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

用于靶向递送多种货物的单分子蛋白质纳米胶囊

• 批准号: 10374167
• 负责人: MALCOLM A LEISSRING
• 金额: $ 23.14万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374167
• 关键词: Adopted; Affinity; Binding; Binding Sites; C-terminal; Calmodulin; Cells; Chemicals; Chimeric Proteins; Confocal Microscopy; Cysteine; Cytosol; Development; Drug Delivery Systems; Encapsulated; Endosomes; Environment; Exposure to; Extracellular Protein; Extracellular Space; Extravasation; Flow Cytometry; HIV; Immobilization; In Vitro; Insulinase; Lead; Mediating; Metalloproteases; Methods; Modification; Molecular Conformation; Mutation; Oligonucleotides; Oxidation-Reduction; Oxides; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Positioning Attribute; Proteins; Resources; Specificity; Structure; System; Therapeutic; Variant; Zinc; base; capsule; controlled release; cytotoxic; disulfide bond; experimental study; extracellular; innovative technologies; nanocapsule; novel; prevent; single molecule; targeted delivery

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.

项目摘要/摘要 该提案探讨了开发基于胰岛素的新型复合输送系统的可行性 降解酶（IDE），一种具有独特的纳米胶囊结构的锌 - 超肽酶。 IDE类似于a 翻盖，完成了两个由“铰链”区域连接的碗形域，这使其可以采用“打开” 和“封闭”构象。关闭后，蛋白酶具有一个大型内室，约有13,000-Å3 体积已完全封装，可以容纳货物大约8000 da。这个建议 有两个主要目标：（1）探索使用IDE封装货物的可行性 通过广泛的体外表征调节释放； （2）开发针对货物的新型系统 专门用于细胞的细胞质，同时明确防止传递到细胞外空间。封装 以可逆和控制的方式货物，我们将生成包含两个半胱氨酸的IDE的变体 （s132c/e817c）定位，以便在蛋白酶在封闭式中时仅形成二硫键 构象。这种双层皮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（例如，细胞外空间）和 仅当暴露于还原环境（例如细胞质）时才会“解锁”。纯化的纳米胶囊会 进行大量的体外实验，以评估可以是的货物范围 成功加载和容纳，加载和卸载速率以及潜在的泄漏，以及 这些初始结果将为构造修改。开发靶向细胞质的货物交付 基于这些纳米胶囊的系统，我们将结合一个特征良好的非可交价细胞渗透 肽（CPP）标签显示出有效地将大蛋白从细胞外空间转移到细胞质的标签 并且至关重要的是，在CPP标记的蛋白质中被困在内体中的趋势。一起 通过其他合适的修改，提出的基于IDE的蛋白质纳米胶囊有望提供 有效的通用系统，用于封装潜水员货物并将其专门运送到细胞质 细胞，同时避免释放到细胞外空间中。如果这些最初的探索性实验是 成功的是，可以想象，这项创新技术可以适应各种应用程序， 有可能导致有力的新方法，用于靶向和受调节的治疗化合物的递送。

项目成果

Correction to: Insulin-degrading enzyme ablation in mouse pancreatic alpha cells triggers cell proliferation, hyperplasia and glucagon secretion dysregulation.

• DOI: 10.1007/s00125-023-06037-9
• 发表时间: 2024-01
• 期刊: DIABETOLOGIA
• 影响因子: 8.2
• 作者: Merino, Beatriz;Casanueva-Alvarez, Elena;Quesada, Ivan;Gonzalez-Casimiro, Carlos M.;Fernandez-Diaz, Cristina M.;Postigo-Casado, Tamara;Leissring, Malcolm A.;Kaestner, Klaus H.;Perdomo, German;Cozar-Castellano, Irene
• 通讯作者: Cozar-Castellano, Irene