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

超稳定单链胰岛素的研究及其治疗意义

• 批准号: 9040780
• 负责人: Michael Glidden
• 金额: $ 4.64万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9040780
• 关键词: Amyloid Fibrils; Biological; Biological Assay; Biological Models; Biological Testing; Cells; Cellular Stress; Chemicals; Chronic Disease; Circular Dichroism; Clinical; Cold Chains; Complex; Culture Media; Data; Development; Diabetes Mellitus; Disulfides; Doctor of Philosophy; Drug Kinetics; Electricity; Elements; Endocrinology; Endoplasmic Reticulum; Exhibits; Exposure to; Formulation; Future; Gel; Gold; Guanidines; Homeostasis; Hormones; Human; Hyperglycemia; Imagery; In Vitro; Indium; Insulin; Insulin, Aspart, Human; Insulin, Lispro, Human; Insulin-Dependent Diabetes Mellitus; Isomerism; Kinetics; Label; Life; Medicine; Mentorship; Metabolic; Methods; Microvascular Dysfunction; Modeling; Molecular Conformation; Monitor; NMR Spectroscopy; Natural History; Neonatal; Non-Insulin-Dependent Diabetes Mellitus; Normal Pressure Hydrocephalus; Nuclear Magnetic Resonance; Patients; Peptides; Pharmacodynamics; Pichia; Prevalence; Proinsulin; Property; Protein Engineering; Proteins; Proteomics; Quality Control; Refrigeration; Regimen; Relaxation; Replacement Therapy; Research; Resistance; Safety; Sampling; Sprague-Dawley Rats; Staging; Streptozocin; Stress; Stress Tests; Structure; Structure of beta Cell of islet; Syndrome; System; Technology; Temperature; Testing; Therapeutic; Thermodynamics; Titrations; Variant; Work; X-Ray Crystallography; Yeasts; analog; biophysical analysis; biophysical model; comparative; design; diabetic; diabetic rat; exhaustion; experience; globular protein; glycemic control; human disease; in vivo; insight; insulin secretion; interest; male; melting; migration; miniproinsulin; monomer; mutant; non-Native; novel; pandemic disease; polypeptide; prevent; programs; protein folding; protein misfolding; protein protein interaction; public health relevance; research study; subcutaneous; thermal stress; thermostability; three dimensional structure; type I and type II diabetes

 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.

 描述(由申请人提供)：单链胰岛素类似物(“迷你胰岛素”；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的兴趣。本申请的重点是包含一个缩短的C结构域(六个残基)的SCI，其设计结合了具有翻译兴趣的新特征。初步研究证实，这种类似物(命名为SCI双相)在高温下表现出极高的稳定性，并具有与预混合的常规/NPH类似产品(礼来公司预混合的75/25胰岛素Lispro或诺和诺德预混合的70/30 Insulin Aspart)相似的两相药效学特性。尽管配方复杂，但这种预混合产品为1型和2型糖尿病患者提供了简化的一天两次的治疗方案，并在发展中国家得到广泛使用。我们预计，在1型和2型糖尿病日益流行的情况下，一种超稳定和直接可溶的SCI双相制剂将具有人道主义价值。我的医学博士项目的研究将集中在SCI-双相的结构、功能和错误折叠上。目的1利用异核多维核磁共振波谱研究该胰岛素类似物在溶液中的结构和动力学。我们试图检验这一假设，即A、B和C结构域中的特定设计元素有助于稳定性，并与构象波动的衰减有关。目的2重点是热稳定性测试，包括在糖尿病大鼠模型中连续测试应激样本。我们试图测试一种假设，即在发展中国家贫困患者经常经历的恶劣条件下(无法获得电力或冰箱)，SCI-BIZIC将在几个月内保持效力和双相药效学，而目前的胰岛素产品在几天内就会降解。目的3利用我们的偶然发现，在巴斯德毕赤酵母(在丰富的生长介质中有效地表达和分泌SCI-双相)中，使用最低限度的培养基会导致这种类似物的两种形式的共分泌：一种是与天然的二硫键配对(如在胰岛素原和胰岛素中)，另一种是与二硫键的重排。非天然折叠为内质网(ER)蛋白质在应激过程中的“隐形错误折叠”提供了一个模型。我们试图鉴定这种非天然的异构体，并探索其3D结构，以此作为2型糖尿病自然病史中发生在人类细胞中的胰岛素原类似的错配和错误折叠的模型。这种错误折叠被认为是在这种慢性病的晚期导致？细胞“衰竭”的原因。据我们所知，这种SCI异构体的结构将首次提供多肽通过内质网氧化蛋白质折叠机械定量插入到动力学陷阱中的可视化。