100 mg/kg of H1.) Serca1 mis-splicing is also partially rescued.
In normal adult mice, the inclusion rate for exon 22 is 99.8
0.1%, while the inclusion rate in the HSALR line is only 30
0.6%. Both dosages of H1, 80 mg/kg and 100 mg/kg, rescue
splicing similarly with inclusion rates of 41 4.0% and 42
3.0%, respectively (Figure 8). The improvement in splicing is
statistically significant when the mice are treated with 100 mg/
kg of H1. (The two-tailed p-values are 0.0522 for treatment
with 80 mg/kg of H1 and 0.0176 for treatment with 100 mg/kg
of H1.)
Selectivity of H1 Binding to DM1 RNA. Because H1
improves splicing defects in cellular and animal models of DM1
and exhibits little toxicity, it must have at least modest
selectivity in vivo. There is little difference in binding affinity or
selectivity of H1 for an RNA with a single copy of the DM1
motif and a fully paired RNA or a mimic of the human A-site
(∼2-fold). However, the selectivity of H1 for RNAs containing
5′CUG/3′GUC improves as the number of repeats increases
(Figure 5). That is, there is only a small difference in the
binding of H1 to an RNA with a single motif and a fully paired
RNA (Figure 5A), while an ∼12-fold difference in binding is
observed between an RNA with six DM1 motifs ((5′CUG/
3′GUC)6) and a fully paired RNA (Figure 5). Thus, H1 could
exhibit some level of specificity for r(CUG)exp in vivo despite
the presence of highly expressed bystander RNAs that also
Figure 7. H1 improves splicing defects and disrupts nuclear foci in a
DM1 cell culture model. (A) H1 improves defects in cTNT pre-
mRNA splicing nearly to wild-type levels. The error bars indicate
standard error. The difference in inclusion percentage for untreated
cells that express r(CUG)exp and treated cells (1 mM and 2 mM of
H1) is statistically significant. The two-tailed p-values are 0.0149 and
0.0192 for cells treated with 1 mM and 2 mM H1, respectively. (B)
Top, H1 disrupts formation of nuclear foci as determined by
fluorescence in situ hybridization; bottom, untreated cells with
multiple nuclear foci. The microscopic images from left to right are
DY-547 fluorescence indicating the presence of CUG repeats, DAPI,
and overlay of DY-547 and DAPI.
Control experiments were also completed in order to
determine if H1 globally affects splicing. First, HeLa cells
were co-transfected with plasmids containing a mini-gene with
only five CTG repeats in the 3′UTR and the cTNT mini-
gene.21 H1 does not affect cTNT splicing in the absence of
CTG repeats (Supporting Information, Figure S-21). We also
investigated whether H1 affects the splicing of pre-mRNAs
whose splicing is not regulated by MBNL1 (Supporting
Information, Figure S-22). A Pleckstrin-2 (PLEKHH2) mini-
gene38 was co-transfected with the DM1 mini-gene, and the
cells were treated with H1. H1 does not affect the splicing of a
PLEKHH2 mini-gene at 2 mM. The splicing of endogenous
genes tetratricopeptide repeat domain 8 (TTC8) and calcium/
calmodulin-dependent protein kinase kinase 2 (CAMKK2) also
was not affected. Thus, H1 does not globally affect RNA
splicing; rather, it affects the splicing of pre-mRNAs controlled
by MBNL1.
The toxicities of the compounds were assessed by flow
cytometry using standard forward and side scatter metrics and
propidium iodide (PI) staining (Supporting Information,
Figure S-26). PI stains DNA in cells that are still grossly intact
but permeabilized; treatment of cells with DMSO results in a
slight increase in the percentage of cells stained with PI. Briefly,
HeLa cells were treated with 500 μM P1, P2, P3, or H1, and
their scatter metrics and PI staining were compared to those of
cells treated with 2% DMSO and those grown in medium
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Figure 8. H1 improves splicing defects in the muscle-specific chloride ion channel (Clcn1) and sarco(endo)plasmic reticulum ATPase (Atp2a1 /
Serca1) pre-mRNAs in a DM1 mouse model. The DM1 mouse model expresses the human skeletal actin (HSA) transgene containing 250 CTG
repeats (HSALR; where LR indicates “long repeats”). WT, or wild-type, refers to FVB mice.22 (A) Top, schematic of Clcn1 alternative splicing in WT
and DM1 mice; bottom, analysis of Clcn1 alternative splicing by reverse transcription-polymerase chain reaction (RT-PCR) when mice are treated
with H1, including a representative gel image and a plot of the corresponding data. The two-tailed p-values are 0.0066 for treatment with 80 mg/kg
of H1 and 0.0016 for treatment with 100 mg/kg of H1. (B) Top, schematic of Atp2a1 (Serca1) alternative splicing in WT and DM1 mice; bottom,
analysis of Atp2A1 alternative splicing by RT-PCR when mice are treated with H1, including a representative gel image and a plot of the
corresponding data. The two-tailed p-values are 0.0522 for treatment with 80 mg/kg of H1 and 0.0176 for treatment with 100 mg/kg of H1.
contain the DM1 motif or a similar one. For example, the
human A-site (Figure 4) is a highly abundant transcript that
contains a single 5′GUC/3′CUG motif (two CUG repeats). At
a first consideration, it would be assumed that it would be the
most occupied binding site in vivo. However, DM1 is caused by
hundreds of copies of the 5′CUG/3′GUC motif. In our cellular
model system, r(CUG)exp contains 479 copies of the 5′CUG/
3′GUC motif ((CUG)960). Thus, the selectivity of H1 for
r(CUG)exp over the A-site could be extrapolated from
competition dialysis experiments by normalizing for the
number of repeats present in vivo, or 479 times greater than
the selectivity of the monomer (∼2-fold) (shaded bars in
Figure 5B).
Implications. Most RNAs that contribute to disease are
unexploited as drug targets. This is generally thought to be due
to a lack of information about the privileged scaffolds that
specifically target RNA. In this study, a lead molecule was
identified by analysis of an RNA motif−ligand database.
Previous studies have shown that Hoechst 3325844 and a
derivative thereof40 bind the regularly repeating 5′CUG/3′GUC
motif that is present in DM1 RNA. By using chemical similarity
searching, a series of related compounds that have improved in
vitro potencies were identified. Importantly, one compound,
H1, is specific for RNAs containing multiple 5′CUG/3′GUC
motifs over mimics of a highly abundant, accessible RNA target
(the human A-site) and a natural MBNL1 substrate (cTNT
pre-mRNA). The compound improves splicing defects in cell
and animal models and disrupts formation of nuclear foci in
cells. Fortuitously, H1 was identified among the ∼80
compounds that were hand-selected from ∼500 virtual
screening hits with known activities. Additional compounds
could have been screened if required.
Perhaps bioactive small molecules that target other RNAs
could be discovered using an approach similar to the one
described herein. Lead small molecules that are not bioactive
due to poor cellular permeability, toxicity, etc. could be
optimized via chemical similarity searching and then tested in
vitro or in vivo. Two key developments bolster the likelihood of
the applicability of the method: (i) a clearer understanding of
the RNA motifs that bind small molecules and the chemical
scaffolds that bind RNA and (ii) annotation of the RNA motifs
present in RNAs that cause disease, including the HIV genome.
By leveraging information in these two areas with compound
optimization as described herein, bioactive small molecules that
target other RNAs could be identified.
SUMMARY AND CONCLUSIONS
■
A computational approach was used to identify compounds
similar to known in vitro inhibitors of a toxic RNA−protein
interaction that have improved potencies and bioactivity. A
bioactive, nontoxic ligand was identified that is similar to the
Hoechst query molecule. Since Hoechst 33258 was identified as
a DM1 RNA binder through analysis of an RNA motif−ligand
database, these studies suggest that the database can provide
lead ligands targeting RNA that can be further improved via
computational screening. Furthermore, the H1 ligand module
can be further improved for targeting the DM1 RNA by using a
modular assembly strategy, which has been shown to improve
the in vitro potency of compounds targeting the DM1 RNA by
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orders of magnitude.40 This approach could be generally
applicable to any RNA of interest.
Quantitative Time-Resolved Fluorescence Resonance En-
ergy Transfer (qTR-FRET) Assay. The qTR-FRET assay used to
identify lead inhibitors of the r(CUG)12-MBNL1 complex is based on
PubChem BioAssay AID 2675 (Figure 2). Briefly, 5′-biotinylated
r(CUG)12 was folded in 1X folding buffer (20 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 110
mM KCl, and 10 mM NaCl) by heating at 60 °C followed by slowly
cooling to room temperature on the benchtop. The buffer was
adjusted to 1X assay buffer (20 mM HEPES, pH 7.5, 110 mM KCl, 10
mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM dithiothreitol, 0.1%
bovine serum albumin (BSA), and 0.5% Tween-20), and MBNL1-His6
was added. The final concentrations of RNA and MBNL1 were 80 nM
and 60 nM, respectively. The sample was allowed to equilibrate at
room temperature for 5 min, and then the compound of interest was
added. After 15 min, strepatividin-XL665 (cisbio Bioassays) and anti-
His6-Tb (cisbio Bioassays) were added to final concentrations of 40
nM and 0.44 ng/μL, respectively, in a total of 10 μL. The samples
were incubated for 1 h at room temperature and then transferred to a
well of a white 384-well plate. Time-resolved fluorescence was
measured on a Molecular Devices SpectraMax M5 plate reader.
Fluorescence was first measured using an excitation wavelength of 345
nm and an emission wavelength of 545 nm (fluorescence due to Tb).
TR-FRET was then measured by using an excitation wavelength of
345 nm, an emission wavelength of 665 nm, a 200 μs evolution time,
and a 1500 μs integration time. The ratio of fluorescence intensity of
545 and 665 nm as compared to the ratios in the absence of ligand and
in the absence of RNA were used to determine IC50’s.
Competition Dialysis. Pierce Slide-A-Lyzer MINI dialyzer units
(Pierce Biotechnology, Inc.) were dialyzed against water for 24 h in
order to ensure that the units did not leak. Competition dialysis was
completed as previously described by Chaires.66 Briefly, RNAs were
folded by heating at 60 °C for 5 min, followed by slowly cooling to
room temperature on the benchtop. Dialysis units containing 0.1 mL
of 1.6 μM RNA or MBNL1 were placed in 200 mL of 1 μM ligand in
1X competition dialysis buffer. The samples were allowed to
equilibrate with the dialysate by stirring the dialysate at 200 rpm for
24 h at room temperature (20−22 °C). Previous studies have shown
that this is sufficient time for the sample to reach equilibrium.66
At the end of the equilibration period, 67.5 μL of each sample was
carefully removed from the dialyzer unit and transferred to a
microcentrifuge tube. To each sample, 7.5 μL of 10% (w/v) sodium
dodecyl sulfate (SDS) was added to give a final concentration of 1%
(w/v) SDS, which is sufficient to dissociate the ligand. The SDS step
was completed to ensure accurate determination of the concentration
of bound ligand, as the spectroscopic properties of the bound ligand
could be different from that of unbound. The total ligand
concentration (Ct) within each dialysis unit was determined
spectrophotometrically using an appropriate absorbance wavelength
and extinction coefficient for each compound. Appropriate corrections
were made for the small dilution resulting from the addition of the 10
μL of SDS.
MATERIALS AND METHODS
■
Chemical Similarity Searching. Chemical similarity searching
was performed on Dell P4 (64-bit) Linux clusters with 32 Xeon
processors using Omega 2.3.2 (version 2.02) and ROCS (version
3.0.0) software from OpenEye Scientific Software (www.eyesopen.
com). As a starting point for the virtual screen, Chem3D (Cambridge
software) was used to energy-minimize (MMFF94 force field) a 3D
conformer of pentamidine and Hoechst 33258. Omega 2.3.2 (version
2.02) was used to generate 100 conformers of pentamidine and
Hoechst 33258, which were submitted as neutral molecules without
any explicit charge. Then, up to 100 conformers of each molecule in
the NCI and eMolecules database were generated. (The number of
conformers generated for some rigid molecules in the database was
less than 100.) The 500 compounds that were the most similar to
pentamidine and the 500 compounds that were most similar to
Hoechst 33258, as determined by the TanimotoCombo scores, were
output in rank order as potential hits.45−47 Of these compounds, 88
were obtained (80 from NCI and 8 from ChemDiv (identified from
eMolecules database)), and 14 were eliminated from screening due to
their insolubility in DMSO or H2O. A complete list of the compounds
screened is available in the Supporting Information (Table S-3).
Three-dimensional shape comparisons, both the shape Tanimoto
coefficients and color scores (chemistry alignment), were completed
using Rapid Overlay of Chemical Structure (ROCS, version 3.0.0)
from the OpenEye software package. The chemistry alignment overlap
(color score) was calculated by using the color force field (explicit or
implicit Mills−Dean as reported in the literature48). The Tanimoto-
Combo score is the sum of the shape Tanimoto coefficient and the
color score.
Mass Spectroscopy and HPLC Purification. Mass spectra were
collected on a Varian 500 MS spectrometer equipped with Varian
Prostar Autosampler 410 and/or on an ABI 4800 MALDI-TOF
spectrometer.
HPLC purifications were completed on a Waters 1525 binary
HPLC pump equipped with a Waters 2487 dual absorbance detector
system. Compounds were purified using a gradient of 5 mL/min, and a
linear gradient of 0% to 100% B in A over 55, 50, or 46 min (A = water
+ 0.1% trifluoroacetic acid (TFA) (v/v); B = MeOH + 0.1% TFA (v/
v)).
The purities of compounds were determined by analytical HPLC
using a Waters 1525 binary HPLC pump equipped with Waters 2487
dual λ absorbance detector system and the following conditions: a
Waters Symmetry C8 5 μm 4.6 × 150 mm column, room temperature,
a flow rate of 2.4 mL/min, and a linear gradient of 0% to 100% B in A
over 45 min.
Oligonucleotide Preparation and Purification. The RNA used
in qTR-FRET assays (5′-biotinylated (CUG)12) was purchased from
Dharmacon. The 2-O-[bis[2-(acetyloxy)ethoxy]methyl] (ACE) pro-
tecting groups were cleaved using Dharmacon’s deprotection buffer.
The sample was lyophilized, dissolved in Milli-Q water, and desalted
using a PD-10 gel filtration column (GE Healthcare). RNAs used in
competition dialysis experiments were prepared via runoff-tran-
scription using synthetic DNA templates (Integrated DNA Tech-
nologies, Inc.) and a Stratagene RNAMaxx transcription kit. They were
purified by denaturing polyacrylamide gel electrophoresis as previously
described62 and dissolved in 1X competition dialysis buffer (8 mM
NaH2PO4, 185 mM NaCl, and 1 mM EDTA; pH 7.2).
The free ligand concentration (Cf) was determined from an aliquot
of the dialysate solution, which did not vary appreciable from the initial
1 μM. The bound ligand concentration (Cb) was then determined
using eq 1:
C = C − C
f
(1)
b
t
where Cb, Ct, and Cf are concentrations of bound, total, and free ligand,
respectively.
Fluorescence Binding Assays. Dissociation constants were
determined using an in-solution, fluorescence-based assay. RNA was
annealed in 1X DNA buffer (8 mM Na2PO4, pH 7.0, 185 mM NaCl,
0.1 mM EDTA) at 60 °C for 5 min and allowed to slowly cool to
room temperature. H1 was then added to a final concentration of 1000
nM. Serial dilutions of the RNA (1:2) were then completed in DNA
buffer containing 1000 nM H1. The samples were incubated for 15
min at room temperature and then transferred to a well of a black 384-
well plate. Anisotropy intensity was measured using a Molecular
Devices SpectraMax M5 plate reader. The change in anisotropy as a
Oligonucleotide concentrations were determined by absorbance at
260 nm (90 °C for the RNA used in qTR-FRET assays; room
temperature for RNAs used in competition dialysis) using a Beckman
Coutler DU800 UV−vis spectrophotometer equipped with a Peltier
temperature controller unit. Oligonucleotide extinction coefficients
were determined using HyTher version 1.0 (Nicolas Peyret and John
SantaLucia Jr., Wayne State University, Detroit, MI).6364 The
parameters used by HyTher were calculated using information on
the extinction coefficients of nearest neighbors in RNA.65
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function of RNA concentration was fit to a standard one-site saturation
ligand binding equation (eq 2):
formamide in 2X saline−sodium citrate (SSC) buffer (30 mM sodium
citrate, pH 7.0, 300 mM NaCl) for 10 min at room temperature.
The cells were incubated in 1X FISH Buffer (30% formamide, 2X
SSC Buffer, 66 μg/mL bulk yeast tRNA, 2 μg/mL BSA, 2 mM vanadyl
complex (New England Bio Laboratories), and 1 ng/μL DY547-
2′OMe-(CAGCAGCAGCAGCAGCAGC)) for 1.5 h at 37 °C. They
were then washed with 30% formamide in 2X SSC for 30 min at 37
°C, 1X SSC for 30 min at room temperature, and 1X DPBS + 0.1%
Triton X-100 for 5 min at room temperature. Finally, nuclei were
stained by incubating the cells with 1 μg/mL 4′,6-diamidino-2-
phenylindole (DAPI) for 5 min at room temperature. The cells were
washed with 1X DPBS + 0.1% Triton X-100, and 100 μL of 1X DPBS
was added to each well. The cells were imaged using an Olympus
FluoView 1000 confocal microscope at 60× magnification.
Treatment in Mice. All experimental procedures, mouse handling,
and husbandry were completed in accordance with the Association for
Assessment and Accreditation of Laboratory Animal Care. A mouse
model for DM1, HSALR in line 20b,22 was used to investigate if H1
improves splicing defects in animals. HSALR mice express human
skeletal actin RNA with 250 CUG repeats in the 3′UTR. Age- and
gender-matched HSALR mice were injected intraperitoneally with 80
or 100 mg/kg H1 in 5% glucose or 5% glucose alone once per day for
7 days. Mice were sacrificed one day after the last injection, and the
vastus muscle was obtained. RNA was extracted from the vastus tissue,
and cDNA was synthesized as previously described.67 PCR
amplification was carried out for 22−24 cycles with the following
primer pairs: Clcn1 forward 5′-TGAAGGAATACCTCACACT-
CAAGG and reverse 5′-CACGGAACACAAAGGCACTG; Atp2a1
forward 5′-GCTCATGGTCCTCAAGATCTCAC and reverse 5′-
GGGTCAGTGCCTCAGCTTTG. The PCR products were separated
by agarose gel electrophoresis, and the gel was stained with SYBR
Green I (Invitrogen). The gel was scanned with a laser fluorimager
(Typhoon, GE Healthcare), and the products were quantified using
ImageQuant. Differences between two groups were evaluated by a t
test.
B
x
max
+ x
y =
K
(2)
d
where y is the observed anisotropy intensity, Bmax is the maximum
anisotropy observed, x is the concentration of RNA, and Kd is the
dissociation constant.
A Bichromatic Reporter System and Flow Cytometry
Analysis To Assess Bioactivity. C2C12 and HeLa cell lines were
maintained as monolayers in 1X DMEM supplemented with 20% or
10% fetal bovine serum (FBS), respectively. Cells were trypsinized
from the surface and plated in 12-well plates in growth medium. While
the cells were adhering to the surface, they were co-transfected with 25
ng of a plasmid encoding the DM1 mini-gene21 and 250 ng of a
plasmid encoding a modified bichromatic fluorescent reporter of
alternative splicing57 using Transit (Mirus). (The bichromatic
fluorescent reporter contains human MBNL1 exon 5 and adjacent
introns in place of cTNT and its adjacent introns.) The compound of
interest was then added (20 μM). After 48 h, the cells were harvested
by trypsinization. The trypsin was inactivated using growth medium,
and the cells were immediately subjected to flow cytometry analysis
using a BD LSR II.
Improvement of Splicing Defects in a Cell Culture Model
Using RT-PCR. In order to determine if H1 improves splicing defects
in vivo, a previously reported method was employed.38 Briefly, HeLa
cells were grown as monolayers in 12-well plates in growth medium
(1X DMEM, 10% FBS, and 1X GlutaMax (Invitrogen)). After the cells
reached 90−95% confluency, they were transfected with 800 ng of
total plasmid using Lipofectamine 2000 reagent (Invitrogen) per the
manufacturer’s standard protocol. Equal amounts of a plasmid
expressing a DM1 mini-gene with 960 CTG repeats21 and a mini-
gene of interest (cTNT21 or PLEKHH260) were used. Approximately
5 h post-transfection, the transfection cocktail was removed and
replaced with growth medium containing the compound of interest.
After 16−24 h, the cells were trypsinized from the surface, and total
RNA was harvested with a Qiagen RNAEasy kit. An on-column DNA
digestion was completed per the manufacturer’s recommended
protocol.
A sample of RNA was subjected to RT-PCR as previously
described,60 except 5 units of AMV reverse transcriptase from Life
Sciences was used. Approximately 300 ng was reverse transcribed, and
150 ng was subjected to PCR using a radioactively labeled forward
primer. RT-PCR products were observed after 25−30 cycles of 95 °C
for 1 min, 55 °C for 1 min, 72 °C for 2 min, and a final extension at 72
°C for 10 min. The products were separated on a denaturing 5%
polyacrylamide gel and imaged using a Typhoon phosphorimager.
Control experiments were also completed in which HeLa cells were
transfected with a plasmid encoding a mini-gene with five CTG
repeats in the 3′UTR or with a mini-gene that encodes a pre-mRNA
whose splicing is not controlled by MBNL1 (PLEKHH2).60 The effect
of the compound on the splicing of endogenous mRNAs not under
the control of MBNL1 (TTC8 and CAMKK2) was also determined as
previously described.38 Differences in alternative splicing were
evaluated by a t test.
Disruption of Nuclear Foci Using Fluorescence in Situ
Hybridization (FISH).38 HeLa cells were grown as monolayers in
Mat-Tak glass-bottomed, 96-well plates. After the cells reached 90−
95% confluency, they were transfected with 100 ng of a plasmid
encoding a DM1 mini-gene21 using Lipfoectamine 2000 per the
manufacturer’s standard protocol. The transfection cocktail was
removed 5 h post-transfection, and the compound of interest was
added in growth medium.
ASSOCIATED CONTENT
■
S
* Supporting Information
Complete ref 14; complete list of all compounds tested in the
original qTR-FRET screen (Table S-3); results of mass
spectroscopy for all compounds identified from the initial
qTR-FRET screen (Tables S-1 and S-2); HPLC retention times
and analytical HPLC chromatograms for all compounds
identified from the initial qTR-FRET screen (Tables S-1 and
S-2 and Figures S-4−S-20); details of the characterization and
synthesis of H1 (Scheme S1, Figures S-1−S-3); representative
autoradiograms of splicing outcomes as determined by RT-
PCR (Figures S-21−S-24); representative binding curves
(Figure S-25); and assessment of toxicity using flow cytometry
(Figure S-26). This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
disney@scripps.edu
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
After 16−24 h, the cells were washed with 1X Dulbecco’s
phosphate-buffered saline (DPBS) and fixed with 4% paraformalde-
hyde in 1X DPBS for 5 min at room temperature. After being washed
with 1X DPBS, the cells were permeabilized with 1X DPBS + 0.1%
Triton X-100 for 5 min at room temperature. The cells were washed
with 1X DPBS + 0.1% Triton X-100 three times and then with 30%
ACKNOWLEDGMENTS
■
We dedicate this work to Douglas H. Turner on the occasion of
his 65th birthday. We thank Sandro Matosevic for assistance
with confocal microscopy, the URMC Center for RNA Biology,
the Rochester Wellstone Muscular Dystrophy Cooperative
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