Engineering cell permeable functional bicyclic peptides by a bismuth-enabled plug-and-display strategy

Bismuth-based bicyclic peptides (Bi-BCPs) have garnered growing interest as a promising drug modality due to their notable conformational rigidity and cell permeability, underscoring their considerable potential for diverse biological applications. However, a generalizable synthetic strategy for producing Bi-BCPs with exceptional cell permeability remains lacking. To address this challenge, we developed a “plug-and-display” strategy that enables the modular synthesis of a novel class of cell-permeable, bifunctional Bi-BCPs. These Bi-BCPs adopt a “cycle A–Bi–cycle B” architecture, in which one cycle serves as a functional motif while the other acts as a cell-penetrating sequence (CPS), termed the “plug.” The two cycles are linked via a bismuth metal ion to form the bicyclic scaffold. Using the integrin-binding RGD peptide as a model functional motif, we conducted comprehensive structure–cell permeability relationship studies by systematically varying the A–B conjugation pattern and CPS parameters—including sequence type, charge number, and chiral configuration. Our results demonstrated that chiral configuration exerts the most pronounced influence on cell permeability, and we identified the iterative (Rr)4 motif as the optimal plug for enhancing this property in Bi-BCPs. Furthermore, the (Rr)4 plug was successfully applied to construct glioma-targeting radioligands, which exhibited significantly improved tumor uptake and retention. Additionally, combining the (Rr)4 plug with hydrophobic tetrapeptides yielded novel super-amphiphilic Bi-BCPs with markedly enhanced anti-tumor and anti-microbial activities. In summary, this study presents a facile and robust “plug-and-display” strategy for constructing unprecedented, multifunctional Bi-BCPs with superior cell permeability, opening new avenues for the development of Bi-BCP–based therapeutics.