Targeted protein degradation using proteolysis-targeting chimeras

Targeted protein degradation (TPD) is a new therapeutic approach used to hijack the cell’s degradation pathways to induce degradation of specific proteins. Protein degradation is a tightly regulated cellular process which eliminates dysfunctional proteins, can modulate levels of protein expression in a cell, and is important for cellular homeostasis.

PROteolysis TArgeting Chimeras (PROTACs®) are small molecule heterobifunctional degraders that coopt the cells ubiquitin-proteasomal degradation pathway (UPP) by linking an E3 ubiquitin ligase to a target protein of interest (POI), prompting transport to the proteasome for degradation. While most of the PROTAC attention has been focused on drug discovery, PROTACs are also proving to be a useful tool in basic biological studies where a fusion-tagged POI can be targeted for degradation allowing interrogation of cellular pathways without the need of genetic manipulation.

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The therapeutic application for using PROTACs was conceptualized by Craig Crews and Raymond Deshaies’ group in 2001 and has been used on multiple targets with varying sub-cellular localization [1, 2]. Initially, the concept of utilizing natural cellular processes for protein degradation for potential therapeutics was developed through virus and plant research.

Viruses are known to harness the human ubiquitin-proteasomal pathway (UPP) by using the cell’s own machinery for efficient replication and survival. For example, the human papillomavirus type 16 (HPV-16) utilizes the E6 protein to recruit the human E3 ligase and ubiquitylate p53, leading to its degradation [1]. In plants, small molecules in addition to proteins are capable of undergoing UPP-mediated protein degradation [1]. The hormone auxin (indole-3-acetic acid; IAA) can impact degradation of the Aux/IAA family of transcriptional repressors that are involved in plant development. These early studies highlighted the possibility of intentionally developing and directing targeted protein degradation using E3 ligases. Newer PROTACs have been designed as small-molecule structures aimed to degrade a wide range of target proteins [1, 3]. Over the past two decades, there is increasing evidence that PROTACs have broad therapeutic potential. Research has moved from basic cellular studies to more animal and in vivo disease models and even recently into clinical trials [1, 4].

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Many small molecule drugs exert their effects by binding to a target protein, leading to inhibition of function. As these small molecule inhibitors need to be bound to the protein to remain active, high concentrations of drug may be required, resulting in unintended off-target effects [1, 2]. Another important limitation of small molecule inhibitors is that it requires them to bind to an active or allosteric site of the target-protein which can be difficult to access on some disease-related proteins.

PROTACs are heterobifunctional molecules that consist of two ligands attached through a linker [1]. One of the ligands is responsible for recruiting and binding the target POI while the other ligand binds an E3 ubiquitin ligase. As the PROTAC molecule links the POI with the E3 ligase, they form a ternary complex which induces the E3 ligase to ubiquitinate the target protein and initiate the degradation process [1, 5]. PROTACs utilize proteolysis mechanisms following the UPP in which intracellular proteins are naturally degraded as a part of cellular maintenance. Upon introduction of the protein into the proteasome, PROTACs are reprocessed and efficiently recycled to be used on other copies of the target protein. The POI is degraded by the UPP within the proteasome, making amino acids and peptide substrates available for future cellular use (Figure 1).

Figure 1. PROTACs in the ubiquitin-proteasome pathway.
 

PROTACs hold several advantages compared to traditional small molecule inhibitors for their use in TPD. First, PROTACs expand the accessibility of druggable proteins by utilizing targets traditionally thought to be undruggable due to the absence of a well-defined binding pocket, curbing off-target effects. Secondly, while small molecule inhibitors act on a specific catalytic function of an enzyme, many enzymes have dual roles in scaffolding which play a critical part in disease. Instead of altering enzymatic activity, PROTACs degrade the POI directly, thereby eliminating all function of the protein. Third, PROTACs are catalytic and do not need to remain bound beyond the ubiquitination process. Once the POI is ubiquitinated, the PROTAC dissociates, and can be recycled for additional rounds of ubiquitination/degradation [1, 4]. This mechanistic capability allows the drug to be used at lower doses, resulting in less off-target effects and reduced drug resistance. With around 600 ubiquitin ligases to explore, the field has a huge potential to develop therapies for many different diseases [1].

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To assess targeted protein degradation functionality and efficiency, one will require sensitive, robust, high-throughput solutions. From E3 ligase binding and ternary complex formation to ubiquitination and degradation, there are multiple steps in which TPD efficiency can be monitored throughout the workflow using a variety of tools and scientific applications (Figure 2).

Figure 2. Common assays for assessing PROTAC efficiency.
 

To create a functioning and efficient degrader, an E3 ligase binding motif must be connected to a POI ligand through a carefully designed linker. Together the POI, PROTAC and E3 ligase form a ternary complex. To determine if an essential E3 ligase binding motif is present on a protein degrader (PROTAC), it is recommended to utilize binding assays such as isothermal calorimetry and/or probe displacement assays utilizing fluorescence polarization or time resolved fluorescence resonance energy transfer (TR-FRET) technology.

PROTACs are heterobifunctional molecules used to recruit an E3 ligase to degrade a protein of therapeutic interest, which is an integral step of the protein degradation process. The formation of the ternary complex triggers the ubiquitination of the POI by the UPP.

One of the most important ways to assess ternary complex formation is to perform proximity-based assays such as TR-FRET or crosslinking mass spectrometry [6]. TR-FRET utilizes long-lived fluorophores and time-gated fluorescence to quantify molecular association and dissociation events. This can be used to study interactions between PROTACs and their POI [6]. Crosslinking mass spectrometry for protein-protein interaction studies enables visualization of the interacting regions by helping identify protein complexes.

To assess if the protein has been degraded, it is important to confirm if the protein has been ubiquitinated. There are many assays to assess protein ubiquitination such as TR-FRET, western blots, ELISA, protein mass spectrometry and protein isolation assays. LanthaScreen TR-FRET assays provide high-throughput screening reagents to monitor PROTAC induced ubiquitination in kinetic or endpoint readouts. Polyubiquitinated proteins can be isolated from cell lysates using a high-binding affinity resin. In this case, the Ubiquitin Enrichment Kit can be used for isolation and analysis of these proteins. Performing protein mass spectrometry analysis can also be an important tool to identify pre- and post-modifications of proteins.

To determine if the POI has been degraded, expression levels of the protein could be assessed through western blotting or performing immunoassays. Large-scale analysis of protein degradation can be achieved through tandem mass spectrometry and tandem mass tag (TMT) systems, which are powerful tools to confirm degradation of the POI and screen for off-target effects of the PROTAC on other proteins. On a smaller scale, single-cell analysis can be utilized. Additionally, TR-FRET assays can determine the lack of interactions between the PROTAC and the respective POI to assess degradation.

In terms of cytotoxicity, it has been shown that PROTAC induced oncogenic protein degradation can lead to cell death through apoptosis [7]. Cellular health after targeted protein degradation can be assessed through a variety of cell viability and function assays including apoptosis assays and cell proliferation assays. Flow cytometry, microplate assays, and high-content screening can also be used to assess cell viability and provide information on the physical health of cells after treatment with PROTAC-based molecules.

With the success of PROTACs, more research is being done into various classes of heterobifunctional molecules that do not rely on the UPP for protein degradation. For example, autophagy-targeting chimeras (AUTACs) work through a guanine derivative that tags the POI for degradation through the autophagy machinery. Autophagosome-tethering compounds (ATTECs) link a ligand that binds to the autophagy protein LC3 (microtubule-associated protein 1 light chain 3a) to the POI, bypassing the UPP by directly tethering the POI to the autophagosome [1]. Molecular glues are another type of protein degrader which constitute an important therapeutic class. Although they are not heterobifunctional like PROTACs, molecular glues stabilize or create novel binding sites between the E3 ligase and neosubstrates leading to the ubiquitylation and degradation via the UPP [1, 8]. While PROTACs and molecular glues continue to be developed to target intracellular proteins, extracellular proteins such as cytokines and growth factors, which make up about 40% of the proteome [1], are inaccessible to the UPP for degradation and require alternative methods for TPD.

Lysosomal-targeting chimeras (LYTACs) work by binding a membrane-bound POI to the extracellular domain of a lysosome-shuttling receptor which internalizes the POI into the lysosome for degradation [1]. Antibody-based PROTACs (AbTACs) on the other hand, are bispecific antibodies that recruit membrane-bound ligases to membrane-bound POIs for degradation through the lysosomal degradation pathway. These novel approaches for targeted protein degradation continue to advance the field with robust therapeutic opportunities.

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