Molecular engineering of complementary glucose-responsive conformational switches in insulin and glucagon

项目摘要 胰岛素和胰高血糖素长期应用于临床，在代谢动态平衡中发挥重要作用 管理糖尿病(DM)。这个MPI应用程序专注于开发葡萄糖响应型 这些激素的类似物。这项拟议的技术有望提高激素的安全性和有效性。 替代疗法，包括在闭环系统中的创新双激素泵。这是一个关键的前沿领域 分子药理学和非标准蛋白质工程。 多学科MPI团队涵盖蛋白质设计、生物物理学、结构生物学、动物生理学、 临床内分泌学和哺乳动物新陈代谢的计算机模拟。动物研究将在 正常和STZ大鼠在F·伊斯梅尔-贝吉教授(转包给CWRU)的指导下；计算机辅助 作为设计周期的一部分，M·斯特拉诺教授将在模拟模型中对这些研究进行解释 和同事(转包给麻省理工学院)。各种胰岛素-胰岛素受体(IR)复合体的冷冻-EM研究将 由M.C.劳伦斯教授执行(转包给澳大利亚墨尔本WEHI)。MPI团队最近联合 出版，包括《自然化学》，J.Biol。化学。和糖尿病。 葡萄糖反应胰岛素(GRI)类似物被认为是一种在 低血糖状态；葡萄糖反应高血糖素(GRG)类似物被认为是一种补充 在高血糖条件下减弱胰升糖素受体(GLR)信号的技术。各自 蛋白质设计基于两个互补的前提： 假设1：适当的葡萄糖结合元件(GBE)的发展将使 无糖封闭(非活性)状态间葡萄糖调控构象开关的构建 和葡萄糖结合的开放(活性)状态，符合WT胰岛素与IR结合和激活的方式；以及 假设2：不同的GBE的发展将使葡萄糖调节的 在葡萄糖结合的非活性状态和无葡萄糖活性状态之间的构象转换 根据WT胰高血糖素与GLR结合和激活的方式。 在每一种情况下，GBE都将利用二元醇的结合特性，即硼酸和苯并氧硼化合物。约束： GRI中的葡萄糖激活荷尔蒙，而GRG中葡萄糖的结合则使激素失活。AIMS 1- 3侧重于GRI，而AIM 4则将我们的方法扩展到GRG。这些技术可能会显著增强 T1D患者和部分T2D患者的长期健康状况。 蛋白质设计将基于胰岛素和胰高血糖素的经典晶体结构，并在最近戏剧性地扩展 IR、GLR及其配体络合物的结构生物学研究进展突出的结构 这些系统之间的差异有望实现相反的交换机的构建。跨学科的 团队方法在综合MPI管理计划中提出。

Project Summary Insulin and glucagon play central roles in metabolic homeostasis with long-standing application to the clinical management of diabetes mellitus (DM). This MPI application focuses on the development of glucose-responsive analogs of these hormones. The proposed technology promises to enhance the safety and efficacy of hormone replacement therapy, including in innovative bihormonal pumps in closed-loop systems. This is a key frontier of molecular pharmacology and non-standard protein engineering. The multidisciplinary MPI team encompasses protein design, biophysics, structural biology, animal physiology, clinical endocrinology, and computer simulations of mammalian metabolism. Animal studies will be performed in normal and STZ rats under the guidance of Prof. F. Ismail-Beigi (Subcontract to CWRU); computer-based interpretation of these studies as part of a design cycle will be undertaken in simulated models by Prof. M. Strano and coworkers (Subcontract to MIT). Cryo-EM studies of variant insulin-insulin receptor (IR) complexes will be performed by Prof. M.C. Lawrence (Subcontract to WEHI, Melbourne AU). The MPI team has recent joint publications, including in Nature Chemistry, J. Biol. Chem. and Diabetes. Glucose-responsive insulin (GRI) analogs are envisioned as a technology to attenuate IR signaling under conditions of hypoglycemia; glucose-responsive glucagon (GRG) analogs are envisioned as a complementary technology to attenuate glucagon-receptor (GlR) signaling under conditions of hyperglycemia. Respective protein design rests upon two complementary premises: Hypothesis 1: That development of an appropriate glucose-binding element (GBE) will enable construction of a glucose-regulated conformational switch between a glucose-free closed (inactive) state and a glucose-bound open (active) state in accord with how WT insulin binds to and activates the IR; and Hypothesis 2: That development of a distinct GBE will enable construction of a glucose-regulated conformational switch between a glucose-bound inactive state and a glucose-free active state in accordance with how WT glucagon binds to and activates the GlR. In each case the GBEs will exploit the diol-binding properties boronic acids and benzoxaboroles. Binding of glucose in a GRI activates the hormone whereas binding of glucose in a GRG inactivates the hormone. Aims 1- 3 focus on GRIs whereas Aim 4 extends our approach to GRGs. These technologies may markedly enhance the long-term health of patients with T1D and a subset of patients with T2D. Protein design will be based on classical crystal structures of insulin and glucagon, extended by dramatic recent advances in the structural biology of the IR, GlR and their respective ligand complexes. Salient structural differences between these systems promise to enable construction of opposing switches. An interdisciplinary team Approach is proposed within integrated MPI Management Plan.