Discovery of Macrocyclic Peptide Inhibitors Targeting MYC Oncoprotein via mRNA Display

Background/Objectives: mRNA display technology has emerged as a powerful platform for discovering macrocyclic peptides against intractable proteins. However, direct screening against the “undruggable” transcription factor MYC using this approach remains largely unexplored. In this study, we aimed to integrate tyrosinase-mediated cyclization with mRNA display to identify novel macrocyclic peptide inhibitors targeting MYC. Methods: We performed mRNA display combined with tyrosinase-mediated cyclization to generate macrocyclic peptides targeting MYC. Antiproliferative activity was assessed in MYC-dependent tumor cells using CCK8 assay. C-terminal fusions with a TAT-derived cell-penetrating peptide were generated to enhance cell membrane permeability. Binding affinities were measured by bio-layer interferometry (BLI). MYC transcriptional activity was evaluated by RNA sequencing (RNA-seq) analysis of canonical MYC target genes. Results: The identified macrocyclic peptides exhibited potent antiproliferative activity against MYC-dependent tumor cells, with half-maximal inhibitory concentration (IC50) values in the micromolar range. Fusion with the TAT peptide improved antiproliferative potency, yielding IC50 values of 1–3 μM in MYC-dependent cell lines. BLI assays confirmed dose-dependent binding of the peptides to MYC, with dissociation constants (Kd) in the micromolar range. Furthermore, RNA-seq analysis revealed significant downregulation of canonical MYC target genes upon treatment with the TAT-fusion macrocyclic peptide, indicating specific suppression of MYC transcriptional activity. Conclusions: This work establishes the feasibility of using mRNA display to target the “undruggable” protein MYC and identifies a panel of macrocyclic peptides as promising lead candidates for further optimization toward targeted therapies for MYC-driven cancers.