Therapeutic Protein Engineering of Single-Chain Insulin Analogs

这项建议是对PAR-16-121(NIDDK感兴趣疾病治疗线索的早期临床前验证；R01机制)的响应，旨在确定用于治疗糖尿病(DM)的超稳定单链胰岛素(SCI)类似物的分子设计原则。这些类似物以前作为结构-活性关系的基本探针被研究过，它们表现出非凡的热稳定性，并有望绕过昂贵的全球胰岛素冷链的输送、分配、储存和使用。我们假设：假设。SCI框架将为开发优化的速效(膳食)、长效(基础)和两相(预混)配方提供一个共享平台。拟议的临床前研究有望实现三项治疗进展：(I)用于一般用途的超稳定耐热快速、两相和长效SCI配方；以及(Ii)扩展到闭环系统中植入的腹膜内胰岛素泵(IIIP)，因为这项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[蒙汀，J.G.，等人]。自然(2013)493、241-5和PNAS-Plus(2014)111、E3395-404]。跨学科方法是由一组分别在每种组件技术方面经验丰富的高级调查人员提出的。这一基本思想利用了荷尔蒙的闭合状态(如在小瓶、笔或泵储存库中)和开放状态(如与模型IR复合体结合时观察到的)之间的构象转换；理想的治疗胰岛素类似物将结合增强的闭合状态的稳定性和在激素-IR复合体中重组(诱导FIT)的天然能力。目标1-3以我们的初步发现为基础，准备并表征一系列合理的SCI，旨在展示SQ和IV注射(包括钳制研究)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。这些目标按可交付成果进行组织：目标1侧重于快速作用的SCI；目标2侧重于基础SCI；目标3侧重于延长保质期和与发展中国家特别相关的两相热稳定SCI。动物研究将使用大鼠(STZ和肥胖的胰岛素抵抗模型)、狗和猪。该申请包含明确的里程碑和时间表，与通过大学技术转让进行的连续退出有关，预计将使准备向FDA提交各自的IND申请成为可能。我们的AIMS的可行性得到了广泛的初步结果的支持，包括在JBC(2017)最后审查中重点讨论SCI的结构和功能的两个团队手稿。我们预计这些关键文章将很快出版，并提供给在线研究科。

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.