Currently, the TPD technology has achieved remarkable success in the development of new therapies. However, the major bottleneck faced by the field is the limited availability of E3 ligases. Current successful TPD applications mostly use the E3 ligase substrate receptors, and the single-subunit E3 ligase is less efficiently ubiquitinated on new targets. Despite the over 600 E3 ligases in the human genome, only 12 have currently been applied to TPD studies, and PROTAC candidates in clinical trials rely mainly on CRBN and VHL ligases. This narrowness of E3 ligase use raises concern because cancer cells may develop resistance through mutation or down-regulation of these E3 ligases. Extending the E3 ligase toolbox not only contributes to broader protein degradation, but may also improve selectivity and overcome drug resistance. The discovery of more E3 ligases will significantly drive the TPD field, and researchers are making a huge effort to do so. Here, we discuss and provide insights into existing approaches to E3 ligases recruited by PROTAC and MGD ligases as well as potential new approaches, and highlight their application and limitations.

Figure 1. Overview of the E3 ligase deconvolution strategy in the field of target protein degradation

01、and CRISPR screening

The CRISPR screen is an effective tool for the identification of the protein targets of drugs or compounds. In the study of TPD, CRISPR-based knockdown screens provided a loss-of-function-based strategy to identify E3 ligases recruited by PROTAC or MGD. There are currently two main CRISPR knockout screening strategies: survival / resistance-based and reporter-based approaches. Survival / resistance-based screening relies on cell survival and drug resistance, applicable to those situations where target proteins are essential for cell survival. Reporter-based methods directly quantify the degradation of POI by fluorescent or luminescent reporter markers and are often used to study degraders (Figure 2).

Figure 2. Two major CRISPR screening strategies were used to identify the E3 ligases for PROTAC or MGD

CRISPR Filening can be in set (pooled) or array (arrayed) formats. The pooled screening introduced the entire mixture of single-directed RNA (sgRNA) libraries into cells, and after applying selection pressure, genes associated with protein degradation were inferred by comparing the abundance of sgRNA with or without selection pressure. This method efficiently screens many genes, but interpreting the results of complex mixtures is challenging. Array screening maintains each sgRNA isolated to facilitate direct determination of each gene function, and data analysis is relatively simple. Although array screening requires specialized equipment and automation, it is more compatible for non-proliferating cells, primary cells, or neuronal cells (Figure 3).

Figure 3. Assembly and array patterns of CRISPR screening

The CRISPR screen also facilitates the identification of essential E3 ligases in disease-associated cells, and is expected to use this information to develop new degraders to overcome the potential resistance of disease cells. Although CRISPR screens have limitations such as redundant functions of E3 ligases and inability of knockout of essential E3 ligases, reporter-based approaches and pooled sublibraries remain efficient and cost-effective strategies to facilitate research and development of new protein degraders.

02、affinity-based proteomics

The affinity-based proteomic approaches have important applications in studying protein interactions and degradation. Through the induction of the E3 ligase to form a ternary complex with the target protein (POI) by PROTAC or MGD, the affinity-based approach enables the effective identification of the recruited E3 ligase. For example, co-immunoprecipitation-mass spectrometry (co-IP-MS) and affinity purification mass spectrometry (AP-MS) techniques are combined with affinity tags or antibodies to analyze protein-protein interactions. In most cases, these methods are applicable to complexes formed through stable interactions, but also face limitations that do not apply to weakly interacting complexes (Figure 4).

Figure 4. General workflow of affinity-based proteomics

Affinity-based proteomics approaches often require relatively strong interactions between two proteins, so that stable ternary complexes are often a prerequisite for rapid and efficient degradation.

03、Activity-based protein analysis (ABPP)

The ABPP method has been used to identify multiple E3 ligases. For example, the binding of nimbolide to the E3 ligase RNF114 was identified by isoTOP-ABPP analysis, and its anticancer activity was verified. In addition, these methods are also used to identify and validate photoaffinity tag-based compounds, such as compounds without intrinsic covalent groups, to enrich and analyze target proteins by forming covalent bonds with nearby proteins through photoactivation (Figure 5).

Figure 5. General workflow of isoTOP-ABPP

With these methods, researchers can more precisely understand how degraders form a complex by inducing E3 ligases with target proteins and then regulate protein degradation. The application of these technologies provides powerful tools for drug discovery and biological research for the deep exploration of molecular mechanisms in complex biological systems.

04、Enzyme-mediated proximity marker (PL)

Enzyme-mediated proximity labeling techniques (PL) are used to identify small molecule targets, especially E3 ligases recruited by PROTACs or MGDs. PL is able to identify strong binding partners while effectively capturing transient and weak protein-protein interactions (PPIs), overcoming the limitations of traditional affinity-based approaches. This technology uses a biotinidase fused to the target protein (POI) to biotinylate adjacent endogenous proteins within a certain range. These biotinylated proteins were identified by enzymatic hydrolysis and subsequent enrichment steps (e. g., streptavidin or anti-biotin antibodies) and finally identified by mass spectrometry (LC-M S/MS) or western blotting (Figure 6).

Figure 6. General workflow of enzyme-mediated proximity labeling

05、thermal proteomics (TPP)

Thermal proteomics (TPP) is an emerging approach that combines multiple mass spectrometry and cellular thermal shift determination (CETSA) for studying E3 ligases recruited by novel PROTACs or MGDs. TPP can monitor the whole proteome during drug treatment and analyze the thermal stability of proteins. CETSA observed the thermal stability of the protein in the presence or absence of the drug by gradually increasing the temperature. Usually, drug-bound proteins exhibit higher thermal stability. This approach is suitable for studying the interaction between E3 ligase and POI caused by PROTACs / MGDs, identified by the enhanced thermal stability of E3 ligase (Figure 7).

Figure 7. General workflow of the TPP

06 、the calculation method

Computational techniques can be used to predict protein – protein interactions (PPI) and then identify E3 ligases that act on the POI through TPD technology. Formation of the ternary complex is critical to achieving rapid and efficient protein degradation. Despite the challenges, several approaches have attempted to predict the ternary complex structure. Currently, identifying E3 ligases by predicting protein-protein interactions requires further investigation. However, the strong interaction between E3 ligase and POI can improve the success rate of PROTACs development and provide an opportunity for the rational design of MGDs. Binding between 600 E3 ligases and a specific POI is predicted by a validated PPI prediction method. Moreover, using protein structure prediction techniques such as AlphaFold, even without experimental structures, the structures of most POIs and E3 ligases today are already available, further facilitating structure-based PPI prediction.

Figure 8. Comparison of the various and different strategies

07 、Application and limitations of various strategies

Developing drugs based on PROTAC and MGD is attracting widespread attention as TPD has become a new strategy to address unmet medical needs. Although various strategies have been used to identify E3 ligases, they still have limitations. For example, CRISPR screening is effective in unbiased E3 ligases, but it is costly, time-consuming and labor intensive; ABPP technology has successfully identified covalent E3 ligase ligand and determined its binding site. However, identifying E3 ligases using chemoproteomics can be challenging, so coco-IP-MS or PL methods are a better option; co-IP-MS is suitable for covalent and non-covalent binders, but requires a stable ternary complex (Figure 8). In summary, there is no general method suitable for all degraders. Method selection should be based on the characteristics of the degrader and the specific experimental conditions. Typically, a combination of multiple methods can more accurately capture the E3 ligase. Furthermore, regardless of the method used, experimental validation of the degradation effects is essential to reveal the E3 ligase.

reference documentation

[1] Ligandability of E3 Ligases for Targeted Protein Degradation Applications

[2] Targeted protein degrader development for cancer: advances, challenges, and opportunities

[3] Targeted Protein Degradation: Current and Emerging Approaches for E3 Ligase Deconvolution

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