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9
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Small Molecules That Degrade RNA
Noah A. Springer
Jessica L. Childs-Disney
1
Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation &
Technology, Jupiter, FL 33458, USA
2
The Scripps Research Institute, Jupiter, FL 33458, USA
1,2
, Samantha M. Meyer
1
, and Matthew D. Disney
1,2
, Amirhossein Taghavi1,
1,2
9.1 Antisense Oligonucleotide Degraders
The most common method to degrade a cellular RNA is by inducing ribonuclease
(RNase H) cleavage upon binding of an antisense oligonucleotide (ASO). ASOs
are modied oligonucleotides that base pair to target RNAs and either facilitate
degradation, [1] act as steric blockers to alter splicing, [2] and/or inhibit translation
[3]. For ASOs that degrade their target, the DNA/RNA heteroduplex is recognized
by endogenous RNase H1 that cleaves the RNA strand of these hybrids, leading to
specic degradation of target RNAs within cells [1, 4]. After cleavage via RNase H1,
the target RNA is then fully degraded by exoribonucleases XRN1 (5
′-3′
clease 1), XRN2 (5
exoribonuclease 2), and the exosome complex [5]. Because
only the RNA strand of the ASO/RNA duplex is cleaved, the ASO can be recycled
and therefore acts sub-stoichiometrically. This process enables potent knockdown
of a gene product in cells.
ASOs have been useful as chemical tools for knocking down targets in cell
culture but are increasingly becoming viable drugs for some targets. The rst ASO
with an RNase H1-mediated mechanism of action, Fomiversen, was originally
approved by the United States Food and Drug Administration (FDA) in 1998 to
treat cytomegalovirus (CMV) retinitis [6], though it was later taken o the market.
More recently, Inotersen was approved by the FDA in 2018 to treat hereditary
transthyretin amyloidosis (hATTR) [7], competing with the small interfering
RNA (siRNA) drug patisiran [8]. Additionally, although it functions via splicing
modulation rather than RNase H1-mediated degradation, the success of nusinersen
[9] demonstrates that ASOs can become blockbuster drugs. The design of potent
ASOs is relatively straightforward when compared to other technologies since
Watson-Crick base-pairing rules allow for rapid ASO design against any known
RNA sequence. Key to the selectivity of an ASO is selection of unique sequences
′-3′
exoribonu-
227
RNA as a Drug Target: The Next Frontier for Medicinal Chemistry, First Edition.
Edited by John Schneekloth and Martin Pettersson.
© 2024 WILEY-VCH GmbH. Published 2024 by WILEY-VCH GmbH.
228 9 Small Molecules That Degrade RNA
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in the RNA target. However, hybridization-free energy, cellular abundance, and
the energetics of on- and o-targets can also aect selectivity; such complexities
should be carefully considered [10, 11]. Nonetheless, various modications to the
nucleotide bases, ribose, and phosphodiester backbone have been identied that
enhance the metabolic stability of ASOs, as well as inuence tissue uptake and
the thermodynamic stability of its interaction with the target RNA [12, 13]. The
success of nusinersen demonstrates the potential of ASOs in the clinic, despite
the challenges associated with the modality in general, centered on its lack of oral
bioavailability and tissue distribution largely limited to the liver and kidney [13].
As exemplied by nusinersen, treatment directly into the cerebrospinal uid can
allow for distribution throughout the central nervous system [9]. Although ASOs
are a promising technology with the potential to transform therapeutics targeting
RNAs, the remainder of this chapter will focus on small molecules that have similar
targeted RNA degradation capabilities.
9.2 Small-Molecule Direct Degraders
Many routes have been explored to identify small molecules capable of inducing
degradation of nucleic acids. These generally fall into three categories: natural products, metal ion-dependent molecules, and metal ion-independent molecules.
Complex natural products that induce nucleic acid degradation include bleomycin
and the enediynes. Bleomycin (described further in Section 9.2.2) induces degradation of DNA and RNA via oxygen activation and hydrogen abstraction [14–16].
The enediyne natural products were initially discovered due to their antibiotic and
antitumor eects [17]. These small molecules undergo a Bergman cyclization upon
binding to DNA [18], resulting in a diradical formation, leading to hydrogen atom
abstraction and DNA double-strand cleavage [19, 20].
Metals and metal complexes have long been used to facilitate DNA degradation,
commonly in DNA ngerprinting analyses. Some of these molecules, including iron
(II) ethylenediaminetetraacetic acid (EDTA) [21] and 1,10-phenanthroline-copper
complexes [22], function by producing hydroxyl radicals, which degrade DNA by
hydrogen atom abstraction. Other examples include photoactivatable compounds
such as rhodium (II) 9,10-phenanthrenequinonediimine complexes [23], among
many others, as reviewed previously [24]. Many metal ions and metal ion complexes, such as tris(1,10- phenanthroline) ruthenium(II) complexes [25, 26], also
show the ability to cleave RNA, as reviewed previously [27, 28]. These complexes
often contain Zn2+, Cu2+, or a lanthanide ion to catalyze the reaction [27, 28]. One
application of these metal ion complexes is for “articial ribonucleases,” where
the metal ion complexes are appended to an oligonucleotide that directs selective
cleavage [29]. Additionally, some metal ion complexes alone can show modest RNA
sequence preference, even in unstructured RNAs [30].
Nonmetal-based small molecules also facilitate cleavage of nucleic acids. These
include photoactivatable DNA cleavers such as anthraquinones, acridine-linked
nitrobenzamides, porphyrins, riboavin, napthalimides, and more, and have been
9.2 Small-Molecule Direct Degraders 229
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previously reviewed [24]. Some of these, such as riboavins [31], can also
lead to RNA degradation. Hydroxyl radical-generating molecules such as
N-hydroxypyridinethiones [32] can also induce both DNA and RNA degradation via hydrogen abstraction [33]. Most of these approaches, however, have not
been used in cells or preclinical animal models of disease.
9.2.1 N-Hydroxypyridine-2(1H)-thione (N-HPT) Conjugates
Early work toward the deliberate design of small molecule RNA-targeted degraders
with activity in cells involved directly appending degrading moieties onto small
molecule RNA binders. The rst example of such a molecule utilized a derivative of
N-hydroxypyridine-2(1H)-thione (N-HPT) [34]. Upon ultraviolet light irradiation,
N-HPT produces hydroxyl radicals that initiate a radical cascade culminating
in cleavage of the phosphodiester bonds of RNA [33]. An N-HPT derivative
(1-hydroxy-6-thioxo-1,6-dihydropyridine-2-carboxylic acid) was coupled to a previously validated dimeric compound (2H-4) that binds the RNA repeat expansion
causative of myotonic dystrophy type 1 (DM1) [34, 35].
DM1 is caused by a trinucleotide repeat expansion in the 3’ untranslated region
(UTR) of the dystrophia myotonica protein kinase (DMPK) gene [36]. The repeat
expansion ranges anywhere from 100 to several thousand r(CUG) repeats [dubbed
r(CUG)
exp
]. This r(CUG)
exp
forms a stable secondary structure comprising 1 × 1
nucleotide UU internal loops. The loops are high anity binding sites for various
RNA-binding proteins such as the muscleblind-like 1 (MBNL1) splicing factor.
The repeats sequester MBNL1 in the nucleus, preventing it from performing its
canonical role as a splicing modulator, thereby leading to deregulation of alternative
pre-mRNA splicing [37–39]. Early studies showed displacing these protein–RNA
interactions with ASOs reversed disease phenotypes, demonstrating that the
r(CUG)
exp
is the toxic entity operating in DM1 [40–42].
The heterobifunctional direct degrader (2H-4-N-HPT) reduced the target RNA
levels in transfected HeLa cells by ∼50%atadoseof5μM after 12–16 h, with degrada-
tion plateauing around 5 h post-photolysis [34]. Additionally, 2H-4-N-HPT, as well
as the irradiation, did not aect the viability of cells, suggesting the compound did
not result in widespread cell toxicity. While this study served as a proof of principle
that small molecules could induce degradation of RNAs in cells, the obvious limitation of this approach was the requirement of irradiation to induce hydroxyl radical
formation and thus its intractability in vivo. Nonetheless, this study was an important proof of concept that small molecules can be programmed to degrade an RNA
target in cells selectively.
9.2.2 Bleomycin
Bleomycins are a group of natural products from Streptomyces verticillus that were
discovered in 1966 and soon thereafter were shown to have anticancer properties [43, 44]. Bleomycin has been used in combination with other drugs to treat a
variety of cancers, including testicular cancer [45], squamous cell carcinoma [46],
230 9 Small Molecules That Degrade RNA
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N
H2N
N
O O
N
H2N
H
NH
2
(a)
O
Proline linker
(b)
Targets r(CCUG)
(c)
HO
OH
HO
N
OH
O
O
NH
O O
OH
O
H
HO
N
HN
N
O
2
HO
O
O
O
NH
N
N
O
HO
HO
2
HN
NH
N
O
O
H
N
O
NH
O
OH
N
H
N
N
N
Targets r(CUG)
N
H
H2N
O
HO
exp
N N
NH
N
Targets pri-miR-96
O
NH
2
H
N
O
R =
S
H
N
N
N
S
S
+
R =
OH
Bleomycin A2
Bleomycin A5
NH
2
O
R
N
H
Metal-binding domain
Carbohydrate domain
Linker domain
DNA-Binding domain
exp
O
N
H2N
O
O
N
N
H2N
O
O
O
N
N
N
O
O
4
O
O
N
N
O
O
3
N
N
Cugamycin
HNH
N
O
H
H
N
N
O
N
H
N
H
Deglycobleomycin
N
N
N
NH
2
O
HO
NH
2
OH
O
HO
O
O
O
N
H2N
O
N
N
N
N
N
O
O
O
4
NH
H
N
N
H
N
N
N
O
N
N
O
O
2
N
H
O
NH
H
N
Figure 9.1 Bleomycin degraders. (a) The natural product bleomycin, with each functional
domain highlighted. (b) Cugamycin, a r(CUG)
with two functional derivatives (bottom left and bottom right). (c) Two other bleomycin
conjugates targeting r(CCUG)
and Hodgkin’s [47] and non-Hodgkin’s [48] lymphoma. Its anticancer properties
are believed to be primarily derived from its ability to induce double-strand breaks
in DNA. Bleomycin is composed of four domains: a metal-binding domain, a carbohydrate domain, a linker domain, and a DNA-binding domain (Figure 9.1a). The
metal-binding domain is responsible for forming a metal ion complex, which can
activate oxygen to extract a hydrogen from the nucleic acid backbone [14, 49–51].
The carbohydrate domain appears to be important for cell recognition and uptake
exp
-targeting bleomycin conjugate (top right)
exp
(top) and pri-miR-96 (bottom).
9.2 Small-Molecule Direct Degraders 231
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as well as DNA binding and cleavage eciency [52–54]. The linker domain length
and composition are both critical for ecient cleavage of DNA [52, 55, 56]. Finally,
the bithiazole and adjacent cation comprise the DNA-binding domain, which
facilitates bleomycin’s anity toward DNA, as well as contributes to its specicity
[57, 58]. These four domains function together to facilitate nucleic acid cleavage.
Moreover, bleomycin also cleaves RNA [15, 16, 59, 60]. After it was initially demon-
strated that high concentrations of activated bleomycin could weakly cleavetRNA
Phe
[16], work by the Hecht group showed that bleomycincleavage of RNA was site selective [15]. Bleomycin eciently and selectively degraded tRNA
any degradation of tRNA
Tyr
precursor RNA [15]. Later, using a series of designed
His
precursor without
RNA constructs, bleomycin A5 was demonstrated to preferentially cleave hairpin
RNAs with longer AU-rich sequences, particularly those rich in purines [60].
To examine whether RNA in cells could be degraded by bleomycin, the microRNA
(miRNA) genome was mined for sequences with the potential to be degraded. One
potential target of bleomycin A5 is the primary transcript of miRNA (pri-miR)-10b,
which contains an AU-rich sequence (AUAUAU). Treatment of both HeLa cells
transfected with pri-miR-10b, as well as a triple-negative breast cancer cell line that
overexpresses miR-10b, with nanomolar concentrations of bleomycin A5, reduced
the abundance of both pri- and mature miR-10b [60]. These studies support that
bleomycin A5 could be useful as part of a heterobifunctional small molecule aimed
at targeted RNA degradation.
9.2.3 Bleomycin Conjugates
To overcome the promiscuous nature of bleomycin to cleave both DNA and RNA
sequences, bleomycin conjugates have been developed to impart selectivity. These
include sequence-selective oligonucleotide-bleomycin conjugates targeting DNA
[61, 62], some of which show RNA degradation capabilities as well [63]. However,
this section will primarily focus on RNA-targeted small-molecule-bleomycin
conjugates that have been recently developed. In these molecules, an RNA-binding
module drives the selective recognition of the target RNA, bringing the bleomycin
proximal to induce direct cleavage. O-target cleavage of DNA is further reduced
if the small molecule is coupled to the free amine in the DNA-binding domain,
removing the positive charge and weakening interactions with DNA [52, 64]. To
date, bleomycin-based direct degraders have been developed to target the RNA
repeat expansions that cause DM1 and myotonic dystrophy type 2 (DM2), as well as
oncogenic pri-microRNAs (Figure 9.1b).
9.2.3.1 Bleomycin Degraders Targeting the r(CUG) Repeat Expansion
That Causes DM1
Since the RNA repeat expansion in DMPK mRNA is the toxic, disease-causing
entity, direct degradation of the RNA is an attractive therapeutic strategy. Coupling the terminal primary amine of bleomycin A5 to a previously validated
r(CUG)
exp
-binding small molecule yielded Cugamycin, a direct degrader of the
RNA repeat expansion [65, 66]. Cugamycin degraded 30–40% of DMPK mRNA
232 9 Small Molecules That Degrade RNA
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in patient-derived broblasts and myoblasts (noting that the mutant r(CUG)
exp
allele accounts for 50–70% of total DMPK in human DM1 skeletal muscle [67])
and ∼40% of the r(CUG)
exp
-containing mRNA in a DM1 mouse model [65, 66]).
In addition to degrading the disease-causing RNA, Cugamycin rescued mis-splicing
events associated with MBNL1 sequestration and decreased myotonia, a hallmark
of DM1, in an in vivo mouse model [66]. Impressively, Cugamycin rescued 97%
of splicing defects in a DM1 mouse model, shifting them toward the splicing
pattern observed in wild-type mice [66]. These promising results demonstrated the
potential and feasibility of bleomycin-conjugated degraders as therapeutics against
disease-associated RNAs.
Two interesting ndings resulted from these studies. First, while acylation of the
DNA-binding domain did not eliminate bleomycin-induced DNA damage, attaching the RNA-binding moieties reduced DNA-double strand breaks (as measured by
γ-H2AX staining) to the level of untreated cells at the eective dose of 1 μM [66].
This supported the hypothesis that converting bleomycin to an RNA degrader by
coupling to its DNA-binding site would enhance the selectivity of bleomycin for
RNA. Second, when treated at doses that nearly equivalently degraded DMPK RNA,
Cugamycin showed increased specicity relative to a locked nucleic acid (LNA)
gap-mer complementary to the repeat expansion. Additionally, the LNA degraded
all mRNAs with short r(CUG) repeats that were examined, as well as DMPK mRNA,
whereas Cugamycin specically degraded the DMPK mRNA. Notably, the short
r(CUG) repeats in various transcripts do not adopt a periodic array of UU internal
loops formed by r(CUG)
exp
, the source of Cugamycin specicity for DMPK [66].
Later studies improved upon Cugamycin by separately optimizing the
RNA-binding and bleomycin moieties. First, bleomycin was exchanged for
deglycobleomycin, where the carbohydrate domain of bleomycin was removed
[68] (Figure 9.1b). As mentioned in Section 9.2.2, the carbohydrate domain contributes to cleavage eciency, cell permeability, and DNA-binding anity via
hydrogen bonding to the DNA backbone [52–54]. While Cugamycin cleaved DNA
in vitro at concentrations above 500nM, its deglycobleomycin derivative showed
no DNA-cleaving ability up to 2 μM [68]. In DM1 myotubes, Cugamycin-induced
DNA damage only at the highest concentration tested, 25 μM, whereas the deglycobleomycin derivative showed no DNA damage at any concentration tested (up
to 25 μM) [68]. Further, this dierence in DNA cleavage activity was not due to
variations in cellular uptake, as Cugamycin showed similar cell permeability to its
deglycobleomycin derivative [68].
In a subsequent study, the dimeric RNA-binding moiety of Cugamycin was optimized by varying the linker composition and length [69]. Changing the linker of the
dimeric RNA-binding moiety from four N-methyl alanine residues in Cugamycin
to two proline residues increased the anity to RNA approximately threefold
in vitro [69]. However, this change in linker composition and length changed the
subcellular localization of the r(CUG)
exp
-binder from primarily nuclear (where the
toxic RNA resides) to both nuclear and cytoplasmic [69]. The resultant bleomycin
conjugate with the short proline linker was approximately vefold more potent
than Cugamycin in DM1 patient-derived myotubes, as measured by the decrease
9.2 Small-Molecule Direct Degraders 233
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in DMPK mRNA abundance and the rescue of the MBNL1 splicing defect [69].
Together, these studies demonstrate that bleomycin-based direct degraders can be
optimized via medicinal chemistry approaches. To date, only the bleomycin sugar
composition and linker length of Cugamycin have been explored. Future medicinal
chemistry investigations around Cugamycin could reveal other modications that
improve potency, cellular uptake, and cleavage eciency.
9.2.3.2 Bleomycin Degraders Targeting r(CCUG) Repeat Expansion
that Causes DM2
DM2 is caused by a tetranucleotide RNA repeat expansion [r(CCUG)
exp
] in intron 1
of CHC-type zinc nger nucleic acid binding protein (CNBP)pre-mRNA[70].Like
the pathology of DM1, the repeat expansion sequesters RNA-binding proteins such
as MBNL1 [71], resulting in nuclear foci and pre-mRNA splicing defects, specically
exclusion of exon 11 in the insulin receptor (IR) pre-mRNA, rendering cells
resistant to insulin [72]. In addition, r(CCUG)
exp
also causes retention of the intron
in which it is harbored [73]. A previously identied dimeric small molecule that
binds r(CCUG)
exp
was coupled to bleomycin A5 in a similar manner as Cugamycin
[74, 75] (Figure 9.1c). The bleomycin conjugatecleaved a r(CCUG) repeat in vitro and
in the CNBP mRNA in DM2 broblasts, without signicantly increasing DNA damage compared to untreated cells [75]. The bleomycinconjugate was more potent than
its parent compound, decreasing CNBP mRNA abundance, improving mis-splicing
of insulin receptor exon 11, and reducing nuclear foci, all by approximately 50%;
the binder alone at the same dose only improved splicing and reduced foci by ∼20%
[75]. Importantly, this r(CCUG)
exp
-targeting bleomycin conjugate was more selective than an ASO targeting the same sequence, as the ASO exacerbated DM2 splicing
defects via o-target degradation of MBNL1 mRNA, which also contains a short
r(CCUG) repeat. However, the short repeat does not form the hairpin structure recognized by the bleomycin conjugate and is thus unaected by the direct degrader
compound [75]. This study demonstrated that bleomycin conjugates can be utilized
against at least two repeat expansion disorders and that bleomycin-conjugated direct
degraders show greater specicity than ASOs targeting the same repeat expansion. It
also highlights more broadly that structure-specic RNA-targeted small molecules
could have advantages over sequence-specic targeting in some cases.
9.2.3.3 Bleomycin Degraders Targeting Oncogenic Precursor microRNAs
MiRNAs are a class of small noncoding (nc)RNAs that modulate the translation of
many genes via base pairing to the 3′UTR of target mRNAs. This base pairing can
lead to either translational repression or directed degradation of the bound mRNA.
Previous work has shown that small-molecule RNA binders can inhibit miRNA processing by the enzymes Dicer and Drosha, aecting the levels of specic mature
miRNAs (see Chapter 6 for more details).
The rst miRNA targeted via bleomycin-based direct degraders was pri-miR-96
[76]. Mature miR-96 inhibits apoptosis in cancer cells by repressing the translation of pro-apoptotic transcription factor Forkhead box protein O1 (FOXO1) [77].
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https://t.me/med1917
Previous studies had identied a compound, Targaprimir-96 (TGP-96), that inhibits
biogenesis of pri-miRNA-96 by binding to the Drosha processing site [78]. Inhibition
of miRNA processing by Drosha resulted in a de-repression of the miR-96 target
FOXO1, thus triggering apoptosis of cancer cells [78]. TGP-96 was converted into
a bleomycin degrader (TGP-96-bleo) via amide coupling to the bleomycin A5 free
amine [76] (Figure 9.1c). Similar to the results seen for Cugamycin, attaching the
RNA binder to the acylated bleomycin conjugate reduced DNA cleavage in vitro and
decreased DNA damage in cells when compared to free bleomycin [76].
Moreover, since TGP-96 inhibits processing of pri-miR-96 by binding to the
Drosha processing site, its mode of action increases pri-miR-96 levels while decreasing mature miR-96 levels. However, because TGP-96-bleo functions via direct
degradation of the RNA, a reduction in both mature and pri-miR-96 levels was
observed upon treatment with 500 nM of TGP-96-bleo [76]. Additionally, miRNA
sequencing revealed that upon TGP-96-bleo treatment, miR-96 was downregulated
to a greater and more statistically signicant extent than the rest of the miRNAs
detected [76].
Importantly, this study developed a method to identify the precise small-molecule
binding sites within RNA targets in cells, dubbed Ribo-SNAP-Map [76]. To do so,
cells were treated with TGP-96-bleo, TGP-96, or an acylated bleomycin compound
alone. RNA was extracted from the cells and subjected to reverse transcription
quantitative polymerase chain reaction (RT-qPCR) using a gene-specic forward
primer for pri-miR-96 and a universal reverse primer. The resulting cDNA was then
subjected to Sanger sequencing. Because TGP-96-bleo functions by cleaving the
pri-miR-96 RNA target, one would expect to identify an increase in cleaved RNA
fragments. Indeed, cDNA corresponding to cleaved RNA fragments was identied
uniquely in TGP-96-bleo-treated samples, corresponding to a cleavage site 5–7
nucleotides away from the predicted and in vitro-validated binding site for TGP-96
[76]. These studies both conrmed target engagement and mapped the binding site
of TGP-96-bleo to pri-miR-96 in cells.
9.2.3.4 Conclusions and Outlook for Bleomycin-Based Direct Degraders
To date, bleomycin conjugates have shown promise as chemical biology tools both
in cells and in vivo [66, 76]. By directly appending RNA-binding moieties to the
DNA-binding domain of bleomycin A5, the nuclease-like ability of bleomycin to
cleave nucleotides can be co-opted to target RNA specically. This has resulted in
the design of heterobifunctional small molecules capable of inducing degradation of
target RNAs in cells, with the most promising degrader, Cugamycin, showing activity in a DM1 transgenic mouse model. Additionally, as a natural product, bleomycin
has been optimized by nature to both enter cells and cleave nucleotides.
However, optimization of bleomycin conjugates by medicinal chemistry is limited. While studies with deglycobleomycin conjugates showed less o-target DNA
cleavage while retaining RNA cleavage, more signicant chemical optimization of
bleomycin is synthetically dicult. While total synthesis of bleomycin, allowing for
alteration of the various domains, is possible, it is realistically impractical for use in
development of mass-produced drugs. For this reason, future endeavors to optimize
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