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.