Beyond Binding: Biologically Active Cyclic Peptides from mRNA Display

Cyclic peptides are a promising class of pharmaceuticals because they can be used to modulate drug targets that are challenging to address with current therapeutics. This thesis specifically explores advances in mRNA display to improve its applicability for the discovery of peptide-based drugs. One of the key challenges of mRNA display is the limited chemical diversity of building blocks that can be used during ribosomal synthesis of peptide libraries. This is addressed in a literature review that discusses strategies to vacate codons in order to increase the efficiency of genetic code reprogramming. Another major challenge is the enrichment of peptides that bind to the target without impacting its biological function. In some cases, preferential propagation of such mere binders complicates identification of interesting bioactive peptides. This work presents three experimental approaches to obtain cyclic peptides that show the desired modulation of their target using mRNA display. In the first approach, mRNA display was used to scan through all possible mutations of an existing peptide that already shows antiviral activity against SARS-CoV-2. This resulted in an optimized lead peptide with up to 49-fold improved potency and 23-fold improved solubility. Trimers and tetramers of this peptide showed further enhanced potency and significant protection against SARS-CoV-2 infection in a hamster model. In the second approach, a de novo mRNA display selection was biased towards enrichment of peptides that bind to a biologically relevant site on an essential malaria protein. Biasing was achieved by alternating protein targets that have mutations outside of the site of interest in each selection round and performing counterselections using target proteins with blocked binding sites. This resulted in cyclic peptides that potently inhibit malaria growth. In the third approach, classical affinity-based mRNA display selection was first used to enrich a library for binding to a target of interest. Next, library members were compartmentalized using hydrogel encapsulated DNA-templated beads and screened for the desired biochemical activity. Altogether, this thesis highlights how more chemically diverse building blocks can be ribosomally incorporated and how mRNA display can be extended beyond the identification of high-affinity binders towards the discovery of biologically active cyclic peptides, both of which improve therapeutic relevance of hits.