Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to α_vβ₃ without Promoting Integrin Allosteric Activation

The isoDGR sequence is an integrin-binding motif that has been successfully employed as a tumor-vasculature-homing molecule or for the targeted delivery of drugs and diagnostic agents to tumors. In this context, we previously demonstrated that cyclopeptide 2, the product of the conjugation of c(CGisoDGRG) (1) to 4-(N-maleimidomethyl)­cyclohexane-1-carboxamide, can be successfully used as a tumor-homing ligand for nanodrug delivery to neoplastic tissues. Here, combining NMR, computational, and biochemical methods, we show that the succinimide ring contained in 2 contributes to stabilizing interactions with αvβ3, an integrin overexpressed in the tumor vasculature. Furthermore, we demonstrate that various cyclopeptides containing the isoDGR sequence embedded in different molecular scaffolds do not induce αvβ3 allosteric activation and work as pure integrin antagonists. These results could be profitably exploited for the rational design of novel isoDGR-based ligands and tumor-targeting molecules with improved αvβ3-binding properties and devoid of adverse integrin-activating effects.isoDGR序列是一类整合素结合基序，已被成功用作肿瘤血管归巢分子，或用于将药物与诊断试剂靶向递送至肿瘤组织。在此研究背景下，我们此前曾证实，环肽2——即c(CGisoDGRG)(1)与4-(N-马来酰亚胺甲基)环己烷-1-甲酰胺偶联得到的产物——可作为肿瘤归巢配体，用于向肿瘤组织递送纳米药物。本研究结合核磁共振（NMR）、计算生物学与生化实验方法，证实了环肽2所含的琥珀酰亚胺环有助于稳定其与肿瘤血管过表达的整合素αvβ3之间的相互作用。此外，我们还证明，多种嵌入不同分子骨架的含isoDGR序列的环肽不会诱导αvβ3发生别构激活，可作为纯整合素拮抗剂发挥作用。上述研究结果可为基于isoDGR序列的新型配体与肿瘤靶向分子的理性设计提供有益参考，助力开发兼具优化的αvβ3结合特性与无不良整合素激活效应的靶向分子。