Overview of 12 Targeted Protein Degradation Technologies
The first era of PROTAC began in 2001 with the publication of a pivotal paper by Sakamoto et al., which validated the concept of PROTAC for the first time in vitro. In 2019, the first PROTAC drug to enter clinical trials, which targeted androgen receptors by recruiting androgen receptors into the CRL4-CRBN ligase complex, ending the fundamental era of targeting protein degradation, and PROTAC entered the era of clinical transformation. On the one hand, a number of PROTACs and molecular gums have entered clinical trials around the world, and some head projects have repeatedly disclosed clinical research data; On the other hand, new technologies are emerging constantly, 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.
Representative events in the TPD development. Purple: technologies related with UPS-based technologies; light blue: technologies related with the endosome-lysosome pathway; dark blue: technologies related with the autophagy-lysosome pathway. (Zhao, L. 2022)
Targeted protein degradation via proteasome
Protein homeostasis maintains the concentration, structure, and location of proteins within the cell, ensuring their normal function. In eukaryotic cells, the proteasome and lysosome are key mechanisms for removing damaged proteins, with the proteasome operating primarily through the ubiquitin-proteasome system (UPS). UPS is a complex protein degradation pathway that is responsible for identifying and eliminating unwanted or misfolded proteins and maintaining protein homeostasis. The system consists of ubiquitin molecules and three enzymes: E1 activating enzyme, E2 conjugating enzyme, and E3 ligase, which can specifically recognize the target protein and label its ubiquitin. The labeled protein is then degraded by the proteasome, releasing amino acids for the cell to recycle. UPS is essential for life activities such as cell cycle, signaling, and gene expression, and plays an important role in diseases such as cancer, where tumor cells may manipulate UPS to promote their growth and survival.
Proteolytic targeting chimera (PROTAC)
PROTACs are used to selectively degrade target proteins. This bifunctional molecule binds to a target protein on one end and E3 ubiquitin ligase on the other. This prompts the E3 enzyme to add the ubiquitin molecule to the target protein and label it for degradation. Subsequently, the target protein of ubiquitination is recognized and degraded by the proteasome, effectively removing the protein from the cell.
Since its conception, PROTAC technology has undergone more than 20 years of development, with more than 160 related projects in research worldwide and nearly 20 drug candidates in clinical trials. Leading companies such as Arvinas, Kymera, C4 Therapeutics, Nurix Therapeutics and others are driving the development of this field, focusing on targets including AR, BTK, BET, ALK and EGFR. It shows the great potential of PROTAC in precision medicine and treatment of refractory diseases.
PROTAC related products at BOC Sciences
Molecular glue degraders are a special class of small molecule drugs whose function is to promote the interaction between E3 ubiquitin ligase and its target protein that it would not otherwise bind to, inducing ubiquitization and subsequent proteasome degradation of the target protein. The representative drug thalidomide compounds showed anti-tumor effects by manipulating CRL4CRBN E3 ligase to trigger the degradation of transcription factors IKZF1 and IKZF3.
PROTAC and molecular glue schematic.
Although both molecular glue and PROTACs are targeted protein degradation technologies using UPS, there are differences in their mechanisms. As a bifunctional molecule, PROTACs binds to both E3 ligase and target protein. The molecular glue only needs to interact with one of the E3 ligases or target proteins to promote their coupling. Because molecular gels do not have the splice structure found in PROTACs, they typically have smaller molecular weight, better oral absorption, and better cell permeability.
Although the research and development of molecular gums is not as extensive as PROTACs, the field is gradually gaining attention, and several companies such as Monte Rosa Therapeutics are actively developing related drugs, some of which have entered clinical trials. Since 2022, there has been an increase in capital inflows in the molecular gum space, with emerging companies and pharmaceutical giants including Plexium, BMS and others increasing their investment and collaboration in the technology.
Molecular glue at BOC Sciences
Chaperone mediated protein degradation (CHAMP)
Chaperones play a central role in protein folding, helping more than half of all proteins fold correctly. These chaperone proteins are composed of multiple families, each with its own unique folding aid mechanism.
In addition to promoting protein folding, molecular chaperones are also tasked with monitoring protein mass. They recognize those misfolded proteins and direct them to the UPS for removal. For example, heat shock protein 90 (HSP90) is involved in the degradation of misfolded proteins by interacting directly with multiple E3 ubiquitin ligases, ensuring that they do not mislocate or affect the normal functioning of cells.
The HSP90 complex exhibits a highly active state in tumor tissues compared to normal tissues, which makes the binding of the small molecule inhibitor to HSP90 tumor specific. Based on this property, Ranok Therapeutics has designed CHAMP, a heteroduplex small molecule targeting BRD4 and HSP90, to achieve selective degradation of specific proteins. In early 2022, BRD4 selective inhibitor RNK05047 received IND approval from FDA and is scheduled to initiate Phase I/II clinical trials.
In cells, three different lysosomal pathways are involved in protein degradation:
1) Cell surface proteins arrive at lysosomes through endocytosis, and then are degraded by lysosomes or transported to other organelles for circulation.
2) In the phagocytosis pathway, cells engulf large extracellular particles, such as pathogens and dead cells, which are subsequently digested in lysosomes.
3) Misfolded proteins, protein aggregates, or damaged organelles are cleared through the autophagolysosome pathway. Autophagy is divided into three types: macroautophagy, microautophagy and chaperon-mediated autophagy (CMA).
In macroautophagy, damaged proteins or organelles are recognized by autophagy receptors and encased in the autophagosome, which then fuses with the lysosome and the substances therein are degraded. Microautophagy involves lysosomes directly engulfing and degrading autophagy cargo. In CMA, chaparonein selects a specific target and helps it to cross the lysosomal membrane for degradation. Different from the other two autophagy, CMA specifically degrades a single protein without the formation of autophagosomes.
Protein degradation via three distinct lysosome pathways. (Zhao, L. 2022)
TPD technologies utilizing lysosomal degradation pathways have developed rapidly in the past few years, including LYTAC, bispecial aptamer chimera, AbTAC, and GlueTAC technologies utilizing endosomal lysosomal pathways to degrade extracellular and membrane proteins. AUTAC, ATTEC and AUTOTAC are used to degrade misfolded proteins, protein aggregates or damaged organelles. Compared with proteasome-dependent degradation technology, lysosomal targeted degradation technology has a wider applicability, not only can degrade intracellular proteins, but also effectively treat protein aggregates, excess or damaged organelles, membrane proteins and even extracellular proteins, so it shows great potential in clinical application.
LYTAC is a bi-functional molecule, similar to PROTAC, but specifically designed to degrade extracellular and membrane bound proteins. One end of LYTAC contains an oligosaccharide peptide that binds to a cation-independent mannose-6-phosphate receptor (CI-M6PR), while the other end binds to a target protein and is linked by linker. By forming a complex with CI-M6PR, LYTAC induces endocytosis and introduces the complex into lysosomes for degradation. Due to LYTAC's ability to reach extracellular and membrane proteins, its therapeutic potential goes far beyond traditional protein degradation techniques, offering new possibilities for treating diseases such as immunity and pain. Lycia Therapeutics, a leader in LYTAC technology, has attracted $120 million in investment since its founding in 2019, and has partnered with Eli Lilly to develop LYTAC-based degraders.
Schematic diagram of LYTAC's mechanism of action.
ASGPR targeting chimera (ATAC)
ATAC is an innovative protein degradation technology that uses the salivary glycoprotein receptor (ASGPR) on the surface of liver cells as a medium to direct the target protein to the lysosome for degradation. The ATAC molecule binds to the target protein and ASGPR to form a complex, which then enters hepatocytes through endocytosis and is degraded in lysosomes. Due to the hepatocellular specificity of ASGPR, ATAC has a potential safety advantage over LYTAC, which is particularly suitable for degrading extracellular liver proteins. Avilar Therapeutics, the company behind the ATAC concept, has raised $60 million in seed funding in 2021.
AbTAC, proposed by scientists at the University of California, San Francisco, is another bispecific molecule that uses antibodies to recruit a membrane-bound E3 ligase (ring finger protein 43) to degrade a cell surface protein (PD-L1). Similar to LYTAC, AbTAC is based on the endosom-lysosome pathway, but its specific mechanisms remain to be clarified, such as the ubiquitination status of POI and the reuse mechanism of RNF43.
AbTAC diagram.
Bi-specific aptamer chimera (BIAC)
BIAC has a similar mechanism of action to LYTAC, mediating POI degradation through the nucleosome-lysosome pathway. The difference is that bispecial aptamer chimeras use DNA aptamers to target CI-MPR and transmembrane POI. In February 2021, researchers from Shanghai Jiao Tong University designed the first BIAC, named A1-L-A2. Studies have shown that A1-L-A2 can transport membrane proteins (such as MET and PTK-7) to lysosomal degradation without having a large impact on non-targeted proteins. Compared with AbTAC, aptamers show promising application prospects because of their simple preparation, precise structure and high stability.
GlueTAC
GlueTAC is a lysosome based degradation strategy consisting of covalently modified nanobodies, cell-penetrating peptides (CPP), and lysosomal sorting sequences for the degradation of cell surface proteins. Nanobodies target POI, CPP promotes endocytosis, and the complex is subsequently degraded in lysosomes. The research team at Peking University developed GlueTAC molecule targeting PD-L1, which experimentally proved to be superior to the PD-L1 antibody atezolizumab in reducing PD-L1 levels and inhibiting tumor growth. However, GlueTAC's safety, half-life and other parameters still need to be further studied.
AUTAC is a technique for targeted protein degradation using the autophagosome lysosome pathway, inspired by the role of 8-Nitro-cGMP in the intracellular regulation of autophagy. The AUTAC molecule is made up of three parts: a cGMP-based degradation tag, a Linker, and a ligand that targets a specific protein. It works by triggering a polyubiquitination event in the K63 link, which then guides the target protein or organelle into the lysosome for degradation.
AUTAC also acts on organelles such as mitochondria. A study published in late 2019 reported that the molecule AUTAC4, a chimera that promotes mitochondrial autophagy, is expected to play a greater role in the degradation of protein aggregates.
ATTEC is a protein degradation technology based on autophagy mechanism, which binds POI to LC3, a key component of autophagosome, thereby guiding POI into the autophagosome and finally fusing with lysosome for degradation. A research team at Fudan University has developed an ATTEC molecule that can specifically bind LC3 and mutant Huntington protein (mHTT) to achieve autophagosome mediated degradation of mHTT. In addition, ATTEC technology has also been shown to degrade lipid droplets, expanding its application in the degradation of non-protein substances. PAQ Therapeutics, Inc., has successfully closed A $30 million Series A funding round using ATTEC technology.
Autophagy targeting chimera (AUTOTAC)
AUTOTAC is another autophagy mediated degradation technology, proposed by Korean scientists in 2022. The AUTOTAC molecule contains modules that interact with the ZZ domain of p62 and a POI targeting module. As an autophagy cargo receptor, p62 binds to the polyubiquitinated protein through its UBA domain, inducing the conformational change of p62, exposing the LIR motif, and promoting the binding to LC3, thus introducing the polyubiquitinated protein into the autophagy. AUTOTAC can not only mediate the degradation of monomer proteins, but also effectively degrade proteins with aggregation tendency. AUTOTACBio is focused on AUTOTAC technology research and development, and its CEO, Dr. Yong Tae Kwon, is the co-corresponding author of the paper on the technology. The company has been deployed in multiple disease areas, including neurodegenerative diseases, cancer, metabolic syndrome and muscular dystrophy, with an extensive product pipeline.
CMA-based degraders
Cma-based degraders consist of three functional domains: cell membrane penetration sequence, POI binding sequence, and CMA targeting motif.
In chaperone mediated autophagy (CMA), heat shock protein 70 (HSP70) uses the KFERQ sequence to recognize soluble protein substrates. Subsequently, the HSP70 substrate complex binds to lysosome associated membrane protein 2A (LAMP2) on the lysosomal membrane, and the substrate is translocated to the lysosomal cavity for degradation.
The technique has been shown to reduce levels of the scaffold proteins PSD-95, DAPK1, and alpha-synuclein. However, to be an effective therapeutic strategy, CMA-based degradants need to overcome at least two major hurdles: first, the stability of the degradants; second, stable process.
Conclusion
TPD has been one of the most dynamic areas of research in science and industry over the past few years, and the proliferation of new technologies has opened new doors for drug development. In the context of today's market of various technologies, analysts have given great expectations to the target protein degradation market, which is expected to reach $480 million in 2024, and is expected to reach $6.94 billion in 2035, with an annual growth rate of 32% during the forecast period.
Several TPD molecules are currently undergoing clinical trials for cancer indications. In addition to cancer, there are also many TPD molecules that show great potential in areas such as neurodegenerative diseases, inflammatory diseases, or viral infections.
In terms of technical types, PROTAC and molecular glue are currently the most advanced TPD technologies (both have multiple projects in clinical development), 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 often faces challenges in terms of cell permeability and oral bioavailability due to its large molecule, while molecular gums are small in size but currently difficult to effectively design.
As a new technology, PROTAC has great potential, but the risk of immature technology is also coexisting, and many mechanisms have not been studied clearly. Nevertheless, with further research and development, it will bring more benefits to mankind.
References:
- Yang, N., et al. Recent advances in targeted protein degraders as potential therapeutic agents. Molecular Diversity. 2024, 28(1): 309-333.
- Zhao, L.,et al. Targeted protein degradation: mechanisms, strategies and application. Signal Transduction and Targeted Therapy. 2022, 7(1): 113.
- Khan, M. Z. I., et al. Targeted protein degradation: A promising approach for cancer treatment. Journal of Pharmaceutical Analysis. 2023.
- Ji, C. H., et al. The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system. Nature Communications. 2022, 13(1): 904.
