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.

构象精度对于肽治疗药物（peptide therapeutic）的设计至关重要，然而交联剂（cross-linker）的几何结构与体内（in vivo）性能之间的定量关联仍不明确。本研究构建了一款可编程平台，采用8种键长、键角、芳香性和对称性各异的双三嗪（bi-triazine）交联剂，以建立多维度的结构-构象-生物学关联。我们以靶向整合素αvβ3（integrin αvβ3）的环状RGD肽，以及靶向网蛋白-1（Plectin-1）的二聚体KTLLPTP肽作为互补模型，整合结合实验、细胞研究与体内镓-68正电子发射断层扫描/计算机断层成像（68Ga-PET/CT），系统评估了交联剂诱导的构象效应。研究发现了两种设计范式：在环状肽体系中，芳香性与对称性决定构象锁定，非镜像对称的萘（naphthalene）基交联剂可提升蛋白亲和力与肿瘤摄取能力；在二聚体体系中，键长与键角可通过‘分子尺效应（molecular ruler）’实现几何匹配，其中120°苯（benzene）环交联剂可实现最优二价结合（bivalent binding）。本研究不仅筛选出具有高肿瘤对比度（约5 每毫升注射剂量百分比（%ID/mL））的先导候选物[68Ga]Ga-8a，还为精准肽工程提供了可推广的通用框架。