A single-molecule protein nanocapsule for targeted delivery of diverse cargo

项目总结/摘要 该方案探讨了开发基于胰岛素的新型复合给药系统的可行性， 降解酶（IDE），一种具有独特的纳米囊状结构的锌金属肽酶。IDE类似于 蛤壳，包括两个由“铰链”区域连接的碗状区域，这使得它能够采用“打开” 和“闭合”构象。当关闭时，蛋白酶具有一个大的内部腔室，约13，000 - 3000英寸 体积，完全封装，可容纳约8000 Da的货物。 有两个主要目的：（1）探讨使用IDE封装货物的可行性， 通过广泛的体外表征调节释放;和（2）开发靶向货物的新系统 排他地递送至细胞的胞质溶胶，同时明确地阻止递送至细胞外空间。来封装 货物在可逆和可控的方式，我们将产生含有两个半胱氨酸的IDE的变体 （S132 C/E817 C）定位成使得当蛋白酶处于封闭的构象中时，它们仅形成二硫键。 构象这种双半胱氨酸突变构成了氧化还原敏感的“闩锁”，如前所述， 在氧化环境中将IDE保持“锁定”在关闭位置（例如，细胞外空间）和 只有当暴露于还原环境时才变得“解锁”（例如，胞质溶胶）。纯化的纳米胶囊将 进行一系列广泛的体外实验，旨在评估可以被运输的货物范围。 成功装载和容纳，装载和卸载速率和潜在泄漏，以及 将根据这些初始结果对构建体进行修改。开发一种细胞溶质靶向的货物输送系统 系统的基础上，这些纳米胶囊，我们将纳入一个良好的特点，非共价细胞穿透 肽（CPP）标签显示出有效地将大蛋白质从细胞外空间转运到细胞质溶胶 并且关键的是克服了CPP标记的蛋白质被困在内体中的倾向。一起 通过其他合适的修饰，预期所提出的基于IDE的蛋白质纳米胶囊提供了 一种有效的通用系统，用于封装不同的货物，并将其专门输送到细胞质中， 细胞，同时避免释放到细胞外空间。如果这些最初的探索性实验 这项创新技术一旦成功，就可以适用于各种各样的应用， 潜在地导致用于治疗化合物的靶向和调节递送的强大的新方法。

PROJECT SUMMARY/ABSTRACT This proposal explores the feasibility of developing a novel compound delivery system based on insulin- degrading enzyme (IDE), a zinc-metallopeptidase with a unique, nanocapsule-like structure. IDE resembles a clamshell, comprising two bowl-shaped domains connected by a “hinge” region, which allows it to adopt “open” and “closed” conformations. When closed, the protease features a large internal chamber, ~13,000-Å3 in volume, that is completely encapsulated and can accommodate cargo as large as ~8000 Da. This proposal has two principal objectives: (1) to explore the feasibility of using IDE for the encapsulation of cargo and for regulated release through extensive in vitro characterization; and (2) to develop a novel system targeting cargo exclusively to the cytosol of cells, while explicitly preventing delivery to the extracellular space. To encapsulate cargo in a reversible and controllable manner, we will generate variants of IDE containing two cysteines (S132C/E817C) positioned such that they form a disulfide bond exclusively when the protease is in the closed conformation. This double-cysteine mutation constitutes a redox-sensitive “latch” that, as shown previously, keeps IDE “locked” in the closed position in an oxidizing environment (e.g., the extracellular space) and becomes “unlocked” only when exposed to a reducing environment (e.g., cytosol). Purified nanocapsules will be subjected to an extensive battery of in vitro experiments aimed at evaluating the range of cargo that can be successfully loaded and accommodated, rates of loading and unloading and potential leakage, and the constructs will be modified as informed by these initial results. To develop a cytosol-targeting cargo delivery system based on these nanocapsules, we will incorporate a well-characterized, non-covalent cell-penetrating peptide (CPP) tag shown to efficiently translocate large proteins from the extracellular space to the cytosol and—crucially—overcome the tendency of CPP-tagged proteins to become trapped in endosomes. Together with other suitable modifications, the proposed IDE-based protein nanocapsules are expected to provide an effective, general-purpose system for encapsulating diverse cargo and delivering it exclusively to the cytosol of cells, while avoiding release into the extracellular space. If these initial, exploratory experiments are successful, this innovative technology can be conceivably be adapted for a wide variety of applications, potentially leading to powerful new methods for the targeted and regulated delivery of therapeutic compounds.