Therapeutic Protein Engineering of Single-Chain Insulin Analogs

该提案响应PAR-16-121 （NIDDK相关疾病治疗先导物的早期临床前验证；R01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。这些类似物之前被研究为结构-活性关系的基本探针，它们表现出显著的热稳定性，有望绕过胰岛素输送、分配、储存和使用的昂贵的全球冷链。我们假设：假设。SCI框架将为开发优化的速效（膳食）、长效（基础）和双相（预混合）配方提供一个共享平台。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；（ii）在闭环（“智能”）系统中扩展到植入式腹腔胰岛素泵（IIIPs），因为该技术目前受到体温下胰岛素降解的限制。分子设计是基于对胰岛素受体（IR）及其激素结合模式的结构理解的最新进展[ming， J.G.，等]。《自然》（2013）493,241-5,PNAS-Plus (2014) 111, E3395-404。跨学科的方法是由一组资深的研究人员提出的，他们分别在每个组成技术方面都有经验。其基本思想是利用激素在封闭状态（如在小瓶、笔或泵储存器中）和开放状态（如与模型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应结合增强的封闭状态稳定性和激素- ir复合物的天然重组能力（诱导契合）。目的1-3建立在我们的初步研究结果的基础上，准备和表征一系列逻辑的SCIs，旨在展示SQ和IV注射（包括钳形研究）后的各种药代动力学和药效学特性（PK/PD）。这些研究将探索生化和细胞机制，包括评估潜在的肝脏和组织特异性代谢选择性。这些目标按可交付性进行组织：目标1侧重于速效SCI；目的2：基础脊髓损伤；目标3是延长保质期的双相热稳定SCI，与发展中国家特别相关。动物研究将采用大鼠（STZ和肥胖胰岛素抵抗模型）、狗和猪。该申请包含明确的里程碑和时间表，与通过大学技术转移的系列退出相关，预计能够准备向FDA提交各自的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SCIs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，并在线提供给研究部。

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.