In recent years, nucleic acid drugs have been developing vigorously with increasing market demand and rapid marketing approval, covering many fields such as cardiovascular and metabolic diseases, liver diseases and tumors. So far, more than 10 nucleic acid drugs have been approved for marketing worldwide, and many nucleic acid drugs are in the stage of clinical trials. Nucleic acid drugs are expected to become the third type of drugs after small molecule drugs and antibody drugs (Figure 1).
Figure 1. Marketed nucleic acid drugs (data source: pharmSnap Global Competitive Intelligence Database for New Drugs)
A growing number of approved nucleic acid therapeutics demonstrate the potential to treat disease by targeting disease-causing genes in vivo. Usually, conventional treatments only produce short-term therapeutic effects because they target proteins rather than the root cause of disease, while nucleic acid drugs directly act on disease-causing target genes or target mRNAs, and play a role in treating diseases at the gene level. Nucleic acid drugs include ASO, siRNA, Aptamer, miRNA, mRNA, saRNA, sgRNA, U1 snRNA, etc. Nucleic acid drugs have the advantages of high therapeutic efficiency, low toxicity, strong specificity and wide application fields, showing their important value in medicine, biological science and other fields.
PROTACs (proteolysis-targeting chimeras) is a drug development technology that utilizes the ubiquitin-proteasome system (UPS) to degrade target proteins. Structurally, PROTACs consists of three parts: an E3 ubiquitin ligase ligand and a target protein ligand, and two active ligands are linked together by a specially designed "Linker" structure to form a ternary complex. The target protein ligand of PROTAC binds to the target protein, and the E3 ubiquitin ligase ligand binds to the substrate-binding region of the intracellular E3 ubiquitin ligase, thereby "pulling" the target protein to the E3 ubiquitin ligase by ubiquitinating the target protein, enabling the UPS system to degrade the target protein (Figure 2).
Figure 2. Protein degradation mechanism mediated by PROTACs
Over the past 20 years, researchers have designed various forms of PROTACs based on peptides and small molecules. However, peptide-based PROTACs have problems such as low activity and immunogenicity, which greatly limit their clinical medical applications. Compared with polypeptide PROTACs, small-molecule PROTACs are smaller, more easily absorbed by the body, and have better druggability, so small-molecule PROTACs are still the mainstream. With the development and progress of science and technology, some new types of PROTACs continue to emerge, and nucleic acid-based PROTACs emerge as the times require.
Functional defects in RNA binding proteins (RBPs) are at the root of many diseases, and targeting RBPs with conventional drugs has proved difficult. RBPs bind to RNA in a dynamic, coordinated, and sequence-selective manner to form ribonucleoprotein (RNP) complexes that play a key role in RNA dependence. Certain diseases are caused by genetic changes in RBP that affect their binding to RNA. In 2021, Jonathan Hall's research group first proposed the design concept of RNA-based PROTACs, and the author successfully constructed RNA-PROTACs targeting RBPs (RNA binding proteins). Using small RNA mimics as targeting groups, they can specifically bind to RBPs RNA binding sites. PROTACs ubiquitinated RBPs and then degraded them by ubiquitin proteasome system. The authors performed a proof-of-concept demonstration of degradation of two RBPs (stem cell factor LIN28 and splicing factor RBFOX1) and demonstrated their use in cancer cell lines.
Transcription factors (TFs) represent an important class of therapeutic targets for the treatment of diseases including cancer. Since TFS lack the active or allosteric sites commonly found in kinases or other enzymes, it is difficult for traditional small-molecule inhibitors to bind to them. Therefore, transcription factors were once considered as "undruggable" targets, presenting an unsurmountable technical bottleneck. Professor Wenyi Wei, Harvard University, and Professor Jian Jin, Icahn School of Medicine, Mount Sinai, reported an oligonucleotide chain-based "TF-PROTAC", which is composed of DNA oligonucleotides and E3 ligand linked by click reaction (Figure 3), and can selectively degradate pathogenic TFs. The selectivity of TF-PROTAC depends on the DNA oligonucleotides used.
Figure 3. Design strategy of TF-PROTAC
The authors successfully developed two series of VHL-based TF-PROTACs: NF-κB-PROTAC (dNF-κB) and E2F-PROTAC (dE2F), which efficiently degrade endogenous p65 and E2F1 proteins in cells, respectively, and showed excellent anti-cell proliferation effect (Figure 4).
Figure 4. TF-PROTACs structure and protein degradation experiments
In 2021, Crews' group also reported oligonucleotide PROTACs targeting transcription factors (TFs): oligoTRAFTACs (Figure 5). OligoTRAFTACs consist of oligonucleotide chains that bind to TFs and E3 ubiquitin ligase ligands.
Figure 5. Design ideas of oligoTRAFTACs
Western blot experiments showed that oligoTRAFTACs successfully degraded two oncogenic transcription factors: c-Myc and brachyury. In addition, the authors found that oligoTRAFTACs could successfully degrade the brachyury of chordoma cell lines, and also showed good degradation activity in the subsequent in vivo zebrafish model experiments (Figure 6).
Figure 6. Degradation experiments of oligoTRAFTACs in a zebrafish model
PROTACs based on Aptamer
An Aptamer is a sequence of oligonucleotides (DNA or RNA). Oligonucleotide fragments are usually obtained from nucleic acid libraries using Systematic evolutionof ligands by exponential enrichment (SELEX) in vitro screening. Nucleic acid adaptors are widely used because they can combine with a variety of target substances with high specificity and selectivity. In 2021, Tan Weihong's research group first designed a PROTAC based on the nucleic acid aptamer AS1411: ZL216 (Figure 7).
Figure 7. Design and synthesis of ZL216
AS1411 can specifically target nucleolin receptors that are highly expressed in tumor cells, and the nucleolin receptors are internalized after binding to their ligands. The authors proved that the PROTAC has high water solubility and serum stability through in vitro experiments. Furthermore, the authors found that ZL216 promoted the formation of the nucleolin receptor-ZL216-VHL ternary complex in breast cancer cells and efficiently induced nucleolin receptor degradation in vitro and in vivo. Subsequent cell proliferation and migration experiments showed that ZL216 also inhibited the proliferation and migration of breast cancer cells (Figure 8).
Figure 8. Results of IP experiments, cell proliferation and migration experiments
Aptamer-PROTAC Conjugates (APCs)
PROTAC is a promising targeted protein degradation strategy. As an effective method for targeted protein degradation, PROTACs significantly outperform traditional small-molecule drugs in terms of catalytic properties, high selectivity, overcoming drug resistance, and effective blocking of non-druggable targets. But PROTACs generally have high molecular weight and high hydrophobicity, and their physicochemical properties largely exceed the "rule of five" (RO5). Therefore, the development of conventional PROTACs into drugs is often limited by their poor cell membrane permeability, poor pharmacokinetic (PK) properties, and lack of tumor-specific targeting.
To this end, Chunquan Sheng's research group proposed the design concept of Aptamer-PROTAC Conjugates (APCs). APC is obtained by coupling the PROTAC targeting BET protein with the Aptamer AS1411 (AS) by a cleavable linker chain ( Figure 9). Among them, the nucleic acid aptamer AS1411 can selectively target the highly expressed nucleolin receptor on the surface of tumor cells. AS itself has good inhibitory activity against nucleolin receptor-overexpressing tumors and is currently being evaluated in a phase II clinical trial. Glutathione is abundant in tumor cells, so the linker chain selects a disulfide bond that can be cleaved by glutathione (GSH), which can selectively respond to the tumor microenvironment and release the active BET degrader after cleavage of the linker chain.
Figure 9. Design strategy for APC
Compared with unmodified BET PROTAC (PRO), the APC conjugate (APR) showed improved tumor-targeting ability in a mouse xenograft model of McF-7 cells, thereby enhancing BET protein degradation in vivo and antitumor potency. Therefore, the APC strategy provides a new design idea for the development of tumor-specific targeting PROTACs.
Figure 10. Protein degradation experiments and in vivo imaging, anti-tumor experiments
The development of Aptamer PROTACs Conjugates (APCs) can be described as "earth-shaking" since it was proposed. Compared with traditional PROTACs, nucleic acid-based PROTACs improve the targeting of traditional small molecule PROTACs, and play an important role in improving water solubility, membrane permeability, and tumor targeting. Since nucleic acid drugs are easily hydrolyzed by nucleases in vivo, the half-life is short, which greatly limits their application in biomedicine. Future research directions should focus on improving the stability of nucleic acid drugs, prolonging half-life, improving pharmacokinetic properties, and solving nucleic acid drug delivery problems.
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. Non-small molecule PROTACs (NSM-PROTACs): Protein degradation kaleidoscope
. Aptamer-PROTAC Conjugates (APCs) for Tumor-specific Targeting in Breast Cancer.
. Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin.
. OligoTRAFTACs: A Generalizable Method for Transcription Factor Degradation.
. RNA-PROTACs: Degraders of RNA-Binding Proteins.
. TF-PROTACs Enable Targeted Degradation of Transcription Factors.
. Future Perspective of PROTAC Combined With CRISPR In Anti-ancer Area
. Four Major Trends In The Development of PROTAC
. PROTAC And Other Protein Degradation Technology
. PROTACs VS. Tranditional Small Molecule Inhibitors
. Focus On PROTAC: Summary Of Targets From 2001 To 2019