Therapeutic Protein Engineering of Single-Chain Insulin Analogs

该提案响应PAR-16-121（NIDDK感兴趣疾病的治疗先导物的早期临床前验证; R 01机制），旨在定义用于治疗糖尿病（DM）的超稳定单链胰岛素（SCI）类似物的分子设计原则。这些类似物以前被研究为结构-活性关系的基本探针，表现出显着的热稳定性，并有望规避昂贵的胰岛素递送、分配、储存和使用的全球冷链。我：假设。SCI框架将为开发优化的速效（餐时）、长效（基础）和双相（预混）制剂提供共享平台。拟议的临床前研究有望实现3项治疗进展：（i）超稳定的耐热速效、双相和长效SCI制剂，用于一般用途;以及（ii）扩展到闭环（“智能”）系统中的植入式腹膜内胰岛素泵（IIIP），因为该技术目前受到胰岛素在体温下降解的限制。分子设计基于对胰岛素受体（IR）的结构理解及其激素结合模式的最新进展[Menting，J.G.，等人Nature（2013）493，241-5和PNAS-Plus（2014）111，E3395-404]。一个跨学科的方法是由一个团队的高级研究人员分别在每一个组件技术经验。基本思想利用了激素的封闭状态（如在小瓶、笔或泵储液器中）和开放状态（如在与模型IR复合物结合时观察到的;“微受体”）之间的构象转换。理想的治疗性胰岛素类似物将结合联合收割机增强的闭合状态的稳定性和天然的在胰岛素-IR复合物中重组的能力（诱导适应）。目的1-3基于我们的初步研究结果，制备和表征一系列逻辑SCI，旨在SQ和IV注射后表现出各种药代动力学和药效学特性（PK/PD）（包括钳夹研究）。这些研究将探索生物化学和细胞机制，包括评估潜在的肝脏和组织特异性代谢选择性。这些目标按可交付成果组织：目标1侧重于速效SCI;目标2侧重于基础SCI;目标3侧重于双相热稳定SCI，具有延长的保质期，特别与发展中国家相关。动物研究将使用大鼠（STZ和肥胖胰岛素抵抗模型）、狗和猪。该申请包含与通过大学技术转让连续退出相关的明确里程碑和时间表，预计将能够准备向FDA提交的相应IND申请。我们的目标的可行性得到了广泛的初步结果的支持，包括JBC（2017）最终审查的两份专注于SCI结构和功能的团队手稿。我们预计，这些关键文章将很快发表，并提供给研究科在线。

This proposal, responsive to PAR-16-121 (Early-Stage Preclinical Validation of Therapeutic Leads for Diseases of Interest to the NIDDK; R01 mechanism) seeks to define principles for molecular design of ultra-stable single-chain insulin (SCI) analogs for the treatment of diabetes mellitus (DM). Previously studied as basic probes of structure-activity relationships, such analogs exhibit remarkable thermal stability and promise to circumvent the costly global cold chain of insulin delivery, distribution, storage and use. We posit: Hypothesis. The SCI framework will provide a shared platform for development of optimized rapid-acting (prandial), long-acting (basal) and biphasic (pre-mixed) formulations. The proposed preclinical studies promise to enable 3 therapeutic advances: (i) ultra-stable heat-resistant rapid-acting, biphasic and prolonged-acting SCI formulations for general usage; and (ii) extension to implanted intraperitoneal insulin pumps (IIIPs) in closed-loop (“smart”) systems as this technology is currently limited by degradation of insulin at body temperature. Molecular design is based on recent advances in the structural understanding of the insulin receptor (IR) and its mode of hormone binding [Menting, J.G., et al. Nature (2013) 493, 241-5, and PNAS-Plus (2014) 111, E3395-404]. An interdisciplinary Approach is proposed by a team of senior investigators respectively experienced in each of the component technologies. The essential idea exploits a conformational switch between a closed state of the hormone (as in a vial, pen or pump reservoir) and an open state (as observed on binding to a model IR complex; “micro-receptor”). An ideal therapeutic insulin analog would combine enhanced stability of the closed state with native competence for reorganization (induced fit) in the hormone-IR complex. Aims 1-3 build on our preliminary findings to prepare and characterize a logical series of SCIs designed to exhibit a variety of pharmacokinetic and pharmacodynamics properties (PK/PD) following SQ and IV injections (including clamp studies). The studies will explore biochemical and cellular mechanisms, including assessment of potential hepato- and tissue-specific metabolic selectivity. These Aims are organized by deliverable: Aim 1 focuses on a rapid-acting SCI; Aim 2 on a basal SCI; and Aim 3 on a biphasic thermally-stable SCI with prolonged shelf-life and of particular relevance to the developing world. Animal studies will employ rats (STZ and an obese insulin-resistant model), dogs and pigs. The application contains explicit milestones and timelines related to serial exits via University Technology Transfer, anticipated to enable preparation of respective IND applications to the FDA. The feasibility of our Aims is supportive by extensive preliminary results, including two team manuscripts focused on structure and function of SCIs in final review at the JBC (2017). We anticipate that these key articles will soon be published and available to the Study Section online.