Enhanced Sampling of G-Protein-Coupled Receptor-G Protein Interactions

项目摘要 G蛋白偶联受体(GPCRs)是人类最大的膜蛋白超家族，具有多种功能 目前市场上约三分之一的药物的主要靶标。腺苷受体的四种亚型，A1，A2A，A2B， 和A3，调节广泛的生理功能。它们已成为重要的治疗靶点。 用于治疗心脏缺血、神经性疼痛和癌症。在功能过程中，A1和A3受体结合Gi/o 蛋白，而A2A和A2B受体结合Gs蛋白。此外，GPCR-G蛋白的相互作用是 受变构配体调节。这些配体结合到腺苷受体的胞外位置，该受体 表现出发散的层序和构象。与传统的激动剂相比，传统的激动剂针对的是高度 由于腺苷结合部位保守且经常引起靶外副作用，变构调节剂应运而生 与选择性GPCR药一样有前途的候选药物。到目前为止，腺苷受体是 具有X射线或冷冻-EM结构的GPCRs，在与不同的G蛋白的复合体中确定。尽管这些 结构提供了有价值的见解GPCR-G蛋白相互作用，它们是相当静态的图像 蛋白质复合体。目前的局限性包括：(1)灵活的GPCRs和G蛋白如何 动态识别彼此。(2)特异性GPCR-G蛋白相互作用的决定因素尚不清楚。 (3)腺苷受体上变构调节剂结合的结构基础和机制尚不清楚。 这些限制极大地阻碍了针对腺苷受体的有效药物设计。为了 克服这些局限性，我们的具体目标包括：(1)开发一种新的计算方法，基于最近的 实现全原子模拟的强健高斯加速分子动力学(GAMD)技术的成功 蛋白质-蛋白质相互作用(PPI)，称为“PPI-GAMD”。(2)在广泛使用的开源平台上实现PPI-GAMD 模拟包。(3)在模拟特定G蛋白与A1和A2A相互作用的过程中测试PPI-GAMD 感受器。(4)应用PPI-GAMD模拟确定变构调节剂与A1的结合机理 以及A2a受体和变构调节的GPCR-G蛋白的相互作用。(5)体外模拟验证 通过突变和结合分析以及通过与领先的 GPCR试验组。反过来，模拟将帮助我们以原子论的方式解释实验数据 水平。我们的长期目标是(1)开发可靠的计算方法来定量描述 疾病相关细胞信号通路中的生物分子识别和(2)设计有效药物 以重要受体为目标的分子。

Project Summary G-protein-coupled receptors (GPCRs) are the largest superfamily of human membrane proteins and serve as primary targets of about 1/3 of currently marketed drugs. Four subtypes of adenosine receptors, the A1, A2A, A2B, and A3, mediate a broad range of physiological functions. They have emerged as important therapeutic targets for treating cardiac ischemia, neuropathic pain and cancer. During function, the A1 and A3 receptors bind the Gi/o proteins, while the A2A and A2B receptors bind the Gs proteins. Moreover, the GPCR–G protein interactions are modulated by allosteric ligands. These ligands bind to a putative extracellular site of adenosine receptors, which exhibit divergent sequences and conformations. In contrast to traditional agonists that target at the highly conserved adenosine-binding site and often cause off-target side effects, allosteric modulators have emerged as promising candidates as selective GPCR drugs. To date, adenosine receptors are the sole subfamily of GPCRs that have X-ray or cryo-EM structures determined in complex with distinct G proteins. Although these structures provide valuable insights into the GPCR–G protein interactions, they are rather static images of the protein complexes. Current limitations include: (1) It remains unknown how the flexible GPCRs and G proteins dynamically recognize each other. (2) The determinants of specific GPCR–G protein interactions remain unclear. (3) The structural basis and mechanism of allosteric modulator binding in the adenosine receptors remain elusive. These limitations have greatly hindered effective drug design targeting the adenosine receptors. In order to overcome these limitations, our specific aims include: (1) Develop a new computational method based on recent success of a robust Gaussian accelerated molecular dynamics (GaMD) technique to enable all-atom simulations of protein-protein interactions (PPIs), called “PPI-GaMD”. (2) Implement PPI-GaMD in widely used open source simulation packages. (3) Test PPI-GaMD on simulations of specific G protein interactions with the A1 and A2A receptors. (4) Apply PPI-GaMD simulations to determine mechanisms of allosteric modulator binding to the A1 and A2A receptors and allosteric modulation of the GPCR–G protein interactions. (5) Validate simulations in vitro by mutagenesis and binding assays and in vivo by cellular functional assays through collaboration with a leading GPCR experimental group. In turn, the simulations will help us to interpret the experimental data at an atomistic level. Our long-term goals are (1) to develop robust computational methodologies to quantitatively characterize biomolecular recognition in disease-associated cellular signaling pathways and (2) to design effective drug molecules targeting important receptors.