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Molecular endocrinology and principles of diabetes therapeutics: application to ultra-stable insulin analogs

分子内分泌学和糖尿病治疗原理：超稳定胰岛素类似物的应用

基本信息

批准号：
10439592
负责人：
Millie M Georgiadis
金额：
$ 61.97万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10439592
关键词：
3-Dimensional Address Advisory Committees Affinity Aliquot American Amyloid Attention Back Binding Biological Biological Assay Biophysics C-Peptide Caring Cell Line Cells Chicago Clinical Code Color Complex Cryoelectron Microscopy Crystallization Crystallography Dependence Diabetes Mellitus Dissection Disulfides Doctor of Medicine Endocrinology Engineering Evolution Female Formulation Gene Expression Heat Stress Disorders High Pressure Liquid Chromatography Hormonal Hormones Human Implant In Vitro Insulin Insulin Receptor Insulin-Dependent Diabetes Mellitus Isoelectric Point Kinetics Label Length Letters Mammalian Cell Measures Medical Membrane Methods Modeling Molecular Molecular Conformation Monitor Nature Non-Insulin-Dependent Diabetes Mellitus Online Systems Phosphorylation Pichia Play Preparation Property Protein Dephosphorylation Protein Dynamics Protein Engineering Proteins Protocols documentation Publications Publishing Pump Rattus Refrigeration Research Resolution Rest Role Safety Sampling Series Ships Signal Transduction Site Spottings Streptozocin Stress Structure Structure-Activity Relationship Surface System Temperature Tertiary Protein Structure Testing Therapeutic Thermodynamics Time Translating Universities alpha helix analog base carcinogenicity chemical stability clinical translation design diabetic diabetic rat dimer global health innovation insight interdisciplinary approach interest intraperitoneal male neoplastic cell pandemic disease pharmacokinetics and pharmacodynamics receptor receptor binding self assembly structural biology three dimensional structure tissue culture

项目摘要

Insulin plays a central role in the treatment of diabetes mellitus (DM). If feasible at the molecular level, ultra-stable insulin analogs could (a) enhance the safety of intraperitoneal pumps for the treatment of Type 1 DM and (b) facilitate global distribution of insulin formulations for the treatment of Type 1 and Type 2 DM. This proposal, in a nutshell, seeks to translate molecular insights to obtain ultra-stable therapeutic insulin analogs. Our approach focuses on the structure and function of ultra-stable single-chain insulin (SCI) analogs. SCIs, containing a shortened connecting peptide (“C domain”) between A- and B chains, also provide unique probes of structure-activity relationships. Protein design rests upon two premises: Hypothesis 1: That the SCI framework provides a “sweet spot” for design of C domains: long enough to enable induced fit on receptor binding yet incompatible with amyloid-associated thermal inactivation. Hypothesis 2: That SCIs can be made ultra-stable via sequence features of the A, B and C domains to damp conformational fluctuations in the free protein and yet permit native biological activity. The proposed studies will provide design rules for clinical translation. Our preliminary results suggest that SCIs can provide a platform for all 3 therapeutic forms of insulin: rapid-acting (prandial), long-acting (basal) and biphasic (pre-mixed) formulations. Attention will be paid to cell-specific signaling properties, including potential mitogenicity relative to WT human insulin and carcinogenic analog X10 (AspB10-insulin). The essential idea underlying our SCI strategy exploits a conformational switch between a closed state of the hormone (as observed in classical structures of insulin) and an open state (as observed on binding of the hormone to a model IR fragment complex; designated the “micro-receptor” (µIR)). Our strategy and its feasibility are described in two recent back-to-back publications: Glidden, M.D., et al. J. Biol. Chem. 293, 47-68 and 69-88 (2018ab). Methods are described in detail in these articles and their web-based Supplements. Protein design will be based both on classical insulin crystal structures (Adams, M.J., et al. Nature (1969) 224, 491-5) and on recent advances in the structural dissection 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], with innovation enhanced via cryo-EM structures of the intact ectodomain containing insulin bound in its signaling conformation [Weis, F., et al. Nature Commun. (2018) 9, 4420]. The Research plan thus exploits the ongoing cryo-EM “resolution revolution” via Subcontract to M.C. Lawrence. An interdisciplinary Approach is proposed by a unique team with integrated MPI Management Plan and Internal Advisory Committee. The significance and innovation are described by American Diabetes Association President Dr. L. Philipson (University of Chicago; letter attached).

胰岛素在糖尿病 (DM) 的治疗中发挥着核心作用。如果在分子水平上可行，超稳定胰岛素类似物可以（a）增强腹膜内泵治疗 1 型糖尿病的安全性，（b）促进用于治疗 1 型和 2 型糖尿病的胰岛素制剂的全球分销。简而言之，该提案旨在转化分子见解以获得超稳定的治疗性胰岛素类似物。我们的方法侧重于超稳定单链胰岛素 (SCI) 类似物的结构和功能。 SCI 在 A 链和 B 链之间含有一个缩短的连接肽（“C 结构域”），也提供了结构-活性关系的独特探针。蛋白质设计基于两个前提：假设 1：SCI 框架为 C 结构域的设计提供了“最佳点”：足够长以实现受体结合的诱导拟合，但与淀粉样蛋白相关的热失活不相容。假设 2：SCI 可以通过 A、B 和 C 结构域的序列特征变得超稳定，以抑制游离蛋白质的构象波动，同时允许天然生物活性。拟议的研究将为临床转化提供设计规则。我们的初步结果表明，SCI 可以为所有 3 种治疗形式的胰岛素提供平台：速效（餐时）、长效（基础）和双相（预混合）制剂。我们将关注细胞特异性信号传导特性，包括相对于 WT 人胰岛素和致癌类似物 X10（AspB10-胰岛素）的潜在有丝分裂性。我们的 SCI 策略的基本思想是利用激素闭合状态（如在胰岛素的经典结构中观察到的）和开放状态（如在激素与模型 IR 片段复合物的结合中观察到的；指定为“微受体”（μIR））之间的构象转换。我们的策略及其可行性在最近的两本连续出版物中进行了描述：Glidden, M.D., et al. J.Biol。化学。 293、47-68 和 69-88 (2018ab)。这些文章及其基于网络的补充中详细描述了方法。蛋白质设计将基于经典的胰岛素晶体结构（Adams, M.J., et al. Nature (1969) 224, 491-5）和胰岛素受体 (IR) 结构剖析及其激素结合模式的最新进展 [Menting, J.G., et al. Nature (1969) 224, 491-5]。 Nature (2013) 493, 241-5, 和 PNAS-Plus (2014) 111, E3395-404]，通过冷冻电镜结构增强创新，完整的胞外域含有结合在其信号构象中的胰岛素 [Weis, F., et al.自然交流。（2018）9，4420]。因此，该研究计划通过向 M.C. 分包，利用了正在进行的冷冻电镜“分辨率革命”。劳伦斯.具有综合 MPI 管理计划和内部咨询委员会的独特团队提出了跨学科方法。美国糖尿病协会主席 L. Philipson 博士（芝加哥大学；附信）描述了其重要性和创新之处。