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Updated: May 16, 2025
High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
Published on: November 9, 2020
Designed peptide binders and nanobodies as PROTAC starting points for targeted degradation of PCNA and BCL6
Shuai Zhao1, Jingwen Luo1, Pingping Xu1
1State Key Laboratory of Biocatalysis and Enzyme Engineering, College of Life Sciences, Hubei University, Wuhan 430062, PR China; Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China.
Abstract:
The efficient degradation of pathogenic proteins, particularly proliferating cell nuclear antigen (PCNA) and B-cell lymphoma 6 protein (BCL6), is crucial for treating various diseases related to cancer. As key biological macromolecules, PCNA plays a critical role in DNA replication and repair, while BCL6 acts as a transcriptional repressor involved in B-cell lymphoma. To enhance the efficiency and specificity of protein degradation, we developed a RS80E-based bioPROTACs system that consists of truncated variants of Ring-B-boxed coiled-coil (RBCC) domains (RS80E) with improved degradation efficiency fused to an AI-driven binder/nanobody targeting specific antigens. Combining state-of-the-art methodologies such as ProteinMPNN, RFdiffusion, AlphaFold3, AlphaFold2, and HADDOCK, we identified binders for PCNA and predicted spatial interrelationships. Employing fragment-based and alanine scanning methods, we designed nanobodies targeting PCNA and BCL6 by combinatorially designing CDR3 and grafting them onto nanobody scaffolds. Significantly, our results demonstrate the utility of bioPROTACs in degrading PCNA and BCL6, thereby activating p53 and promoting apoptosis. This highlights the therapeutic potential of targeting PCNA and BCL6 degradation and lays the groundwork for developing PCNA and BCL6-degrading therapeutics. In summary, our system offers a modular and rapid pathway for exploration other intractable therapeutic targets, and emphasizes the importance of interdisciplinary methods in advancing therapeutic interventions.
Insights
We developed a novel bioPROTAC system using AI to efficiently degrade cancer-driving proteins like PCNA and BCL6, paving the way for new cancer therapies.
Area of Science:
- Biochemistry and Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- Efficient degradation of pathogenic proteins, including proliferating cell nuclear antigen (PCNA) and B-cell lymphoma 6 protein (BCL6), is vital for cancer treatment.
- PCNA is critical for DNA replication and repair, while BCL6 is a transcriptional repressor involved in B-cell lymphoma.
Purpose of the Study:
- To develop an RS80E-based bioPROTACs system for enhanced and specific degradation of PCNA and BCL6.
- To explore the therapeutic potential of targeting PCNA and BCL6 degradation in cancer treatment.
Main Methods:
- Utilized AI-driven methodologies including ProteinMPNN, RFdiffusion, AlphaFold3/2, and HADDOCK for binder identification and spatial relationship prediction.
- Employed fragment-based and alanine scanning methods to design nanobodies targeting PCNA and BCL6.
- Developed a modular RS80E-based bioPROTACs system by fusing RBCC domains with AI-designed nanobodies.
Main Results:
- Successfully demonstrated the degradation of PCNA and BCL6 using the developed bioPROTACs system.
- Observed activation of p53 and promotion of apoptosis, indicating therapeutic efficacy.
- Validated the utility of bioPROTACs for targeting intractable therapeutic targets.
Conclusions:
- The RS80E-based bioPROTACs system offers a modular and rapid approach for targeting disease-causing proteins.
- Targeting PCNA and BCL6 degradation holds significant therapeutic potential for cancer treatment.
- Interdisciplinary methods are crucial for advancing therapeutic interventions in oncology.
