Stepwise Evolution Improves Identification of Diverse Peptides Binding to a Protein Target

Victor I Lyamichev,Joerg Benz,Lauren E Goodrich,Eric H Sullivan,Ryan M Bannen,Jigar J Patel,Thomas J Albert

doi:10.1038/s41598-017-12440-1

Abstract

Considerable efforts have been made to develop technologies for selection of peptidic molecules that act as substrates or binders to a protein of interest. Here we demonstrate the combination of rational peptide array library design, parallel screening and stepwise evolution, to discover novel peptide hotspots. These hotspots can be systematically evolved to create high-affinity, high-specificity binding peptides to a protein target in a reproducible and digitally controlled process. The method can be applied to synthesize both linear and cyclic peptides, as well as peptides composed of natural and non-natural amino acid analogs, thereby enabling screens in a much diverse chemical space. We apply this method to stepwise evolve peptide binders to streptavidin, a protein studied for over two decades and report novel peptides that mimic key interactions of biotin to streptavidin.

Highlights

A significant number of peptidic compounds with favorable therapeutic properties are currently on the market
Despite improvements to classical display techniques such as the Flexizyme[7,8] and RaPID9 technologies, the need still exists for (1) more diverse libraries containing modifications (e.g., N-alkylation, D-stereochemistry, and cyclization) that are commonly found in natural bioactive peptide molecules, (2) methods that select for molecules with inherent properties such as proteolytic stability and cell permeability, and (3) the ability to rapidly iterate and rationally mature a “hit” to a “lead” by exploring a pre-defined chemical space to further optimize therapeutic characteristics
To identify hotspot sequences that bind to streptavidin (SA), we bound Cy5-SA to an array library of 2,476,099 5-mer L-peptides synthesized with 19 of 20 natural amino acids

Summary

Introduction

A significant number of peptidic compounds with favorable therapeutic properties are currently on the market. Despite improvements to classical display techniques such as the Flexizyme[7,8] and RaPID9 technologies, the need still exists for (1) more diverse libraries containing modifications (e.g., N-alkylation, D-stereochemistry, and cyclization) that are commonly found in natural bioactive peptide molecules, (2) methods that select for molecules with inherent properties such as proteolytic stability and cell permeability, and (3) the ability to rapidly iterate and rationally mature a “hit” to a “lead” by exploring a pre-defined chemical space to further optimize therapeutic characteristics. We report a digital light-directed array technology[10,11,12] to synthesize arrays containing peptides on an amine-functionalized slide This technology uniquely combines four characteristics that could facilitate its use as a novel binder-discovery platform: (1) high feature density, resulting in 2.9 million unique molecules per array; (2) ability to accommodate broad chemical diversity, e.g., non-natural amino acids; (3) digitally controlled www.nature.com/scientificreports/. The obtained results were confirmed by SPR analysis and co-crystallization with streptavidin

Methods

Results

Conclusion