Synthetic Cell Surface Receptors for Anticancer Drug Delivery

描述(申请人提供)：药物输送中尚未解决的主要问题之一是如何通过膜屏障运送渗透性差的分子。自然界解决这个问题的方法是通过膜转运；细胞不透水的配体，如携带铁的转铁蛋白蛋白，与同源受体结合，在这种情况下，转铁蛋白受体驻留在动态的膜转运途径中。转铁蛋白受体所占据的途径涉及细胞表面和早期/再循环内小体之间的快速循环，将转铁蛋白输送到脊椎动物的每一个细胞，是一种非凡的自然输送工具。在上一个资助周期中，我们证明了合成膜锚定N-烷基-3？-胆固醇胺的衍生物具有独特的生物活性：当它们加入哺乳动物细胞时，它们有效地参与了转铁蛋白受体占据的膜转运途径，并在质膜和早期/再循环内小体之间快速循环。通过这种方式，N-烷基-3？-胆固醇胺可以作为人工细胞表面的小受体发挥作用；当它们与药物结合基序相连时，它们可以通过内吞作用将药物输送到细胞和组织中。这些化合物似乎在功能上模拟了游离胆固醇，这是血浆和内膜的关键成分。它们结合了富含胆固醇的低密度脂蛋白和高密度脂蛋白颗粒，并可能参与了天然的胆固醇摄取和运输机制。由于转铁蛋白受体介导的转铁蛋白的传递是如此高效，胆固醇胺有效地参与相同的膜转运途径的能力为创造变革性的新的药物传递工具提供了前所未有的潜力。这项建议的重点是阐明控制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膜锚定近端的连接区具有结构多样性的化合物的生物学性质，我们将获得定量结构-性质关系(QSPR)，旨在预测细胞的摄取、运输、外排、代谢和毒性性质。结构相关的光交联剂、RNAi方法和蛋白质过表达将被用来识别参与细胞摄取和外排的受体和配体。这些生物活性介体将对N-烷基-3-胆固醇胺在体内的药代动力学产生深远的影响。我们进一步建议构建第一个抗癌药物传递系统，旨在通过进入一个明确的膜转运途径来跨越膜屏障。为了获得转移性癌细胞相对于正常细胞的特异性，肿瘤特异性蛋白酶的多肽底物将被用来将胆固醇胺与抗癌药物阿霉素联系起来。当这种药物被转移性癌细胞表达的基质金属蛋白酶-2酶切割时，将被选择性地释放。我们将优化阿霉素和相关分子探针在体外对癌细胞的传递，以验证一个全新的概念：参与特定膜转运途径的化合物为选择性传递抗癌药物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。在过去的资助周期中，我们发现被称为N-烷基-3？-胆固醇胺的合成化合物参与了转铁蛋白受体所占据的特定的膜转运途径。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，我们建议创造抗癌药物输送的创新工具。

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.