为了实现精确性和选择性，抗癌化合物和纳米颗粒 (NP) 可以通过与血管中的恶性肿瘤相关分子结合的亲和配体为目标。虽然肿瘤穿透性 C 端规则 (CendR) 肽有望实现精准肿瘤递送，但 C 端暴露的 CendR 肽可能会在表达 Neuropilin-1 (NRP-1) 的非恶性组织（例如肺部）中积聚。此类混杂肽的一个例子是 PL3（序列：AGGRRLVR），这是一种通过其 C 端 CendR 元件 RLVR 与 NRP-1 结合的肽。在这里，我们报告了仅在蛋白水解后才与 NRP-1 结合的 PL3 衍生物的开发通过尿激酶型纤溶酶原激活剂 (uPA) 进行加工，同时保持与肽的另一个受体（腱蛋白-C (TNC-C) 的 C 结构域）的结合。通过合理的设计方法和筛选重组 NRP-1 上的 uPA 处理的肽噬菌体文库（PL3 肽，后接四个随机氨基酸），PL3 肽的衍生物仅在 uPA 处理后才能与 NRP-1 结合均被成功识别。体外裂解、结合和内化测定，以及原位胶质母细胞瘤小鼠的体内生物分布研究，证实了两种新型肽 PL3uCendR (AGRGRLVR↓SAGGSVA) 和 SKLG (AGRGRLVR↓SKLG) 的功效，这两种肽表现出 uPA 依赖性与 NRP-1 结合，减少与健康 NRP-1 表达组织的脱靶结合。我们的研究不仅揭示了新型 uPA 依赖性 TNC-C 靶向 CendR 肽，还引入了更广泛的范式并建立了筛选蛋白水解激活的肿瘤穿透肽的技术。© 2024。作者。

To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report the development of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.© 2024. The Author(s).