Synthetic Cell Surface Receptors for Anticancer Drug Delivery

描述（由申请人提供）：药物递送的主要未解决问题之一是如何通过膜屏障运输渗透性差的分子。大自然解决这个问题的方法是通过膜的运输；细胞不渗透配体，如载铁转铁蛋白结合同源受体，在这种情况下，转铁蛋白受体，存在于动态膜运输途径。转铁蛋白受体所占据的途径涉及细胞表面和早期/循环内体之间的快速循环，将转铁蛋白传递到脊椎动物的每个细胞，是一种非凡的天然传递载体。在上一个授权周期中，我们证明了合成膜锚的衍生物n -烷基-3？-胆固醇胺表现出独特的生物活性：当添加到哺乳动物细胞中时，它们有效地参与转铁蛋白受体占据的膜运输途径，并在质膜和早期/循环内体之间快速循环。这样n -烷基-3？-胆固醇胺可以作为小的人造细胞表面受体；当与药物结合基序连接时，它们可以通过内吞作用将药物运送到细胞和组织中。这些化合物似乎在功能上模仿游离胆固醇，游离胆固醇是血浆和内体膜的关键成分。它们结合含有胆固醇的低密度脂蛋白和高密度脂蛋白颗粒，可能参与天然胆固醇摄取和运输机制。由于由转铁蛋白受体介导的转铁蛋白递送非常有效，胆固醇胺有效参与相同的膜运输途径的能力为创造变革性的药物递送新工具提供了前所未有的潜力。本研究的重点是阐明控制n -烷基-3?-胆固醇胺独特生物活性的机制。通过合成和评估一组在靠近该膜锚点的连接区域具有结构多样性的化合物的生物学特性，我们将获得定量结构-特性关系（QSPR），旨在预测细胞摄取、运输、外排、代谢和毒性特性。结构相关的光交联剂、RNAi方法和蛋白质过表达将用于鉴定参与细胞摄取和外排的受体和配体。这些生物活性介质将对n -烷基-3的药代动力学产生深远影响。-体内胆固醇胺我们进一步建议构建第一个抗癌药物传递系统，通过进入一个确定的膜运输途径来穿越膜屏障。为了获得对转移性癌细胞的特异性，肿瘤特异性蛋白酶的肽底物将用于将胆固醇胺与抗癌药物阿霉素连接起来。该药物被转移性癌细胞表达的MMP-2蛋白酶裂解后选择性释放。我们将优化阿霉素和相关分子探针在体外对癌细胞的递送，以验证一个基本的新概念：参与确定的膜运输途径的化合物为抗癌药物的选择性递送提供了一种新的策略。公共卫生相关性：这里提出的研究与人类健康的进步高度相关。在过去的拨款周期中，我们发现n -烷基-3？-胆固醇胺参与转铁蛋白受体占据的特定膜运输途径。这种生物活性使这些化合物能够在哺乳动物细胞中定义跨越膜屏障的新途径。通过阐明这些化合物的生物学机制，我们建议创造新的抗癌药物传递工具。

DESCRIPTION (provided by applicant): One of the major unsolved problems in drug delivery is how to transport poorly permeable molecules across membrane barriers. The way that Nature solves this problem is through membrane trafficking; cell impermeable ligands such as the iron-carrying transferrin protein bind cognate receptors, in this case the transferrin receptor, that reside in dynamic membrane trafficking pathways. The pathway occupied by the transferrin receptor involves rapid cycling between the cell surface and early/recycling endosomes, delivers transferrin to every cell of vertebrate animals, and represents a remarkable natural delivery vehicle. In the last grant cycle, we demonstrated that derivatives of the synthetic membrane anchor N-alkyl-3?-cholesterylamine exhibit a unique biological activity: when added to mammalian cells, they efficiently engage the membrane trafficking pathway occupied by the transferrin receptor and rapidly cycle between the plasma membrane and early/recycling endosomes. In this way, N-alkyl-3?-cholesterylamines can function as small artificial cell surface receptors; when linked to drug-binding motifs, they can shuttle drugs into cells and tissues via endocytosis. These compounds appear to functionally mimic free cholesterol, a key component of plasma and endosomal membranes. They bind cholesterol-laden LDL and HDL particles and presumably participate in natural cholesterol uptake and trafficking mechanisms. Because delivery of transferrin mediated by the transferrin receptor is so efficient, the ability of cholesterylamines to efficiently engage the same membrane trafficking pathway offers unprecedented potential to create transformative new tools for drug delivery. This proposal is focused on elucidating mechanisms that control the unique biological activities of N-alkyl-3?-cholesterylamines. By synthesizing and evaluating the biological properties of a panel of compounds bearing structural diversity in the linker region proximal to this membrane anchor, we will obtain quantitative structure-property relationships (QSPR) designed to predict cellular uptake, trafficking, efflux, metabolism, and toxicity properties. Structurally related photocrosslinkers, RNAi methods, and protein overexpression will be used to identify receptors and ligands involved in cellular uptake and efflux. These mediators of biological activity will have a profound effect on the pharmacokinetics of N-alkyl-3?-cholesterylamines in vivo. We further propose to construct the first anticancer drug delivery systems designed to cross membrane barriers by accessing a defined membrane trafficking pathway. To gain specificity for metastatic cancer cells over normal cells, peptide substrates of tumor-specific proteases will be used to link cholesterylamines to the anticancer drug doxorubicin. This drug will be selectively released when cleaved by the MMP-2 protease expressed by metastatic cancer cells. We will optimize the delivery of doxorubicin and related molecular probes into cancer cells in vitro to validate a fundamentally new concept: compounds that engage a defined membrane trafficking pathway offer a new strategy for the selective delivery of anticancer agents. PUBLIC HEALTH RELEVANCE: The research proposed here is highly relevant to the advancement of human health. In the past grant cycle we discovered that synthetic compounds termed N-alkyl-3?-cholesterylamines engage the specific membrane trafficking pathway occupied by the transferrin receptor. This biological activity enables these compounds to define new pathways across membrane barriers in mammalian cells. By elucidating biological mechanisms of these compounds, we propose to create innovative new tools for anticancer drug delivery.