Supporting data for "CONSTRUCTION AND APPLICATION OF PHAGE DISPLAYED SUPER DIVERSE MINI-PROTEIN LIBRARY FOR HIGH THROUGH SCREENING"

Peptide drugs are being extensively discussed in the field of drug development due to their combination of high targeting and specificity akin to antibody drugs, as well as low immunogenicity and manufacturing cost like small molecule drugs. The utilization of high-throughput screening libraries has significantly expedited the progress of peptide drug development. Among these methods, phage display peptide technology has emerged as a popular approach for identifying novel peptide drugs, offering advantages such as simplicity, low-cost manufacture, large library capacity, and diverse screening methods. In this thesis, the long peptide library that exhibit miniprotein-like properties characterized was constructed using phage display technology. The library was screened against α-synuclein and PD-1 to identify long peptide binders.

For the first project, the aggregation of α-synuclein, a hallmark of Parkinson's disease (PD), has presented a challenge in developing effective therapies due to the intrinsic disorder of α-synuclein. Previous research has demonstrated that the truncated SUMO1 (15-55) peptide, derived from the SUMO1 protein, exhibits micromolar affinity for α-synuclein and can impede its aggregation. To enhance peptide affinity for α-synuclein, a 41-amino acid long peptide library was engineered based on the mini-protein characteristics of the SUMO1 scaffold using phage display technology. Through this approach, the peptide PD-6 was identified, which targets α-synuclein with nanomolar affinity. PD-6 has demonstrated the ability to inhibit α-synuclein aggregation and exhibits promising therapeutic effects in an in vivo C. elegans model. Analysis via NMR spectroscopy has unveiled interactions between PD-6 and α-synuclein residues involving hydrogen bonding and stacking interactions.

In the second project, the focus on PD-1/PD-L1-based immune checkpoint inhibitors has become pivotal in cancer immunotherapy. While antibody-based treatments have shown efficacy, they also come with certain side effects, prompting the exploration of peptide-based alternatives for anti-cancer immunotherapy. Through the utilization of mirror-image phage display technology, Some D-long peptide binders were identified targeting PD-1 with high affinity and displaying in vitro resistance to hydrolysis. The anti-tumor efficacy of the identified peptide, PDI-49, was validated in a humanized mouse tumor model, positioning it as a promising lead compound for further optimization and potential application in cancer treatment.