Identification, Characterization, and Optimization of Integrin α_vβ₆-Targeting Peptides from a One-Bead One-Compound...

The current translation of peptides identified through the one-bead one-compound (OBOC) technology into positron emission tomography (PET) imaging agents is a slow process, with a major delay between ligand identification and subsequent lead optimization. This work aims to streamline the development process of ¹⁸F-peptide based PET imaging agents to target the integrin α_vβ₆. By directly identify α_vβ₆⁻targeting peptides from a 9-mer 4-fluorobenzoyl peptide library using the on-bead two-color (OBTC) cell-screening assay, a total of 185 peptide beads were identified and 5 beads sequenced for further evaluation. The lead peptide 1 (VGDLTYLKK(FB), IC₅₀ = 0.45 ± 0.06 μM, 25% stable in serum at 1 h) was further modified at the N-, C-, and bi-termini. C-terminal PEGylation increased the metabolic stability (>95% stable), but decreased binding affinity (IC₅₀ = 3.7 ± 1 μM) was noted. C-terminal extension (1i, VGDLTYLKK(FB)KVART) significantly increased binding affinity for integrin α_vβ₆ (IC₅₀ = 0.021 ± 0.002 μM), binding selectivity for α_vβ₆-expressing cells (3.1 ± 0.8:1), and the serum stability (>99% stable). Our results demonstrate the challenges in optimizing OBOC-derived peptides, indicate both termini of 1 are sensitive to modifications, and show that further modification of 1 is necessary to demonstrate utility as an ¹⁸F-peptide imaging agent.