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Protein degradation is a fundamental process for the regulation of cellular homeostasis and the removal of misfolded or damaged proteins. However, traditional approaches for protein degradation, such as small molecule inhibitors or RNA interference, have limitations such as low selectivity, off-target effects, or inability to target intracellular proteins. A new class of molecules called PROTACs has emerged as a promising alternative for protein degradation. BioPROTAC is an engineered fusion protein that is a complementary approach to small molecule-based PROTACs and could serve as a potential therapeutic approach and research tool for targeted protein degradation.
The term PROTACs stands for Proteolysis-Targeting Chimeras, which refers the bifunctional small molecules that recruit E3 ubiquitin ligases to specific target proteins, leading to their polyubiquitination and subsequent degradation by the proteasome. The BioPROTAC (biological PROTACs) technology is based on the same principles as traditional PROTACs, but with some modifications.
BioPROTACs (biological PROTACs) are a complementary approach to targeted protein degradation that involve reengineering the E3 ligase by directly replacing its natural substrate recognition domain with a ligand that binds a POI. Unlike small molecule-based degraders, which use small molecules to bridge the substrate and the E3 ligase, bioPROTACs are composed of a fusion protein that combines the POI-binding peptide or miniprotein with the E3 ligase, which are expressed in cells to drive targeted degradation of POIs. BioPROTACs is a biologic equivalent of small molecule-based PROTACs, which can be serve both as a biological tool and as a potential therapeutic approach.
BioPROTACs are engineered fusion proteins consisting of a target binding domain and an E3 ligase, and the activity of BioPROTACs can be validated using GFP-tagged proteins as their substrates. there is considerable flexibility in the choice of substrate binders (binding sites, scaffold classes) and E3 ligases for BioPROTACs. For example, regions of the structural domain of the E3 ligase can be N-terminal fused to high-affinity anti-GFP nanobodies targeting POI. BioPROTACs can effectively deplete GFP signal strength when expressed in certain cell lines bearing GFP fusion protein knockouts. However, the formation and ubiquitination efficiency of BioPROTAC-POI complexes may be affected by species-related differences.
After transient transfection and expression of BioPROTACs in target cells and stable integration of GFP-POI, GFP fluorescence can be measured by flow cytometry. In cells expressing BioPROTAC, GFP intensity would be reduced. Interestingly, the degradation of GFP-POI is attenuated as BioPROTAC expression increased, which is consistent with the hook effect of small molecule-based PROTACs. Specifically, above a threshold concentration of PROTAC molecules, degradation is reduced because of the reduced likelihood of forming the prerequisite substrate:PROTAC:E3 ternary complex.
The adaptability of BioPROTACs to GFP ligands or E3 ligases can support GFP binders based on different protein scaffolds.
By fusing substrate binders directly to E3 ligases, one can recruit any E3 of interest using the BioPROTAC method.
In contrast to the not always obvious correlation between ligand binding affinity and degradation efficiency of small molecule PROTACs, the degree of degradation of BioPROTACs should be proportional to the substrate binding affinity, which helps to simplify the rational design and guide the optimization of bioPROTACs.
BioPROTAC is a powerful tool for studying degradation methods, target protein biology and potentially therapeutic impact. BioPROTAC has the potential to be used in a wide range of diseases caused by abnormal protein function, including cancer, neurodegenerative diseases, autoimmune diseases and infectious diseases. The BioPROTAC technology, a complementary approach of PROTAC, enables the degradation of disease-causing proteins that were previously considered undruggable.
BioPROTACs can be designed to induce the degradation of oncogenic proteins that drive cancer progression, such as BCL-2, KRAS, or MYC.
BioPROTACs can be designed to induce the degradation of disease-causing proteins involved in neurodegenerative diseases, such as tau or alpha-synuclein.
BioPROTACs can be designed to induce the degradation of viral proteins that are essential for the replication of viruses, such as HIV or SARS-CoV-2.