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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)的治疗中起着核心作用。如果在分子水平上可行，超稳定的胰岛素类似物可以(A)提高治疗1型糖尿病的腹膜内泵的安全性，以及(B)促进治疗1型和2型糖尿病的胰岛素制剂的全球分布。简而言之，这项提议旨在将分子洞察力转化为获得超稳定的治疗性胰岛素类似物。我们的研究重点是超稳定单链胰岛素(SCI)类似物的结构和功能。SCIS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。蛋白质的设计基于两个前提：假设1：SCI框架为C结构域的设计提供了一个“甜蜜点”：足够长，能够诱导FIT与受体结合，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结构域的序列特征而变得超稳定，以抑制游离蛋白质的构象波动，同时允许天然的生物活性。拟议的研究将为临床翻译提供设计规则。我们的初步结果表明，SCI可以为所有三种治疗形式的胰岛素提供一个平台：速效(餐后)、长效(基础)和两相(预混合)配方。将关注细胞特有的信号特性，包括相对于WT人胰岛素和致癌类似物X10(AspB10-胰岛素)的潜在有丝分裂活性。我们SCI策略的基本思想利用了荷尔蒙的关闭状态(如在胰岛素的经典结构中观察到的)和开放状态(如荷尔蒙与模型IR片段复合体结合时观察到的)的构象切换，称为“微受体”(µIR)。我们的战略及其可行性在最近的两份背靠背出版物中进行了描述：Glidden，M.D.等人。J.Biol.化学。293、47-68和69-88(2018ab)。这些文章及其基于Web的补充资料中详细描述了这些方法。蛋白质设计将基于经典的胰岛素晶体结构(Adams，M.J.等人)。自然(1969)224,491-5)，以及胰岛素受体(IR)的结构解剖及其激素结合模式的最新进展[Monting，J.G.，et al.自然(2013)493,241-5和PNAS-Plus(2014)111，E3395-404]，通过在其信号构象中包含结合胰岛素的完整胞外结构域的冷冻EM结构来增强创新[Weis，F.，et al.自然界的普通人。(2018)9，]。因此，研究计划通过将合同转包给M.C.劳伦斯，利用了正在进行的低温电磁“解决革命”。一个独特的团队提出了一种跨学科的方法，该团队整合了MPI管理计划和内部咨询委员会。美国糖尿病协会主席L.Philipson博士描述了该研究的意义和创新之处(芝加哥大学，信附后)。