Screening of one-bead one-compound cyclic peptide libraries using unpurified proteins from the wheat germ cell-free system to identify protein-protein interaction inhibitors

Abstract

Protein-protein interactions (PPIs) are recognized as both attractive and challenging therapeutic targets. Cyclic peptides are particularly well-suited for PPI inhibition due to their ability to effectively interfere with the extensive surface areas involved in these interactions. One-Bead One-Compound (OBOC) libraries have been widely utilized in affinity-based on-bead screening approaches to identify cyclic peptides targeting PPIs. However, a major bottleneck in OBOC library screening is the requirement for purified proteins to ensure screening accuracy. In this study, we present a novel screening platform that integrates an OBOC cyclic peptide library with crude bait proteins produced using a wheat germ cell-free system, eliminating the need for a purification step. This approach facilitates drug discovery for biologically relevant target proteins and those that are difficult to purify. To demonstrate the effectiveness of this method, we selected the p53-murine double minute 2 (MDM2) interaction as a model target and performed a large-scale on-bead binding assay using MDM2. The primary hits identified through this screening exhibited PPI inhibitory activity in the AlphaScreen assay, and docking simulations further verified their binding mode to MDM2. This method offers an efficient strategy for screening cyclic peptides against challenging drug targets, expanding opportunities for PPI-targeted drug discovery.