Yeast Display Technology Enables Rapid Discovery of Low-Nanomolar Macrocyclic Peptide Inhibitors of Human Angiotensin-Converting Enzyme 2

Macrocyclic peptides (MPs) are valuable molecular formats for drug development, bridging small molecules and larger biologics due to their favorable pharmacological properties. Here, we describe the discovery of low-nanomolar inhibitors of human angiotensin-converting enzyme 2 (hACE2) by quantitatively screening millions of structurally diverse disulfide-cyclized peptide ligands using yeast display technology. The most potent yeast-encoded "one-ring" and "two-ring" MP inhibit hACE2 with <i>K</i>_i values of 1.9 and 1.5 nM, respectively. These inhibitory potencies are comparable to those of other cyclic peptides discovered using well-established <i>in vitro</i> display technologies. Crystal structures of the two MPs in complex with hACE2 reveal the adoption of either a rigid β-hairpin or a cysteine-stabilized α-helix/α-helix motif. Both MPs exhibit binding modes distinct from those of previously reported inhibitors. Thus, yeast display is a valid technology to rapidly generate MPs with desired binding properties for the development of potential therapeutics.