by on Designed Ankyrin Repeat Proteins (DARPins) are synthetic binding proteins that offer a powerful alternative to antibodies. Although antibodies have conventionally been preferred for selective and high-affinity binding, their drawbacks, including their large size, dependence on disulfide bonds, and complex production, have led to the exploration of alternative scaffolds for binding proteins. Based on naturally occurring ankyrin repeat proteins, DARPins consist of 2-4 repeat modules of 33 amino acids each and two capping repeats that shield the hydrophobic core. Overall, they assemble into a right-handed solenoid structure providing an extensive solvent-accessible surface for interaction with target proteins. They can be rapidly selected from vast libraries using ribosome or phage display. Compared to antibodies, DARPins are much smaller, highly stable, easy to produce, and ideally suited for use inside living cells. These features have enabled their adoption as versatile tools in research, diagnostics, and emerging therapeutic strategies. DARPins can recognize specific protein conformations or post-translational modifications, function as potent intracellular inhibitors, and serve as biosensors. Our lab has developed DARPins targeting the folded domains of the p53 family members to investigate their function and modulate their activity. Recent highlights include:
- Intracellular Inhibition of the p53 family using DARPins binding to the DNA-binding domains of p63 and p73
- Targeted Protein Degradation: By fusing DARPins to the E3-ligase adaptor SPOP, we engineered bioPROTACs that selectively degrade p63 and p73, providing a new approach to modulate their cellular levels.
- Conformation specific DARPins: We developed DARPins that selectively bind the hetero-tetramer formed by p63 and p73, providing the first direct experimental confirmation of these complexes in epithelial tissues and demonstrating their upregulation in squamous cell carcinoma
- Reactivating p53 in cancer: We characterized a DARPin that binds to the same site on p53 as the HPV E6 protein, displacing the E6/E6AP complex. This restores p53 activity and reactivates its transcriptional program in HPV-positive cancer cells, highlighting a potential therapeutic avenue against HPV-driven cancers.
Currently we are focusing on developing strategies to deliver DARPins and DARPin-based bioPROTACs into cells for therapeutic applications, with a particular interest in mRNA-lipid nanoparticle (LNP) technology. In addition, we are expanding our toolkit by generating DARPins and Sybodies against other target proteins, including the Atg8 family, kinases, and conformation-specific binders for the dimeric state of TAp63α as well as for certain p53 mutants.
