Structure and function of PTH class B GPCR

PTH B 类 GPCR 的结构和功能

• 批准号: 10657916
• 负责人: Ivet Bahar
• 金额: $ 65.1万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657916
• 关键词: 25-hydroxyvitamin D; Address; Affect; Affinity; Allosteric Site; Binding; Biological Assay; Blood; Bone Diseases; Bone remodeling; CYP27B1 gene; Calcium; Cell physiology; Cells; Communication; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cyclic AMP; Data; Databases; Defect; Disease; Elasticity; Endocrine System Diseases; Feedback; Fracture; Future; G alpha q Protein; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic Polymorphism; Goals; Homeostasis; Hyperactivity; Hypercalcemia; Hypercalcemia of Malignancy; Hyperparathyroidism; Hypocalcemia; Individual; Ions; Libraries; Ligand Binding; Ligands; Location; Mechanics; Mediating; Medical; Methods; Minerals; Modeling; Modification; Molecular Conformation; Organic Chemistry; Osteoblasts; Osteoporosis; PTH gene; Parathyroid Hormone Receptor; Pathology; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology Study; Polypeptide Hormones; Property; Public Health; Receptor Signaling; Recombinants; Recycling; Research; Resolution; Second Messenger Systems; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Testing; Therapeutic; Time; Vitamin D; bone; bone turnover; computational pipelines; computer studies; conformer; design; enzyme biosynthesis; experimental study; extracellular; hormonal signals; human disease; improved; in silico; inorganic phosphate; mineralization; molecular dynamics; multidisciplinary; network models; novel; parathyroid hormone-related protein; particle; peptide hormone; pharmacologic; pharmacophore; pilot test; programs; protein activation; protein complex; receptor; receptor function; receptor internalization; response; screening; simulation; small molecule; structural biology; trafficking; validation studies; virtual model; virtual screening

Summary The objective of this project is to identify small non-peptidic molecule modulators of the parathyroid hormone type 1 receptor (PTHR), a medically important G protein-coupled receptor (GPCR) regulating blood calcium and phosphate homeostasis, and bone remodeling in response to PTH. Detrimental hypercalcemia caused by excess of blood PTH level, or polymorphisms of PTHR resulting in receptor hyperactivity, osteoporosis, and hypocalce- mia caused by defective PTHR signaling, underlie numerous bone and mineral-ion pathologies affecting public health. Previous support via R01DK-116780 has solved the near-atomic structure of PTHR in complex with Gs and made discoveries that led us to formulate the hypothesis that integrated computational, and pharmacological studies, using high-resolution structures of the PTHR and medicinal chemistry methods, may address the chal- lenge of discovering and developing small molecules capable of allosterically modulating PTHR signaling. Aim 1 will use a combination of computational approaches, including molecular dynamics (MD) simula- tions coupled to elastic network model (ENM)-based methods, to predict PTHR sites that are simultaneously druggable and allosteric, and identify small molecules that can potentially serve as allosteric modulators. These compounds will be iteratively refined and validated with feedback from experiments conducted in Aims 2 and 3. Aim 2 will characterize and optimize (via synthesis and/or modifications using advanced medicinal chem- istry methods) the pharmacological profile and cellular impact of these hit compounds by determining the binding, signaling, and trafficking properties of PTHR in the presence of these compounds. Cell studies will test the func- tional actions of compounds with most interesting pharmacological profiles on osteoblast mineralization and expression of 25-hydroxyvitamin D 1-a-hydroxylase, rate limiting enzyme for the biosynthesis of vitamin D). Aim 3 will carry out single particle cryo-EM studies to solve the structure of PTHR in complex with Gq, the G protein that mediates PTH signaling through Ca2+ and PKC signaling pathways. The new structure(s) will permit us to determine the mechanistic basis by which different G proteins –Gq and Gs– reciprocally couple to the PTHR. Structural data will be further used for simulating the collective dynamics of the complex and the mechanism of allosteric communication between the Gq protein and the orthosteric and allosteric ligand-binding pockets. The significance of this research program lies in its first of its kind computational pipeline integrated with multidisciplinary experimental characterization and validation studies, permitting a precise identification of drug- gable allosteric sites in the PTHR to identify novel small molecules with potential therapeutic utility for the treat- ment of bone and mineral ion diseases. Our research may ultimately prove useful to other peptide hormone GPCRs to identify small molecules as future treatments human diseases.

概括 该项目的目的是鉴定甲状旁腺激素的小非肽分子调节剂 1 型受体 (PTHR)，一种医学上重要的 G 蛋白偶联受体 (GPCR)，调节血钙和 磷酸盐稳态和骨重塑，以应对由过量 PTH 引起的有害高钙血症。 血液 PTH 水平或 PTHR 多态性导致受体过度活跃、骨质疏松和低钙血症 由 PTHR 信号传导缺陷引起的 mia 是影响公众的众多骨和矿物质离子病理的基础 之前通过 R01DK-116780 的支持已经解决了 PTHR 与 G 复合物的近原子结构。 并做出的发现使我们提出了整合计算和药理学的假设 使用 PTHR 的高分辨率结构和药物化学方法进行的研究可能会解决这一问题 一系列发现和开发能够变构调节 PTHR 信号传导的小分子。 目标 1 将结合使用计算方法，包括分子动力学 (MD) 模拟 与基于弹性网络模型（ENM）的方法相结合，以预测同时发生的 PTHR 位点 可药物化和变构，并鉴定可潜在用作变构调节剂的小分子。 化合物将根据目标 2 和 3 中进行的实验的反馈进行迭代完善和验证。 目标 2 将表征和优化（通过使用先进的药物化学合成和/或修饰） istry方法）通过确定结合来了解这些命中化合物的药理学特征和细胞影响， 细胞研究将测试这些化合物存在下 PTHR 的信号传导和运输特性。 具有最有趣药理学特征的化合物对成骨细胞矿化的作用 25-羟基维生素 D 1-α-羟化酶（维生素 D 生物合成的限速酶）的表达。 目标 3 将进行单粒子冷冻电镜研究，以解析 PTHR 与 Gq 复合物的结构， 通过 Ca2+ 和 PKC 信号通路介导 PTH 信号传导的 G 蛋白将具有新的结构。 使我们能够确定不同 G 蛋白（Gq 和 Gs）相互偶联的机制基础 PTHR 结构数据将进一步用于模拟综合体和区域的集体动态。 Gq 蛋白与正构和变构配体结合之间的变构通讯机制 口袋。 该研究计划的意义在于它是第一个与 多学科实验表征和验证研究，允许精确鉴定药物 PTHR 中的 gable 变构位点，以识别具有潜在治疗效用的新型小分子 我们的研究最终可能对其他肽激素有用。 GPCR 识别小分子作为未来治疗人类疾病的方法。