Genetically encoded bicyclic peptide libraries for the discoveryof novel antiviral agents

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PROJECT SUMMARY Bicyclic peptides are conformationally constrained peptides comprised of two macrocyclic rings. Owing to their increased rigidity, bicyclic peptides can bind to protein targets with greater affinity and selectivity than their linear and monocyclic counterparts. As a result, these molecules are highly desirable scaffolds for the development of peptide-based therapeutics. Phage display is a laboratory evolution technique that enables the discovery of high- affinity peptide ligands from large, combinatorial peptide libraries. Although initially limited to linear peptides, this technique has been adapted for the discovery of bicyclic peptide ligands. Most often, phage-displayed bicyclic peptides are generated by chemically modifying linear peptides using cysteine-reactive small molecules; how- ever, this method is technically challenging. As a result, progress in this field has been limited. Recently, several studies have used genetic code expansion to co-translationally install cysteine-reactive noncanonical amino acids (ncAAs) into phage-displayed peptides to produce libraries of cyclic peptides. This strategy has significant advantages over the chemical cyclization approach, but is currently limited to monocyclic peptides. The over- arching objective of this proposal is to develop technology that enables phage display of bicyclic pep- tides using genetic code expansion. Our central hypothesis is that bifunctional ncAAs, i.e. ncAAs containing two cysteine-reactive functional groups, can be used to generate ribosomally synthesized bicyclic peptides by intramolecular reaction with cysteine residues. To realize our objective, we will pursue three Specific Aims. In Aim 1 (K99 Phase) we will engineer an aminoacyl-tRNA synthetase that recognizes bifunctional ncAAs contain- ing two cysteine-reactive moieties. This will be accomplished using traditional and state-of-the-art methods of directed evolution. In Aim 2 (K99/R00 Phase) we will develop a phage display system that is compatible with co- translational installation of bifunctional ncAAs and we will optimize this system for bicyclic peptide formation. We will then validate this system by selecting and characterizing ligands for model targets. In Aim 3 (R00 Phase) we will use phage display to identify bicyclic peptides that bind to the spike protein of human coronaviruses and inhibit virus-host membrane fusion. By targeting proteins from various coronaviruses, we will strive to identify inhibitors with broad-spectrum antiviral activity. The proposed work will provide a facile route for generating bi- cyclic peptide libraries thereby greatly accelerating the discovery of therapeutic peptide leads. The Candidate, Dr. Jeffery Tharp’s long-term career goal is to establish an independent research program that uses genetic code expansion and phage display to develop antiviral peptides for the diagnosis, treatment, and prevention of infectious diseases. Herein we propose a detailed five-year Career Development Plan supervised by mentors Drs. Dieter Söll and Craig Wilen, and a team of subject-matter experts. This plan will augment previous training and address key training gaps to prepare Dr. Tharp for accomplishing his long-term career goal.