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

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

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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).

胰岛素在治疗糖尿病（DM）中起着核心作用。如果在分子水平上可行，超稳的胰岛素类似物可以（a）增强腹膜泵在1型DM治疗1型的治疗方面的安全性，（b）促进胰岛素配方的全球分布，用于处理1型和2型DM。简而言之，该提议试图翻译分子见解以获得超稳的治疗性胰岛素类似物。我们的方法集中在SCI上，其中包含A-和B链之间的缩短连接肽（“ C域”），还提供了结构活性关系的独特问题。蛋白质设计基于两个前提：假设1：SCI框架为C域设计提供了一个“最佳点”：足够长的时间可以使诱导的受体结合且与淀粉样蛋白相关的热灭活不相容。假设2：SCI可以通过A，B和C结构域的序列特征使SCI超稳，以潮湿自由蛋白质中的构象波动，但可以允许天然的生物学活性。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性胰岛素形式提供一个平台：快速作用（促进），长效（基底）和双相（预混合）公式。将注意细胞特异性信号传导特性，包括相对于WT人类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策略基础的基本思想探索了马烯的封闭状态（如在胰岛素的经典结构中观察到的）和开放状态（如hors烯与模型IR片段复合物结合时所观察到的；设计的“微受体”（µIR））。我们的战略及其可行性在最近的两个背靠背出版物中进行了描述：Glidden，M.D。等。 J. Biol。化学293、47-68和69-88（2018AB）。这些文章及其基于网络的补品中的方法进行了详细描述。蛋白质设计将既基于经典的胰岛素晶体结构（Adams，M.J。等人（1969）224，491-5），以及胰岛素受体（IR）的结构解剖及其同种结合模式的最新进展[Menting，J.G.等。 Nature（2013）493、241-5和PNAS-Plus（2014）111，E3395-404]，通过完整的胞过域的冷冻EM结构增强了创新，该结构包含胰岛素，该结构包含胰岛素在其信号构型中结合的胰岛素[Weis，F。等。大自然社区。（2018）9，4420]。因此，研究计划通过分包合同利用了正在进行的低温EM“解决革命”。劳伦斯。一个跨学科的方法是由一个综合MPI管理计划和内部咨询委员会的独特团队提出的。美国糖尿病协会主席L. Philipson博士（芝加哥大学；附带的信）描述了意义和创新。