# Genetically encoded bicyclic peptide libraries for the discovery of novel antiviral agents

> **NIH NIH R00** · INDIANA UNIVERSITY INDIANAPOLIS · 2024 · $44,016

## Abstract

PROJECT SUMMARY
Bicyclic peptides are conformationally constrained peptides comprised of two macrocyclic rings. Owing to their
conformational rigidity, bicyclic peptides are highly resistant to proteolysis, and can bind to protein targets with
antibody-like affinity and selectivity. 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 originally limited to
linear peptides, phage display was recently adapted for the discovery of novel bicyclic peptide ligands. Most
often, phage-displayed bicyclic peptide libraries are prepared by chemically modifying linear peptides using
cysteine-reactive small molecules; however, this method is time consuming and technically challenging. As a
result, phage-displayed bicyclic peptide technology has not been widely adopted. Recently, several studies have
used genetic code expansion to 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 overarching objective of this
proposal is to develop technology that enables phage display of bicyclic peptides 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 two cysteine residues. To realize our objective, we will pursue three Specific Aims. In Aim 1 we will engineer
an aminoacyl-tRNA synthetase that recognizes bifunctional ncAAs containing two cysteine-reactive moieties.
This will be accomplished using traditional and state-of-the-art methods of directed evolution. In Aim 2 we will
develop a phage display system that is compatible with co-translational installation of bifunctional ncAAs and we
will optimize this system for efficient bicyclic peptide formation. We will then validate this system by selecting
and characterizing bicyclic peptide ligands for two model targets. In Aim 3 we will use our phage-displayed
bicyclic peptide libraries to identify peptides that bind to the spike protein of human coronaviruses and inhibit
virus-host membrane fusion. By targeting spike proteins from diverse 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.

## Key facts

- **NIH application ID:** 10939563
- **Project number:** 3R00GM141320-03S1
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** Jeffery Micheal Tharp
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $44,016
- **Award type:** 3
- **Project period:** 2021-09-02 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10939563

## Citation

> US National Institutes of Health, RePORTER application 10939563, Genetically encoded bicyclic peptide libraries for the discovery of novel antiviral agents (3R00GM141320-03S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10939563. Licensed CC0.

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