Cyclic peptides as ligands for competition binding assays

Cyclic peptides are a class of ligands readily discovered from nucleic acid-encoded libraries by affinity-based screening methodologies developed in recent years. Advancing these cyclic peptide ligands into drug candidates has been limited, often due to the peptide's poor pharmaco-dynamic and -kinetic properties such as cell permeability and metabolic stability. However, their broad protein surface complementarity and straightforward discovery methods make cyclic peptides ideal assay ligand probes to enable high throughput screening (HTS) assays against protein targets that were traditionally difficult or prohibitive to screen. In this chapter, we will discuss the discovery and characterization of macrocyclic peptide ligands and their development and utilization as assay probes in a variety of ligand-displacement assays. We will cover two examples where prior assay designs were either inefficient or intractable for HTS, co-factor independent phosphoglycerate mutases (iPGM) and the secreted Mycobacterium tuberculosis chorismate mutase (*MtbCM). In the context of these enzymes, we will describe eight different ligand-displacement assay formats, including direct 'one-component' assays such as fluorescence binding and fluorescence polarization assays as well as more complex 'two-component' assays such as laser scanning cytometry, homogeneous time-resolved fluorescence (HTRF), and NanoLuc luciferase bioluminescence resonance energy transfer (NanoBRET).