article

published online: 1 june 2015 | doi: 10.1038/nchembio.1837

2015 Nature America, Inc. All rights reserved.

SMN2 splice modulators enhance U1pre-mRNA

association and rescue SMA mice
James Palacino1,3, Susanne E Swalley1,3*, Cheng Song1,3, Atwood K Cheung1, Lei Shu1, Xiaolu Zhang1,
Mailin Van Hoosear1, Youngah Shin1, Donovan N Chin1, Caroline Gubser Keller2, Martin Beibel2,
Nicole A Renaud1, Thomas M Smith1, Michael Salcius1, Xiaoying Shi1, Marc Hild1, Rebecca Servais1,
Monish Jain1, Lin Deng1, Caroline Bullock1, Michael McLellan1, Sven Schuierer2, Leo Murphy1,
Marcel J J Blommers2, Cecile Blaustein1, Frada Berenshteyn1, Arnaud Lacoste1, Jason R Thomas1,
Guglielmo Roma2, Gregory A Michaud1, Brian S Tseng1, Jeffery A Porter1, Vic E Myer1,
John A Tallarico1, Lawrence G Hamann1, Daniel Curtis1, Mark C Fishman1, William F Dietrich1,
Natalie A Dales1 & Rajeev Sivasankaran1*
Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene,
represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing
a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that
elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small
nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5 splice site is increased in a sequenceselective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule
mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

MA is a debilitating motor neuron disease with an estimated

worldwide incidence of 1 in 11,000 live births1. SMA is the
most common genetic cause of mortality in children, with
more than 50% of the affected patients dying before the age of two.
The fundamental pathogenesis of SMA results from deficiency
in SMN (survival of motor neuron) protein, which causes death
of -motor neurons in the spinal cord. Humans have two SMN
genes, SMN1 and SMN2, which produce SMN protein. Full-length
SMN (FL-SMN) protein is primarily a product of the SMN1 gene.
Roughly 95% of SMA cases are due to a homozygous deletion of the
SMN1 gene, and the remaining cases are attributed to point mutations in this gene24. The SMN2 gene can partially offset the loss
of SMN1. However, a single nucleotide transition5,6 in the SMN2
gene causes altered splicing and exclusion of exon 7, which in turn
results in only 1020% production of full-length SMN mRNA and
protein. The remaining mRNA encodes a truncated and unstable
protein712. Phenotypically, there is an inverse relationship between
disease severity and SMN2 copy number in SMA patients, leading
to degrees of severity and differences in age of onset ranging
from the most common, infantile onset (type I) form to a rare, adult
(type IV) form13.
Splicing is a robustly conserved mechanism of high-precision
processing of pre-mRNA into a mature mRNA. Proper assembly of
nearly all 213,400 exons in the human genome requires the U1-U2
major spliceosome. The spliceosome acts via a stepwise process
to achieve catalytic ligation of the exons and release of an intron
lariat structure (reviewed in ref. 14). Although the system must
function with precision to ensure proper assembly of transcripts,
there is also dynamic regulation of transcript assembly, with

estimates that ~90% of all multiexon genes are assembled into

more than one splice isoform15. This process is regulated through
the action of a large collection of splice enhancer and suppressor
element cofactors that influence the recruitment of the spliceosome
to alternatively spliced exons (reviewed in ref. 16).
Selective enhancement or alteration of exon splicing could
be therapeutically beneficial in a broad spectrum of genetic disorders, as aberrant splicing is associated with a large percentage of disease-causing mutations (reviewed in ref. 17). For SMA,
efforts to provide therapies have been focused on approaches
to enhance expression of the SMN2 gene or correct aberrant
splicing, for instance, by blocking a suppressor element with
antisense oligonucleotides18,19. Additionally, a recent report
described small molecules that enhance exon 7induced SMN2
levels, both in vitro and in vivo, albeit without providing information detailing the mechanism of action20.
In this report, we describe the discovery and advancement of
a pyridazine class of orally active, small-molecule enhancers of
SMN exon 7 inclusion. These splicing modulators elevate FL-SMN
protein and in turn extend survival in a severe SMA mouse model.
We show that these molecules function via stabilization of the
transient double-strand RNA (dsRNA) structure formed between
the SMN2 pre-mRNA and U1 snRNP complex, a key component
of the spliceosome14,16,21. Furthermore, these compounds act
by increasing the binding affinity of U1 snRNP to the 5 splice
site (5ss) in a sequence-selective manner that is discrete from
constitutive recognition. Specific mutations of this sequence
ablate enhancement of exon inclusion without adversely affecting
baseline splicing efficiency. These studies reveal a new mechanism

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA. 2Novartis Institutes for Biomedical Research, Forum 1, Basel, Switzerland.
These authors contributed equally to this work. *e-mail: susanne.swalley@novartis.com or rajeev.sivasankaran@novartis.com

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Nature chemical biology doi: 10.1038/nchembio.1837

demonstrating the feasibility of using small molecules to attain

sequence-selective splice modulation, opening the possibility of
using similar strategies for other splicing diseases2224.

2015 Nature America, Inc. All rights reserved.

RESULTS
Identification of small-molecule SMN2 splicing modulators

To identify small-molecule modulators of SMN2 exon 7 inclusion,

we used an NSC34 motor neuron cell line expressing an SMN2 minigene reporter for high-throughput screening (HTS). The designed
pair of SMN2-matched reporter constructs indicated either exon 7
inclusion (full-length reporter) or exon 7 exclusion (7 reporter)
based on in-frame luciferase expression (Fig. 1a). A screen of the
Novartis compound library (~1.4 106 compounds) resulted in a
hit rate of <1% (Supplementary Results, Supplementary Table 1).
We selected hits on the basis of complementary changes in forward
and reverse reporter activity, relative to control, that fit the anticipated profile for SMN2 splicing modulators (Fig. 1b). Molecules that
scored positively in the HTS and elicited dose responsiveness were
assessed via qPCR to confirm the desired splicing activity and by
ELISA to confirm increased SMN protein levels in SMN7 mouse
myoblasts, indicating efficacy on the endogenous gene.
One of the scaffolds identified from the HTS efforts contained a pyridazine core group as the core functionality (Fig. 1c).
Compound optimization aimed at maximizing cellular potency
guided by the SMN ELISA assay in both mouse myoblasts and SMA
patient fibroblasts led to the identification of compounds NVS-SM1
(1) and NVS-SM2 (2) as well as inactive analogs NVS-SM3 (3) and
NVS-SM4 (4) (Fig. 1d and Supplementary Fig. 1 a,b). We observed
favorable pharmacokinetics profiles of the two active compounds
in rodents. Both NVS-SM1 and NVS-SM2 showed high plasma
exposure, good bioavailability and, notably, good distribution to the
brain, a primary target tissue (Supplementary Fig. 1c).

Full-length reporter

Exons 16 Exon 8 Luciferase

Compound

Exon 7 Exon 8 Luciferase

Exon 7

Compound

Exons 16

Exons 16

Exons 16 Exon 8 Luciferase

Exon 8 Luciferase

Exon 7

7 reporter

Exon 8 Luciferase

Exons 16

Exon 7 Exon 8 Luciferase

Full-length reporter
7 reporter

5,000
4,000

3,000

linker

2,000
1,000
0

A
9

7
6
5
log[compound]

N
Hit scaffold

NVS-SM1
ELISA EC50: 20 nM

d
N

NH

OH

N
HN

N
NVS-SM2

ELISA EC50: 5 nM

NH

OH

In vivo efficacy of SMN splicing modulators

HN

As NVS-SM1 and NVS-SM2 were suitable in vivo tools, we

sought to explore whether the cellular potency we observed translated to in vivo activity. For this purpose, we used the C/+ SMA
mouse model25. After oral administration, both compounds
produced dose-dependent elevations of SMN2-FL transcript and
SMN protein in brain and spinal cord (Fig. 2a,b), thus establishing
translatability and a pharmacokinetic-pharmacodynamic relationship. Although both compounds showed dose-responsive activity
in the C/+ model (Fig. 2a,b), NVS-SM1 exhibited efficacy at lower
doses and exposures, and thus this analog was studied further
in additional cellular and animal models. NVS-SM1 showed
robust activity across disease-relevant induced pluripotent stem
cell (iPSc)-derived neurons (Supplementary Fig. 2a,b). We also
demonstrated the desired transcript response in SMA-type III
PBMCs after NVS-SM1 treatment, suggesting that this readout
could serve as a peripheral pharmacodynamic marker in the clinic
(Supplementary Fig. 2c).
In the cellular setting, we observed that SMN protein levels
remained elevated after compound treatment, which is in agreement with published findings26 and suggests that SMN protein has
a long half-life. We sought to confirm this finding in the C/+ in vivo
model, as this could affect the design of longer-term efficacy studies.
In fact, a single, 30 mg per kg body weight oral dose of NVS-SM1
resulted in significant (P < 0.05) and durable SMN protein elevation
in brain for up to 160 h (Fig. 2c).
To evaluate the efficacy of NVS-SM1, we used the SMN7
mouse model, which displays a severe phenotype. In the specific
colony used for this study, death typically occurs before postnatal
day 15 (ref. 27). We were pleased to observe that, in addition to a
dose-dependent elevation of SMN protein in the brain (Fig. 2d),
oral administration of NVS-SM1 improved body weight (Fig. 2e)
and extended lifespan, with 50% of the animals in the 1 mg per kg
2

Exons 16

Relative light units

article

NVS-SM3
ELISA EC50: >10 M

NH

OH

NVS-SM4

ELISA EC50: >10 M

N
HO

NH

Figure 1 | Screening paradigm and molecules. (a) Schematic

representation of SMN minigene constructs. Full-length construct (top)
expresses luciferase in frame when exon 7 is included in transcript.
7 construct (bottom) expresses luciferase in frame when exon 7 is
skipped in transcript. Asterisks indicate insertion of C nucleotides to adjust
reading frame for constructs and to ensure that luciferase is in frame
for the appropriate splice variant. (b) Data from example hit from the
high-throughput screen showing elevation of full-length reporter luciferase
signal (black) and a concomitant reduction in 7 reporter luciferase
signal (red). Data represent mean s.e.m. (n = 2). (c) Hit scaffold
structure for pyridazine molecules. (d) Chemical structures of NVS-SM1,
NVS-SM2, NVS-SM3 and NVS-SM4 with SMN ELISA half-maximum
effective concentration (EC50) values.

body weight group and 62% of animals in the 3 mg per kg body

weight group showing increased survival (Fig. 2f).
Next, we evaluated whether NVS-SM1 elicited sustained efficacy
by comparing two additional cohorts; the first cohort continued with
1 mg per kg body weight daily dosing of NVS-SM1 for an additional
two weeks until day 49, whereas the second cohort received only

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Nature chemical biology doi: 10.1038/nchembio.1837

******

10
1

100

****

200

***

160

1,000
100

***

10

120

0.1

0.
1
0.
3

80

0
0.
03

NVS-SM1
(mg per kg
body weight)

20

10
5
0

NVS-SM1 (mg per kg

body weight)

2015 Nature America, Inc. All rights reserved.

3
1
0.3
0.1
0.03
0

****
***
**

15
Weight (g)

240

10

15 20
Age (d)

25

30

1,000

**** ***

120

35

100
10
1

100

0.1
0.01

Time (h)

NVS-SM1 (nM)

SMN protein elevation

NVS-SM1
NVS-SM2
(mg per kg
(mg per kg
body weight) body weight)

SMN protein
Total plasma exposure

**** ****

140

80
NVS-SM1 + + + + +

NVS-SM1
NVS-SM2
(mg per kg
(mg per kg
body weight) body weight)

100
10

0
3
10
30

0
3
10
30

*****
****

0
3
10
30

1,000

SMN protein elevation

100

10

150

***
***

SMN protein
Total plasma exposure

24

48

88

120

0.001

160

NVS-SM1
(mg per kg
body weight)

100

**
*

50

NVS-SM1 (nM)

1,000

10,000

****
****

Compound (nM)

15

200

0
3
10
30

10,000

****

Compound (nM)

Exon 7 inclusion ratio

20

SMN protein
Total plasma exposure

Percentage survival

Exon 7 inclusion/exclusion
Total plasma exposure

SMN protein elevation

3
1
0.3
0.1
0.03
0

10 15 20 25 30 35
Age (d)

Figure 2 | Small molecules modulate SMN levels in vivo. (a,b) Ratio of exon 7 included/excluded SMN2 transcripts (a) and SMN protein increase in
brains of C/+ mice (n = 7) (b). (c) SMN protein increase in brains of C/+ mice (n = 8) dosed orally and killed at indicated time points. (d) SMN protein
increase in SMN7 mice (n = 8). For ad, right (red) axis and red circles represent total plasma exposure of indicated compound, and data represent
mean s.d. Differences relative to vehicle were determined by Students t-test (Online Methods). (e) Body weights of SMN7 mice (n = 12) after
treatment with varying oral doses of NVS-SM1. Data represent mean s.d., and differences relative to vehicle were determined by multiple
Students t-tests (Online Methods). (f) Survival curves of SMN7 mice (n = 8) after treatment with varying oral doses of NVS-SM1. Log-rank
(Mantel-Cox) test was used for pairwise comparisons. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All data presented in this
figure are derived from a single in vivo study.

vehicle on days 3649. Markedly, body weight and survival data for
the two cohorts were indistinguishable (Supplementary Fig. 3).
Thus, NVS-SM1 exerted durable beneficial effects in SMN7
mice after oral administration early in postnatal development. The
sustained effect on survival following withdrawal of NVS-SM1 was
consistent with a recent study28, which demonstrated that early
postnatal induction (SMN transgene expression induced by doxycycline addition) followed by removal of induction at age 28 d
results in long-lasting rescue in the same SMN7 mouse background. Furthermore, an independent study showed that mice
with normal SMN levels from fertilization to age 21 d followed by
reduction of SMN to type 1 SMA levels show no marked phenotype29. These data support that treatment of SMA patients must be
started before large-scale neuronal death has taken place to achieve
pronounced and sustained efficacy.

NVS-SM1 regulates a discrete set of splice variants

Given the potent regulation of SMN2 exon 7 inclusion, we were

interested to learn whether this activity was specific to SMN2 or
whether large-scale changes in gene expression levels, splicing or
both occurred. First, we measured global gene expression changes
by RNAseq triggered by NVS-SM1 or an inactive analog (NVSSM3). For transcript abundance, we considered genes exhibiting an
absolute fold change > 2 with a multiple testing correction (MTC)adjusted P value < 0.05 to be significantly altered in expression.
NVS-SM1 treatment induced changes in the levels of 175 genes,
whereas NVS-SM3 significantly altered expression of 23 genes
(Fig. 3a). Close evaluation of known splicing factors and RNA binding proteins in the data set revealed little to no changes induced
by NVS-SM1. These data suggested that the compound does not
work via changes in transcript abundance and instead works via
alterations in splicing activity.
To explore the direct impact of NVS-SM1 on splicing, we evaluated the RNAseq data for splicing alterations by scanning for both
exons and exon-exon junctions that were differentially regulated

across conditions when compared to overall expression of all genes

using the HG19 RefSeq assembly (Fig. 3b)30. Filtering for transcripts
where the event represented an exon change with a corresponding
change in junction read revealed 39 candidate events where splicing changed in response to NVS-SM1 treatment (Supplementary
Table 2). These 39 events were present in 35 genes, of which only
five (ADAM12, ANXA11, APPL2, RCC1 and SREK1) also exhibited
gene-level changes in expression.
The changes in splicing did not seem to be biased toward either
the 5 or 3 end of transcripts. For example, inclusion of AXIN1 exon 9
and skipping of ATG5 exon 3 were enhanced following NVS-SM1
treatment (Fig. 3c). Furthermore, the expected changes were seen
in the corresponding junctions for the transcripts (Supplementary
Fig. 4). The splicing changes were further validated for both AXIN1
and ATG5 using qPCR probe sets targeting exon-included or
skipped transcripts (Fig. 3d). Of the 39 candidates, we correlated
11 transcript-level events from RNAseq data in the qPCR experiments (Supplementary Fig. 5). On the basis of the limited number
of splicing changes observed, we concluded that NVS-SM1 exhibits
fairly selective splicing modulation.

Sequences within the 5ss correlate with NVS-SM2 activity

The limited number of observed splicing changes suggested that

compound-mediated splicing occurred via interaction with a cis
element common among these genes. The polymorphism in SMN2
that induces exon skipping occurs at the 5 end of exon 7 (refs. 5,6).
We explored this site as a possible cis element using a BRCA1 minigene containing a similar mutation also described to induce exon
skipping31. For this work, we chose to use NVS-SM2 owing to its
excellent in vitro potency. We found that mutant BRCA1 was not
responsive to NVS-SM2 (Supplementary Fig. 6), suggesting that
the molecular mechanism was separable from the SMN2 polymorphism and thus not operating via the cis element common to BRCA1
and SMN2. However, we hypothesized that we could identify the
compound-responsive cis element by making a series of chimerae

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0.6
0.8

1.0
2
4
6
logfold change variation

NVS-SM1

2 3 4 5 6 7 8 9 10 11

0
NVS-SM3

de

2 3 4 5 6 7 8 9 10 11

d
1

****

clu

Exon

**

2
1

NVS-SM3

4
3

de

10

ATG5

In

4
6
8
logfold change variation

****

AXIN1

NVS-SM1

ATG5
0

RQ

de

2.5

AXIN1
2

Exon
log2 relative
fold change

clu

log2 relative
fold change

2.5

2.5
logfold change

RQ

0.6
0.8

1.0
2.5

0.4

clu

5
0

2015 Nature America, Inc. All rights reserved.

0.4

de

10

0.2
FDR P value

15

Atg5 Axin1

0.2
FDR P value

log adjusted P value

20

Atg5 Axin1

In

Up

NVS-SM3 versus DMSO

clu

No change

Ex

Down
NVS-SM1 versus DMSO

Ex

Nature chemical biology doi: 10.1038/nchembio.1837

Figure 3 | Gene- and transcript-level changes in response to NVS-SM1. (a) Volcano plots for changes in gene expression following treatment of human
fibroblasts with NVS-SM1 (left) and NVS-SM3 (right). (b) Exon-level (left) and junction-level (right) changes following treatment of human fibroblasts
with NVS-SM1. The horizontal axis shows variation (log2-fold) in expression between compound and DMSO for all exons and exon-exon junctions of a
given gene. The vertical axis refers to the false discovery rate (FDR)-adjusted 2 test significance (inverted scale). The test is run on a table with counts
for all exons and exon-exon junctions of a given gene following comparison between compound and DMSO. Axin1 (blue) and Atg5 (red) are marked.
(c) AXIN1 and ATG5 exon plots. Top panels (red) show NVS-SM1 treatment. Bottom panels (blue) show NVS-SM3 treatment. Changes shown are
represented as log2-fold increases (up) or decreases (down) in exon reads relative to DMSO. (d) qPCR validation of AXIN1 and ATG5 exon exclusion
or inclusion for DMSO-treated (black bars) and NVS-SM1-treated (gray bars), normalized to DMSO values. RQ, relative quantitation (here, relative to
GusB). Data represent mean values s.e.m. (n = 8, from two independent experiments with four samples each). Subsequent analysis used two-way
ANOVA, followed by the Tukey post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

between SMN2 and BRCA1 (Fig. 4a). A number of chimeric constructs exhibited near-complete exon inclusion or skipping (Chi1
and Chi3, respectively, shown as representative examples). One
chimera, incorporating the 3 end of SMN2 exon 7 and the 5 end
of intron 7 (Chi2), resulted in a dose-dependent exon inclusion
comparable to the full-length SMN2 minigene (Fig. 4b), suggesting
that compound responsiveness required the exon-intron junction.
By progressively deleting sections from the SMN2 sequence, we
discovered that a 21-nt sequence containing only the 5ss and part of
the Tsl2 region was sufficient for compound-dependent splicing
activity (Fig. 4c,d)32.
Prior work reported that destabilization of Tsl2 (Fig. 4e) or
enhancement of U1 snRNP base pairing to the SMN2 exon 7
5ss enhances production of full-length SMN2 (ref. 32), and thus
our observation that the 21-nt sequence covering this region was
sufficient for compound-dependent splicing activity was particularly notable. To dissect this sequence further, we destabilized Tsl2
at nucleotides either within the 5ss or at the converse face of the
stem (Fig. 4e), taking care to select residues that do not alter basal
splicing and to ensure similar base-pairing and thermal stability
(Supplementary Fig. 7)32. Somewhat unexpectedly, we found
that 1AC or 2GC mutations selectively disrupted NVSSM2mediated splicing but not basal splicing of the exon (Fig. 4f).
In contrast, mutations to corresponding residues on the opposite
end of the stem (15CG or 16UA), similar to mutations tested
in another small-molecule splicing modulator report20, did not alter
NVS-SM2mediated splicing (Fig. 4f). These data suggested that
our molecule was acting at the 5ss rather than through a mechanism to destabilize Tsl2. The location of the mutations within the
5ss suggested a mechanism of action consistent with either directly
or indirectly affecting recruitment of U1 snRNP.
4

One question that arose from the chimera data was whether
there are similar motifs, in or around the 5ss, of the NVS-SM1
sensitive splicing events. We addressed this question by performing
a motif enrichment analysis using sequence constraints flanking the
5ss (10 nt to +10 nt) of affected exons in the RNAseq data33,34.
In contrast to the consensus 5ss motif from RefSeq data (HG19;
Fig. 4g), NVS-SM1responsive exons were enriched for an nGA
motif at the exon portion of the predicted 5ss junctions (Fig. 4g),
a motif represented in only 2.6% of the 213,400 exons annotated
within RefSeq. The presence of an enriched sequence motif directly
at the 5ss again suggested a possible role for U1 snRNP. We could
not rule out an as-yet-uncharacterized cis suppressor element;
however, in silico analysis showed no common suppressor binding
sites in the affected exon regions35. Further analysis by RNA immunoprecipitation, using SMN2 and BRCA1 as NVS-SM2responsive
and nonresponsive sequences, respectively, failed to demonstrate
any sequence-selective enrichment of suppressor proteins.
Although the RNAseq data revealed several exon inclusion
events, we identified a small subset of events where NVS-SM1 triggered enhanced exon skipping (Supplementary Table 2). These
skipping events brought into question the validity of the U1 target
hypothesis. We observed that one of the skipped exons, ATG5 exon
3, does not have an nGA motif at the 5ss but does have a predicted
nGA-like U1 5ss sequence less than 30 nt from the 3ss (Fig. 4h and
Supplementary Fig. 8). This particular sequence could compete for
the U1 site recognition on the basis of similar in silico predictions
for U1 affinity36. To determine whether exon skipping was mediated
by this nGA-like internal 5ss sequence, we made ATG5 minigenes
containing either a wild-type or a 1C mutated sequence, analogous
to the compound-insensitive SMN2 sequence. The mutation blocked
the dose-dependent enhancement of exon skipping (Fig. 4i), which

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Chi2
Exon 17

Exon 19

Exon 7

Chi3
Exon 17

Exon 19

Exon 18

40
20

21 nt
Intron 7

auuccu UAAAUCUAAGGA GUAAGUCUG

5 ss

60

15G
16A

A
U
U
C
C
U
U
A
5

U
A
A
G
G
A
G
U
A

Tsl2

50

+
+

NVS-SM2

GGAGUAAGUC
U
G
A
GUCCAUUCAU

23

40

20
0.01
0.1
NVS-SM2 (M)

7.2
C20

7.6

U22

U5

SMN2
SMN2(1C)
SMN2(2C)
SMN2(15G)
SMN2(16A)

80
60
40
20
0.01
0.1
NVS-SM2 (M)

G U
C U
3

U1 binding site
Exon 2

Exon 3

E
Exon 4

5 ss

ATG5-WT:

AGA GUAAGUUA

ATG5(1C): AGC GUAAGUUA

Percentage exon
inclusion

100
RNA-seq enriched
3 2 1 1 2 3 4 5 6 7

1C

7.8

150
100
50
0
2C
150
100
50

0
0.001

Genome wide
3 2 1 1 2 3 4 5 6 7

NVS-SM4

C21

0
0.001

+
+

100

2C
1C

7.4

f
Percentage exon
inclusion

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A U

100

Compound

80

Tsl2

150

40

RNA

WT

80

f1 (p.p.m.)

Exon 7

100

Percentage exon
inclusion

Exon 19

0.01
0.1
NVS-SM2 (M)

120

0
U1 snRNP

0
0.001

d
Exon18

Chi1
Chi2
Chi3

60

Exon 19

c
Exon 17

80

Percentage exon
inclusion

Exon 7

100

Response (RU)

Chi1
Exon 6

Compound
detected (nM)

80
60
40
20
0

0.01

0.1
1
NVS-SM2 (M)

10

Figure 4 | NVS-SM2 sequence selectively alters exon splicing.

(a) Schematic of SMN2 (blue)BRCA1 (white) chimerae. (b) RT-PCR for
chimerae. (c) Schematic of 21-nt fragment (blue) of SMN2 sequence,
with Tsl2 domain (gray bar) and 5ss (white bar) shown below.
(d) RT-PCR for 21-nt SMN2 chimera. (e) Schematic of the Tsl2 stem-loop.
Mutated nucleotides are shown with arrows, and numbers represent
distance from the 5ss based on the minigene sequence. (f) RT-PCR for
Tsl2 mutations. (g) Schematic of 5ss from exons in HG19 (top) and 35
events from RNAseq data (bottom). (h) Schematic of ATG5 illustrating the
cryptic U1 binding site within exon 3. (i) RT-PCR for ATG5-WT (wild type)
and ATG5(1C) minigenes, with the predicted U1 site and the 1AC
mutation shown. Graphs show mean values s.e.m. (n = 3, representative
of three independent experiments). Schematics are not drawn to scale.

would be unlikely if the mechanism involved competition between

U1 snRNP and a splice suppressor protein. This finding provided
further support for the hypothesis that the molecules act via a sequenceselective interaction with the U1 snRNPpre-mRNA complex.

NVS-SM2 stabilizes the U1 snRNP5ss RNA complex

To establish whether compound activity is directly mediated via an

interaction with the U1 snRNPpre-mRNA complex, we enriched

8.0
5.8

5.6

5.4
5.2
f2 (p.p.m.)

5.0

0
0

100 200 300 400 500

Time (s)

Figure 5 | NVS-SM2 binds to and stabilizes the U1 snRNP:5ss RNA

complex. (a) Binding of NVS-SM2 and NVS-SM4 to RNA, U1 snRNP or the
U1 snRNPRNA complex, as measured by size exclusion chromatography.
Data represent mean values s.e.m. for two technical replicates.
(b) SPR showing specific effect of 1 M NVS-SM2 (blue) versus DMSO
(fuchsia) on U1 snRNP binding to three 5ss RNAs (mutations as in
Fig. 3). Representative sensorgrams are shown (n = 2). WT, wild type;
RU, resonance units. (c) TOCSY spectra of RNA-23 (red) and NVS-SM2
bound RNA-23 (black). Discrete spectral peaks are seen for spin-coupled
C5-C6 protons on C and U residues. Schematic of RNA-23 illustrates base
pairing and residues exhibiting TOCSY resonance shifts (yellow boxes)
with NVS-SM2. The SMN2 RNA sequence is shown in blue, and the
U1 snRNA sequence is shown in purple.

U1 snRNP from compound-responsive, splicing-competent HeLa

nuclear extracts (Supplementary Fig. 9). Using size-exclusion
chromatography with MS detection37, we found that NVS-SM2
bound only U1 snRNP in the presence of SMN2 exon 7 5ss RNA
and did not bind either component alone (Fig. 5a). In contrast, an
inactive analog, NVS-SM4, did not bind any components in combination or alone. To understand this binding in greater depth, we
conducted surface plasmon resonance (SPR) studies, performed
in screening mode owing to the uncertain concentration of the
enriched U1 snRNP, which showed that NVS-SM2 enhanced the
association of U1 snRNP to the SMN2 RNA, whereas NVS-SM4
had no effect (Supplementary Fig. 10). When 1 M NVS-SM2
was present in both kinetic phases, we observed a marked slowing
of U1 snRNP dissociation from the SMN2 5ss sequence (approximately fourfold), with no observable effect on U1 snRNP binding to
either 1AC or 2GC mutant sequences (Fig. 5b). These data
supported our hypothesis that the compounds act by stabilizing
the interaction of U1 snRNP with the SMN2 pre-mRNA in a
sequence-selective manner.
As NVS-SM2 bound neither the U1 snRNP nor 5ss RNA alone,
we postulated that the compound acts at the snRNApre-mRNA
interface. To test this hypothesis, we performed one-dimensional
1
H NMR with NVS-SM2 or NVS-SM4 and observed chemical shift
changes as well as resonance line broadening in only the NVS-SM2
spectra upon addition of dsRNA (Supplementary Fig. 11). To
determine which residues interact with NVS-SM2, we performed
TOCSY NMR spectroscopy using dsRNA and detected shifts in the
spin resonances of U5, C20, C21 and U22, all of which are residues
proximal to the nGA motif (Fig. 5c). As we observed binding to

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article

Nature chemical biology doi: 10.1038/nchembio.1837

compounds NVS-SM1 and NVS-SM2 bind along the major groove
of the dsRNA between the U1 snRNA and the 5ss in the region
proximal to the nGA motif. Next, we overlaid the NVS-SM2U1
RNASMN2 RNA model with a recently published 3.3- crystal
structure of U1 snRNP38 and observed a good overall fit, thus giving
increased confidence in our computational model (Fig. 6a).

DISCUSSION

b
SMN1
Exon 6

Exon 7

Exon 8

Exon 7

Exon 8

Exon 7

Exon 8

2015 Nature America, Inc. All rights reserved.

C
SMN2
Exon 6
T

SMN2
Exon 6
U1 snRNP

?
T

U2 snRNP
Compound

U1 snRNP complex

CAUUCAUApppG

SMN2 exon 7

SMN2 intron 7

Figure 6 | Computational model and schematic of mechanism of action.

(a) Computational model illustrating the binding mode of NVS-SM2
with U1 snRNPSMN pre-mRNA superimposed on the published
U1 snRNP crystal structure. The SMN2 RNA sequence is shown in green,
the U1 snRNA sequence is shown in gold, the RNA-contacting part of
the U1C protein is shown in red, and the compound is shown in turquoise.
(b) Schematic model of mechanism of action. SMN1 and SMN2 transcripts
are shown with splicing occurring through recruitment of U1 and U2 snRNP
complexes flanking the exons.

RNA at high concentrations in NMR, we attempted to evaluate the

effect of NVS-SM2 on the transient dsRNA structure that forms
between U1 and the 5ss of SMN2 exon 7 by SPR. We were able
to test up to 10 M NVS-SM2 and observed no effect on either
association or dissociation kinetics of the U1 RNA5ss RNA
interaction (Supplementary Fig. 12a). Although NVS-SM2 interacts with dsRNA, as observed by NMR, our potency and selectivity
data suggest that U1 protein components are required for full
biological activity.
Building upon the chimera and mutation studies, biophysical
data and insights from NMR, we sought to generate a computational
model. Several feasible RNA models were generated, and incorporation of SMN cellular activity allowed us to identify a preferred
model of the binding mode that was consistent with compound
activities (Supplementary Fig. 12b). According to the model, active
6

SMA is a devastating disorder with no approved therapies. Recently,

the field has been bolstered by encouraging preclinical and clinical data from a number of research groups20,39. Potential therapies
under clinical evaluation include SMN1 gene therapy, an intra
thecally delivered antisense oligonucleotidebased therapy39 and a
small-molecule modulator of SMN2 exon 7 inclusion20 similar to
the one we describe here.
To our knowledge, the work described here represents the first
mechanistic elucidation of sequence-selective small-molecule
splicing modulators. Previous reports detail oligonucleotide-based
approaches with a known mechanism of action; however, these
therapies are known to have delivery challenges39. Our results offer
new insights into selective SMN2 splicing modulation, and we demonstrate sustained in vivo efficacy in a severe preclinical model of
SMA with NVS-SM1, an orally bioavailable molecule that is currently in a clinical trial for treatment of type I SMA (ClinicalTrials.
gov code NCT02268552).
In our proposed mechanism, small-molecule SMN2 splicing
modulators act by enhancing the binding of U1 snRNP to the SMN2
exon 7 5ss. Analogous to the pladienolides, which disrupt SF3B1 in
a global context40, a nonspecific alteration in binding of U1 snRNP
would most likely alter splicing in a global fashion. This alteration
was not observed in our RNAseq analysis. In fact, we detected a
discrete subset of altered genes and exons, and we attribute the
observed sequence selectivity to the rarity of the target nGA motif.
Our cell biology and biophysical data point to a mechanism by
which our compounds stabilize the interaction between U1 snRNP
and the SMN exon 7 5ss, specifically at this motif. Combining our
data with a recently published high-resolution crystal structure
of U1 snRNP38, we propose a binding mode whereby compound
binding occurs in the major groove proximal to the nGA site and
in turn stabilizes the interaction of the U1 snRNP with the 5ss
RNA (Fig. 6a). A key feature in the crystal structure is that the U1C
protein supports the dsRNA interaction along the minor groove,
illustrating the role of the U1C protein. On the basis of our model, we
hypothesize that the active compounds enhance the stabilization of
the duplex in the 1A bulge major groove in the presence of the U1C
protein, consistent with our biophysical data. In the crystal structure, we noted a buffer component molecule (2-[4-(2-hydroxyethyl)
piperazin-1-yl]ethanesulfonic acid) bound proximal to the interface
of the snRNA and the 5ss RNA.
We present a schematic representation (Fig. 6b) whereby compound action leads to enhanced splicing of the exon 7exon 8 junction. In the absence of compound, SMN2 transcripts are poorly
recognized at exon 7 by U1-U2, resulting in a high frequency of
exon skipping. NVS-SM1 and NVS-SM2 enhance affinity of U1 at
the 5ss of SMN exon 7, facilitating inclusion of exon 7. This model
is consistent with previous literature reports, demonstrating that
improved use of the exon 7 5ss through either SMN2 sequence
mutations or modified U1 snRNA resulted in enhanced production
of FL-SMN32. The work presented here highlights unique opportunities to use small molecules for selectively targeting splicing disorders previously considered accessible only by protein-based and
oligonucleotide-based approaches while offering the bioavailability
and delivery advantages of small molecules.
Received 5 December 2014; accepted 6 May 2015;
published online 1 June 2015

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Nature chemical biology doi: 10.1038/nchembio.1837

Methods

Methods and any associated references are available in the online

version of the paper.
Accession codes. NCBI Short Read Archive: The raw RNA sequencing reads are available under accession code SRP055454.

2015 Nature America, Inc. All rights reserved.

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Acknowledgments

The authors wish to acknowledge members of the Novartis Institutes for BioMedical
Research (NIBR) Leadership Spinal Muscular Atrophy Advisory Board (J. Hastewell,
J. Bell, K. Briner, P. Bouchard, E. Beckman and G. Kwei), NIBR Project Management
(D. Silva and C. Gauthier) and NIBR Translational Medicine (R. Roubenoff) for their
advice and contributions to the drug discovery efforts; J.R. Kerrigan, D. Glass, P. Manos,
F. Harbinski, C. Mickanin, R.E.J. Beckwith, R. Sun, W. Broom, S.J. Luchanksy, L. Murphy,
M. Schirle, J. Duca, R. Chopra and K. Clark for their contributions to experimental
efforts and insights on the manuscript; S.J. Burden (NYU School of Medicine) for his
gift of SMN7 mouse myoblasts; K. Mineev and A.S. Arseniev for their assistance
with NMR peak assignments; A. Abrams for his artwork in the schematic diagram;
the SMA Foundation (K. Chen, D. Kobayashi, S. Paushkin and L. Eng) for their advice
and contributions to the drug discovery efforts; Psychogenics (S. Ramboz and K. Cirillo);
and PharmOptima (D. Decker, R. Poorman and P. Zaworski) for their contributions
to in vivo studies.

Author contributions

C.S., T.M.S. and R.S. performed the high-throughput screen. A.K.C., L.S., L.G.H. and
N.A.D. performed the chemical synthesis. C.S., M.V.H., Y.S., C. Blaustein, F.B., A.L. and
R.S. performed cell-based structure-activity experiments. M.V.H., Y.S., R.S., M.J., L.D.,
C. Bullock, M.M., W.F.D. and R.S. performed in vivo experiments. J.P., C.G.K., M.B.,
N.A.R., X.S., M.H., S.S., L.M., G.R. and R.S. performed the RNAseq experiments.
J.P. and C.S. performed the chimera experiments. S.E.S., M.S. and J.R.T. performed
the biochemistry and biophysics experiments. X.Z. and M.J.J.B. performed the NMR
experiments. D.N.C. performed the computational modeling. L.G.H. and N.A.D.
supervised the medicinal chemistry experiments. M.J., B.S.T., W.F.D. and R.S. provided
intellectual input to the in vivo mouse biology experiments. B.S.T., J.A.P., D.C., M.C.F.
and R.S. provided intellectual input to the overall drug discovery studies. G.A.M., J.A.P.,
V.E.M. and J.A.T. provided intellectual input to the mechanism of action studies. N.A.D.
and R.S. directed the drug discovery experiments. J.P. and S.E.S. directed the mechanismof-action experiments. J.P., S.E.S., J.A.T., N.A.D. and R.S. prepared the manuscript.

Competing financial interests

The authors declare competing financial interests: details accompany the online version
of the paper.

Additional information

Supplementary information, chemical compound information and chemical probe table

is available in the online version of the paper. Reprints and permissions information is
available online at http://www.nature.com/reprints/index.html. Correspondence and
requests for materials should be addressed to S.E.S. or R.S.

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ONLINE METHODS

2015 Nature America, Inc. All rights reserved.

Minigene constructs. The SMN2 minigene construct was constructed via

overlapping PCR according to previous methods41. BRCA1 mutant and
wild-type minigenes as well as BRCA1-SMN2 chimeric constructs were
constructed similarly. The fragment of the 3 end of intron 18 and exon 19
were fused with Luciferase cDNA by overlapping PCR and cloned into
pcDNA3.1(+) at BamHI and NotI sites. PCR primers: hBRCA1 intron 18 fw: 5-CG
GGATCCAATCGCTGACCTCTCTATCT-3; hBRCA1 exon 19 rs: 5-ctcattcag
catttttctttctttaatagac-3; hBRCA1 exon 19-luc fw: 5-gtctattaaagaaagaaaaatgc
tgaatgagTGGAAGACGCCAAAAACATAAAG-3; Luc rs: 5-ATAAGAATG
CGGCCGCttacacggcgatctttccg-3. The 5 fragments of BRCA1 mutant/wild
type chierae and all chimerae containing the first two exons and introns were
synthesized by GeneArt (Invitrogen). The 5 fragments then were ligated to
the 3 fragment fused with firefly luciferase at the BamHI site. The resulting
minigene constructs were sequence confirmed.
Cell culture, transfection and RT-PCR assay. Human fibroblasts from SMA
patient (151) and NSC34 cells were cultured in DMEM with 10% FBS. For
transfection with minigene constructs, NSC34 cells were plated in 12-well
plates at 4 105 cells per well and transfected using Lipofectamine 2000
according to the manufacturers protocol (Life Technologies). Cells were then
plated to 96-well plates 5 h after transfection. At 24 h after transfection, cells
were treated with compounds for 24 h, as indicated in figure legends (Figs. 1
and 4 and Supplementary Fig. 6). Cells then were lysed in Cells-to-CT lysis
buffer (Life Technologies) according to manufacturer protocol. 1.5 l lysate
was used for cDNA synthesis in a 10-l reaction, using Cell-to-CT 2RT reagent (Life Technologies) at 37 C for 60 min and then 95 C for 5 min. 1 l of
the cDNA was then used in a 20-l PCR reaction using Takara hot start Ex
Taq polymerase (Clontech) with corresponding primers specific for each gene
or minigene. PCR cycles: 2 min at 94 C, then 30 s at 94 C, 30 s at 60 C and
30 s at 72 C for 35 cycles, followed by 10 min at 72 C. DNA fragments representing exon included and/or excluded forms from the PCR products were
separated and quantified using Caliper LabChip GX II (PerkinElmer) with
HT DNA extended Range LabChip (Part number: 760517). Percentage exon
inclusion was calculated to represent the percentage of exon included form
in total mRNA transcript (exon included plus exon excluded).
Primary high-throughput screen. The HTS was run on a HTS-3 automated
system (Thermo). The entire Novartis deck of compounds was tested as
singlicates in the SMN RGA assay at a final concentration of 10 M. The active
control on the plate, 8 M of resveratrol, was used to define the +100% control.
The neutral control, 0.45% DMSO, was defined as 0%. About 130 1,536-well
plates were processed each day, which allowed for the entire screen to be
finished in 2 weeks. The average Z values and average RZ for the primary
screen were 0.59 and 0.71, respectively. With a cutoff of >50% activity
relative to resveratrol active control, with the exception of the PURE collection
(hits selected at > 70%), the overall hit rate was < 1.0%.
Compound synthesis. NVS-SM1 and NVS-SM2 were prepared as described
in US Patent 8,729,263 (May 2014), publication number WO2014028459
(ref. 42). Analytical data for NVS-SM1 and NVS-SM2 are shown below.
Synthesis and analytical data for NVS-SM3 and NVS-SM4 are shown below. All
materials used the studies were >99% pure, as assessed by analytical methods
including NMR, HPLC and LC/MS.
NVS-SM1: 5-(1H-pyrazol-4-yl)-2-(6-((2,2,6,6-tetramethylpiperidin-4-yl)oxy)
pyridazin-3-yl)phenol. 1H NMR (400 MHz, DMSO-d6) 9.52 (d, J = 12.0 Hz,
1H), 8.69 (d, J = 12.0 Hz, 1H), 8.51 (d, J = 9.6 Hz, 1H), 8.23 (s, 2H), 7.95 (d, J = 8.2
Hz, 1H), 7.50 (d, J = 9.5 Hz, 1H), 7.32 7.23 (m, 2H), 5.76 5.63 (m, 1H), 2.31
(dd, J = 13.2, 4.0 Hz, 2H), 1.93 1.82 (m, 2H), 1.54 (d, J = 2.6 Hz, 12H). 13C NMR
(101 MHz, DMSO) 162.03, 158.09, 155.79, 135.77, 131.01, 128.83, 127.80,
120.16, 120.02, 116.07, 114.80, 113.11, 67.75, 56.32, 28.85, 24.70 (Supplementary
Fig. 13). HRMS (ESI, M+H) calculated for C22H28N5O2 394.2238, found 394.2243.
Purity (Method A): Tr = 2.36 min, 100% at 214 nm and 254 nm.
NVS-SM2: 2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin3-yl)-5-(1H-pyrazol-4-yl)phenol. 1H NMR (400 MHz, DMSO-d6) 13.82
(s, 1H), 12.98 (s, 1H), 8.21 (d, J = 10.0 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.35
(d, J = 9.9 Hz, 1H), 7.24 7.15 (m, 2H), 5.01 4.90 (m, 1H), 2.95 (s, 3H),
1.58 1.39 (m, 4H), 1.27 (s, 6H), 1.10 (s, 6H). 13C NMR (101 MHz, DMSO)
nature chemical biology

158.49, 157.61, 151.04, 134.86, 126.27, 125.21, 120.63, 115.96, 115.29, 114.94,
113.40, 47.36, 40.55, 34.46, 28.97, 28.51 (Supplementary Fig. 14). HRMS
(ESI, M+H) calculated for C23H31N6O 407.2554, found 407.2540. Purity
(Method A): Tr = 1.38 min, 100% at 214 nm and 254 nm.
Synthesis of NVS-SM3: 4-((1H-imidazol-1-yl)methyl)-2-(6-(methyl(2,2,6,6tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol. Step 1: 1-(3-bromo4-methoxybenzyl)-1H-imidazole. A mixture of 2-bromo-4-(chloromethyl)-1methoxybenzene (0.5 g, 2.12 mmol) and imidazole (0.43 g, 6.40 mmol, 3 eq)
in acetonitrile (4.2 ml) was heated at 80 C overnight. After cooling to room
temperature, the mixture was concentrated in vacuo. The resulting solid was
taken up in dichloromethane, washed with aqueous saturated Na2CO3, dried
over Na2SO4 and concentrated to afford crude 1-(3-bromo-4-methoxybenzyl)1H-imidazole. This material was taken on without purification.
Step 2: (5-((1H-imidazol-1-yl)methyl)-2-methoxyphenyl)boronic acid. A mixture of 1-(3-bromo-4-methoxybenzyl)-1H-imidazole (567 mg, 2.12 mmol),
bis(pinacolato)diboron (809 mg, 3.18 mmol, 1.5 eq), potassium acetate (667 mg,
6.79 mmol, 3.2 eq), 1,1-bis(diphenylphosphino)ferrocene (118 mg, 0.212 mmol,
10 mol%) and PdCl2(dppf) (155 mg, 0.212 mmol, 10 mol%) in dioxane (10.6 ml)
was heated at 80 C for 2 d. To the mixture was added bis(pinacolato)diboron (809 mg, 3.18 mmol, 1.5eq), potassium acetate (667 mg, 6.79 mmol,
3.2 eq), 1,1-bis(diphenylphosphino)ferrocene (118 mg, 0.212 mmol, 10 mol%)
and PdCl2(dppf) (155 mg, 0.212 mmol, 10 mol%) with continued heating at
80 C overnight. After cooling to room temperature, the suspension was
diluted with ethyl acetate, filtered through Celite and concentrated. The crude
material was taken up in methanol and loaded onto 10G Bond Elute SCX-BSA
resin (Agilent). The resin was washed with methanol, and then the product
was eluted with 2N NH3 in methanol and concentrated in vacuo to afford
crude product (5-((1H-imidazol-1-yl)methyl)-2-methoxyphenyl)boronic acid,
which was taken on without further purification.
Step 3: 6-(5-((1H-imidazol-1-yl)methyl)-2-methoxyphenyl)-N-methyl-N(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine. A mixture of 6-chloroN-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine (250 mg,
0.88 mmol), (5-((1H-imidazol-1-yl)methyl)-2-methoxyphenyl)boronic acid
(308 mg, 1.33 mmol, 1.5 eq), sodium carbonate (281 mg, 2.65 mmol, 3.0 eq)
and PdCl2(dppf).CH2Cl2 adduct (72 mg, 0.088 mmol, 10 mol%) in dimethoxyethane (3.3 ml) and water (1.1 ml) was degassed with nitrogen for 5 min
and then heated at 80 C overnight. After cooling to room temperature, the
mixture was diluted with methanol and filtered through Celite. The filtrate
was acidified with acetic acid and concentrated, then taken up in methanol
and loaded onto 5G Bond Elute SCX-BSA resin (Agilent). The resin was
washed with methanol, and the product was eluted with 2N NH3 in methanol
and concentrated in vacuo to afford crude product 6-(5-((1H-imidazol-1-yl)
methyl)-2-methoxyphenyl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)
pyridazin-3-amine, which was taken on without further purification.
Step 4. 4-((1H-imidazol-1-yl)methyl)-2-(6-(methyl(2,2,6,6-tetramethylpiperidin4-yl)amino)pyridazin-3-yl)phenol. To a mixture of 6-(5-((1H-imidazol-1-yl)
methyl)-2-methoxyphenyl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)
pyridazin-3-amine (350 mg, 0.81 mmol) in dichloromethane (2.7 ml) at 78 C
was added 1 M boron tribromide in dichloromethane (2.0 ml, 2.01 mmol,
2.5 eq). The suspension was removed from the bath and stirred for 2 h, then
cooled to 0 C, quenched with excess methanol and concentrated in vacuo. The
resulting solid was purified by HPLC to afford 4-((1H-imidazol-1-yl)methyl)2-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol
(51.7 mg, 0.122 mmol, 15%). 1H NMR (400 MHz, methanol-d4) p.p.m. 8.09
(d, J = 10.04 Hz, 1H), 7.78 (s, 1H), 7.75 (d, J = 2.01 Hz, 1H), 7.32 (d, J = 10.04
Hz, 1H), 7.21 (dd, J = 8.41, 2.13 Hz, 1H), 7.16 (t, J = 1.25 Hz, 1H), 7.01 6.92
(m, 2H), 5.20 (s, 2H), 5.17 5.05 (m, 1H), 3.02 (s, 3 H), 1.77 1.65 (m, 2H)
1.66 1.52 (m, 2H) 1.40 (s, 6H) 1.25 (s, 6H). 13C NMR (101 MHz, MeOD)
159.49, 159.45, 152.34, 138.40, 131.07, 129.30, 128.75, 126.71, 126.66, 120.71,
119.44, 119.29, 116.08, 53.06, 51.31, 41.84, 34.19, 29.74, 27.91 (Supplementary
Fig. 15). HRMS (ESI, M+H) calculated for C24H33N6O 421.2710, found
421.2705. Purity (Method A): Tr = 2.30 min, 100% at 214 nm and 254 nm.
Synthesis of NVS-SM4: 3-fluoro-4-(6-(methyl(2,2,6,6-tetramethylpiperidin4-yl)amino)pyridazin-3-yl)phenol. Step 1: 6-(4-(benzyloxy)-2-fluorophenyl)N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine.
6-chloro-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine
(350 mg, 1.238 mmol), (4-(benzyloxy)-2-fluorophenyl)boronic acid (487 mg,
1.980 mmol), Na2CO3 (328 mg, 3.09 mmol), and Pd(Ph3P)4 (143 mg, 0.124 mmol)
doi:10.1038/nchembio.1837

 2015 Nature America, Inc. All rights reserved.

combined in dimethoxyethane (Volume: 4950 L, Ratio: 4) and water

(Volume: 1238 L, Ratio: 1.000). The reaction mixture was degassed via 3x
vacuum/purge with N2 (g), then heated in microwave reactor at 100 C for 3 h.
LC-MS showed that reaction was complete. The mixture was filtered through
a Celite column, washed with methanol, and concentrated in vacuo. The crude
was diluted with dichloromethane/methanol acidified to ~pH 4, loaded onto
10G Bond Elute SCX-BSA (Agilent), and washed with methanol; then the
product was eluted with 2N NH3 in methanol. The compound was of sufficient
purity and taken on to the next reaction without additional purification. The
reaction provided the title compound as a white solid (540 mg, 1.204 mmol,
97% yield). 1H NMR (400 MHz, Chloroform-d) 8.09 (t, J = 9.0 Hz, 1H), 7.66
(dd, J = 9.6, 2.2 Hz, 1H), 7.49 7.30 (m, 5H), 6.90 (dd, J = 8.8, 2.5 Hz, 1H), 6.82
(d, J = 9.6 Hz, 1H), 6.76 (dd, J = 13.1, 2.5 Hz, 1H), 5.21 5.08 (m, 3H), 2.98
(s, 3H), 1.71 (dd, J = 12.5, 3.3 Hz, 2H), 1.36 (s, 8H), 1.19 (s, 6H). 13C NMR
(101 MHz, CDCl3) Carbon fluorine coupling observed. 162.01, 160.19,
160.08, 159.54, 158.22, 146.75, 146.73, 136.28, 130.62, 130.57, 128.67, 128.24,
128.19, 128.14, 127.51, 117.99, 117.87, 111.30, 111.27, 111.13, 102.91, 102.64,
70.32, 51.48, 47.45, 41.75, 35.17, 29.17, 28.65. HRMS (ESI, M+H) calculated
for C27H34FN4O 449.2711, found 449.2676. Purity (Method A): Tr = 2.59 min,
100% at 214 nm and 254 nm.
Step 2: 3-fluoro-4-(6-(methyl(2,2,6,6-tetramethylpiperidin-4-yl)amino)pyridazin-3-yl)phenol. In a 40 ml vial, 30% Palladium on carbon (71.2 mg, 0.067
mmol) was added to a solution of 6-(4-(benzyloxy)-2-fluorophenyl)-N-methyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)pyridazin-3-amine (300 mg, 0.669
mmol) in ethyl acetate (Volume: 3344 L, Ratio: 1.000)/methanol (Volume:
3344 L, Ratio: 1.000) at room temperature. The vial was equipped with a rubber septa and then H2 was bubbled into solution for 5 min. The reaction mixture was then stirred under H2 atmosphere (balloon) for 4 h. LC-MS showed
that the reaction was complete. Celite was added, and the mixture was filtered
through a Celite plug, carefully washing with ethyl acetate and dichloromethane without allowing the filter cake to dry. The filtrate was concentrated
in vacuo. The residue was purified by prep-HPLC (Gradient 1540% acetonitrile,
3.5 min gradient on X-Bridge 30 x 50 mm 5 m column acetonitrile/H2O
w/ 5 mM NH4OH 75 ml/min). The product fractions were partially concentrated, and residue was placed on a freeze dryer. The residue was placed in
a drying oven to provide the title compound (117 mg, 0.326 mmol, 48.8%
yield) as a slightly yellow solid. 1H NMR (400 MHz, Methanol-d4) 7.70
7.57 (m, 2H), 7.09 (d, J = 9.6 Hz, 1H), 6.70 (dd, J = 8.6, 2.4 Hz, 1H), 6.58
(dd, J = 13.3, 2.3 Hz, 1H), 5.28 5.15 (m, 1H), 2.95 (s, 3H), 1.69 (dd, J = 12.8,
3.6 Hz, 2H), 1.57 (t, J = 12.5 Hz, 2H), 1.38 (s, 6H), 1.24 (s, 6H). 13C NMR
(101 MHz, MeOD) Carbon - fluorine coupling observed. 163.63, 162.66,
162.54, 161.17, 159.70, 148.90, 148.88, 131.54, 131.49, 130.33, 130.26, 116.44,
116.32, 113.95, 113.71, 113.68, 104.47, 104.23, 53.70, 48.33, 41.68, 33.92, 29.69,
27.71 (Supplementary Fig. 16). HRMS (ESI, M+H) calculated for C20H28FN4O
359.2241, found 359.2206. Purity (Method A): Tr = 1.02 min, 100% at
214 nm and 254 nm.
Transcript analysis. Type I SMA fibroblast cells (Coriell) were seeded at
10,000 cells per well in 96 well microtiter plate in 100 l of culture medium
(complete DMEM, Life Technologies) and incubated overnight. Cells were
treated with either 100 nM NVS-SM1 or 5 M NVS-SM3 at a final concentration of 0.1% DMSO for 24 h. Total RNA was isolated as per Turbo 96 RNEasy
Plus kit instructions (Qiagen). Reverse transcription was performed as per
High Capacity cDNA kit instructions (Life Technologies). Human SMN2
transcript was quantified by TaqMan real-time PCR (Life Technologies).
Human SMN2 exon 7 exclusion was quantified using forward primer 5-CA
TGGTACATGAGTGGCTATCATACTG-3, reverse primer 5-TGGTGTCA
TTTAGTGCTGCTCTATG-3 and FAM-MGB-labeled probe 5-FAM-CCAGC
ATTTCCATATAATAGC-3 (Life Technologies). Human SMN2 exon 7 inclusion was quantified using forward primer 5-CAAAAAGAAGGAAGGTG
CTCACATT-3, reverse primer 5-GTGTCATTTAGTGCTGCTCTATGC-3
and FAM-MGBlabeled probe 5-FAM-CAGCATTTCTCCTTAATTTA-3
(Life Technologies). SMN transcripts were normalized to human GusB endogenous control (Life Technologies, Hs00939627_m1), and compound-induced
fold changes were analyzed relative to DMSO treatment.
Protein analysis. SMN protein analysis was performed as per SMN ELISA
(Enzo Life Sciences, ADI-900-209) kit instructions. Plate was read on a
Paradigm plate reader (Molecular Devices), and 4-parameter logistic data
analysis was performed by SoftMax Pro 6 software.
doi:10.1038/nchembio.1837

Generation of hiPSC lines and hiPSC-derived neurons. Dermal fibroblasts from

healthy donors and patients affected by SMA were obtained from Coriell Cell
Repositories (GM00232 and GM09677). HiPSC lines were generated by expressing reprogramming factors Oct4, Klf4, Sox2 and c-Myc using lentiviral vectors43
or transposons44. Immunostaining analyses confirmed that all hiPSC lines were
positive for Tra-1-60, Tra-1-81, SSEA-3, SSEA-4, Oct4 and Nanog. qRT-PCR
analyses further confirmed that all hiPSC lines were positive for markers associated with the pluripotent state, including Nanog, Sox2, Lin28 and Ddmt3b.
Pluripotency was finally confirmed by a teratoma formation assay showing that
all hiPSC lines were able to differentiate into representatives of all three germ
layers. qRT-PCR was used to verify that the four vector-encoded transgenes
were silenced in all hiPSC lines. Normal karyotypes were confirmed, and DNA
fingerprinting analysis was used to verify that each patient-specific hiPSC line
was genetically matched to the corresponding donor fibroblasts from which it
was derived. hiPSCs were maintained on Matrigel-coated plates (growth factor
reduced; BD Biosciences) with mTeSR1 medium (Stemcell Technologies) according to WiCell protocols. Cells were passaged every 46 d. In order to induce
the differentiation of hiPSC into neural progenitors, undifferentiated hiPSC
cells were transferred to knockout serum replacement medium and exposed to
Noggin (500 ng/ml) and TGF- inhibitor SB431542 (10 M) to block SMAD
signaling45. The TGF- inhibitor was withdrawn after the fifth day of differentiation. Noggin was maintained and increasing amounts of N2 medium (25%, 50%,
75%) were added to the knockout serum replacement medium every 2 d. After
10 d, neural progenitors were expanded for 24 weeks in DMEM/F-12 with B27
and N2 supplements (Life Technologies), 10 ng/ml human epidermal growth
factor and 10 ng/ml human basic fibroblast growth factor (Life Technologies).
Neural progenitors were finally differentiated into neurons.
Animals and in vivo studies. Founder FvB.129(B6)-Smn1<tm5(Smn1/SMN2)
Mrph>/J animals (Jackson Laboratory Stock no. 008604) were bred and provided water and chow ad libitum. Animals that were heterozygous (C/+),
homozygous (C/C) or wild type (+/+) at the C allele were identified with an
allele-specific PCR from ear biopsies. The primers used for the PCR assay were:
5-TAC CCA GAT GCA GTG CTC TTG TAG-3 (mutant forward), 5-CCT
TAT GGC ATA GAC ACC AAC TTC T-3 (mutant reverse), 5-GTC CCT
GGT CGA CAA GAA CAG-3 (wild type forward) and 5-ACG CTC TGC
TGC TGA CTT AGG-3 (wild type reverse).
Heterozygote animals (C/+) were crossed with +/+ segregants from the
colony to maintain a supply of breeding and experimental animals. Female
C/+ animals, age-matched at 810 weeks of age, were used in the pharmacology experiments described here. All experiments were conducted under the
IACUC-approved protocol 12 DMP 013.
Statistical methods for in vivo studies. For body weight estimation (Fig. 2e and
Supplementary Fig. 3) multiple t-tests, as enabled in GraphPad Prism (version
6.04 for Windows), were used. The weight data were analyzed within each day
after birth. The P values were corrected for multiple comparisons using the
Holm-Sidak method, with = 5%. The group sizes for all the drug-treated groups
were n = 8 at the beginning, although mortality among the animals reduced
those numbers at the later times after birth. For survival curves (Fig. 2f), the
Mantel-Cox test was used in GraphPad Prism (version 6.04 for Windows).
RNAseq. A normal human fibroblast line (HD1994) was treated with NVS-SM1
(100 nM), NVS-SM3 (5 M) or DMSO for 24 h. Total RNA was isolated using
the RNeasy Mini isolation kit (Qiagen). RNAseq libraries were prepared using
the TruSeq RNA Sample Prep kit v2 (Illumina) and sequenced using the Illumina
HiSeq2500 platform. Each sample was sequenced on four different lanes belonging to the same flow cells to a length of 2 76 bp. A total of 499 million 76-bp
paired-end reads were mapped to the Homo sapiens genome (HG19)46 (release
59, May 3, 2013) and a custom junction database by using an in-house gene and
exon quantification pipeline based on the aligner Bowtie47, version 2.0.2.
Purification of U1 snRNP. HeLa nuclear extracts (Accurate Chemical and
Scientific Corp) were tested for compound-responsive splicing activity
using previously published methods48. U1 snRNP was purified from the
most active extracts according to previously described methods with some
modifications49,50. The extracts were applied to K121 antibody-linked resin
(Calbiochem) as described49 and eluted in steps, first with 6 column volumes
of trimethylguanosine (Biolog) followed by 50 column volumes of 7-methylguanosine (Sigma). The U1 snRNP was further purified using a Mono Q 5/50
nature CHEMICAL BIOLOGY

 2015 Nature America, Inc. All rights reserved.

GL column (GE Healthcare), and the complex was eluted via a KCl gradient50.
The purified U1 snRNP was flash frozen in its anion exchange buffer (20 mM
Tris-HCl pH 7.0, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF and ~370 mM
KCl). The final concentration was estimated as 930 nM, as described50. Western
blot detection was performed with 1:1,000 dilution U1-70k antibody (rabbit,
Millipore, 06-1297), 0.5 g/mL U1-A antibody (mouse, Abcam, Ab55751) or
1 g/mL U1-C antibody (rat, Sigma, SAB4200188) followed by 1:10,000 dilution
DyLight 680 anti-rabbit (Rockland, 611-144-002), anti-mouse (Rockland, 810644-002) or DyLight 800 anti-rat (Rockland, 612-132-003) secondary antibodies.
Fluorescence was detected with an Odyssey scanner (LICOR Biosciences).
Compound binding to U1 snRNPRNA complexes by size-exclusion chromatography (SEC). Experiments were performed essentially as described37.
SEC plates were prepared using SEC buffer (38 mM HEPES, pH 7.6, 60 mM
KCl, 0.12 mM EDTA, 3.2 MgCl2). RNA was diluted in SEC buffer with no KCl,
heated to 90 C for 5 min, placed at 37 C for 15 min and then stored at room
temperature. Final concentrations were as follows: 1 M biotinylated SMN2
RNA with a TEG spacer (WT: 5-biotinTEG/UCUAAGGAGUAAGUCUGCC
AG-3, IDT), 10 M compound and 1:1 dilution of U1 snRNP. Mock buffer
(20 mM Tris pH 7.0, 1.5 mM MgCl2, 0.5 mM DTT, 370 mM KCl) was used
to replace the U1 snRNP in the conditions lacking it. Similarly, SEC buffer
without KCl was used for conditions without RNA. Compounds were analyzed
using an Agilent 1100 binary pump (Agilent Technologies) coupled to a CTC
HTC pal auto-sampler (Leap Technologies) and a Quattro Premier mass spectrometer (Waters). Chromatography consisted of betabasic C8 javelin guard
columns (Thermo Scientific) eluted with a gradient of 0.1% formic acid in
water versus 100% methanol at a 1 ml/min flow rate. The peak area from a
standard curve of compounds was used to generate a linear regression curve,
and the peak areas from experimental wells were converted to nM compound
detected using the resulting equation from the linear regression. These values
represent the amount of small molecule that co-elutes with targets.
SPR analysis of U1 snRNP binding to RNA. Biotinylated RNAs (WT as in
SEC-TID, 1C: 5-biotinTEG/UCUAAGGCGUAAGUCUGCCAG-3, 2C:
5-biotinTEG/UCUAAGCAGUAAGUCUGCCAG-3,) were synthesized by
Integrated DNA Technologies. Initial SPR studies with compound only in
the association phase were performed on a Biacore T100 at 25 C. RNA was
diluted into SPR buffer (38 mM HEPES, pH 7.6, 60 mM KCl, 0.12 mM EDTA,
3.2 MgCl2, 0.05% P20), heated to 90 C, slowly cooled to room temperature and
centrifuged for 10 min at 14,000g, and a target level of 110 relative units (RU)
was captured onto a streptavidin-coated SA chip (GE Healthcare). U1 snRNP
was diluted 1:50 with SPR buffer containing either DMSO or compound. Final
DMSO concentration was 0.5%, and the running buffer was adjusted to the
same percentage. The surface was regenerated with 1 M NaCl, 10 mM NaOH.
Co-injection experiments were performed under the same buffer conditions
on a ProteOn XPR36 at 25 C using a NLC chip (Bio-Rad) with a minimum of
25 RUs of target RNA loaded on the surface. The ProteOns co-inject function
allowed testing of NVS-SM2 or DMSO in both the association and dissociation
phases. Dissociation rate constants are independent of analyte concentration
and were measured using the ProteOn software from two duplicate injections.
All data were double referenced to a protein-only surface as well as a buffer
injection, and a DMSO correction for excluded volume was performed51.
SPR analysis of U1 snRNA binding to RNA. SPR studies were performed on
a ProteOn XPR36 at 20 C using a NLC chip (BioRad) with a minimum of 300
RUs of target RNA loaded on the surface. U1 snRNA (5-AUACUUACCUG-3)
was diluted to 1 M with SPR buffer containing either DMSO or compound.
The co-inject feature was used so that the association and dissociation phases
contained either DMSO or compound. Surface regeneration and referencing
were performed as above.
NMR preparation of RNA and RNAcompound complex samples.
RNA-11, SMN ssRNA (5-GGAGUAAGUCU), RNA-12, U1 snRNP RNA
(5-GAUACUUACCUG) and RNA-23, SMN ssRNA/U1 snRNP-linked RNA
(5-GGAGUAAGUCU-GAUACUUACCUG) were synthesized by TriLink
BioTechnologies or Integrated DNA Technologies. The dsRNA was prepared
by mixing equimolar concentrations of RNA-11 and RNA-12 in NMR buffer
(20 mM potassium phosphate, pH 6.2, 100 mM KCl and 0.1 mM EDTA). The
mixture was heated to 60 C for 5 min and then cooled to room temperature.
The samples for one-dimensional NMR binding studies were made with 100 M
nature chemical biology

compound and 5 M dsRNA in D2O buffer. RNA-23 was used for the computational modeling structure determination after confirmation that the stemloop base pairing patterns were the same as those of the SMN ssRNA/snRNP
RNA dsRNA by TOCSY. The samples for TOCSY with RNA-11 and RNA-12
in D2O or H2O buffer were heated to 85 C for 5 min and then cooled to room
temperature. The RNA-11RNA-12NVS-SM2 complex was prepared by adding 10 mM DMSO-d6 stock solution of NVS-SM2 to 350500 M of dsRNA
(RNA-11 or RNA-12) until the compound concentration reached saturation.
NMR experiments. All NMR experiments were performed on AVANCE III
600 MHz or 800 MHz spectrometers (Bruker). The sample temperature was
20 C for binding experiments with the dsRNA and 537 C for structure
determination experiments including 1D 1H, and 2D COSY and TOCSY
with RNA-11 and RNA-12. The model was assembled from a data set that
included analysis of TOCSY spectra.
Computational model generation. The transient dsRNA sequence was folded
into a population of 50 secondary structures using MC-Fold52, scoring on the
basis of free energy minimization and both canonical and noncanonical base
paring. A base-paring pattern from MC-sym that was suggested by NMR was
used for further 3D structure predictions. MC-Sym52 was used to predict 150
diverse 3D structures (0.5- resolution) of the selected secondary structure
for analysis with the NOE data. The structures from MC-Sym are assembled
from a database of nucleotide cyclic motifs of experimentally derived RNA
structures. Each structure from MC-Sym was refined using constrained energy
minimization using simulated annealing as part of the MC-Sym workflow.
The population of 3D structures was then used as input to the Alibero
method to select the minimal subset of dsRNA structures that best discriminate the actives (NVS-SM1 and SM2) from inactives (NVS-SM3 and SM4)53.
This subset was found by docking all compounds against all 3D dsRNA models
using the ICM method54 and then selecting the best subset of models that
separate actives from inactives. The broad docking location scanned by Alibero
was the entire region proximal to the nGA of dsRNA as suggested by the
TOCSY shift data. The metric used in the optimization by Alibero was the area
under the log2 ROC curve for actives and inactives. The final conformation of
dsRNA-compounds was used as the model and then compared to the recently
published 3.3- crystal structure of the U1 5 splice site complex.
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44. Lacoste, A., Berenshteyn, F. & Brivanlou, A.H. An efficient and reversible
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45. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS
cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275280 (2009).
46. Pruitt, K.D., Tatusova, T. & Maglott, D.R. NCBI reference sequences (RefSeq):
a curated non-redundant sequence database of genomes, transcripts and
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49. Krainer, A.R. Pre-mRNA splicing by complementation with purified human
U1, U2, U4/U6 and U5 snRNPs. Nucleic Acids Res. 16, 94159429 (1988).
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