Proteolysis targeting chimeras (PROTACs) has come a long way since Crews first reported in 2001. At present, various degradation technologies based on PROTAC have been successfully developed for the degradation of kinases, nuclear receptors, epigenetic proteins, misfolded proteins and RNA. These technologies have greatly broadened the range of targets and clinical applications for diseases such as cancer, neurodegenerative diseases and viral diseases. To date, more than 15 PROTAC molecules have entered clinical trials. In this article, we summarize various targeted degradation strategies and their respective advantages and disadvantages, hoping to provide guidance value for the development of targeted protein degradation drugs.
Traditional small molecule inhibitors play a therapeutic role by interfering with protein function, while protein-targeted degraders play a role by proteasomal degradation of pathogenic target proteins, resulting in different biological effects, so they have higher selectivity and efficacy. Several targeted protein degradation strategies have been reported, among which the most famous is the proteolysis targeting chimera (PROTAC). In addition, researchers have also developed other types of degraders one after another, including intracellular click-formed proteolysis-targeting chimeras (CLIPTACs), photochemical targeting chimera (PHOTAC), semiconducting polymer nano-PROTAC (SPNpro), floate-PROTAC, antibody-PROTAC conjugate, antibody-based PROTAC (AbTAC), ribonuclease targeting chimera (RIBOTAC), transcription factor PROTAC (TF-PROTAC), chaperone-mediated protein degradation (CHAMP), biological PROTAC (bioPROTAC) and molecular glue, etc.
Figure 1. Summary of different targeted protein degraders
Targeted Protein Degradation Technologies
1. Proteolysis Targeting Chimeras (PROTACs)
Compared with other targeted protein degradation technologies, PROTAC has been more widely and deeply studied. It is composed of E3 ubiquitin ligase ligand, protein of interest (POI) ligand and linker. The formation of the POI-PROTAC-E3 ligase ternary complex can trigger the ubiquitination and degradation of POI through the ubiquitin-proteasome system. The concept of PROTAC was first proposed by Craig M. Crews in 2001. Many companies are involved in PROTAC, including Arvinas, Nurix Therapeutics, Kymera Therapeutics, C4 Therapeutics, Bristol-Myers Squibb and Novartis. Currently, at least 15 PROTACs have entered the clinic (Figure 2). Among them, the most advanced ones are the androgen receptor (AR) degrader ARV-110 for prostate cancer and the estrogen receptor (ER) degrader ARV-471 for breast cancer, which have entered phase II clinical trials in 2021.
Figure 2. Summary of PROTACs entering clinical trials
PROTACs have successfully degraded various POIs, including kinases, nuclear receptors, epigenetic proteins, misfolded proteins, etc. According to statistics, about 3939 PROTACs have been reported so far, including 981 POI ligands, 74 E3 ligase ligands and 1100 linker chains. A variety of kinase degraders have been developed, such as the degradation of BCR-Abl, BTK, IRAK4 and EGFR. In addition, nuclear receptor degraders have been developed, such as the degradation of AR, ER and CRABPs (cellular retinoic acid binding proteins). There are also many cases of PROTACs targeting epigenetic proteins, such as: BET, STAT3, Trim24, BRD7/9 IKZF, HDAC6, Sirt2, PCAF/GCN5, etc. PROTAC technology has also been used to degrade pathogenic misfolded proteins, such as Tau protein and alpha-synuclein. Studies have shown that PROTACs can cross the blood-brain barrier for the treatment of neurodegenerative diseases.
In addition to cancer and neurodegenerative diseases, PROTACs can also treat immune diseases by targeting IRAK4, sirtuin and PCAF/GCN5, as well as viral infections by targeting HCV NS3/4A protease. For viral infectious diseases, viral RNA replication requires RNA-dependent RNA/DNA polymerases (RdRp/RdDp), none of which is present in the human body, and which possess highly conserved catalytic domains, making them important targets for a wide range of antiviral drugs. Therefore, PROTACs targeting RdRp/RdDp will have great clinical potential as pan-viral antivirals.
One of the greatest advantages of PROTAC is that it can degrade "non-drugable" targets that lack suitable drug-binding sites, such as STAT3 and KRAS. However, due to the high structural homology of STAT3 with other STAT family members, it is difficult to find highly selective STAT3 inhibitors. The STAT3 PROTACs SD-36 developed by Wang's group can selectively degrade STAT3 protein and have excellent anticancer activity in vitro and in vivo. Another example is the so-called non-drugable target KRAS, mutations of which have been linked to various cancers. The Crews research group reported in 2020 that the KRAS-targeting PROTAC molecule LC2 can rapidly and persistently degrade the G12C mutant KRAS protein. In addition, PROTAC also provides a new way to solve the problem of cancer drug resistance. For example, the androgen receptor AR-based degrader ARD-61 developed by Xu's group can overcome the resistance caused by enzalutamide in vitro and in vivo. In addition, PROTACL18I developed by Yu's group can also effectively overcome resistance caused by various Bruton's tyrosine kinase (BTK) mutations produced by ibrutinib. One of the disadvantages of PROTAC is that it can only degrade intracellular targets, another is poor permeability and oral bioavailability due to its large molecular weight.
2. Intracellular Click-formed Proteolysis-targeting Chimeras (CLIPTACs)
PROTACs have limitations due to their poor solubility and poor cell penetration in living organisms. By utilizing the PROTAC principle and dynamic combinatorial chemistry, the Heightman team developed a novel PROTAC technology called CLIPTAC (intracellular click-formed protein hydrolysis-targeted chimeras) to address these issues. CLIPTAC consists of two fragments, a tetrazine-labeled thalidomide derivative and a trans-cyclooctene (TCO) -labeled POI ligand. This structure has a lower molecular weight and better penetration, which can self-assemble and form functional PROTAC molecules in the cell by rapid click reaction. Because of the high efficiency and specificity, the reaction does not cross react with other groups. Two CLIPTACs that degrade BRD4 and ERK1/2 have been successfully developed (Figure 3). These results provide an advanced strategy for the design of targeted protein depressors. However, a potentially significant drawback of CLIPTAC is that the bioorthogonal combination of two click reaction pairs may occur outside the cell, thus preventing CLIPTAC with high molecular weight and poor membrane permeability from entering the cell.
Figure 3. Schematic diagram of the mechanism of action of CLIPTAC and the chemical structure of CLIPTAC based on BRD4 and ERK1/2
3. Photochemical Targeting Chimera (PHOTAC)
Another limitation of PROTAC is that, when administered systemically, it may act in both tumor and normal cells, leading to off-target effects and non-tumor-specific toxicity. Photochemically targeted chimeras (PHOTACs), also known as photo-PROTACs or opto-PROTACs, offer us an excellent solution. PHOTAC is composed of a PROTAC and a photoremovable group (eg, nitrovaleryloxycarbonyl) or a photoswitchable group (eg, azobenzene) (Fig. 4). These molecules are inactive in the dark, and upon UV or visible light irradiation, the photoremovable groups will be separated from PHOTAC, and the photoswitchable groups will be isomerized, inducing the formation of active PROTAC and promoting POI degradation. Compared with conventional PROTACs, PHOTACs showed a more controlled degradation effect in time and space, which may reduce the adverse toxicity of PROTACs. Various phoTACs have been reported, and many targets such as BRD4, FKBP12, IKZF1/3, ALK and BTK have been successfully degraded. However, UV radiation is difficult to penetrate deep into the body because it induces DNA damage and is less penetrable. Therefore, the development of PHOTAC with longer wavelength excitation and higher security and penetration is one of the future development directions.
Figure 4. Mechanism of action and chemical structure of PHOTAC
4. Semiconducting Polymer Nano-PROTAC (SPNpro)
SPNpro is another strategy to address the PROTAC off-target problem. Developed by Kanyi Pu's group in 2021, it is a ternary complex consisting of a semiconducting polymer core, a cancer biomarker cleavable peptide, and a traditional PROTAC molecule (Figure 5). On the one hand, SPNpro can produce singlet oxygen under NIR (near infrared) irradiation to kill tumor cells by inducing a series of cancer immune responses. On the other hand, SPNpro molecules can be cleaved by cancer biomarkers such as cathepsin B and release active PROTAC molecules in situ to trigger the degradation of target proteins. SPNpro shows high cancer tissue specificity and low off-target effects. It provides us with a new approach for cancer treatment by synergistically combining photochemotherapy and protein degradation technology. A disadvantage of this approach is the high molecular weight of SPNpro, resulting in low oral bioavailability.
Figure 5. The mechanism of action and design ideas of SPNpro
Folate receptor alpha (FOLR1) is highly expressed in various cancer cells such as ovarian, lung, and breast cancers, but low in normal cells. In order to improve the targeting efficiency of PROTAC and reduce its off-target effect, the folic acid group is attached to the traditional PROTAC molecule, so Floate-PROTAC was developed. Floate-PROTAC can be selectively transported into cancer cells through FOLR1, and the active PROTAC molecules will be released by cleavage of endogenous hydrolase in cancer cells, thereby inducing POI ubiquitination and degradation. Jin's group has developed three Floate-PROTACs (floate-ARV-771, floate-MS432, floate-MS99) targeting BRD, MEK or ALK (Fig. 6). This strategy can significantly improve the cell selectivity of PROTACs for selectively degrading proteins in cancer cells. There are currently no in vivo data on Flot-PROTACs, and their pharmacokinetic and pharmacodynamic properties remain unknown.
Figure 6. Mechanism of Floate-PROTAC and chemical structure of Floate-PROTAC
6. Antibody-PROTAC Conjugates and Antibody-Based PROTACs (AbTACs)
Based on the high selectivity of antibodies, researchers have developed antibody-PROTAC conjugates and antibody-based PROTAC (FIG. 7) to address problems such as poor cell and tissue selectivity of PROTACs. For example, trastuzumab (Herceptin) is a monoclonal antibody used to treat HER2-positive (HER2+) breast cancer. Trastuzumab-PROTAC conjugate induces degradation of BRD4 protein in HER2-positive breast cancer cells. Another strategy, called antibody-based PROTAC (AbTAC), is to combine two antibodies, one linked to an E3 ligase (such as RNF43) and the other to a targeting protein (such as the immune checkpoint protein PD-L1) (eg AC-1). Both strategies can overcome the cell and tissue selectivity issues of PROTACs. However, due to the properties of antibodies, AbTACs need to be administered by injection.
Figure 7. Mechanism of action of AbTACs
7. Ribonuclease Targeting Chimeras (RIBOTACs)
RNA was previously considered an undruggable drug target due to its small size and lack of stability. Scientists have recently developed ribonuclease targeting chimeras (RIBOTACs) by linking RNA-binding molecules to ligands of ribonuclease (RNase L) to induce RNA degradation (Figure 8). Compared with related inhibitors, RIBOTACs can degrade RNA in a more efficient and selective manner without binding to the functional site of RNA. The Disney group developed two RIBOTACs that recruit active RNase L to oncogenic miRNA-96 or miRNA-210 precursors, respectively, leading to their degradation, thereby inducing apoptosis in breast cancer cells. In addition, they developed a RIBOTACs called C5-RIBOTAC that targets the degradation of SARS-CoV-2 FSE (frameshift element) RNA for SARS-CoV-2 therapy. The disadvantages of RIBOTACs are due to their negatively charged structure and large molecular weight (>2000), resulting in poor cellular uptake and permeability. In addition, RIBOTACs are expected to function in the cytoplasm since RNase L is predominantly distributed in the cytoplasm.
Figure 8. The mechanism of action of RIBOTAC and the schematic diagram of the structure of C5-RIBOTAC
8. Transcription Factor-PROTAC (TF-PROTAC)
Transcription factors (TFs) are important targets for cancer therapy. However, the lack of active sites and allosteric binding pockets makes the development of TFs-based small molecule inhibitors difficult. Kim's group developed a targeted protein degradation strategy called TF-PROTAC in 2021 to selectively degrade TFs (FIG. 9). Firstly, based on the fact that TFs can bind specific DNA-binding sequences or motifs, they modified the DNA oligonucleotide chain (ODN) that can bind TFs by azide group to obtain ODN (N3-ODN). After that, N3-ODN binds to bicyclic octylene (BCN) -modified E3 ligase ligand through click reaction to synthesize target TF-PROTAC, which is then transfected into cells to induce specific degradation of target TFs. So far they have developed two series of TF-PROTAC, NF-κB-Protac (dNF-κB) and E2F-PROTAC (dE2F), and shown efficient degradation of p65 and E2F1 proteins in cells. These results indicate that TF-PROTAC is an effective strategy to achieve selective degradation of TFs.
F9 Mechanism of action of TF-PROTAC
9. Chaperone-mediated Protein Degradation (CHAMP)
It has been found that some chaperone proteins such as heat shock protein 90 (HSP90) are highly activated in tumor cells and can directly interact with various E3 ligases. They are involved in protein degradation processes to prevent misfolded proteins from interfering with normal cell function. Based on these functions of chaperones, researchers have developed a molecular chaperon-mediated protein degradation agent (CHAMP) consisting of a ligand for POI, a ligand for chaperones, and a linker (FIG. 10). It was found that CHAMP based on BRD4 exhibited excellent BRD4 degradation and anti-proliferation effects both in vitro and in vivo. Compared with traditional PROTAC, CHAMP has higher tumor selectivity.
Figure 10 Mechanism of action of CHAMP
10. Biological PROTACs (bioPROTACs)
There are more than 600 E3 ligases in the human body, of which less than 2% are used in PROTAC research. To expand the E3 ligase toolbox of PROTACs, the researchers developed bioPROTACs, an engineered fusion protein consisting of a target protein-binding domain and an E3 ubiquitin ligase-binding domain (Figure 11). At present, researchers have developed two bioPROTACs, Con1-SPOP167-374 and K27-SPOP, which show degradation effects on proliferating cell nuclear antigens PCNA and KRAS, respectively. However, since this method relies on genetic coding, it can only be used as a biological tool.
Figure 11 Mechanism of action of bioPROTAC and bioPROTAC based on PCNA and KRAS
Molecular glue is a small molecule compound that can induce PPI (protein-protein interaction) between E3 ligase and POI, resulting in POI degradation. Unlike PROTACs, molecular glues have smaller molecular weights and more drug-like properties, making them a potential cancer treatment strategy. Typical examples of molecular glues include immunomodulatory drugs (IMiDs), such as thalidomide, which induce the degradation of casein kinases CK1α, GSPT1 and IKZF 1/3. In addition, arylsulfonamide anticancer drugs, such as indisulam, can form ternary complexes with DCAF15 and splicing factors RBM23 or RBM39 and induce their degradation. The molecular glue CR8 can also bind CDK12-cyclin K and the CUL4 adaptor protein DDB1, resulting in the degradation of cyclin K (Figure 12). However, all molecular glues reported so far have been discovered by chance, and there is no strategy for rationally designing molecular glue degraders.
Figure 12. The mechanism of action of molecular glues and the structures of several molecular glues
In addition to the above strategies, there are various other strategies for targeted protein degradation, such as Trim-Away protein degraders using TRIM21 as E3 ligase and specific antibodies as POI ligands, lysosome-targeted chimeras (LYTAC), autophagy-targeted chimeras (AUTAC), and autophagosome-bound compounds (ATTEC). These technologies provide new ideas for the research and development of targeted protein degradation drugs, broaden the scope of drug targets, and greatly promote the development of targeted therapeutic drugs. It has potential application value in other aspects.
However, there are challenges and opportunities, and each strategy has its limitations. Some have cell and tissue selectivity problems (such as PROTAC, CLIPTAC, CHAMP and molecular glue), some have poor cell permeability (such as PHOTAC), and some have solubility and absorption problems due to special structure or high molecular weight (such as SPNPRO, Floate -PROTAC, AbTAC, RIBOTAC, TFTAC, bioPROTAC). In addition, less than 2% of E3 ligases are used for targeted degradation. Therefore, the discovery of novel E3 ligases that can be used for targeted degradation is one of the future research directions for PROTAC.
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