Linearizable Macrocyclic Peptide Libraries for Affinity Selection-Mass Spectrometry

Macrocyclic peptides are attractive for therapeutic development but have been limited in their application to combinatorial library selection from synthetic libraries. Here, we establish a synthetic approach based on split-pool chemistry to produce 100-million membered macrocyclic libraries containing natural and non-natural amino acids. Near-quantitative intramolecular disulfide formation is facilitated by rapid (<10 minute) oxidation by iodine to prepare macrocyclic synthetic libraries in solution. Treatment with dithiothreitol post-affinity selection enables near-quantitative reduction of the library members, rendering the linear analogs amenable to standard tandem mass spectrometry sequencing. We demonstrate the utility of these libraries to discover novel macrocyclic binders to cadherin-2 and the anti-hemagglutinin antibody clone 12ca5. The lead cadherin-binding peptide (CBP) is endowed with nanomolar binding affinity measured by biolayer interferometry (BLI, apparent dissociation constant KD = 53 nM). Structure-activity relationship (SAR) studies including alanine and D-amino acid scans reveal the amino acids responsible for driving affinity (hot-spots) and the positions tolerating mutagenesis (cold-spots). Informed by SAR data, two new macrocyclic libraries are designed to derivatize these positions with a variety of abiotic amino acids based on the hot- and cold-spots. Following affinity selection and experimental validation by BLI, 10 high-affinity ligands out of 10 discovered were identified from the library that derivatized the CBP cold-spots, while zero of the two peptide ligands discovered from the hot-spot library were high-affinity binders. Of these noncanonical CBPs (NCBP), NCBP-4 demonstrates improved affinity to cadherin-2 (KD = 29 nM). Overall, we expect that this work will pioneer the use of large-scale macrocyclic libraries to further catalyze therapeutic peptide discovery and development.