Protein-protein interfaces play a central role in biology, and the majority of these interfaces include an alpha helix, the most common protein secondary structure motif. Such interfaces have posed attractive drug targets due to their importance in a wide range of diseases, in which inhibition, by blocking interactions, or activation, by triggering function, can result in therapeutic applications. However, due to the large size of protein-protein interaction surface areas, small molecules have seen little success in drug development against these targets. Here, we describe an application of bacteria surface display using non-natural amino acid incorporation and bio-orthogonal chemistry for directed evolution of potent stabilized peptide binders. By incorporating azidohomoalanine (AHA) residues spaced 7 residues apart in chemical synthesis, we and others have demonstrated stabilization of peptides using a bifunctional alkyne-containing linker. Such design allows for the inclusion of fluorophores for imaging applications or pharmacologic enhancers like PEG chains. Our lab and others have demonstrated that click-stabilized peptides have increased protease resistance, subcutaneous bioavailability, and binding affinities. Taking advantage of the ability of methionine auxotrophic E. coli to efficiently incorporate AHA as a methionine surrogate, we constructed libraries of randomized peptides containing two AHA residues for on-surface stabilization. We demonstrate efficient display of AHA incorporated peptides using the eCPX scaffold and robust reaction yields and specificity. Following surface display and chemical double-click stabilization, the resulting peptides show increased protease resistance and are available for surface binding and screening. To show this method’s potential for diverse applications, we screened a stabilized peptide library with a diversity of 4 x 108 sequences based on the original p53-MDM2 binding motif and obtained several unique sequences demonstrating significantly increased affinity over the native analog. Furthermore, we isolated sequences whose binding was enhanced upon stabilization, indicating the effectiveness of chemical stabilization prior to the selection process. We also screened a naïve library against extracellular targets to isolate stabilized peptide binders for imaging applications. This screening approach demonstrates the ability to affinity mature or select novel sequences with stabilized alpha helix peptide display.

Surface display-enabled selection of bioorthogonally stabilized alpha helices using non-natural amino acid incorporation

255th National Meeting (2018)

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