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7.3 Pharmacological Mechanisms of Splicing Modulation 175
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NH
O
N
O
N
H
H
O
O
Helicase IC50: 1,300 nM
N
N
ATPase IC50: 79 nM
N
Cmpd 9
Brr2 inhibitor
O
N
N
S
O
F
Cmpd 32a
Brr2 inhibitor
ATPase IC50: 21 nM
Helicase IC50: 480 nM
N
N
O
N
H
O
NH
Staurosporine
Posited helicase inhibitor
Figure 7.17 Examples of inhibitors of splicing-associated RNA helicases.
target class has been severely neglected. To-date, only two examples of
small-molecule inhibitors of splicing-related RNA helicases have been described –
both targeting the ATPase domain of Brr2, a key driver of U4 snRNP dissociation – but
the applicability of this strategy remains unconrmed as neither compound was
assessed for splicing modulatory activity (Cmpd 9 and Cmpd 32a, Figure 7.17)
[140, 141]. Nonetheless, RNA helicases are highly homologous, so Cmpd 9 and
Cmpd 32a are noteworthy for their excellent inhibitory selectivity for Brr2 over
closely related helicases. Both compounds were developed from hits originally
identied in high-throughput screening for recombinant Brr2 ATPase activity – not
helicase activity – as it is well documented in the DNA helicase eld that assays
measuring helicase activity are exceptionally susceptible to false positivity arising
from interactions of small molecules with the DNA substrate [142]. An additional
advantage of the ATPase tactic is that it aords an opportunity for focused library
screening around kinase inhibitors. Indeed, it has been posited that the splicing
activity of staurosporine (a nonselective kinase inhibitor) in yeast may be due to
RNA helicase ATPase inhibition [143].
7.3.5 Drugging the Spliceosome
An assortment of small molecules has been found to inhibit spliceosome assembly:
Madrasin, discovered in 2014, appeared to stall assembly at complex A [144], and
isoginkgetin was reported in 2008 to interfere with recruitment of the U4/U5/U6
tri-snRNP complex, resulting in complex A accumulation (Figure 7.18) [145]. The
mechanisms of these and other compounds are not well understood, but direct interaction with the spliceosome is unlikely for most. For example, quinone-containing
inhibitors – e.g. NSC659999 – and other redox-active small molecules indirectly
inhibit assembly through reactive oxygen species [146, 147]. Compound-induced
oxidative stress has also been implicated in inhibition of splicing catalysis (BN82685,
Figure 7.18) [148]. Nevertheless, there exist well-characterized small molecules
that specically target the spliceosome, and they represent the most successful
modulators of pre-mRNA splicing to-date, with multiple disclosed clinical agents
and one FDA-approved drug as of 2023.
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O
NN
N
O
NHN
H
O
H
N
S
N
N
O
Madrasin
Assembly inhibitor
O
OH
Br
N
OH
O
NSC659999
Assembly inhibitor
HO
N
OH
O
BN82685
Catalysis inhibitor
O
O
OH
HO
O
Isoginkgetin
Assembly inhibitor
O
O
O
OH
Figure 7.18 Examples of spliceosome assembly and splicing catalysis inhibitors.
Redox-active quinone functionality highlighted in red.
7.3.5.1 Inhibition of U2 snRNP Recognition of the 3′-Splice Site
The rst spliceosome-directed splicing modulator was discovered in 1996 but was
not recognized as such until 2007 – FR901464, a natural secondary metabolite
isolated from bacteria [149]. This was followed by herboxidiene in 2002 and
spliceostatin A in 2007 [150, 151], and today many families are known, including
the meayamycins,sudemycins, thailanstatins, and pladienolides (Figure 7.19)
[152–154]. All of these compounds bind the interface of SF3B1 and PHF5A – two protein components of U2 snRNP – in a pocket that recognizes the branch point adenosine and facilitates recruitment of U2 snRNP to the pre-mRNA [155]. Compound
binding simultaneously locks SF3B1 in an inactive conformation and physically
blocks pre-mRNA binding, arresting early spliceosome assembly [153, 156–158].
7.3.5.2 E7107
The rst recognized small-molecule splicing modulator to enter clinical trials
was the SF3B1 inhibitor E7107, a semi-synthetic macrolide derived from the
pladienolide family of secondary metabolites (Figure 7.20) noted for potent antitumor activities [159–162]. In 2007, E7107 entered two Phase I, open-label, single-arm
clinical trials for patients with solid tumors. The decision to pursue cancer indications was based not only on the excellent growth inhibition potency of E7107
but also on selectivity for cancer cells over normal cells. The mechanistic basis of
this preferential killing is still not fully understood, but several hypotheses have
been proposed. First, many cancers suer function-compromising spliceosomal
mutations that render them exceptionally dependent on remaining wild-type
spliceosomes [163]. Second, SF3B1 splicing modulators suppress expression of
some tumorigenic genes and enhance expression of apoptotic factors; sudemycin,
for example, inhibits MDM2 oncogene expression via exon skipping [152, 164].
7.3 Pharmacological Mechanisms of Splicing Modulation 177
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Figure 7.19 Examples of SF3B1 inhibitors. The shared pharmacophore (polyketide tail) is
colored blue.
Finally, this compound class has been shown to enhance immune response to
cancer cells through several mechanisms, including (i) expression of immunogenic
alternatively spliced neoantigens and (ii) activation of antiviral signaling due to
accumulation of mis-spliced double-stranded RNA [165, 166]. Unfortunately,E7107
human trials were discontinued in 2009 due to dose-limiting adverse events [167],
but interest in pladienolide-derived splicing modulators continued. In 2010, Eisai
established H3 Biomedicine, who developed a remarkable successor to E7107 called
H3B-8800 (Figure 7.20).
7.3.5.3 H3B-8800
The most notable feature distinguishing H3B-8800 from E7107 is the tail, which
is distinctly synthetic in the former. All natural SF3B1 splicing modulators share
a highly variable polyketide “tail” pharmacophore, excision of which aords
analogs completely lacking in binding anity, cytotoxic activity, and splicing ability
(Figure 7.19) [168, 169]. The tolerance for diversely substituted tails is consistent
with cryo-EM models in which the tail is disordered and solvent exposed, particularly near the tip (Figure 7.21); even the conspicuous oxirane is an unreactive
and dispensable component [170]. However, all SF3B1 inhibitors require a notably
conserved diene; consistent with this, H3B-8800 retains the diene but substitutes
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OH
OH
O
O
O
O
OH
O
Pladienolide B
• First isolated 2004
OH
OH
O
N
N
O
O
O
HO
OH
O
E7107
• First SF3B1 inhibitor in clinical trials (2007)
• Trials suspended due to adverse events (2009)
• Not orally bioavailable
OH
OH
O
O
N
O
N
O
N
H3B-8800
• Entered Phase I trials (2016)
• Currently in Phase I/II
• Orally bioavailable
Figure 7.20 Development and
evolution of pladienolide-derived
SF3B1 inhibitors. Critical
pharmacophore features (red);
potency-enhancing substitutions
colored (green); feature essential for
sequence-specificity/gene selectivity
(blue).
Figure 7.21 Cryo-EM model of E7107 bound to a SF3B1-PHF5A complex. Solvent-exposed
polyketide tail depicted. PDB code 5ZYA.
7.3 Pharmacological Mechanisms of Splicing Modulation 179
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the lenient tail tip with pyridine. H3 Biomedicine has not explicitly disclosed the
reason for this substitution, but one explanation may be stability as many naturally
occurring pladienolides are susceptible to tail-promoted enolide hydrolysis [171].
However – whatever the original reason – the pyridyl tail was discovered to
confer sequence specicity toward weak branch points (i.e. weak splicing events),
rendering H3B-8800 a more selective (and potentially safer) splicing modulator
than E7107.
In a panel of variable-3′ss minigenes, H3B-8800 not only enhanced intron retention at weak 3′-splice sites relative to E7107 but it also exhibited reduced splicing
activity at canonical sites (Table 7.1) [172]. Transcriptome-wide RNA-sequencing
revealed that weak branch point preference was not the only factor contributing
to H3B-8800’s selectivity; retained introns were generally (i) short, (ii) GC-rich at
the 3′-ss, and (iii) adjacent to a GC-enriched exon. (In contrast, the spliceosome
itself conventionally prefers long introns with pronounced dierences between
intron and exon GC content.) [173] These properties of H3B-8800 were manifest
in cellular and mouse xenograft models, where it preferentially killed cancer lines
carrying splicing mutations over wildtype cells; E7107, on the other hand, killed
both cell lines equally.
Table 7. 1 Splicing selectivity and sequence specificity of H3B-8800 vs. E7107. Splicing
was measured in HeLa nuclear extracts.
OH
OH
O
N
O O
N
O
HO
OH
O
O
N
O
N
OH
OH
O
O
H3B-8800E7107
Ad2
a)
BPS
b),c)
PYT
d)
3′ss
Ty p e
E7107
IC50(nM)
H3B-8800
IC50(nM)
Ad2.1 UACUAAUCC CCCUUUUUUUUCCA Strong 9.1 >25,000
Ad2.2 UACUACUCA CCCUUUUUUUUCCA Weak 9.9 NA
Ad2.14 UACUUAUCC CCCUUUUUCCCCCA Strong 12.2 1,000
Ad2.17 UAGUUAUCC CCCUUUUUCCCCCA Weak 4.7 30
Ad2.12 UACUUAUCC CCCCCCCCCCCCCA Strong 3.8 1,838
Ad2.15 UAGUUAUCC CCCCCCCCCCCCCA Weak 4.8 55
Ad2.13 UACUUAUCC CCCCCCCCCCCCCA Strong 5.1 347.2
Ad2.16 UAGUUAUCC CCCCCCCCUUUUUA Weak 3 37.3
a) Adenovirus Ad2-derived mini-mRNA.
b) BPS, branch point sequence.
c) Branch point adenosine is bold and underlined, while noncanonical point mutation is bold
only.
d) PYT, polypyrimidine tract.
Source: Adapted from Buonamici et al. [172]
N
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In 2016, H3B-8800 entered open-label Phase I clinical studies in patients with
myeloid and myelomonocytic leukemias, and, in stark contrast to E7107, presented
no to mild adverse eects. Although clinical responses did not meet threshold criteria (despite the presence of splicing mutations in 88% of the enrolled patients), the
results helped identify potentially responsive patients for future studies. In 2022,
H3B-8800 was expanded into Phase II for a select patient cohort, where it remains
under parallelassessment with the ongoing Phase I study. These achievements teach
that (i) it is possible to develop sequence-specic, gene-selective splicing modulators
and (ii) these selectivity properties are critical to drug safety.
7.3.5.4 Stabilizers of U1 snRNP Recognition of the 5′-Splice Site
The most lucrative class of spliceosome splicing modulators are the U1 snRNP
stabilizers, which promote splicing by strengthening the interaction of U1 snRNP
with weak, noncanonical 5′-splice sites (Figures 7.22 and 7.23). Examples include
(i) PTC Therapeutic’s and Homan-La Roche’s risdiplam – Evrysdi® – the rst
FDA-approved small-molecule splicing modulator, indicated for SMA; (2) Novartis’
branaplam – a clinical agent which achieved Phase II clinical trials for SMA and
Huntington’s disease; and (3) PTC Therapeutic’s PTC518 –anundisclosedagent in
Phase II clinical trials for Huntington’s disease.
Both risdiplam and branaplam were discovered during target-agnostic
high-throughput screening for SMN2 splicing modulators; consequently, their
mechanisms of action were initially unknown [174, 175]. Evidence for U1
snRNP/5′ss stabilization was rst inferred from RNA-seq, in which SMN2 exon
7-like 5′ss sequences were observed enriched among modulated genes [176].
Both compounds favor weak, noncanonical −2G/−1A 5′ss (the canonical 5′ss is
−2A/−1G, Figure 7.23b), and it has been proposed on the basis of NMR modeling
that this specicity results from compound-mediated stabilization of a bulged
register; the noncanonical U1 snRNA/pre-mRNA duplex is deformed by base-pair
exclusion of −1A, a situation rectied by compound binding (Figure 7.24) [177].
Interestingly, despite ostensibly similar splicing proles, deeper inspection revealed
that the two compounds in fact selectively inuence dierent gene subpopulations.
To explain this, Allain et al. proposed a two-site binding model in which risdiplam’s
selectivity arises from a dual requirement for both a cis-regulatory ESE sequence
and an SMN2-like 5′ss, whereas branaplam’s only precondition is the latter [178].
However, comprehensive whole-genome RNA-seq investigations from PTC Therapeutics discerned discrete sequence specicities at the 5′ss itself: (i) risdiplam
exhibits an additional pronounced preference for −4A (Figure 7.23c), (ii) but
branaplam has a strong preference for −3A (Figure 7.23d) [179]. Unfortunately,
the molecular etiology underlying these specicities is as yet unknown.
7.3.5.5 Introduction to Spinal Muscular Atrophy (SMA)
SMA is an often-lethal genetic disorder aecting 1 in 10,000 live births in the
United States and 1 in 100,000 worldwide [180]. It is caused by mutation-linked
loss of function of the SMN1 gene and consequent deciency of the protein SMN
(survival of motor neurons), which normally mediates the assembly and biogenesis
7.3 Pharmacological Mechanisms of Splicing Modulation 181
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OH
N
NH
N
N
N
HN
O
Risdiplam
U1/pre-mRNA stabilizer
N
N
N
HN
N
N
O
Branaplam
U1/pre-mRNA stabilizer
(a)
U mutation
C
U1
Exon 6
Exon 6
5’ss 3’ss
Exon 8 Exon 6 Exon 7 Exon 8
Translated to degraded protein Translated to functional protein
(b)
Exon 7 Exon 8
5’ss
3’ss
+ Small molecule
U1
Exon 49
5’ss
psi-3’ss
Psi49
psi-5’ss
3’ss
Exon 50
+ Small molecule
Exon 49
Translated to toxic protein
Exon 50 Exon 50
Exon 49 Psi49
Not translated; mRNA degraded
(c)
Figure 7.22 (a) Structures of risdiplam and branaplam. (b) Illustration of SMN2 alternative
splicing. A single C-to-U mutation promotes exon 7 skipping. The Δ7isoformmRNA
encodes SMN protein that is rapidly degraded. The full-length isoform mRNA encodes
stable, functional SMN protein. (c) Illustration of HTT-induced splicing. Small-molecule
stabilization of U1 snRNP and pre-mRNA at pseudo-5′ss promotes inclusion of a
pseudoexon, psi49. The inclusion mRNA is rapidly degraded by nonsense-mediated decay.
of nuclear ribonucleoproteins [181, 182]. SMA is categorized by severity from
most severe (Type 0, prenatal) to least (Type IV, adult) and is dened by the age
of symptom onset. Type I is the most prevalent (∼60%); diagnosed within the rst
six months of birth, it is marked by a survival probability of 18% by 20 years of age
[183]. Symptoms include neurodegeneration and loss of muscle function, including
muscles that regulate essential functions like breathing.
Most humans have two to three copies of a homologous gene called SMN2 that
could compensate for the loss of SMN1 if not for one aw: a single C-to-U nucleotide
182 7 Pre-mRNA Splicing Modulation
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U1
Exon
(a)
5’-splice site recognition sequence
G
C
C
Canonical pre-mRNA 5’-splice site
(b)
GUC C A U U C A
G
CAGGUA AGU
N N
–5
–4 –3 –2 –1 +1 +2 +3 +4 +5 +6
5’ss3’ss
U1 snRNA
G
C
G
C
(c)
G
(d)
G
U
C
C
N
AGGAGUA A GU
–5 –4 –3 –2 –1 +1 +2 +3 +4 +5 +6
C
C
G
G
U
C
C
CAGAN
–5 –4 –3 –2 –1 +1 +2 +3 +4 +5 +6
GUAA GG
AUUCA
AUUCA
Figure 7.23 (a) Illustration of U1 snRNP interacting with the 5′-splice site. (b) The snRNA
component of U1 snRNP recognizes the conserved 5′ss via a complementary sequence.
(c) SMN2 exon 7 weak 5′ss; several exonic nucleotides differ from the canonical sequence.
Risdiplam exhibits a strong preference for −4A (red). (d) HTT exon pseudoexon 49 weak
5′ss; exonic nucleotides differ from the canonical sequence. Branaplam displays a strong
preference for −3A (red).
U1 snRNA U1 snRNA U1 snRNA
CCAU U C CAUU C CAUU
GAG
UAA GAGUA A GAGUA A
+2 +3 +4 +2+1–1–2 +3 +4 +2+1–1–2 +3 +4
–2
(a)
(b)
–1
–3G
–2G
–1A
–3G
–2G
–1A
+1G
+1G
+1U
+1U
Figure 7.24 SMN2 weak 5′ss stabilization model. (a) −1A adenosine bulges out of register
due to absence of a complementary base (PDB: 6HMI); risdiplam binds the U1
snRNA/pre-mRNA interface to permit −1A incorporation (PDB: 6HMO).
7.3 Pharmacological Mechanisms of Splicing Modulation 183
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dierence near the 3′ss of exon 7 (Figure 7.22b). This point mutation renders SMN2
pre-mRNA susceptible to exon 7 skipping, resulting in translation of an unstable,
truncated SMN protein [184]. However, the SMN2 gene is not completely disabled,
and low level inclusion produces a small quantity of full-length SMN. Clearly, there
is some degree of spliceosome recognition, and 1990s researchers recognized the
opportunity this presented – that splicing might be rescued by small molecules capable of strengthening the existing interaction between the spliceosome and SMN2
pre-mRNA [185]. A burst of SMN splicing modulators were reported between 2001
and 2010, but few were drug-like and none were particularly potent [186–192].
With the underwhelming performance of small molecules, this was a popular
time for anti-sense oligonucleotides (ASOs) [193–195]. Though optimization can be
complicated, ASO design is conceptually straightforward: (i) identify a pre-mRNA
sequence and (ii) synthesize a complementary ASO. Although there are several
mechanisms through which ASOs may alter splicing, the most popular approach
to SMN2 involved physically blocking splicing suppressor cis-regulatory sequences.
Many varieties featuring stabilized carbohydrate backbones were developed;
Krainer et al., for example, spearheaded ASOs that obstructed binding of hnRNPs
A1 and A2 to splicing silencer sequences in SMN2 intron 7 [196]. These eorts
would culminate in 2016 in the FDA approval of Biogen and Ionis Pharmaceutical’s
nusinersen (Spinraza®) for the treatment of SMA [197]. However, ASOs have
several disadvantages compared to small molecules. First, they are generally not
orally bioavailable; nusinersen, for instance, is administered to the central nervous
system by intrathecal injection into the spine (known also as a lumbar puncture or
spinal tap) [198]. Second, they do not exhibit the same broad systemic distribution
of small molecules; ASOs delivered to the central nervous system do not readily
proportionate to peripheral tissues (and vice versa), which is of consequence to
diseases like SMA that suer both central and peripheral neurodegeneration [199].
7.3.5.6 Risdiplam (Evrysdi®)
Attentive to ASO deciencies in the treatment of SMA, PTC Therapeutics
launched a discovery program in 2007 to identify and develop small-molecule
splicing modulators with support from the SMA Foundation. Aiming for
(i) excellent signal-to-noise and (ii) dynamic range for the detection of weak
hits, a target-agnostic minigene-based reporter assay was developed suitable for
high-throughput screening [175]. The minigene contained all of SMN2 exon 7,
downsized exons 6 and 8, and a rey luciferase region (Figure 7.25). Exon 7 skipping aorded an out-of-frame sequence and therefore no translation of the luciferase
reporter, whereas exon 7 inclusion produced an in-frame luciferase gene encoding
photocatalytically competent luciferase. However, rey luciferase reporters suer
a well-documented susceptibility to false positivity, so a quantitative PCR (qPCR)
analysis of minigene transcripts was incorporated into the screening triage [200].
>200,000 compounds were screened and the weak hit 21 was identied and
conrmed, after which hit expansion quickly established a strict requirement
for a basic amine, although no structural or mechanistic insight was yet available at the time, this is consistent with NMR models in which the amine is
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C
U mutation
Exon 6
5’ss 3’ss 5’ss 3’ss
– Active compound
Exon 6 Exon 8 Exon 6 Exon 7 Exon 8
Figure 7.25 SMN2 minigene construct for high-throughput screening.
f-Luciferase
Out of frame In frame
Exon 7 Exon 8 f-Luciferase
+ Active compound
f-Luciferase
observed near the negatively charged phosphate backbone (Figure 7.24b). Major
development milestones are illustrated in Figure 7.26. Lead optimization was
primarily driven by minigene assay qPCR quantitation, but – as compound potency
improved – an HTRF-based assay was included to assess full-length SMN protein in
disease-relevant cells.
Select compounds were promoted into advanced studies to determine their
functional disposition. Motor neuron cultures (prepared from SMA patient-derived
induced pluripotent stem cells – IPSCs) produced >2-fold more protein when treated
with RG7800 (EC
mini
= 23 nM, EC
1.5x
SMN
= 87 nM) and >3-fold more full-length SMN
1.5X
from an SMA mouse model following oral administration [201]. Importantly,
increased protein was apparent in both CNS and peripheral tissue as expected
from a broadly distributable small molecule. Cross-section immunohistological
assessment of SMA mouse muscle and spine revealed dose-dependent arrest of both
neurodegeneration and muscle atrophy. It is particularly noteworthy that while
SMA model mice do not typically survive beyond 20 days after birth, once-daily
RG7800 administration enhanced survival and tness such that the animals were
nearly indistinguishable from healthy wild-type mice.
By this time, PTC Therapeutics and Homann-La Roche were collaboratively
joined and together launched RG7800 into human clinical trials in 2014, where
it demonstrated a clinically signicant increase of full-length SMN in SMA
patients [202]. Unfortunately, RG7800 was discontinued due to unacceptable
risks, including (i) hERG inhibition, (ii) phospholipidosis, and (iii) photoxicity.
However, these specic events were not inherent to splicing activity, although
scaold hopping programs were ultimately unsuccessful, RG7800 itself proved
receptive to physicochemical tuning [203]. Incorporation of a cyclopropyl group sufciently lowered amine pKa such that hERG inhibition and phospholipidosis were
simultaneously eliminated (but not so low that potency was impaired), and substitution of 2-pyrazolpyrazine with 6-imidazopyridazine cleared phototoxicity risk
(Figure 7.26). This new compound, risdiplam (EC
mini
= 4 nM, EC
1.5x
SMN
1.5X
= 29 nM),
entered clinical trials in 2015 and exited in 2020 as the FDA-approved Evrysdi®.
It is noteworthy that risdiplam continues to exhibit outstanding long-term
clinical safety [204–206]. This has been attributed in part to its sequence specicity
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