Targeted Protein Degradation

Figure 1. Schematic showing the catalytic mode of action of Degrader molecules.

What are Small Molecule Protein Degraders?

Protein Degraders are bifunctional compounds comprising 3 components: an E3 ubiquitin ligase ligand, a linker, and a ligand for a target protein of interest. They induce the formation of a ternary complex by simultaneously binding to both an E3 ligase and target protein. Ternary complex formation effectively ‘hijacks’ the E3 ligase to polyubiquitinate a target of interest, inducing subsequent degradation by the 26S proteasome.

The PROTAC^® concept was first described in 2001. The first generation of Degraders synthesized were peptide-based and were not cell-permeable, however they demonstrated the principle that it is possible to direct an E3 ligase to degrade a protein of interest. The first small molecule protein Degraders were also reported by Craig Crews’ group in 2008. Since then, a wide range of degraders have been synthesized, recruiting different E3 ligase enzymes, including Cereblon, von Hippel Lindau (VHL) and inhibitor of apoptosis (IAP), and targeting a variety of protein targets, such as enzymes, receptors, bromodomains and transcription factors. 

How do Degrader molecules work?

Targeted Protein Degradation is a technique that uses heterobifunctional small molecule Degraders to bring about efficient and highly selective degradation of a target protein of interest. Targeted protein degradation enables investigation of the downstream consequences of protein knockdown in a reversible and tuneable manner, without the requirement for genetic modification to cells, and provides an easy-to-use method for target validation. Degraders also have therapeutic potential as an approach to target the ‘undruggable’ proteome and overcome common resistance mechanisms to current therapies.

R&D Systems small molecules provides Protein Degraders, including PROTAC (PROteloysis TArgeting Chimera) molecules and TAG Degraders (dTAG/aTAG/BromoTag). PROTAC molecules comprise a ligand that binds a target of interest joined by a linker to an E3 ligase ligand. A ternary complex is formed by the simultaneous binding of the Degrader to the target protein and the E3 ligase, which initiates ubiquitination of the target protein and its subsequent destruction by the proteasome. TAG Degraders work on a similar principle, but comprise an E3 ligase ligand linked to a compound that binds a fusion of a TAG degron with a target protein of interest. Degraders enable the knockdown of proteins for which there are no known ligands, enabling targeting of previously undruggable targets.

The R&D Systems brand supplies a range of other products and services to support Targeted Protein Degradation studies, including Degrader Building Blocks to enable you to create your own Degraders, as well as Ubiquitin-Proteasome System (UPS) Proteins and Assays, Assays for Protein Degradation, and Custom Degraders Services.

Why Use Degraders?

Small molecule Degraders offer several advantages over gene editing methods such as CRISPR and RNAi for target protein knockdown:

• Degraders are cell-permeable, are suitable for use in vivo and in vitro, and can be applied directly to cells with no need for transfection or expression vectors.
• They are applicable to multiple cell lines with no requirement that cells are easily transfectable
• The duration of effect is adjustable and reversible on compound washout
• The level of protein degradation can be easily tuned by altering the dose of Degrader used
• They have a catalytic mode of action repeatedly engaging and directing ubiquitination of target molecules, allowing use at sub-stoichiometric concentrations.

Using Protein Degraders

The activity of Degraders is represented by the DC₅₀ and D_max values. The DC₅₀ is a measure of potency and is the concentration at which 50% of the target protein is degraded; it is equivalent to the IC₅₀ for an inhibitor. The D_max is the maximum level of degradation achievable.  These values are useful guides to determine the appropriate concentration of Degrader to use in your experiment.  The timescale to achieve maximum degradation varies significantly between different Degraders and should also be taken into consideration when planning your experiment.

Effective knockdown of target proteins can be achieved with very low Degrader concentrations. In some cases, Degraders exhibit the “hook effect” at higher doses. This effect refers to the reduced degradation efficiency resulting from the increased formation of binary complexes at higher Degrader concentrations, which compete with formation of ternary complexes.

Molecular Glues 

Molecular Glues are small molecule protein dimerizers that bring about targeted protein degradation via a similar mechanism to PROTAC^® Degraders. However, molecular glues are not chimeric molecules.  Molecular glues act by forming a ternary complex with a target protein and an E3 ligase leading to ubiquitination of the protein and its subsequent destruction by the proteasome. The interaction of the molecular glue with the E3 ligase may result in allosteric modification of the E3 ligase binding site, such that it is unable to bind its native substrate, enabling ternary complex formation with a protein of interest (or neosubstrate). Like other small molecule Protein Degraders, molecular glues act catalytically.

Figure 2: Wes data showing knockdown of both CDK9 isoforms after THAL SNS 032 (Cat. No. 6532) treatment (200 or 500 nM) of MOLT-4 cells. Protein quantification (relative to DMSO-only control) is shown beneath the corresponding lane.

Experiments were performed by the Simple Western™ applications science team. Simple Western fully automated western blotting systems provide quantitative data, which allows for an accurate determination of DC₅₀ and D_max values for Protein Degraders.

Figure 3: The so-called “hook effect” is demonstrated in this western blot, which shows Degrader-mediated polyubiquitination of recombinant BRD4. CRBN E3 ligase complex (Cat. No. E3-650) was used to investigate Degrader-induced in vitro polyubiquitination of recombinant FLAG-tagged BRD4 (Cat. No. SP-600). Results were analyzed using an anti-FLAG Western Blot. The degree of substrate ubiquitination varies with the concentration of Degrader used in the reaction.

Types of Degrader

• LYTAC ( (LYsosome TArgeting Chimera molecules): LYTACs direct an extracellular protein of interest to the lysosome for degradation and were invented at the University of Wisconsin
• PROTAC® (PROteolysis TArgeting Chimeric molecules): this term was coined in a paper from Craig Crews’ lab (Yale University) in a 2001 paper and is now widely used to refer to protein degrader molecules.
• SNIPERs (Specific and Nongenetic Inhibitor of apoptosis protein (IAP)-dependent Protein ERasers): SNIPERs specifically recruit the E3 ligase IAP and were developed at the University of Tokyo.
• PHOTAC (PHOtochemically TArgeting Chimeras): these are photoswitchable degraders that can be activated by different wavelengths of light and were originated at New York University.
• uSMITE™ (Ubiquitin-mediated, Small Molecule-Induced Target Elimination technology): a type of Degrader developed by Cullgen