Rapid discovery of cyclic peptide protein aggregation inhibitors through continuous selection HTS data

We report a new platform for the rapid phenotypic selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in E. coli based on phage-assisted continuous evolution (PACE). Here, we developed a new PACE-compatible selection for protein aggregation inhibition and employed it to identify cyclic peptides that suppress amyloid-ß42 (Aß42) and human islet amyloid polypeptide (hIAPP) aggregation. Additionally, we integrated a negative selection that removes false positives and off-target hits, significantly improving cyclic peptide selectivity. We show that selected inhibitors are active when chemically re-synthesized in in vitro assays. Our platform provides a powerful new approach for the rapid discovery of cyclic peptide inhibitors of protein aggregation and may serve as the basis for the future evolution of cyclic peptides with a broad spectrum of inhibitory activities. Data deposited here are HTS data critical to the conclusions of this study. Overall design: Samples analyzed here are post-selection M13 phage pools. Gene of interest encoding cyclic peptide sequence in those samples were amplified via standard PCRs using primers included in this study. The resulted PCR products were pooled together based on library ID. Pooled libraries were subjected to NGS using Illumina NovaSeq platform. Raw sequencing data were processed using a customized script included in this study to calculate the read frequency of cyclic peptide sequence to discover enriched peptide sequence after selection.