One-bead-one-compound (OBOC) combinatorial chemistry library technology enables the parallel synthesis of a vast number of structurally diverse compounds on solid-phase resin beads, enabling high-throughput screening from purified proteins to in vivo models at the single-bead level. It has become an important platform for peptide drug discovery and functional probe development. This review provides a systematic overview of recent advances and key challenges of OBOC technology from three perspectives: (i) library construction, (ii) screening methodologies, and (iii) targeting strategies. In library construction, cyclic and bicyclic peptides have significantly enhanced target affinity and metabolic stability through conformational constraint, while mixed peptide libraries combined with nanoflow mass spectrometry and de novo sequencing enable high-throughput, bead-separation-free decoding of peptide mixtures. In screening methodologies, high-throughput strategies for self-assembling peptides, anti-adhesive peptides, and non-inhibitory enzyme ligands continue to expand the boundaries of OBOC technology. In targeting screening, the transition from protein targets to cellular and in vivo levels has provided preliminary experience for improving the in vivo translatability of screening results. Although OBOC technology still faces challenges such as false-positive control, sequencing complexity of constrained peptides, in vivo reproducibility, and translational efficiency, future integration of artificial intelligence, automated synthesis, and function-oriented phenotypic screening is expected to drive the technology toward intelligent and ultra-high-throughput platforms.

Contents

1 Introduction

2 Construction Strategies and Sequencing of OBOC Combinatorial Peptide Libraries

2.1 Construction Strategies of OBOC Peptide Libraries

2.2 Sequencing and Decoding of Cyclic Peptide Libraries

2.3 Mass Spectrometry Sequencing of Mixed Peptide Libraries

3 Screening Methods for Novel Functional Peptides

3.1 Screening of Self-Assembling Peptides

3.2 Screening of Anti-Adhesion Peptides

4 Screening Based on Target Types

4.1 Protein-Targeted Screening

4.2 Cell-Based Screening

4.3 In Vivo Targeted Screening

5 Summary and Outlook