Ansamer‐Controlled Bicyclic Peptides as Integrin αvβ6 Targeting Agents

Bicyclic peptides are promising candidates for peptide-drug conjugates due to their structural rigidity and high target specificity. Recently, it became more apparent that some peptides form nonclassical conformational isomers, termed ansamers. These conformational isomers designated as Pansa and Mansa are non-interconvertible and separable, thus broaden the chemical space of the peptide template. In this study, we incorporated the RGD motif into a bicyclic peptide and systematically assessed the inhibitory activities of the Pansa and Mansa isomers against various integrins. The molecule 10-Mansa exhibited high selectivity and potent inhibitory activity against the αvβ6 integrin, whereas 10-Pansa, its conformational counterpart, lacked such activity. The cryo-EM structure of αvβ6 bound to 10-Mansa shows, that it adopts a conformation with the cyclohexane sidechain of the amino acid Chg engaging in hydrophobic interactions with Ile183 and the disulfide bond within the β6 SDL2 loop. Compared to the linear peptide A20FMDV, a broadly applied αvβ6 inhibitor, 10-Mansa displays faster cellular internalization and also sustains prolonged enrichment within tumor tissues. Moreover, employing 10-Mansa a designed toxin-drug conjugate exhibits remarkable tumor-suppressing effects in vivo. These findings reveal how conformational isomers of ansamers affect biological activity-and highlight their potential for the identification of new bioactive molecules.