The Discovery of Peptide Macrocycle Rescuers of Pathogenic Protein Misfolding and Aggregation by Integrating SICLOPPS Technology and Ultrahigh-Throughput Screening in Bacteria.

The phenomenon of protein misfolding and aggregation has been widely associated with numerous human diseases, such as Alzheimer's disease, systemic amyloidosis and type 2 diabetes, the vast majority of which remain incurable. To advance early stage drug discovery against these diseases, investigation of molecular libraries with expanded diversities and ultrahigh-throughput screening methodologies that allow deeper investigation of chemical space are urgently required. Toward this, we describe how Escherichia coli can be engineered so as to enable (1) the production of expanded combinatorial libraries of short, drug-like, head-to-tail cyclic peptides and (2) their simultaneous functional screening for identifying effective inhibitors of protein misfolding and aggregation using a genetic assay that links protein folding and misfolding to cell fluorescence. In this manner, cyclic peptides with the ability to inhibit pathogenic protein misfolding and/or aggregation can be readily selected by flow cytometric cell sorting in an ultrahigh-throughput fashion. This biotechnological approach accelerates significantly the identification of hit/lead molecules with potentially therapeutic properties against devastating diseases.