Integrating SICLOPPS screening with next-generation sequencing for the discovery of cyclic peptide modulators of...

Protein-protein interactions (PPI) are central to cellular regulation, yet remain difficult to target due to their flat, large, and dynamic interfaces. Cyclic peptides offer a promising solution, combining conformational rigidity, high affinity, and cell permeability. Split Intein Circular Ligation of Peptides and Proteins (SICLOPPS) enables intracellular generation of genetically encoded cyclic peptide libraries and functional selection for PPI inhibitors. Here, we detail an integrated SICLOPPS workflow that uses next-generation sequencing (NGS) in combination with iterative biopanning for high-throughput, large-scale discovery of PPI-disrupting cyclic peptides. The method involves transformation of a reverse two-hybrid system (RTHS) with SICLOPPS libraries, followed by functional selection under pre-defined conditions, pooled plasmid recovery, NGS-based quantification of peptide sequences. Generation of a new library from these hit sequences, followed by RTHS-based selection enables hit-enrichment via biopanning and tracking hit enrichment across selection rounds. This strategy enables real-time monitoring of library evolution, precise identification of enriched sequences, and effective filtering out of false positives. SICLOPPS-NGS pipeline offers a scalable, reproducible, and broadly applicable platform for the discovery of intracellular cyclic peptide inhibitors, substantially expanding the toolkit for modulating diverse PPI.