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.

High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR.

We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a Bio-protocol that describes gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains.

Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation.

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer's and Parkinson's diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-ß peptide (Aß42), linked to Alzheimer's disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aß42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.