Since the first PROTAC molecule entered clinical development in March 2019, Targeted Protein Degradation (TPD) technology has developed rapidly. On the one hand, several PROTAC and molecular glues have entered clinical trials worldwide, and some leading projects have disclosed clinical investigation data several times; on the other hand, new technologies have emerged, such as LYTAC, ATTEC, ATAC, AUTOTAC, etc. In this article, we list the targeted protein degradation technologies that have made important research progress in recent years, mainly including mechanism of action, representative companies, global R&D status, etc.
Figure 1. Representative events in the TPD development. Image source: reference 
Targeted protein degradation via proteasome
Protein homeostasis refers to the highly complex and interrelated processes that cells use to maintain protein concentration, conformation and subcellular localization. It consists of a series of pathways that control protein synthesis, folding, transport, and disposal. In eukaryotic cells, damaged proteins or organelles are able to be cleared by the proteasome or lysosome. These two pathways are independent but interconnected with each other. In general, the proteasome eliminates short-lived proteins and soluble misfolded proteins through the ubiquitin-proteasome system (UPS). In contrast, lysosomes are responsible for the degradation of long-lived proteins, insoluble protein aggregates, and even whole organelles, macromolecular compounds, and intracellular parasites (e.g., certain bacteria) via the endocytosis, phagocytosis, or autophagy pathways. First, we present targeted protein degraders (TPDs) developed based on proteasomes.
Figure 2. Different TPD technologies, source: reference 
PROTAC (Proteolysis-Targeting Chimeras)
PROTACs, known as Proteolysis-Targeting Chimeras, are heterobifunctional molecules consisting of an E3 ligase binder, a Linker, and a target protein binder. Specifically, one end of the PROTACs molecule is bound to the target protein and the other end is bound to the E3 ubiquitin ligase. The E3 ubiquitin ligase marks the target protein as defective or damaged by attaching a small protein called ubiquitin to it. Afterward, the proteasome degrades the tagged target protein.
By the way, PROTAC linker plays a vital role in the efficient ubiquitination of the target protein and its ultimate degradation. PEG linkers are the most common motifs incorporated into PROTAC linker structures. Biopharma PEG is dedicated to the R&D of PROTAC Linker, providing high purity PEG linkers with various reactive groups to continuously assist your project development.
Figure 3. PROTAC Global R&D Overview and Hot Targets, source: reference 
PROTAC has been in development for more than 20 years. According to statistics, there are currently over 160 PROTAC projects in development worldwide, and nearly 20 projects are already in clinical development, with companies in the first tier including Arvinas, Kymera, C4 Therapeutics, Nurix Therapeutics, Haisco Pharmaceutical, Kintor Pharma-B, BeiGene, etc. Currently, the more competitive targets include AR, BTK, BET, ALK, EGFR, etc.
Figure 4. PROTACs in Pipeline
Molecular glue degraders are a class of small molecules that induce novel interactions between E3 ubiquitin ligase substrate receptors and target proteins, leading to the degradation of the target protein. A notable example of molecular glue is the thalidomide anticancer drugs, which redirect the E3 ubiquitin ligase CRL4CRBN, thereby polyubiquitinating the transcription factors IKZF1 and IKZF3, leading to the degradation of IKZF1 and IKZF3 by the proteasome. Similarly, the anticancer sulfonamide drug indisulam directs the E3 ubiquitin ligase CRL4DCAF15 to degrade the splicing factors RBM23 and RBM39.
Figure 5. Molecular Glues vs PROTACs
Although both molecular glues and PROTACs utilize UPS for protein degradation, they differ in several ways. First, PROTAC is a heterobifunctional degradant that interacts with both E3 ligase and protein of interest (POI), compared to molecular glue degraders that can only interact with E3 ligase (more frequently) or POI and then induce/stabilize E3 ligase interaction with POI. Second, compared to PROTACs, molecular glues do not contain a linker and therefore have a smaller molecular weight, higher oral bioavailability, and better cell permeability. Finally, although rational design strategies are emerging, molecular glues are currently more difficult to design.
Examples of molecular glue degraders include thalidomide, lenalidomide, and pomalidomide. Interestingly, they have been approved by the FDA for the treatment of various types of tumors long before their functional mechanisms were elucidated.
Although the research and development of molecular glues have not yet formed the same scale as PROTAC, several companies around the world have joined the research and development, and the fastest progressing projects are in Phase I/II clinical, with representative startups such as Monte Rosa Therapeutics and Gluetacs Therapeutics, etc.
It is worth mentioning that in 2022, the financing fever in the molecular glue field has increased, with several companies dedicated to molecular glue development announcing financing, such as Plexium, TRIANA Biomedicines, Ambagon Therapeutics, etc.; in addition, pharmaceutical giants such as BMS and Merck have announced large molecular glue-related collaborations. It is expected that the molecular glue field will receive more support in the coming years.
CHAMP (Chaperone-mediated Protein Degradation/Degrader)
Chaperones are involved in the folding of more than half of all mammalian proteins. Chaperones contain many different families, such as the HSP60 family, the HSP70 family, and the HSP90 family, each of which helps protein folding in a different way.
In addition to participating in protein folding, in some cases, chaperones can also recognize misfolded proteins and guide them through the ubiquitin-proteasome system (UPS) for degradation. Some chaperones, such as heat shock protein 90 (HSP90), interact directly with many different E3 ubiquitin ligases, thereby assisting in the completion of protein degradation and preventing the normal localization of misfolded proteins by preventing them from interfering with normal cellular function.
Figure 6. CHAMP (Chaperone-mediated Protein Degradation/Degrader), source: Ranok Therapeutics
It was found that the HSP90 complex is highly activated in tumor tissues relative to normal tissues, which leads to small molecule compounds that bind HSP90 to display unique tumor-selective pharmacokinetics.
Using these properties of HSP90, Ranok Therapeutics developed a heterobifunctional small molecule that targets both BRD4 and HSP90, also known as CHAMP. In January 2022, the US FDA has approved the IND application for RNK05047, a small molecule BRD4 selective degradation agent, and patient enrollment for Phase I/II clinical trials is expected to begin in the first half of 2022.
Targeted protein degradation via lysosome
In cells, three different lysosomal pathways allow protein degradation: 1) cell surface proteins reach the lysosome via endocytosis, after which they can be degraded by the lysosome or transported to the plasma membrane or other organelles for recycling. 2) in the phagocytic pathway, cells phagocytose large extracellular particles, such as invading pathogens and dead cells, which are then degraded via the lysosome. 3) Misfolded or aggregated proteins, damaged organelles, and intracellular pathogens are removed via the autophagy-lysosome pathway. Among them, there are three different forms of autophagy, including macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA).
Figure 7. Protein degradation via three distinct lysosome pathways. source: reference 
In macroautophagy, dysfunctional proteins or organelles are recognized by autophagic receptors and selectively enclosed in autophagosomes, after which the autophagosomes fuse with lysosomes and their contents are degraded. In microautophagy, the lysosome directly swallows the autophagic cargo and causes its degradation. In CMA, chaperones select target proteins and directly span the lysosomal membrane for degradation. there are two unique features of CMA: first, CMA only degrades certain proteins and not organelles; second, autophagosome formation is not necessary in CMA.
Figure 8. Summary of lysosome-dependent protein degradation strategies. source: reference 
With the in-depth study of different lysosomal degradation pathways, scientists have developed a variety of novel protein degradation techniques, including LYTAC, ATAC, AbTAC, GlueTAC, AUTAC, ATTEC, AUTOTAC, etc. It is worth mentioning that compared to proteasome-based targeted protein degradation technologies that can only degrade certain intracellular proteins, lysosome-based targeted protein degradation technologies have the potential to remove protein aggregates, damaged excess organelles, membrane proteins and extracellular proteins, and therefore, the clinical development of these technologies is highly anticipated.
LYTAC (Lysosome-Targeting Chimaeras）
LYTAC, similar to PROTAC, is a bifunctional molecule with two binding domains (below), one end carrying an oligoglycopeptide moiety that binds to the cell surface transmembrane receptor CI-M6PR (cation-independentmannose-6-phosphatereceptor), and the other end carries an antibody or small molecule that binds to the target protein. These two binding domains are linked by a chemical linker. The trimeric CI-M6PR-LYTAC-target protein complex formed on the plasma membrane is "engulfed" by the cell membrane and forms a transport vesicle. The vesicle transports the complex to the lysosome, where the target protein is degraded.
Figure 9. The mechanism of LYTAC, source: reference 
Unlike PROTAC (which degrades intracellular proteins), LYTAC is mainly used to degrade extracellular and membrane-bound proteins. As many therapeutic targets (e.g. growth factors, disease-associated receptors, cytokines) actually belong to secreted (extracellular) and membrane-associated proteins, the LYTAC technology has a very promising therapeutic future as an effective tool capable of expanding the range of degradable proteins.
A representative company developing LYTAC is Lycia Therapeutics, a San Francisco-based company founded in 2019 and driven by Versant Ventures, which has raised $120 million in cumulative funding. In August 2021, Lycia announced a multi-year research collaboration and licensing agreement with pharmaceutical giant Eli Lilly, under which the two companies will utilize Lycia's proprietary LYTAC technology to develop and commercialize novel degradation agents against up to five targets, with a focus on disease areas including immunity and pain. Under the terms of the agreement, Lycia will receive an upfront payment of $35 million, in addition to being eligible for more than $1.6 billion in potential milestone payments and a share of future sales. At this time, Lycia has not disclosed any pipeline and is looking forward to specific projects to be announced in 2022.
ATAC (ASGPR Targeting Chimeras)
ATAC is known as ASGPR Targeting Chimeras, or a sialoglycoprotein receptor (ASGPR) targeting chimera, an endocytic surface receptor that plays a key role in the natural process by which endogenous proteins are internalized into hepatocytes and degraded.
Figure 10. ASGPR Targeting Chimeras, source: Avilar
Overall, ATAC is similar to LYTAC and acts as a bifunctional molecule, binding to the target protein on one end and to ASGPR on the other. The resulting ASGPR-ATAC-target protein complex is "engulfed" by the cell membrane, forming a transport vesicle, which transports the complex to the hepatocyte lysosome, where the target protein is degraded. Since ASGPR is a liver-specific lysosomal targeting receptor, the ATAC technology has a potential safety advantage over LYTAC (CI-M6PR is widely expressed in a variety of cells) in that it can degrade extracellular proteins in a cell-type restricted manner.
The company that came up with the ATAC concept, called Avilar Therapeutics, announced in November 2021 that it had received $60 million in seed funding from founding investor RA Capital Management. Avilar's goal is to target disease-causing extracellular proteins and broaden the target boundaries of protein degraders.
It is worth mentioning that technologies such as GalNAc-LYTAC (developed by the team of LYTAC pioneer Prof. Carolyn R. Bertozzi) and MoDE-A have the same mechanism as ATAC and are named differently by different research teams. Based on the design ideas of LYTAC and ATAC, it is worth looking forward to whether scientists can develop new degradation technologies based on other lysosomal targeting receptors in the future.
Figure 11. GalNAc-LYTAC and MoDE-A, source: reference 
BIAC (Bispecific Aptamer Chimera)
The mechanism of action of bispecific aptamer chimeras is similar to that of LYTAC, mediating POI degradation via the nucleosome-lysosome pathway. The difference is that bispecific aptamer chimeras use DNA aptamers to target CI-MPR and transmembrane POI. In February 2021, researchers from Shanghai Jiao Tong University and other institutions designed the first bispecific aptamer chimera molecule, named A1-L-A2, where A1 and A2 specifically bind the CI-MPR and a POI, and L stands for linker DNA. it was shown that A1-L-A2 can transport membrane proteins, such as the receptor tyrosine kinases MET and PTK-7, to the lysosome for degradation. transported to the lysosome for degradation. At the same time, this aptamer chimera had no significant effect on the level of non-targeted proteins. Compared with antibodies, nucleic acid aptamers have the advantages of simple preparation, precise synthesis, and good stability, and are therefore a promising class of technologies.
Figure 12. Bispecific Aptamer Chimera, source: reference 
AbTAC (Antibody-based PROTAC)
In a study published in January 2021 in the Journal of the American Chemical Society, a team of scientists from the University of California, San Francisco proposed the antibody-based PROTAC (AbTAC) technology. AbTAC, essentially a bispecific antibody, recruits membrane-bound E3 ligases to degrade cell surface proteins. AbTAC has been shown to degrade PD-L1 by recruiting the membrane-bound E3 ligase RNF43 (transmembrane E3 ligase).
Figure 13. Antibody-based PROTAC, source: reference 
Although the name is related to PROTAC, AbTAC is actually more similar to LYTAC because both are based on the endosomal-lysosomal pathway to induce cell surface POI degradation. AbTAC connects RNF43 on one end and POI on the cell surface on the other, forming a complex that is internalized into the cell, after which POI is degraded by the lysosome. However, the mechanism of action of AbTAC is not as clear as that of LYTAC. For example, it is not clear whether the intracellular region of POI is ubiquitinated prior to endocytosis; if so, the contribution of ubiquitination to the internalization of the complex is yet to be investigated. In addition, whether RNF43 can be recycled and reused like the receptors CI-MPR and ASGPR of LYTAC/ATAC needs to be further investigated. In the future, AbTAC technology may need to work on finding other membrane receptors for better development.
GlueTAC is another lysosome-based degradation strategy developed to degrade cell surface proteins and consists of a covalently modified single-domain antibody (nanobody), a cell-penetrating peptide (CPP) and a lysosome-sorting sequence. The single-domain antibody (nanobody) is responsible for targeting POI, CPP-induced endocytosis of the GlueTAC-POI complex and subsequent lysosomal degradation.
Figure 14. GlueTAC, source: reference 
The collaborative team of Peng Chen and Jian Lin from the School of Chemical and Molecular Engineering, Peking University, published research results related to GlueTAC technology in the Journal of the American Chemical Society in October 2021. The study developed a GlueTAC molecule that targets PD-L1 and demonstrated that compared to the PD-L1 antibody atezolizumab, the GlueTAC molecule was more effective in reducing PD-L1 levels in cells and inhibiting tumor growth in immunodeficient mice. Although GlueTAC represents another exciting approach to degrade cell surface proteins, there are still some issues to think about, such as safety, half-life, etc.
AUTAC (Autophagy-Targeting Chimera)
In addition to the nucleosomal-lysosomal pathway, the autophagosomal-lysosomal pathway has also been used for the development of targeted protein degraders. The nucleotide 8-nitro-cGMP (8-nitrocyclic guanosine monophosphate) is an important signaling molecule for intracellular regulation of autophagosome recruitment. this property of 8-nitro-cGMP has been used for the development of autophagy-targeting chimeras (AUTAC). the AUTAC molecule consists of three components: a cGMP-based degradation tag, a linker, and a small molecule ligand for a protein of interest (POI) or organelle. AUTAC molecules trigger K63-linked polyubiquitination and subsequent lysosome-mediated degradation. In contrast, PROTAC molecules induce K48-linked polyubiquitination and proteasome-mediated degradation.
Figure 15. Autophagy-Targeting Chimera, source: reference 
In addition to cytoplasmic proteins, organelles such as mitochondria can also be degraded by AUTAC. Mitochondrial dysfunction is associated with many aging-related diseases, and removal of dysfunctional or damaged mitochondria can ameliorate these diseases. In a study published in Molecular Cell in December 2019, a team of scientists developed an AUTAC4 molecule that promotes fragmented mitochondrial autophagy. AUTAC4 uses 2-phenylindole derivatives, transporter ligands on the outer mitochondrial membrane, as mitochondrial binder. These results suggest that AUTAC has a wide range of applications and is expected to play a greater role in the degradation of protein aggregates, among others.
ATTEC (Autophagosome-Tethering Compound)
Similar to the autophagy-based AUTAC technology, ATTEC works by binding POI to the autophagosome. Specifically, ATTEC binds to both LC3, a key protein of autophagosomes, and POI. During autophagy, the key protein LC3 is lipidated and then polymerized and amplified to form a membrane structure, and wraps proteins, lipids, organelles and other degradation targets in it to form a complete autophagosome, which is fused with lysosomes to degrade the substances wrapped in it.
Figure 16. Autophagosome-Tethering Compound, source: reference 
In a study published in Nature in October 2019, Professor Lu Boxun's team at Fudan University developed an ATTEC molecule that binds to both LC3 and mutant Huntington protein (mHTT), thereby binding the target protein to autophagic vesicles for degradation. In 2021 another paper published in the journal Cell Research paper confirmed that ATTEC technology is also able to degrade lipid droplets, an intracellular organelle that stores fat (excessive storage of lipid droplets may be associated with a variety of diseases, such as obesity, NAFLD, neurodegenerative diseases, etc.), achieving a breakthrough in targeted degradation technology from protein to non-protein substances.
Based on ATTEC technology, a company called PAQ Therapeutics has been formed with Professor Lu Boxun as one of the co-founders. In July 2021, PAQ Therapeutics closed a $30 million Series A round of funding. The company is initially focused on developing therapeutics for inherited central nervous system diseases, with subsequent plans to expand to other protein and non-protein targets for the treatment of oncology, metabolic diseases, and other major diseases.
ATUOTAC (Autophagy-Targeting Chimera)
The autophagic cargo receptor p62/SQSTM1 acts as a bridge between polyubiquitinated cargoes and autophagosomes. The polyubiquitinated cargo binds to the UBA structural domain of p62, leading to a conformational change in p62. This conformational change exposes the LIR motif of p62 and facilitates its interaction with LC3 at the autophagic membrane.In February 2022, a paper published in Nature Communications by a team of scientists from South Korea proposed the AUTOTAC technology. The AUTOTAC molecule consists of a module that interacts with the ZZ domain of p62 and a POI-targeting module. AUTOTAC acts to link POI and p62 independent of POI ubiquitination. AUTOTAC promotes the oligomerization and activation of p62, leading to POI degradation via the autophagy-lysosome pathway.
AUTOTAC mediates the targeted degradation of not only monomeric proteins, but also proteins with aggregation prone proteins. Using mouse models expressing human pathological tau mutants, researchers have demonstrated that AUTOTAC is effective in removing misfolded tau. In contrast, proteasome-based techniques, such as PROTAC and molecular glue, are generally ineffective in dealing with misfolded proteins. In addition to Tau, AUTOTAC is also effective in removing a variety of oncoproteins, such as degrading the androgen receptor (AR).
The representative company developing AUTOTAC technology is AUTOTACBio, headquartered in Seoul, Korea. The company's CEO, Dr. Yong Tae Kwon, is the co-corresponding author of the above paper. Currently, the company has laid out a variety of disease areas such as neurodegenerative diseases (e.g. AD), cancer, metabolic syndrome, and muscular dystrophy, and has built a rich pipeline.
In chaperone-mediated autophagy (CMA), heat shock protein 70 (HSC70) recognizes soluble protein substrates with KFERQ sequences. Subsequently, the HSC70-substrate complex binds to lysosome-associated membrane protein 2A (LAMP2) on the lysosomal membrane and the substrate translocates to the lumen of the lysosome for degradation.
Figure 17. CMA-based degrader, source: reference 
CMA-based degrader consists of three functional domains: a cell membrane penetrating sequence, a POI binding sequence, and a CMA targeting motif. Upon use of CMA-based degrader, such degrader first enters the cell, then binds the target protein via the POI binding sequence, and finally is transported to the lysosome for degradation. This technique has been shown to reduce the levels of scaffold proteins PSD-95, DAPK1, and α-synuclein. However, to be an effective therapeutic strategy, CMA-based degradants need to overcome at least two major hurdles, firstly, the stability of the degradant; and secondly, effective delivery.
Targeted protein degradation (TPD) has arguably been one of the most dynamic areas of research in both science and industry over the past few years, with a proliferation of new technologies opening new doors for drug development.
In terms of technology types, PROTAC and molecular glues are the most advanced TPD technologies (both have several projects in clinical development), both are based on the ubiquitin-proteasome system and are mainly used to degrade intracellular proteins. However, the development of these two types of technologies still faces many challenges, such as PROTAC, which often faces challenges in cell permeability and oral bioavailability due to its large molecule, and molecular glue, which is small but currently difficult to design effectively.
TPD technologies using lysosomal degradation pathways have also been rapidly developed in the past few years, including LYTAC, Bispecific Aptamer Chimeras, AbTAC and GlueTAC technologies that degrade extracellular and membrane proteins using the endosomal-lysosomal pathway, and autophagy-lysosomal pathways to degrade misfolded proteins, protein aggregates or damaged organelles with technologies such as AUTAC, ATTEC, and AUTOTAC. The development of TPD technologies based on the lysosomal degradation pathway is still in its infancy compared to PROTAC and molecular glues. There is still much to learn about the specific mechanisms of each technology. As an important intracellular organelle, lysosomes regulate many important cellular and physiological functions, such as metabolism and homeostasis, in addition to protein degradation. It is unclear whether "hijacking" the lysosomal degradation pathway affects the whole organism, therefore, further characterization of TPD technologies based on the lysosomal degradation pathway is necessary.
Currently, several TPD molecules are in cancer clinical trials. In addition to cancer, many TPD molecules show great potential in areas such as neurodegenerative diseases, inflammatory diseases or viral infections. Although there are many challenges to overcome, there is no doubt that TPD technology will hold great promise for future drug development.
 Zhao, L., Zhao, J., Zhong, K. et al. Targeted protein degradation: mechanisms, strategies and application. Sig Transduct Target Ther 7, 113 (2022). https://doi.org/10.1038/s41392-022-00966-4
 He, M., Cao, C., Ni, Z. et al. PROTACs: great opportunities for academia and industry (an update from 2020 to 2021). Sig Transduct Target Ther 7, 181 (2022). https://doi.org/10.1038/s41392-022-00999-9
 Whitworth C, Ciulli A. New class of molecule targets proteins outside cells for degradation. Nature. 2020;584(7820):193-194. doi:10.1038/d41586-020-02211-w
 Caianiello, D.F., Zhang, M., Ray, J.D. et al. Bifunctional small molecules that mediate the degradation of extracellular proteins. Nat Chem Biol 17, 947–953 (2021). https://doi.org/10.1038/s41589-021-00851-1
 Miao Y, Gao Q, Mao M, et al. Bispecific Aptamer Chimeras Enable Targeted Protein Degradation on Cell Membranes. Angew Chem Int Ed Engl. 2021;60(20):11267-11271. doi:10.1002/anie.202102170
 Daiki Takahashi, Jun Moriyama, Tomoe Nakamura, Erika Miki, Eriko Takahashi, Ayami Sato, Takaaki Akaike, Kaori Itto-Nakama, Hirokazu Arimoto, AUTACs: Cargo-Specific Degraders Using Selective Autophagy, Molecular Cell, Volume 76, Issue 5, 2019, Pages 797-810.e10, ISSN 1097-2765, https://doi.org/10.1016/j.molcel.2019.09.009.
PROTACs and Targeted Protein Degradation
PROTACs VS. Traditional Small Molecule Inhibitors
Four Major Trends In The Development of PROTAC
Molecular Glues: A New Dawn of Small Molecule Drugs After PROTAC