Identification of an αvβ3-targeting bicyclic peptide with atypical norArg-Gly-Asp sequence

Bicyclic peptides, which integrate the advantageous properties of small molecules and antibodies, have emerged as a promising class of therapeutic candidates. In particular, integrin αvβ3 serves as a critical molecular target for cancer diagnosis and therapy. However, the development of bicyclic peptide ligands specifically targeting this integrin remains inadequately explored. To address this gap, we designed and synthesized a series of RGD-containing bicyclic peptides featuring a tryptathionine bridge. Notably, bicyclic peptide 5j incorporates the non-canonical sequence norArg-Gly-Asp, exhibiting high affinity and selectivity toward integrin αvβ3. Molecular dynamics simulations provided insights into the conformational preferences and demonstrated that norArg plays a critical role in determining the selectivity between αvβ3 and αIIbβ3. Employing peptide 5j as the targeting ligand, the peptide drug conjugates P1 showed significant inhibitory effects on the A549 cell line in both, in vitro and in vivo experiments. These data provide important theoretical foundations for the development of αvβ3-targeting bicyclic peptides and offer new options for αvβ3-targeted tumor therapy. Bicyclic peptides targeting integrin are promising drug candidates, yet achieving high selectivity for specific integrins remains challenging. Here, the authors design RGD-containing bicyclic peptides with a tryptathionine bridge, demonstrating that peptide 5j selectively targets integrin αvβ3, and induces αvβ3-targeted cytotoxicity by drug-peptide conjugates.