Abstract

Integrin-subtype selectivity remains a challenging for RGD-based imaging probes because of conserved binding interfaces among integrins. Here, we developed a conformational tuning strategy for cyclic RGD peptides by varying the carbon-spacer length and turn-inducing motifs in a 25-member library. Biolayer interferometry screening identified subtype-preferred ligands with nanomolar-level apparent affinities toward αvβ6, αvβ3, and α5β1, including 3ba/3bb/3bd , 3be, and 3bc/3dc , respectively. Circular dichroism suggested that cross-linker geometry and turn motifs modulate the RGD peptides’ secondary structure. FITC-labeled probes showed receptor-associated cellular uptake in BxPC3, A549, and U87MG cells, supporting integrin-subtype-preferred recognition at the cellular level. Furthermore, [ 68 Ga]Ga-8ba showed enhanced αvβ6-associated tumor uptake in BxPC3 xenografts, whereas [ 68 Ga]Ga-8bc and [ 68 Ga]Ga-8dc exhibited favorable α5β1-targeted imaging in U87MG tumors. The αvβ3-targeted probe retained subtype-associated recognition in vitro but showed limited tumor accumulation in vivo . These findings support conformational regulation as a viable strategy for designing subtype-selective integrin PET probes.