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.