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

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，超稳定胰岛素类似物可以（a）增强用于治疗1型DM的腹膜内泵的安全性和（B）促进用于治疗1型和2型DM的胰岛素制剂的全球分布。简而言之，该提案旨在将分子见解转化为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的单链胰岛素（SCI）类似物的结构和功能。在A链和B链之间含有缩短的连接肽（"C结构域"）的SCI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一个“最佳点”：足够长的时间以使受体结合诱导适合，但与淀粉样蛋白相关的热失活不相容。假设二：可以通过A、B和C结构域的序列特征使SCI超稳定，以抑制游离蛋白质中的构象波动，但仍允许天然生物活性。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。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）的潜在促有丝分裂性。我们的SCI策略的基本思想是利用激素的闭合状态（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片段复合物结合时观察到的;称为"微受体"（µ IR））之间的构象转换。我们的策略及其可行性在最近的两篇背靠背的出版物中进行了描述：Glidden，M.D.，等人J. Biol. Chem. 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（亚当斯，M.J.，等. Nature（1969）224，491 - 5）和胰岛素受体（IR）的结构解剖及其激素结合模式的最新进展[Menting，J.G.，等人Nature（2013）493，241 - 5和PNAS-Plus（2014）111，E3395 - 404]，其中通过含有以其信号传导构象结合的胰岛素的完整胞外域的冷冻-EM结构增强了创新[Weis，F.，等人Nature Commun.（2018）9，4420]。因此，研究计划通过分包给M.C.，利用正在进行的冷冻EM "分辨率革命"。劳伦斯一个跨学科的方法是由一个独特的团队与综合MPI管理计划和内部咨询委员会。美国糖尿病协会主席L. Philipson（芝加哥大学;附信）。

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).