Leveraging Diverse Bi-Triazine Cross-linkers for Modulating Conformation and Biological Activity of Cyclic and Dimeric Peptides

Conformational precision is critical for peptide therapeutic
design,
yet the quantitative link between cross-linker geometry and in vivo performance remains unclear. We present a programmable
platform using eight bi-triazine cross-linkers with varied bond length,
angle, aromaticity, and symmetry to establish multilevel structure–conformation–biology
relationships. Using cyclic RGD peptides targeting integrin αvβ3
and dimeric KTLLPTP peptides targeting Plectin-1 as complementary
models, we integrated binding assays, cell studies, and in
vivo 68Ga-PET/CT imaging to systematically evaluate
linker-induced conformational effects. Two design paradigms emerged:
in cyclic peptides, aromaticity and symmetry govern conformational
locking, with nonmirror-symmetric naphthalene linkers enhancing protein
affinity and tumor uptake. In dimeric systems, bond length and angle
enable geometric matching via the “molecular ruler”
effect, with a 120° benzene linker enabling optimal bivalent
binding. This work not only identifies the lead candidate [68Ga]Ga-8a with high tumor contrast
(∼5 %ID/mL) but also provides a generalizable framework for
precision peptide engineering.