Studies of an ultra-stable single-chain insulin and its therapeutic implications

 描述（由申请人提供）：单链胰岛素类似物（“微型胰岛素原”; SCI）作为蛋白质设计研究的模型系统和治疗糖尿病的治疗平台具有互补性。本申请的重点是一个SCI含有缩短的C结构域（6个残基），其设计采用了新的功能的翻译的兴趣。初步研究已经确定，该类似物（指定为SCI-双相）在高温下表现出非凡的稳定性，并且具有与预混常规/NPH类似物产品（礼来预混75/25赖脯胰岛素或Novo-Nordisk预混70/30门冬胰岛素）相似的双相药效学特性。尽管其配方复杂，但这种预混合产品为1型和2型糖尿病患者提供了一天两次的简化方案，并在发展中国家广泛使用。我们设想SCI-双相的超稳定和直接的可溶性制剂将具有与1型和2型糖尿病的日益增长的患病率相关的人道主义价值。我的MD/PhD研究计划将专注于SCI双相的结构，功能和错误折叠。目的1采用异频多维核磁共振波谱研究胰岛素类似物在溶液中的结构和动力学。我们试图测试的假设，在A，B和C域的特定设计元素有助于稳定性，并与构象波动的阻尼。目标2侧重于热稳定性测试，包括在糖尿病大鼠模型中连续测试应激样品。我们试图检验这样一个假设，即在发展中国家贫困患者经常经历的恶劣条件下（无法获得电力或制冷），SCI-双相将保持效力和双相药效学数月，而目前的胰岛素产品在几天内降解。目的3利用了我们的偶然发现，即在酵母巴斯德毕赤酵母（其在丰富的生长培养基中有效表达和分泌SCI-双相）中，使用基本培养基导致两种形式的这种类似物的共分泌：一种具有天然二硫键配对（如胰岛素原和胰岛素），另一种具有二硫键重排。非天然折叠提供了一个模型的“隐形错误折叠”的蛋白质在内质网（ER）在应激。我们试图识别这种非天然异构体，并探测其三维结构，作为2型糖尿病自然史中人类β细胞中胰岛素原发生类似错配和错折叠的模型。已经提出这种错误折叠有助于在这种慢性疾病的晚期阶段的β-细胞“衰竭”。据我们所知，这种SCI异构体的结构将提供第一个可视化的多肽定量插入到一个动力学陷阱的ER氧化蛋白折叠机械。

 DESCRIPTION (provided by applicant): Single-chain insulin analogs ("mini-proinsulins"; SCIs) are of complementary interest as a model system for studies of protein design and as a therapeutic platform for the treatment of diabetes mellitus. This application focuses on an SCI containing a foreshortened C domain (six residues) whose design incorporates novel features of translational interest. Preliminary studies have established that this analog (designated SCI- biphasic) exhibits extraordinary stability at elevated temperatures and has biphasic pharmacodynamics properties similar to those of pre-mixed regular/NPH analog products (Lilly Pre-Mixed 75/25 Insulin lispro or Novo-Nordisk Pre-mixed 70/30 Insulin Aspart). Despite the complexity of their formulation, such pre-mixed products provide Type 1 and Type 2 diabetes patients with a simplified twice-a-day regimen and are in broad use in the developing world. We envisage that an ultra-stable and straightforward soluble formulation of SCI- biphasic would be of humanitarian value in relation to growing prevalence of both Type 1 and Type 2 diabetes. My MD/PhD program of research will focus on the structure, function, and misfolding of SCI-biphasic. Aim 1 employs heteronuclear multidimensional NMR spectroscopy to investigate the structure and dynamics of this insulin analog in solution. We seek to test the hypothesis that specific design elements in the A, B, and C domains contribute to stability and are associated with the damping of conformational fluctuations. Aim 2 focuses on tests of thermal stability, including successive testing of stressed samples in a rat model of diabetes. We seek to test the hypothesis that under the harsh conditions often experienced by underprivileged patients in the developing world (lacking access to electricity or refrigeration) SCI-biphasic will retain potency and biphasic pharmacodynamics for several months whereas current insulin products degrade within days. Aim 3 exploits our serendipitous finding that in the yeast Pichia pastoris (which in rich growth medium efficiently expresses and secretes SCI-biphasic) use of minimal medium leads to the co-secretion of two forms of this analog: one with native disulfide pairing (as in proinsulin and insulin) and the other with a rearrangement of the disulfide bridges. The non-native fold provides a model of the "stealth misfolding" of a protein in the endoplasmic reticulum (ER) during stress. We seek to identify this non-native isomer and probe its 3D structure as a model for the analogous mispairing and misfolding of proinsulin that occurs in human ß-cells in the natural history of Type 2 diabetes. Such misfolding has been proposed to contribute to ß-cell "exhaustion" in the late stages of this chronic disease. To our knowledge, the structure of this SCI isomer will provide the first visualization of a polypeptide quantitatively inserted into a kinetic trap by the ER oxidative protein-folding machinery.