New Progress of Targeted Degradation Based On Nucleic Acid

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New Progress of Targeted Degradation Based

On Nucleic Acid

Targeted protein degradation (TPD) is a promising strategy in the field of drug
discovery. Proteolysis targeting chimera (PROTAC) is the most representative
technology of TPD strategy, which uses the ubiquitin-proteasome system (UPS) to
degrade target proteins containing cytosolic domains [1]. In addition
to PROTAC, lysosome-targeting chimaeras (LYTAC), antibody-based PROTAC
(AbTAC), and autophagy targeting chimeras (AUTOTAC) have been successful in
degrading membrane proteins, extracellular proteins, and protein aggregates, thus greatly
expanding the range of target proteins.

Figure 1: Ubiquitin-Proteasome System (UPS) [1]

However, the application of PROTAC is limited to protein targets containing cytosolic
domains that can be linked to sites. The ligands that bind the target proteins are usually
determined by high-throughput screening and design. For proteins lacking a clear active
site, the development of small molecule ligands is difficult and time-consuming [2].
Encouragingly, nucleic acid-based strategies have opened up new ways to degrade
protein targets recently.

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Nucleic acid-based TPD has the following advantages: First, it expands the range of
intracellular target proteins. PROTAC, which uses nucleic acid motifs as warheads, has
been successfully used to degrade proteins that lack active ligand-binding sites, including
RNA-binding proteins (RBPs), transcription factors (TFs), and G-quadruplex (G4) -binding
proteins. Second, it can be used to develop platforms for targeted degradation of
membrane proteins (e.g., bispecpecic aptamer chimeras). Nucleic acid aptamers can also
be used as targeted delivery tools for tumor-specific targeted degradation. Third, nucleic
acid motifs can be used as substrates for targeted degradation in the treatment of RNA
diseases. An emerging RNA degradation technology—ribonuclease targeting chimeras
(RIBOTAC)—suggests that the chimeric degradation principle of PROTACs has been
extended to the RNA realm. This review introduces the newly emerging TPD
strategies based on nucleic acids as well as new strategies for targeted
degradation of nucleic acid (RNA) targets[3].

Newly Emerging TPD Strategies Based On

Nucleic Acids

1. Targeting RNA-binding proteins

RNA-binding proteins (RBPs) belong to a special class of nucleic acid interactors
involved in various important cellular processes. However, most RBPs lack traditional
enzyme pockets that can efficiently bind small molecules, and the presence of their
homologous protein domains can also lead to limited intracellular activity of small
molecules.

To address these issues, Hall's team developed an RNA-PROTAC that utilizes short RNA
mimics -- homologous to the RNA common binding element (RBE) of RBP -- as warheads
that bind RBE and E3 to recruit peptides that mediate proteasome degradation [4] (FIG. 2).
The first RNA-PROTACs were designed to degrade two RBPs: stem cell factor LIN28
(Lin28A) and splicing factor RBFOX1. Although RNA-PROTAC is an exciting method
for RBPs degradation, it still has some drawbacks, such as the instability of RNA
oligomers in vivo and the need for RNA secondary structure to properly interact with
RBPs.

Figure 2: Schematic diagram of the RNA-PROTACs design strategy

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2. Targeting transcription factors

Transcription factors (TFs) are DNA-binding proteins that interact with specific DNA
sequences to control chromatin and transcription and are important drivers of many
diseases, especially cancer. Nearly 300 oncogenic TFs account for approximately 20% of
all known oncogenes. However, the majority of oncogenic TFs do not have a natural
ligand-binding pocket and have long been considered undruggable targets. Although TPD
technologies targeting TFs have recently been developed, traditional inhibitor-based
degradation strategies are not applicable to all TFs targets. To address this issue, a
variety of nucleic acid-based degraders targeting TFs have emerged, as shown in the
table below (Table 1).

Table 1: Examples of small molecule and nucleic acid-based degraders of TFs

Crews' team developed transcription factor targeting chimeras (TRAFTACs) that
induce targeted TF degradation by selecting cellular degradation
mechanisms. TRAFTACs are heterobifunctional chimeric oligonucleotides consisting of
two parts: double-stranded DNA that binds the target TF and CRISPR-RNA that binds the
dCas9-Halotag7 fusion protein. TRAFTAC recruits the VHL E3 ligase complex via
dCas9HT7 to the vicinity of target TFs, which are then labeled with ubiquitin and degraded

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by UPS. Wei and Jin's team developed the TF-PROTACS universal platform (Figure 3A)
to assemble azide modified DNA oligomers (N3-ODN) with various linkers and bicyclic
octane modified VHL ligand (VHLL-XBCN) for selective TF degradation (Figure 3B).

Figure 3: Design strategy (A) and representative chemical structure (B) of TF-PROTACs

3. Targeting G4-binding proteins

G-quadruplex (G4s) is a special four-stranded nucleic acid structure rich in guanine
bases, which is formed by Hoogsteen-hydrogen bonding to form a square planar structure
of tetrads, while two or more guanine tetrads are formed by π-π stacking in the presence
of cations (especially potassium) coordination. It has been reported that G4s exists in the
genome of many eukaryotic cells, is involved in many key biological processes and a
variety of human genetic diseases, and is also an important target of newly discovered
anticancer drugs in recent years [5].

Phan's team designed the first G4-PROTAC, using G4 as a warhead, to target the
degradation of the G4-binding protein RHAU (FIG. 4A), a potential therapeutic target for
amyotrophic lateral sclerosis (ALS). Given that RHAU preferentially binds to the
high-affinity all-parallel chain G4, the sequence TT (GGGT)4 (called T95−2T) with the
all-parallel chain G4 topology was used as the warhead. The azide-modified E3 ligase
recruiter was ligated to T95−2T at the 5′ end by a click reaction to assemble G4-PROTAC
(Fig. 4B).

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Figure 4: Design strategy (A) and representative chemical structure (B) of G4-PROTACs

4. Targeting membrane-associated proteins

Membrane-associated proteins can be targeted for degradation by bispecific aptamer
chimeras. Aptamers, also known as chemical antibodies, are single-stranded DNA or
RNA oligonucleotides with unique three-dimensional structures that can specifically bind
to homologous molecular targets. Compared with other targeting vectors, nucleic acid
aptamers have the advantages of simple preparation, precise synthesis, easy chemical
modification, good stability, high specificity, good physicochemical properties, and good in
vivo safety, but their immunogenicity is limited. In recent years, aptamers have emerged
as efficient recognition elements and delivery tools for therapeutic drugs, usually via the
reticulin-mediated endocytic pathway.

Han's group developed bispecific aptamer chimeras, becoming the first aptamer-based
technology to target degradation of membrane-associated proteins. The bispecific
aptamer chimera is a bifunctional molecule with the basic structure "aptamer
1-Linker-aptamer 2" (A1-L-A2), in which A1 and A2 specifically bind to IGFIIR and
membrane-associated proteins, respectively (Figure 5A). The widely expressed IGFIIR is
a typical lysosome-targeted receptor on the cell surface, and when the dual-specific
aptamer chimera binds, the IGFIIR is endocytosed with its cargo, mediating the transfer of
membrane-associated proteins to the lysosome for degradation. This technique has been
effectively validated at two membrane receptors - the mesenchymal epithelial transition
(Met) receptor and tyrosine kinase-like protein 7 (PTK-7) - (Figure 5B).

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Figure 5: Design strategy (A) and representative chemical structure (B) of bispecific aptamer chimera

5. Specific targeting of tumors

Aptamers have been widely used as tumor recognition elements for targeted cancer
therapy [6].Among them, the nucleolar aptamer AS1411, a synthetic single-stranded DNA
oligonucleotide rich in 26-base guanine, has been intensively studied and proven to have
good tumor targeting and safety profiles, making it suitable as a tumor targeting element
for biochemical studies and drug development. Wang's team
developed aptamer-PROTAC conjugates (APCs), which improved the specificity and
antitumor effect of traditional PROTACs by taking advantage of the good physicochemical
properties and high specificity of aptamers. APCs consist of three elements: PROTAC
elements (mediating E3 ligase and protein interaction, catalyzing protein ubiquitination
degradation); aptamer elements (serves as delivery vehicles for targeting
tumors); cleavable linker (enables targeted intracellular release of the original PROTAC).

In addition to serving as a tumor-specific recognition element and a delivery vehicle
for PROTACs, AS1411 is also capable of acting as a warhead for targeting
nucleophosmin (Figure 6). Nucleolin on the cell surface has been found in a variety of
cancers and plays an important role in regulating cancer progression. Tan's team
developed the first nucleolin degrader by linking DBCO-tagged AS1411 to an
azide-modified VHL-binding ligand.

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Figure 6: Design strategy for aptamer-PROTACs

New Progress of Targeted Degradation Based

On Nucleic Acids

The vast majority of targeted degradation technologies target proteins. Encouragingly,
Disney's team has developed several targeted RNA degradation approaches, including a
new strategy called RIBOTAC[7]. RIBOTAC consists of an RNA-binding small molecule
and a ribonuclease (RNase) L-recruiting structure (FIG. 7). RNase L is ubiquitously and
minimally expressed in cells as an inactive monomer. When the immune system is
activated, latent RNase L is upregulated and activated by self-dimerization, followed by
cleavage of cytoplasmic RNA containing UU, UA, AU, AA and UG dimers. RNA
degradation leads to translational arrest, which inhibits protein synthesis and viral
replication. In conclusion, RIBOTAC is a novel chemical program that achieves selective
cleavage and degradation of target RNAs by mobilizing local innate immune responses.
Compared with TPD, nucleic acid-targeted degradation is still in its infancy.

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Figure 7: RIBOTAC Design Strategy

Conclusion

At present, nucleic acid-based TPD strategies have attracted extensive attention, greatly
expanding the scope of target proteins, and have the advantages of simple preparation,
precise synthesis, high specificity, high efficiency, and low toxicity. Several technical
platforms have been developed, as shown in the following table (Table 2).

Table 2: Examples of TFs degradation agents based on small molecules and nucleic acids

Despite rapid progress in nucleic acid-based TPD strategies, several challenges remain.
Firstly, the instability of oligonucleotide may limit the application of corresponding
chimeras in vivo. Second, the instability and negative charge of oligonucleotides require
other delivery methods to enter cells. Third, rapid degradation of oligonucleotides may

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lead to adverse pharmacokinetic disadvantages. In conclusion, there is a long way to go,
and the clinical application of nucleic acid-based chimeric degraders still needs more
in-depth exploration and research.

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References:
[1] Gu S, Cui D, Chen X, Xiong X, Zhao Y. PROTACs: An Emerging Targeting Technique
for Protein Degradation in Drug Discovery. Bioessays. 2018;40(4):e1700247.
doi:10.1002/bies.201700247.
[2] Samarasinghe KTG, Jaime-Figueroa S, Burgess M, et al. Targeted degradation of
transcription factors by TRAFTACs: TRAnscription Factor TArgeting Chimeras. Cell Chem
Biol. 2021;28(5):648-661.e5. doi:10.1016/j.chembiol.2021.03.011
[3] Wang W, He S, Dong G, Sheng C. Nucleic-Acid-Based Targeted Degradation in Drug
Discovery. J Med Chem. 2022;65(15):10217-10232. doi:10.1021/acs.jmedchem.2c00875.
[4] Ghidini A, Cléry A, Halloy F, Allain FHT, Hall J. RNA-PROTACs: Degraders of
RNA-Binding Proteins. Angew Chem Int Ed Engl. 2021;60(6):3163-3169.
doi:10.1002/anie.202012330.
[5] Balasubramanian S, Hurley LH, Neidle S. Targeting G-quadruplexes in gene
promoters: a novel anticancer strategy?. Nat Rev Drug Discov. 2011;10(4):261-275.
doi:10.1038/nrd3428.
[6] Nimjee SM, White RR, Becker RC, Sullenger BA. Aptamers as Therapeutics. Annu
Rev Pharmacol Toxicol. 2017;57:61-79. doi:10.1146/annurev-pharmtox-010716-104558.
[7] Costales MG, Matsumoto Y, Velagapudi SP, Disney MD. Small Molecule Targeted
Recruitment of a Nuclease to RNA. J Am Chem Soc. 2018;140(22):6741-6744.
doi:10.1021/jacs.8b01233.

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[1] Overview of New Targets And Technologies of PROTAC
[2] Summary of PROTAC Degraders in Clinical Trials
[3] Several Types of PROTACs Based On Nucleic Acids
[4] PROTAC And Other Protein Degradation Technology
[5] Focus On PROTAC: Summary Of Targets From 2001 To 2019
[6] PROTACs and Targeted Protein Degradation