Small Molecule RNA Degraders

Abstract

RNA is a central molecule in life, involved in a plethora of biological processes and playing a key role in many diseases. Targeting RNA emerges as a significant endeavor in drug discovery, diverging from conventional protein‐centric approaches to tackle various pathologies. Whilst identifying small molecules that bind to specific RNA regions is the first step, the abundance of non‐functional RNA segments renders many interactions biologically inert. Consequently, small molecule binding does not necessarily meet stringent criteria for clinical translation, calling for solutions to push the field forward. Converting RNA‐binders into RNA‐degraders presents a promising avenue to enhance RNA‐targeting. This mini‐review outlines strategies and exemplars wherein simple small molecule RNA binders are reprogrammed into active degraders through the linkage of functional groups. These approaches encompass mechanisms that induce degradation via endogenous enzymes, termed RIBOTACs, as well as those with functional moieties acting autonomously to degrade RNA. Through this exploration, we aim to offer insights into advancing RNA‐targeted therapeutic strategies.

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RNA Degraders
Small Molecule RNA Degraders
Javier Bonet-Aleta,* Tomoaki Maehara, Benjamin A. Craig, and Gonçalo J. L. Bernardes*
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Abstract: RNA is a central molecule in life, involved in a plethora of biological processes and playing a key role in many
diseases. Targeting RNA emerges as a significant endeavor in drug discovery, diverging from conventional protein-
centric approaches to tackle various pathologies. Whilst identifying small molecules that bind to specific RNA regions is
the first step, the abundance of non-functional RNA segments renders many interactions biologically inert.
Consequently, small molecule binding does not necessarily meet stringent criteria for clinical translation, calling for
solutions to push the field forward. Converting RNA-binders into RNA-degraders presents a promising avenue to
enhance RNA-targeting. This mini-review outlines strategies and exemplars wherein simple small molecule RNA binders
are reprogrammed into active degraders through the linkage of functional groups. These approaches encompass
mechanisms that induce degradation via endogenous enzymes, termed RIBOTACs, as well as those with functional
moieties acting autonomously to degrade RNA. Through this exploration, we aim to offer insights into advancing RNA-
targeted therapeutic strategies.
1. Introduction
Nucleic acids are essential components that underpin all life
processes. While DNA encodes the information to build and
maintain an organism, RNA functions as both a messenger
between DNA and proteins and a regulator of genome
organization and gene expression.[1] RNA occupies the
pivotal position in the so-called “central dogma of molecular
biology,” which describes the flow of information within a
living system, from DNA to the final protein. The central
role of RNA in cell biology[2] makes this intermediary
molecule a unique target with immense potential to target
numerous diseases, as defects or dysregulation of certain
RNAs are implicated in many pathologies.[3] Targeting RNA
will also expand the existing druggable genome (Figure 1a).
This stems from the extensive transcription (~70 %) of the
human genome into non-coding RNA (i. e. RNAs without
protein-coding regions) with only 1.5 % encoding proteins,[4]
which are the target of most of small molecule drugs. Given
that, out of ~20,000 human proteins, fewer than 700 have
been targeted by approved drugs,[5] this indicates that only
0.05 % of the human genome has been drugged so far.[6]
Drugging RNA can be conventionally achieved either
through use of synthetic oligonucleotides which base pair to
a target sequence, used in siRNA and antisense oligonucleo-
tide (ASOs) strategies, or by binding its folded tertiary
structures, usually with small molecules.[7,8] By 2023, up to
eight ASOs and five siRNA are currently approved by the
FDA for the treatment of a diverse range of diseases,
including spinal muscular atrophy, atherosclerotic cardiovas-
cular disease or acute hepatic porphyria, among others.[9]
Although binding to target RNA is usually highly specific,
this strategy faces important challenges. First, the highly
negatively charged backbone present in most synthetic
oligonucleotides typically entails poor cell and/or tissue
permeability and, ultimately, delivery issues.[8,10] Addition-
ally, therapeutic oligonucleotides are prone to be degraded
by endogenous RNases intracellularly or by serum
nucleases,[9] leading to low metabolic stability.[11] Finally,
clinical trials have revealed myriad adverse reactions, renal
clearance, and immunogenicity issues.[11]
Targeting RNA with small molecules may negate many
of these limitations. In general, the use of small molecules
improves pharmacokinetic properties, especially in terms of
metabolic stability and cell permeability.[12] The first exam-
ple of targeting RNA with this approach dates from 1940
with the discovery of streptomycin, whose antimicrobial
activity was derived from its binding to the prokaryotic
ribosomal RNA.[13] Since then, important milestones were
reached in the 60s, 2000 and 2020 with the discovery of
RNA binders, such as macrolides, oxazolidinones or pre-
mRNA splicing modulators, respectively.[14] Small molecules
which bind to a functional site of RNA aim to disrupt
downstream biology in a controlled way, ultimately leading
to a therapeutic benefit (Figure 1b).
The discovery of small molecule binders can be challeng-
ing due to RNA’s inherent properties. Firstly, its negatively
charged sugar-phosphate backbone restricts the number of
compatible structures,[15] and the number of possible combi-
nations of the four nucleobases that comprise RNA (A, U,
G, C) is considerably lower than the 20 major amino acids
that form proteins.[16] Despite this, the combination of
computational tools, large databases gathered from high-
throughput experimentation, and novel screening assays
have driven the discovery of new RNA-small molecule
binders.[17] Secondly, the binding event must occur at a
functional RNA site; otherwise, it will be biologically silent,
and no phenotypic effect is expected to be observed. For
some small molecule binders, it has been predicted that
70 % of binding sites are non-functional.[18] Converting RNA
binders into degraders allows the exploitation of these non-
functional binding sites, facilitating therapeutic targeting.[19]
Considerable efforts have been directed towards degrading
RNA instead of merely binding to it since the early 1990s,
particularly using ASOs.[20] Recent findings in the field
suggest that the use of small molecule RNA degraders will
have an even stronger effect on the downregulation of
disease-causing RNAs.[18,21]
[*] Dr. J. Bonet-Aleta, Dr. T. Maehara, B. A. Craig,
Prof. G. J. L. Bernardes
Yusuf Hamied Department of Chemistry
University of Cambridge, CB2 1EW Cambridge, United Kingdom
E-mail: gb453@cam.ac.uk
jb2648@cam.ac.uk
© 2024 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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All known small molecule degraders have a common
architecture (Figure 1c). This involves the utilization of
bifunctional molecules comprising three separate parts: (1) a
small molecule moiety that recognizes the target RNA,
referred to as the binder; (2) another moiety which triggers
—directly or indirectly—the degradation of the targeted
RNA; and (3) a linker between the binder and the degrader.
This approach encompasses sequential two-stage RNA
targeting: first, the binder attaches to a specific region of an
RNA target (Figure 1c-1); subsequently, the degrader moi-
ety promotes the cleavage of RNA (Figure 1c-2). Degrada-
tion of RNA has been successful using three different
elements so far: (i) a RNase recruiter, or (ii–iii) an organic
cleavage unit, termed as warhead, which act through (ii) a
base-mediated mechanism (particularly using imidazole) or
(iii) through metal-mediated catalysis. Based on this, our
review aims to summarize and categorize the existing small
molecule RNA degraders, highlighting their current advan-
tages and limitations to provide a motivation for further
development of the field.
2. Ribonuclease Targeting Chimeras
RNases are ubiquitous enzymes found in every organism,
which catalyze the degradation of RNA and can be
exploited for targeted RNA degradation, a strategy termed
Ribonuclease Targeting Chimeras (RIBOTACs). This ap-
proach draws lessons from Proteolysis Targeting Chimera
(PROTACs) technology, initially conceptualized in 2001 by
Craig M. Crews[26] group. In PROTACs, a bifunctional
molecule induces proximity between the targeted protein
and an endogenous ubiquitin E3 ligase to trigger protein
ubiquitination, ultimately leading to protein degradation.[27]
RIBOTACs work in an analogous fashion, using bifunc-
tional molecules to sequentially i) bind the targeted RNA
and ii) recruit an endogenous RNase, mostly RNase L, to
degrade the target (Figure 2a). RNase L is part of our innate
immune defense, predominantly against viruses. Typically, it
is found as two inactive monomers (Figure 2a). Upon viral
infection, a short oligonucleotide sequence is produced, 2’, 5
oligoadenylate (2’-5’ poly(A)), which interacts with RNase
L monomers and fosters their dimerization into an active
dimeric complex.[28] The activated RNase L finally cleaves
the viral RNA to inhibit further infection. RNase L also
cleaves other substrates such as rRNA, mRNA, or tRNA,
aiding regulation of their diverse biological functions.[29] The
first example of in vitro recruitment of RNase L for targeted
RNA degradation dates to 1993,[20] but used an ASO rather
than a small molecule. A tail of 2’-5’ poly(A) was covalently
linked to an ASO, allowing recognition of a specific region
of HIV RNA while recruiting endogenous RNase L,
mimicking the natural response mentioned above. These
conjugates even achieved positive results in African Green
Monkeys infected with respiratory syncytial virus,[37] reduc-
ing viral replication.
However, these ‘protoRIBOTACs’ had to deal with the
limitations of using oligonucleotides as binding ligands,[8,10]
as mentioned in Section 1. The first use of RIBOTACs was
reported in 2018 by the M. Disney group,[30] targeting
Javier Bonet-Aleta is a Herchel–Smith
postdoctoral fellow at the Yusuf-Hamied
Department of Chemistry at the Univer-
sity of Cambridge. He obtained his BSc
in Chemistry (2018) with honors and his
MS in Nanotechnology (2019) from the
University of Zaragoza, Spain. He com-
pleted his Ph.D. at the Institute of Nano-
science and Materials of Aragon (2023)
under the supervision of Prof. Jesus
Santamaria and Dr. José L. Hueso. His
research focuses on finding synergies
between small molecule drugs and met-
al nanoparticles to improve the efficacy
of cancer treatment.
Tomoaki Maehara is a visiting research-
er at the Yusuf-Hamied Department of
Chemistry at the University of Cam-
bridge. He studied chemistry at the
University of Tokyo and received his
Ph. D. in 2017 under the guidance of
Prof. Tohru Fukuyama at Nagoya Uni-
versity, focusing on total synthesis of
natural products. He then joined Kyowa
Kirin Co., Ltd. as a medicinal chemist,
working on several oncological and
immunological projects. His current re-
search interest is to develop chemistry-
driven novel therapeutics, such as tar-
geted RNA degradation.
Benjamin Craig is a PhD student at the
Yusuf-Hameid department of Chemistry
in Cambridge. He obtained his BA and
Msci in Natural sciences, specialising in
Chemistry, at the Univeristy of Cam-
bridge, graduating in 2022. His master’s
project was undertaken under the super-
vision of Professor Erwin Reisner, study-
ing electrochemical, biohybrid ap-
proaches to CO2reduction. His current
research area is in the development of
novel RNA degrading warheads, and in
better understanding their mechanism
of action.
Gonçalo Bernardes is a Professor of
Chemical Biology at the University of
Cambridge. After completing his D. Phil.
degree in 2008 at the University of
Oxford, UK, he then performed postdoc-
toral work at the Max-Planck Institute of
Colloids and Interfaces, the ETH Zürich.
His research group interests focus on the
use of chemistry principles to provide
new biological insights and derive new
targeted therapeutics. He is a first gen-
eration high-school and university grad-
uate in his family. He has recently
received the 2024 Corday-Morgan Prize
for Chemistry.
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oncogenic miRNA-96, which plays important roles in
breast[38] and pancreatic[39] cancers with functions including
regulation of apoptotic response and cell proliferation. They
combined the 2’-5’ poly(A) recruiter (Figure 2b) with two
binders (Figure 2c, RIBO-1) reported to target the miR-96
hairpin precursor.[40] In vitro treatment of MDA-MB-231
with 200 nM (48 h) of RIBOTAC RIBO-1+R1 led to 2-fold
increased expression of FOXO1, a protein repressed by
miRNA-96 involved in promoting apoptosis via Bclxl
proteins.[41] Comparing the biological activity of this RIBO-
TAC with the binder alone, the authors concluded that
recruitment of RNase L led to a 5-fold increased activity.[30]
The use of 2’-5’ poly(A) recruiter (Figure 2b) has also been
combined with a single ligand (Figure 2b, RIBO-2) to target
pre-miR-210,[42] a ncRNA overexpressed in hypoxic tumors
which, essentially, represses Glycerol-3-phosphate dehydro-
genase 1-like (GDP1L) mRNA. GDP1L encodes a protein
that binds to prolyl hydroxylase, an enzyme which triggers
hydroxylation of the Hypoxia-Inducible factor 1-alpha
(HIF1α) and its ultimate polyubiquitination for proteaso-
mal-induced degradation.[43] Therefore, overexpression of
miR-210 yields reduced degradation of HIF1α, and the
expression of HIF-related genes related to proliferation and
metastasis.[44] By using a ligand with high affinity for pre-
miR-210 (Figure 2b, RIBO-2), other RNAs present in the
cell remained unchanged upon the treatment with
Figure 1. Drugging RNA with small molecules. (a) The potential to combat diseases increases significantly when RNA is targeted instead of
proteins. In essence, only a small fraction (~1.5 %) of the human genome contains the information to synthesize coding RNA, which is ultimately
translated into a protein. Additionally, only a small percentage of the approximately 2,000–3,000 proteins associated with a disease[22] have been
successfully targeted with drugs.[6] (b) Small molecule binders exert their action by binding to a specific region of an RNA target. Example shown
consists of a benzimidazole[23] binding to the internal ribosomal entry site in the Hepatitis C virus RNA (PDB: 3tzr)[24] to inhibit translation.
(c) Small molecule degraders consist of a binder group, which recognizes a structural site of RNA, and a degrader domain, which triggers the
degradation of the targeted RNA either by recruiting an endogenous RNase or by the action of a cleavage unit. This can be mediated by an organic
base, such as an imidazole- or by a metal-binding unit which enables oxidative cleavage, such as bleomycin. Represented RNA is pre-miR-31, an
oncogenic RNA[25] (PDB:8fcs).
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RIBOTAC.[42] An alternative to 2’-5’ poly(A) was reported
in 2020 by the M. Disney group to recruit an endogenous
RNase L.[31] The structure (see Figure 2b, Recruiter 1) was
previously identified in 2007 to activate RNase L.[45] Interest-
ingly, the introduction of Recruiter 1 in the structure of the
final RIBOTAC (R2 +RIBO-4) did not negatively affect
drug uptake by the MDA-MB-231 cells,[46] whilst the use of
2’-5’ poly(A) as an RNase recruiter decreased RIBOTAC
uptake by the same cell line.[30,42] Reflecting this, the RNase
recruiter of most RIBOTACs reported to date is Re-
Figure 2. Targeted RNA degradation by exploiting endogenous nucleases. (a) Overview of RIBOTAC strategy. RIBOTACs utilize bifunctional small
molecules, composed of a binding ligand with a high affinity for a specific structure of a particular RNA, and an RNase recruiter to target and
degrade RNA. After the RIBOTAC binds to the RNA through the ligand, the free recruiter moiety interacts with endogenous RNase, ultimately
degrading the RNA. (b) Summary of the discovered RNase recruiters linked to small molecule binders to date. References for the first use of R1,
R2, and R3 linked to small molecules are,[30,31] and,[32] respectively. (c) Outline of the binders tested in RIBOTAC strategy along with their Kdfor the
targeted RNA, mentioned in parentheses. References for RIBO-6, RIBO-7, RIBO-8 and RIBO-12 are,[33,34,35] and,[36] respectively.
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cruiter 1. Diseases such as SARS-CoV-2 infection have also
been approached through RIBOTAC strategy,[47] even using
non-biological RNase recruiters (for example, Recruiter 1+
RIBO-5[47b]), demonstrating the broad applicability of this
new family of therapeutics.
A breakthrough for RIBOTACs came in 2023. Tong
et al.[18] identified a novel class of compounds, mainly
containing azolium groups (Figure 2c, RIBO-11), which bind
to a 3D-fold RNA library by using a high-throughput
screening approach. However, only ~30 % of the targetable
sites within screened RNAs are functional, reducing signifi-
cantly the potential biological effect of the small molecule-
RNA interaction.[18] Transforming the binders into RIBO-
TACs by including Recruiter 1 into their molecular structure
can overcome the above limitation: three oncogenic RNAs
were targeted using this strategy, including c-MYC (RIBO-
9), JUN (RIBO-10), and pre-miR 155 (RIBO-11).[18] Addi-
tionally, Zhang et al.[48] improved the selectivity of a
RIBOTAC which targets G-quadruplex enriched oncologic
mRNAs by caging a RNase recruiter, similar to Recruiter 1,
through different strategies. By masking the RNase recruiter
with different functional groups which can be in situ
activated by NQO1, an enzyme biomarker in tumors, or
tumor overexpressed metabolites such as H2O2or selenocys-
teine, it is possible to tune the selectivity of the RIBOTAC
towards a specific cell line under or towards specific
chemical conditions. Preliminary therapeutic results ob-
tained with RIBOTACs are promising, as this technology
has the potential to transform an inactive RNA-ligand
interaction into active degradation, improving the final
biological effect. RIBOTACs’ main limitation lies in their
dependence on intracellular RNase L levels, which are
typically low[49] and some studies suggest depending on cell
type.[50] Thus, some cell types may not be compatible with
RIBOTAC approach. In addition, although transforming a
small molecule binder into a RIBOTAC partially overcomes
the non-functional binding site issue, binding must take
place close to an RNA position susceptible to RNase L
cleavage. Taken together, these caveats can limit the
therapeutic efficacy of RIBOTACs in some scenarios.
3. Mimicking Natural RNases using
Imidazole-Based Warheads
Achieving an autonomous RNA degradation is highly
attractive to remove the dependency of endogenous ele-
ments such as RNase L. Many artificial approaches at RNA
cleavage are inspired by the mechanism of natural RNase A
(Figure 3a), a strategy pioneered by Breslow et al. in the
1970 s.[51] The active site is composed of two histidine
residues, His12 and His119, whose side chains containing
imidazole rings act as acid-base catalysts,[52] in addition to
Lys41, which stabilizes the increasing negative charge on
nonbridging phosphoryl oxygen in both transition
structures.[52a] In the first step (Figure 3b, (1)), the pyrrolic N
of His12 abstracts an H from the 2’-OH subsequently
forming a 2’,3’-cyclic phosphodiester. The acidic pyrrolic N
of His119 facilitates the displacement of the reaction by
Figure 3. Autonomous RNA degradation using imidazole-based warheads. (a) Illustration of the active site in RNase A, highlighting the catalytic
role of the histidine residues His12 and His119 (PDB: 7rsa). (b) Mechanism of catalytic cleavage of RNA assisted by His12 and His119 residues of
RNase A, involving step (1) where the pyrrolic N of His12 abstracts the H from the 2’ OH group, complemented by the acidic properties of the
pyrrolic NH of His119[52a] and leading to the cleavage of RNA. In a step (2), the His119 catalyzes the hydrolysis of the cyclic phosphodiester to
recover the active catalytic species.[52a] (c) Summary of the chemical structures of RNA degraders based on imidazole warheads and their target
RNA.
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protonating the 5’-oxygen of the next nucleoside, ultimately
leading to the cleavage of two nucleoside units (Figure 3b).
Finally, the basic imidazole from His119 catalyzes the
hydrolysis of the cyclic phosphodiester to recover the active
catalytic species (i. e. a deprotonated pyrrolic N in His12 and
an acidic pyrrolic N in His119, Figure 3b).[52a]
Following this strategy, small molecule RNA degraders
can be synthesized by linking an RNA binder (Figure 3c,
highlighted in red) with an imidazole containing warhead
(Figure 3c, highlighted in blue). The mechanism of action
works in a sequential manner: first, the binder recognizes a
specific region of the targeted RNA; then, induced prox-
imity allows the imidazole warhead to cleave the RNA.[53]
Numerous attempts have been made to cleave RNA with
metal free organic molecules based on imidazole[54] or
guanidium,[55] but these required a large concentration and
led to inefficient degradation in vitro. This is probably due
to the lack of a binding ligand incorporated in the molecule
structure, thus emphasizing the importance of the proximity
induced effect.[56] The first use of a liganded imidazole-based
RNA degrader was reported in 2015 by Nguyen et al.[57]
(Figure 3c, Imi-1) for targeting myotonic dystrophy type 1
(DM1). In this disease, an abnormal expansion of CTG
trinucleotide repeats (referred to as (CTG)exp) occurs in
the 3’-untranslated region of the DMPK gene.[58] This
expansion is transcribed into a (CUG)exp RNA transcript,
which sequesters the alternative-splicing regulator muscle-
blind-like protein (MBNL), ultimately leading to disease
symptoms. Developing binding competitors of (CUG)exp
with oligonucleotides, peptides or small molecules has been
demonstrated to improve DM1-associated defects.[59] In their
work, Nguyen et al. designed bifunctional molecules by
combining a binder with a high affinity for (CUG)exp,
previously identified by the same research group,[60] with a
cleavage subunit containing an imidazole.[57] Treatment with
50 μM of Imi-1 for 72 h in DM1 model cells led to a
reduction of the 60–70 % of CUGexp mRNA levels in cell
culture, and in Drosophila disease phenotypes. Similar to
RIBOTACs, imidazole-based degraders have also been used
to degrade viral RNA. In 2022, M. Duca’s group reported a
small molecule degrader to target the trans-activating region
(TAR) of the HIV-1 virus RNA[61] (Figure 3c, Imi-2) based
on a neomycin binder, reported to recognize the TAR
region of HIV-1 RNA.[62] They connected a histidine residue
via Copper-catalyzed Azide-Alkyne Cycloaddition
(CuAAC) to the neomycin unit to form Imi-2 compound.
The small size of the degradation warhead (i. e., histidine)
yielded a negligible effect on the Kdvalue for the TAR
region of HIV-1 RNA (1.7 μM for neomycin vs. 1.4 μM for
Imi-2, respectively[61]), while providing the cleavage of the
target RNA, achieving a IC50 value for the inhibition of Tat/
TAR interaction below 0.5 μM in vitro. Improved activity
was observed at basic pH, suggestive of histamine acting as a
general base.
Two further examples of targeting viral RNA were
reported by our laboratory and collaborators in 2023.[21] Two
small molecules, termed Proximity Induced Nucleic Acid
Degraders (PINADs), were validated to target two key
structures found in SARS CoV-2 genome:[63] G-quadru-
plexes (Imi-3, Figure 3c) and betacoronaviral pseudoknots
(Imi-4, Figure 3c). These RNA structures play key roles in
stabilizing viral proteins[63a] and producing vital enzymes for
viral replication,[64] respectively, and are therefore appealing
targets for novel antiviral therapeutics. The selection of Imi-
3 binder was motivated by its high affinity for G-quad-
ruplexes, with a Kdof 26 nM,[65] while Imi-4 binder was
previously demonstrated to bind SARS-CoV-2
pseudoknot.[66] As proof of its good performance, the
incubation of Imi-4 with pseudoknot oligomers resulted in a
~75 % degradation after 3 h, without affecting levels of a
mutated control strand, demonstrating the absence of off-
targeting. In vivo experiments with Imi-4 showed a signifi-
cant reduction of the lung viral load, and a marked
reduction in the expression of p38 protein, a biomarker of
SARS-CoV-2 infection and replication.[67] PINAD-assisted
RNA degradation may take place following two different
mechanisms: (1) a general base mechanism, mimicking
RNase A activity (Figure 3b) and (2) a copper-mediated
mechanism, where the imidazole warhead is prone to
coordinate copper ions that may ultimately cleave the RNA
through the production of ROS.[53] This latter mechanism is
further discussed in the next section.
4. Transition-Metal Catalysis for RNA Cleavage
In fact, the combination of transition-metal catalysis and
organic molecules has been an area of exploration for RNA
degradation since the early ‘80s. Several molecules contain-
ing a metal-coordinating group such as amines[68] or
pyridines[69] have been demonstrated to cleave RNA in a
test tube but rarely in cells. Again, for an efficient RNA
degradation it is imperative to use a binder to achieve
proximity. Prior to small molecules, the design was based on
a metal-coordinating group attached to an oligonucleotide
which provides the recognition, and ultimately the proxim-
ity-induced effect. Some examples of metal coordinating
groups utilized include phenanthroline-copper,[70] pyridine-
zinc[71] or amine crown-zinc system,[72] as well as various
lanthanide chelates,[73] to the corresponding antisense oligo-
nucleotide.
Joyner et al. conducted a systematic analysis of different
metal-coordinating ligands (DOTA, EDTA or NTA, among
others) linked to a Rev peptide,[74] known for binding to
HIV-1 RNA,[75] with different metal ions such as Cu2+, Co2+,
Fe2+or Ni2+. The presence of the oxidative system with
ascorbate/H2O2promoted a fast cleavage of RRE RNA,
especially in the Cu-NTA-Rev system. The authors explain
this trend by analyzing the reduction potential, E0, of the
conjugates. The most active complexes had their E0between
380 and 66 mV which matched with the E0values of H2O2/
HO*and ascorbyl/ascorbate reactions,[76] so multiple-
turnover single-electron oxidation and reduction reactions
are thermodynamically favored for the complexes with their
E0in this range. Further investigations by the same research
group concluded that the RNA cleavage was through 5’-
hydrogen abstraction, but hydrolysis and 2’-OH endonu-
cleolysis also contributed to degradation.[77]
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Transforming an RNA small molecule binder into an
active, autonomous degrader by using imidazole-containing
or metal-containing warheads has the potential to provide a
high turnover in terms of therapeutic action. Ideally, a
complete turnover cycle would involve (i) selective recog-
nition and interaction with the desired RNA target through
the binder, (ii) the irreversible degradation of RNA via
either general acid/base or transition-metal catalysis, (iii)
dissociation from the degraded RNA and (iv) regeneration
of the active catalytic species to repeat the cycle. This
‘catalytic’ mechanism of action could offer important
advantages over traditional drug-binding systems. Apart
from the improvement entailed by the irreversible RNA
degradation, a cyclic oxidative cleavage of RNA has the
potential to reduce the amount of drug required to reach the
target organ/tissue, as one molecule can cleave multiple
RNAs. Moreover, no mechanisms of repair of mRNA are
known,[78] thereby reducing the possibility of chemoresist-
ance by target cells, especially in cancer disease. An
important challenge in this perspective is achieving fast
reaction kinetics of RNA cleavage to provoke a strong
biological response. The use of transition metal catalysis in
cells is sometimes restricted by the precise regulation cells
exert on metals, ultimately leading to their intracellular
deactivation. Glutathione (GSH) is the main antioxidant
molecule present in mammalian cells, with very large intra-
cellular concentrations in the range of 2–10 mM.[79] Apart
from preventing oxidative damage via GPX4 enzyme, GSH
is a major compound in regulating the trafficking of intra-
cellular metal ions due to its strong coordinating
properties,[80] transferring the captured metal to metallothio-
nein for further disposal.[81] Therefore, GSH is mostly
responsible for the deactivation of transition metal catalysis
within cells.[82] In this way, significant attention should be
paid to synergies of PINADs or metal-coordinating war-
heads with GSH-depleting systems to drive efficient RNA
degradation.
Naturally produced by the fungus Streptomyces verticil-
lus, bleomycin is a drug currently used to treat various
cancers such as testicular or ovarian cancer, or Hodgkin
lymphoma.[83] Bleomycin’s primary mechanism of action
involves inducing cleavage of DNA strands in a process
dependent on iron and oxygen.[84] This CP450-like activity
led to the exploration of bleomycin for targeted RNA
degradation in the early ‘90 s.[85] The structure of bleomycin-
based RNA degraders consist of three domains (Figure 4a).
The carbohydrate moiety facilitates cell internalization[86]
(Figure 4a). Then, the metal-binding domain can interact
with iron ions via N-coordination.[84a,87] The bleomycin-Fe
complex can activate molecular O2to produce oxidative
cleavage of RNA through formation of FeV
=O centers.[84] To
achieve the proximity induced effect, a third domain (Fig-
ure 4a, highlighted in red), was linked to bleomycin through
one of its terminal amines, consisting of an RNA binder.
The first example of a Bleomycin A5-binder conjugate was
reported by M. Disney group in 2017 for treatment of DM1
by cleaving r(CUG)exp (see Section 2).[59] Interestingly, small
molecules (Figure 4b, Bl-1) showed a 100-fold increase in
potency when compared to antisense oligonucleotides.
Apart from lower cell uptake, oligonucleotides are prone to
suffering slow kinetics of binding to structured RNAs[88]
highlighting the advantages of using small molecules over
ASOs. Moreover, no cleavage of r(CUG)exp RNA was
observed in the presence of bleomycin alone, showcasing
again the pivotal importance of a binder to achieve a
proximity-induced effect[59] and target selectivity. It is also
possible to tune the chemical structure of bleomycin to gain
selectivity and reduce off-targeting.[89] As mentioned above,
bleomycin has partial affinity by DNA that may provoke
undesired damage.[84a] By removing the carbohydrate do-
main of bleomycin-A5, the amount of phosphorylated
histone H2 A variant H2AX (γ-H2AX), which forms foci in
response to DNA damage, was reduced significantly.[89] In
terms of the linker between the binder and the bleomycin
unit, a peptoid linker containing proline units performed
better in RNA degradation than others containing alanine,
tyrosine or hydroxylated proline, and reduced the off-target
DNA cleavage.[90] Although the efficacy of using bleomycin
as a warhead has been demonstrated, these types of RNA
degraders depend on endogenous iron and oxygen levels,
which may be a limitation depending on the targeted
disease. For example, numerous tumors are hypoxic,[91]
where the efficacy of bleomycin may be limited due to
oxygen scarcity. Consequently, we believe it is imperative to
consider optimizations not only in terms of the chemical
structure, but also the endogenous fuel availability (i. e. iron
ions and oxygen) of the targeted cell type to achieve the
desired reaction kinetics and, thus, the successful therapeutic
outcome.
5. Conclusions and Outlook
Recent breakthroughs in the discovery and design of small
molecule RNA binders pave the way to developing active
RNA degraders by adding small functional groups which
induce RNA cleavage. Depending on the degradation
mechanism and the nature of the functional group, we have
classified the existing small molecule degraders into three
categories: (1) RIBOTACs, where the functional group is a
RNase L recruiter which drives RNA degradation through
dimerization of an endogenous RNase L; (2) base-mediated
warheads and (3) metal-based which degrade RNA through
a specific mechanism involving metal coordination to the
warhead and subsequent ROS production. When targeting
RNA, the advantages of using small molecule ligands over
traditional oligonucleotides are significant, especially in
terms of pharmacokinetic properties and cell permeability,
as well as due to the high stability of duplexed RNA.
Furthermore, recent in vitro and in vivo experimental results
strongly suggest the necessity of transforming RNA binders
into RNA degraders for maximum functionality[18,21]—ideally
one would be able to cleave the targeted RNA specifically
and catalytically. This would result in maximum therapeutic
efficacy using low amounts of a small molecule drug. We
anticipate that the insights described at the end of each
section of this minireview will foster further developments
of small molecule warheads that add function—degradation
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—to neutral RNA binders, especially if these novel
technologies are well integrated with existing research using
ASOs to direct RNA cleavage. The discovery of novel RNA
binders, especially aided by high-throughput experimenta-
tion and tools as ML/AI has the potential to expand the
number of targets exponentially. Such research will assist
the development of small molecule RNA degraders as
valuable drugs.
Acknowledgements
J.B.-A. acknowledges Herchel–Smith Fund for financial
support. T. M. acknowledges Kyowa Kirin Co., Ltd for
support. B. A. C acknowledges the Yusuf-Hamied Depart-
ment of Chemistry for financial support.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
Keywords: RNA ·small-molecule ·degradation ·
proximity-induced ·therapy
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Manuscript received: July 9, 2024
Accepted manuscript online: August 20, 2024
Version of record online: October 17, 2024
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