Targeted protein degradation and next-generation degraders

June 17, 2024

Innovation for targeted protein degradation and next-generation degraders is gathering pace. This blog introduces some of the different approaches that act via the lysosome or proteasome.

Dr Barry Whyte

Protein degradation in the cell

Research over many years helped build a comprehensive picture of protein synthesis in the cell. However, knowledge of how proteins are broken down and how degradation contributes to cellular regulation is more recent. The half-lives of proteins in cells are known to span from minutes to several days and differences in the rates of protein degradation contribute significantly to cell regulation. Regulatory proteins like transcription factors get degraded, other proteins like those involved in cell proliferation are dismantled in response to specific signals, and faulty proteins are recognized and removed to avoid damage to the cell. ¹

Today two major pathways are known to be involved in protein degradation in eukaryotic cells: the ubiquitin-proteasome pathway and the lysosomal proteolysis pathway. ¹ The discovery of ubiquitin tagging of proteins and their cyclical degradation via the proteasome heralded an appreciation for the importance of protein degradation pathways to the overall homeostasis of the cell. In addition to the proteasome, lysosomes are the other major end destination for many proteins (Fig.1). These membrane-bound organelles have roles in cell metabolism, which includes the degradation of extracellular proteins taken up by endocytosis as well as the steady turnover of cytoplasmic organelles. Different routes to the lysosome exist. Autophagy is one example. Here autophagosomes are formed to enclose unwanted entities and these membrane-bound structures eventually fuse with lysosomes to ensure degradation. Other examples are endocytosis and phagocytosis which are processes that can engulf larger particles into vesicles that subsequently fuse with lysosomes for degradation.

Knowledge of the cell’s pathways and capabilities to remove proteins is now being harnessed for therapeutic applications and drug discovery. Targeted protein degradation induced by small molecules offers opportunities to fine control the levels of unwanted proteins. Other degraders are under investigation as ways to remove damaged organelles or other molecules.

This blog serves as an entry point for targeted protein degradation and emerging degraders, describes some of the different types of approaches under development, and gives a glimpse of applications in different disease areas. Improvements to our understanding of the way cells do their own housework is triggering further innovation in drug development.

Different types of targeted protein degradation

Targeted protein degradation offers ways to remove disease-related proteins from the cell using small molecules known as degraders^.2,3 This approach can seal the fate of unwanted proteins in the cell including many previously undruggable protein targets.

Until recently, research on targeted protein degradation mainly focused on disease-related proteins that end up being dismantled in the proteasome. However, new approaches are emerging that degrade other disease targets including DNA/RNA molecules, protein aggregates, peroxisomes, ribosomes, damaged mitochondria, and even microbial pathogens.⁴ This diversity is being driven by innovation that looks beyond the proteasome to new “end destinations” in the cell like the lysosome opening up new possibilities for drug discovery.

Proteasomal and lysosomal degradation: some distinctions

In the world of targeted protein degradation, PROTACs and molecular glues have mostly been in the spotlight.^2,3,5 Both types of degraders use the ubiquitin-proteasome system to achieve targeted protein degradation (Fig. 2). However, inspired by progress with PROTACs and molecular glues, new degraders are emerging that make use of endocytosis and autophagy to send a broader range of targets to the lysosome, an alternative location within the cell for destruction of disease-related entities (Fig. 3).

Targeted degradation by small molecules can thus be achieved via many different routes that ultimately lead to either the proteasome or lysosome (Fig. 4). This expansion is extending the repertoire of degraders from intracellular soluble proteins and membrane proteins to new horizons that include nucleic acids, dysfunctional organelles, infectious organisms, extracellular proteins, lipid droplets and even aggregated proteins. These advances promise to unlock new therapeutic opportunities for a wide range of diseases including but not limited to cancer, neurodegenerative disorders and autoimmune conditions.

The ubiquitin-proteasome system

PROTACs

PROteolysis TArgeting Chimeras or PROTACs are the most well-known class of molecules in the targeted protein degradation portfolio.² PROTACs are bifunctional molecules that consist of two essential components: a ligand for the target protein and another ligand for an E3 ubiquitin ligase enzyme. The target ligand binds the protein of interest while the E3 ubiquitin ligase ligand recruits an E3 ligase enzyme. The recruited ligase brings the target protein into proximity with the ubiquitin-proteasome system where it is degraded (Fig. 2B).

Researchers are looking at different ways to advance the use of PROTACs for targeted protein degradation as part of their drug discovery efforts. A significant number of PROTACs are now in clinical trials which underlines the potential of targeted protein degradation to bring new solutions for unmet medical needs. Most of the early candidates being tested relate to different forms of cancer but new applications in different disease areas are steadily increasing. You can read more about this technology and targeted protein degradation in the BMG LABTECH blog PROTACs: Proteolysis targeting chimeras.

Molecular glues

Molecular glues are a class of small molecules that also facilitate targeted protein degradation via the ubiquitin-proteasome system. Unlike PROTACs, molecular glues do not have a linker molecule and interact with the ligase (most frequently) or the protein of interest. The absence of a linker molecule contributes to the smaller molecular weight of molecular glues which can translate into improved cellular permeability and bioavailability versus PROTACs.2 Compared with conventional occupancy-driven inhibitors used in drug discovery and development they may offer longer-lasting effects for targeted protein degradation, improved toxicity, and higher efficacy that is less likely to trigger drug resistance.² You can read more about molecular glues and targeted protein degradation in the BMG LABTECH blog Molecular glues: new solutions for undruggable proteins.

The lysosomal system

As pointed out in the introduction, new degraders are emerging that harness endocytosis and autophagy to seal the fate of a wider range of targets via the lysosome (Fig. 2B, Fig. 4). ⁶ The lysosome is a membrane-bound organelle found in eukaryotic cells that serves as a location for the controlled intracellular degradation of macromolecules. Lysosomes contain a variety of hydrolytic enzymes capable of breaking down various macromolecules including proteins, lipids, nucleic acids, and carbohydrates. The new generation of degraders thus avoid the ubiquitin-proteasome system and make use of cellular pathways that deliver a wider range of targets to the lysosome. The lysosome might be described as the cell’s “garbage disposal system” with capacity for a wider range of incoming macromolecules compared to the proteasome. The degradative enzymes within the lysosome function best under acidic conditions, which are maintained by proton pumps on the lysosomal membrane. The acidic environment is crucial for the efficient activity of the hydrolytic enzymes of the lysosome.

There are different pathways that lead to the lysosome which offer innovative approaches for targeted degradation of a broad range of unwanted cargo. Most of the next generation degraders function either through the endosome-lysosome pathway or the autophagy-lysosome pathway. Here we look at both routes and some of the emerging degraders in each class.

Endosome-lysosome pathway

Endosomes are membrane-bound organelles found in eukaryotic cells that play an important role in the sorting, processing, and trafficking of molecules within the cell. Endosomes operate as part of the endocytic pathway, which involves the internalization of extracellular material into the cell through the formation of vesicles (Fig. 2B). They function in coordination with other organelles including lysosomes and the Golgi apparatus.

Some of the new degraders under investigation can enter cells through endocytosis and traffic through the endosome-lysosome pathway to reach the lysosome where they deliver the target for degradation. In theory, this offers access to the degradation of most extracellular and membrane proteins which collectively amount to around 40% of the proteome.^6,7

LYTACs

LYTACs or LYsosome Targeted Chimeras are perhaps the most well-known of the endosome-lysosome degraders. LYTACs can bind the extracellular domain of a membrane protein (or an extracellular protein) and a lysosome-targeting receptor (LTR; also referred to as a lysosomal shuttling receptor; see Fig. 5) that resides on the cell surface. A ternary complex is formed which leads to protein internalization via clathrin-mediated endocytosis. The endosomes eventually fuse with the lysosome and the target of interest is subsequently degraded in the lysosome.

GlueTACs

GlueTACs or Glue Targeted Chimeras are another type of endosome-lysosomal degrader that can target cell-surface proteins. GlueTACs combine the use of nanobodies, antigens as well as a cell-penetrating peptide and lysosome-sorting sequence (CPP-LSS) to promote internalization and lysosomal degradation of the target. The nanobodies replace conventional antibodies to achieve cell penetration. The nanobodies and antigens interact via covalent interactions to overcome low binding affinities. GlueTACs are in the early stages of development and further studies are underway to define fully their mechanism of action and therapeutic potential.

AbTACs

AbTACs or Antibody-based PROTACs are an emerging type of endosome-lysosomal degrader that can act on extracellular or membrane proteins. While “PROTAC” is in-built to their full name, AbTACs bear more similarities to LYTACs. AbTACs make use of bispecific antibodies. One of the arms of the antibody targets the cell-surface protein of interest. The other arm targets a transmembrane E3 ligase. The AbTAC molecule induces the internalization of the cargo and subsequent degradation of the protein of interest in the lysosome. Further work is in progress to understand the precise mechanism of action including the exact details of the ubiquitination steps.

Autophagy-lysosome pathway

Autophagy is a cellular process involved in the degradation and recycling of cell components, including damaged organelles and proteins. It is required to maintain the homeostasis or effective functioning of the cell. The word “autophagy” originates from the Greek word meaning “self eating” which reflects the way cells engulf and degrade their own components (Fig.2B).

Autophagy kicks off when the phagophore embraces cell contents. The phagophore seals its cargo into a double-membrane-bound autophagosome vesicle which can fuse with the lysosome and deposit its contents for degradation.⁶

AUTACs

AUTACs or AUtophagy-TArgeted Chimeras are autophagy-lysosome degraders that consist of three parts: a cGMP-based degradation tag; a linker molecule; and a small molecule ligand suitable for the protein of interest or an organelle. The cGMP degradation tag is based on nucleotide 8-nitrocyclic guanosine monophosphate which is a signaling molecule in the cell and can recruit autophagosomes (Fig.2A). AUTACs are capable of degrading organelles such as mitochondria in addition to cellular proteins.

Mitochondrial dysfunction is linked to several age-related and other diseases and the removal of damaged organelles is an attractive strategy to meet unmet clinical needs. AUTACs potentially have a wide range of applications beyond proteins of interest per se including the ability to degrade protein aggregates.

ATTECs

ATTECs or AuTophagosome TEthering Compounds are autophagy-lysosome degraders that tether or link a protein of interest to the autophagosome (Fig.6).⁸ ATTECs bind to LC3 which is a mammalian homolog of yeast Atg8 (autophagy-related protein 8) and a key protein that is unique to the autophagosome. Autophagosomes are double membrane vesicles containing cytoplasmic material destined for degradation. In autophagy, autophagosomes fuse with lysosomes to form autolysosomes (Fig.6). The fusion process allows the contents of autophagosomes to be degraded by lysosomal enzymes including any protein of interest bound to LC3 by the action of an ATTEC. ATTECs have also been reported to target lipid droplets. Some small molecule ATTECs bind LC3 protein as well as lipid droplets. Thus, ATTECs can harness the autophagy-lysosome pathway for the degradation of non-protein materials as well as protein molecules.

ATTECs have been reported to recognize mutant huntingtin protein, specifically the polyglutamine stretch of the defective protein that leads to Huntington disease. They may therefore prove useful as new therapeutic approaches to neurodegenerative disease.

AUTOTACs

AUTOTACs or AUTOphagy-TArgeting Chimeras also work by recruiting specific proteins to autophagosomes for ultimate degradation within lysosomes. AUTOTACs bind p62 and a protein of interest. p62 (or SQSTM1 as it is also known) bridges polyubiquitinated cargo and autophagosomes. The p62 protein is a multifunctional protein involved in various cellular processes including selective autophagy. p62 contains multiple protein-protein interaction domains which allow it to interact with ubiquitinated cargo and LC3, the same protein involved in the mechanism of ATTECs. The addition of AUTOTACs therefore bridges a protein of interest and p62 in a way that is independent of ubiquitin on the target protein. An ATTEC molecule simultaneously binds LC3 and a protein of interest. However, AUTOTACs bind p62 and a protein of interest but the LC3 interaction proceeds as a next step after a conformational change in p63.

Future developments

Today most targeted protein degraders in clinical trials are for cancer applications. However, the diversity of the existing and new approaches offers great promise for the treatment of neurodegenerative diseases, inflammatory and autoimmune diseases, viral infections as well as other disease areas where work is just getting underway.

New targeted degrader concepts appear almost daily. ⁶ The world of targeted protein degradation and other innovative targeted degraders is rapidly expanding, and new acronyms are being coined on a regular basis. As shown in Fig.3, ATTECs, AUTACs, LYTACs, AbTACs, GlueTACs and AUTOTACs are very much new kids on the block compared with the targeted protein degradation options offered by PROTACs and molecular glues. Research in these exciting new areas has just started and much work lies ahead in the drug development space.

Microplate readers in degrader research

Microplate readers are versatile tools for protein degrader research and offer an inclusive and cost-effective option to look at the complete process from target engagement to degradation. Moreover, microplate readers help increase throughput and decrease the need for manual intervention, facilitating an efficient screening for degrader candidates.

What is the preferred BMG LABTECH microplate reader for specific needs and applications related to targeted degrader research? BMG LABTECH offers a range of detection devices for sensitive absorbance, fluorescence and luminescence measurements.

The PHERAstar FSX was specifically conceived for screening campaigns and is your go-to reader for high-performance high-throughput investigations in targeted degrader research.

Both the VANTAstar^® and CLARIOstar Plus allow for wavelength scanning and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options. In addition, they can be equipped with the Atmospheric Control Unit for live cell-based assays.

All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus and PHERAstar FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection.

Collectively, these multi-mode readers combine high performance with miniaturized assays, short measurement times, and offer considerable savings on materials and other resources.

You can read more about assay options for targeted protein degradation in the BMG LABTECH blog Cell-based protein degrader assays for microplates

For more information on targeted protein degradation experiments and more ways to evaluate molecular glues, PROTACs and other degraders check out this scientific talk:

Knowledge of PROTACs and molecular glues has been built over decades. New technologies for targeted protein degraders are emerging at pace along with approaches targeting different degradation routes and even ways to degrade larger components of cells including defective organelles. The field has come a long way since the Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avrim Hershko and Irwin Rose in 2004 for the discovery of regulated protein degradation. Not many people were interested in how proteins were broken down at that time. But the discovery of ubiquitin tagging of proteins and their cyclical degradation in the proteasome not only improved understanding of how processes like cell division, DNA repair, quality control of proteins and immunology are governed but also strengthened the idea that useful drugs could arise from looking at the native degradation systems of the cell.

BMG LABTECH is ideally placed to support existing and future needs in this promising area of research that offers new opportunities to reach previously undruggable protein targets.

What is the preferred BMG LABTECH microplate reader for specific needs and applications related to molecular glue degrader research? BMG LABTECH offers a range of detection devices for sensitive absorbance, fluorescence and luminescence measurements.

The PHERAstar FSX was specifically conceived for screening campaigns and is your go-to reader for high-performance high-throughput investigations in molecular glue research.

Both the VANTAstar^® and CLARIOstar Plus allow for wavelength scanning and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options. In addition, they can be equipped with the Atmospheric Control Unit for live cell-based assays.