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 该项目的目的是识别甲状旁腺同性子的小型非肽分子调节剂 1型受体（PTHR），一种医学上重要的G蛋白偶联受体（GPCR），可调节血液钙和 磷酸盐稳态，并响应于PTH的骨骼重塑。过量引起的有害高钙血症 血液PTH水平或PTHR的多态性导致受体过度活跃，骨质疏松和低聚 由PTHR信号不良引起的MIA，无数骨骼和影响公众的矿物离子病理的基础 健康。先前通过R01DK-116780的支持已解决了与GS复合物中PTHR的近原子结构 并进行了发现，使我们提出了整合计算和药物的假设 研究使用PTHR和医学化学方法的高分辨率结构，可以解决CHAL- 发现和开发能够变构调节PTHR信号传导的小分子的借助。 AIM 1将使用计算方法的组合，包括分子动力学（MD）模拟方法 与基于弹性网络模型（ENM）的方法结合在一起的方法，以预测简单的PTHR位点 可吸毒和变构，并鉴定有可能用作变构调节剂的小分子。这些 通过在AIM 2和3中进行的实验的反馈，将对化合物进行迭代完善和验证。 AIM 2将表征和优化（通过使用先进的医学化学 - 通过合成和/或修改 - ISTRY方法）通过确定结合，这些命中化合物的药物剖面和细胞影响 在这些化合物存在下PTHR的信号传导和运输特性。细胞研究将测试功能 具有成骨细胞矿化和最有趣的药物剖面的化合物的典型作用 25-羟基维生素D 1-A-羟化酶的表达，速率限制维生素D的生物合成酶。 AIM 3将进行单个粒子冷冻EM研究，以解决与GQ复合物中PTHR的结构 通过Ca2+和PKC信号通路介导PTH信号传导的G蛋白。新结构将 允许我们确定不同的G蛋白（GQ和GS）相互互惠的机械基础 PTHR。结构数据将进一步用于模拟复合物的集体动力学和 GQ蛋白与直角和变构结合之间变构通信的机制 口袋。 该研究计划的重要性在于与与 多学科的实验表征和验证研究，允许对药物进行精确识别 PTHR中的山墙变构位点，以鉴定具有潜在治疗效用的新型小分子，用于治疗 提及骨骼和矿物质离子疾病。我们的研究最终可能证明对其他肽恐怖症有用 GPCR将小分子识别为未来疗法的人类疾病。