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

描述(申请人提供)：本申请建议高通量筛选模仿内化和组织穿透性多肽基序R/KXXR/K(R=精氨酸；K=赖氨酸；X=任何氨基酸)的小分子化合物。含有该基序的多肽和蛋白质通过与细胞表面受体--神经粘连蛋白-1结合，激活胞饮转运途径进入细胞和通过组织。R/KXXR/K基序必须位于C-末端才能与神经粘连蛋白结合；即使封闭精氨酸残基的C-末端羧基也会消除活性。由于这一特征，该主题已被指定为C-End规则或CENDR主题。肿瘤特异性归巢多肽在细胞内和肿瘤组织内扩散，包含特定的肿瘤归巢序列和一个隐蔽的cendr基序，该基序在肿瘤中通过蛋白分解处理而被激活。有效载荷可以共价偶联到cendR多肽上，也可以简单地与其共同给药以实现组织渗透。因此，cendr途径提供了一种克服癌症化疗的主要局限性之一的方法，即抗癌药物对肿瘤组织的渗透性差。这种途径还可以运输纳米颗粒，使纳米颗粒有效载荷的深层组织输送成为可能。许多炎性蛋白、感染剂和毒液都含有cendr元件的蛋白质，这些蛋白质可能介导进入细胞并在组织中传播。与Sanford-Burnham化学基因组中心合作，开发了基于cendR多肽与神经毛细蛋白-1片段结合的新型高通量分析，并成功地进行了中试筛选。现在，有人提议用更大的屏幕来组装一个“热门”面板。这些分子将被确认为cendr途径的特异性调节剂，它们作为cendr途径的激动剂或拮抗剂的生物活性将被确定和评估。这项工作产生的化合物在活性上与cendr肽相似，将成为将材料引入细胞和组织以及探索cendr组织渗透现象的分子基础的有用工具。它们还可以作为开发新的诊断和治疗传递系统的先导化合物。这些筛子还可能产生cendr系统的拮抗剂，这可能有助于抑制毒素和感染性物质通过cendr运输系统的运输。

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.