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: 9244802
• 负责人: Michael Glidden
• 金额: $ 4.9万
• 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/9244802
• 关键词: Amyloid Fibrils; Amyloid beta-Protein; Beta Cell; Biological; Biological Assay; Biological Models; Biological Testing; Biophysics; Cellular Stress; Chemicals; Chronic Disease; Circular Dichroism; Clinical; Cold Chains; Complex; Crystallization; Data; Development; Diabetes Mellitus; Disulfides; Doctor of Philosophy; Drug Kinetics; Electricity; Elements; Endocrinology; Endoplasmic Reticulum; Exhibits; Exposure to; Formulation; Foundations; Future; Gel; Gold; Growth; Guanidines; Homeostasis; Hormones; Human; Hyperglycemia; Imagery; Impairment; In Vitro; 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; 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; cost; design; diabetic; diabetic rat; exhaustion; experience; experimental study; globular protein; glycemic control; human disease; in vivo; insight; insulin secretion; interest; male; melting; migration; miniproinsulin; misfolded protein; monomer; mutant; non-Native; novel; pandemic disease; polypeptide; prevent; programs; protein folding; protein misfolding; protein protein interaction; public health relevance; subcutaneous; thermal stress; thermostability; three dimensional structure

 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（通过应用程序提供）：单链胰岛素类似物（“迷你 - 胰岛素”； SCIS）是蛋白质设计研究的模型系统，并且是治疗糖尿病的治疗平台的模型系统。该应用程序着重于包含概要的C域（六个残差）的SCI，其设计结合了转化兴趣的新特征。 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).尽管它们的配方很复杂，但此类预混合产品提供了1型和2型糖尿病患者，其简化为每天两次，并且在发展中国家中广泛使用。我们设想，与1型和2型糖尿病的越来越多的患病率有关，科学的超稳且直接的解决方案将具有人道主义价值。我的MD/PHD研究计划将重点介绍科学的结构，功能和错误折叠。 AIM 1员工异核多维NMR光谱法研究了溶液中这种胰岛素类似物的结构和动力学。我们试图检验以下假设：A，B和C域中的特定设计元素有助于稳定性，并且与构象波动的阻尼有关。 AIM 2专注于热稳定性的测试，包括在糖尿病大鼠模型中成功测试应力样品。我们试图检验以下假设：在发展中国家中贫困患者经常经历的HARMH条件下（缺乏电力或制冷）科学双性异体将保留几个月的效力和双相药效学，而当前的胰岛素胰岛素在几天之内降低。 AIM 3利用了我们的偶然发现，即在酵母菌中，Pichia Pastoris（在丰富的生长培养基中有效地表达并分泌科幻媒介）对最小培养基的使用导致这种类似物的两种形式的分泌：一种与天然二硫化物配对（在proinsulin and胰岛素和胰岛素中一样），而另一种是与一个重新分析的杂物。非本地褶皱提供了在压力期间内质网（ER）中蛋白质的“隐身错误折叠”模型。我们试图识别这种非本地异构体，并将其3D结构探测为在2型糖尿病的自然历史上发生在人ß细胞中的类似错误折叠和错误折叠的模型。在这种慢性疾病的后期，这种错误折叠被提议为ß细胞“精疲力尽”做出贡献。据我们所知，该SCI异构体的结构将通过ER氧化蛋白折叠机器定量地插入动力学陷阱的多肽的首次可视化。