High-throughput screen to identify modulators of CendR-mediated cellular uptake

描述（由申请人提供）：本申请提出高通量筛选模拟内化和组织穿透肽基序R/KXXR/K（R=精氨酸; K=赖氨酸; X=任何氨基酸）的小分子量化合物。含有该基序的肽和蛋白质通过与细胞表面受体神经纤毛蛋白-1结合来激活进入细胞并通过组织的胞饮转运途径。R/KXXR/K基序必须位于C-末端位置才能与神经纤毛蛋白结合;即使阻断精氨酸残基的C-末端羧基也会消除活性。由于这一特征，该基序已被指定为C-末端规则或CendR基序。内化到细胞中并在肿瘤组织内扩散的肿瘤特异性归巢肽含有特异性肿瘤归巢序列和隐蔽的CendR基序，其在肿瘤中通过蛋白水解加工活化。有效载荷可以共价偶联至CendR肽或简单地与其共同施用以实现组织渗透。因此，CendR途径提供了一种克服癌症化疗的主要限制之一，即抗癌药物向肿瘤组织中的渗透性差的方法。该途径还可以运输纳米颗粒，使纳米颗粒有效载荷的深部组织递送成为可能。许多炎性蛋白、感染因子和毒液具有CendR元件的蛋白质，其可以介导进入细胞并通过组织扩散。与Sanford-Burnham化学基因组学中心合作开发了基于CendR肽与neuropilin-1片段结合的新型高通量测定，并成功进行了使用该测定的中试筛选。更大的屏幕现在被提议组装一个面板的“命中”。这些分子将被验证为CendR途径的特异性调节剂，并且将确定和评估它们作为CendR途径的激动剂或拮抗剂的生物活性。从这项工作中产生的化合物，类似于CendR肽在其活性将是有用的工具，用于将材料引入细胞和组织，以及用于探索CendR组织渗透现象的分子基础。它们也可以作为开发新型诊断和治疗传递系统的先导化合物。筛选还可产生CendR系统的拮抗剂，其可用于抑制毒素和感染剂通过CendR转运系统的转运。

DESCRIPTION (provided by applicant): This application proposes high-throughput screening for small molecular weight compounds that mimic the internalizing and tissue-penetrating peptide motif R/KXXR/K (R=arginine; K=lysine; X=any amino acid). Peptides and proteins containing this motif activate a pinocytotic transport pathway into cells and through tissues by binding to a cell surface receptor, neuropilin-1. The R/KXXR/K motif has to be in a C-terminal position to bind to neuropilin; even blocking the C-terminal carboxyl group of the arginine residue eliminates the activity. Because of this feature, the motif has been designated the C-end Rule or CendR motif. Tumor- specific homing peptides that are internalized into cells and spread within tumor tissue contain both a specific tumor-homing sequence and a cryptic CendR motif, which is activated in tumors by proteolytic processing. A payload can either be covalently coupled to a CendR peptide or simply co-administered with it to achieve tissue penetration. Thus, the CendR pathway provides a way of overcoming one of the main limitations of cancer chemotherapy, poor penetration of anticancer drugs into tumor tissue. This pathway can also transport nanoparticles, enabling deep tissue delivery of nanoparticle payloads. Many inflammatory proteins, infectious agents and venoms possess proteins with CendR elements that may mediate entry into cells and spreading through tissues. Novel high-throughput assays that are based on the binding of a CendR peptide to a fragment of neuropilin-1 have been developed in collaboration with the Sanford-Burnham Center for Chemical Genomics, and a pilot screen using the assay has been successfully carried out. Larger screens are now proposed to assemble a panel of "hits". These molecules will be validated as specific modulators of the CendR pathway and their biological activity as agonists or antagonists of the CendR pathway will be determined assessed. The compounds resulting from this work that resemble the CendR peptides in their activity will be useful tools for introducing materials into cells and tissues, as well as for exploring the molecular basis of the CendR tissue penetration phenomenon. They may also serve as lead compounds in the development of novel diagnostic and therapeutic delivery systems. The screens may also yield antagonists of the CendR system, which could be useful in inhibiting the transport of toxins and infectious agents through the CendR transport system.