Structure and function of PTH class B GPCR

总结 本项目的目的是确定小的非肽类分子的甲状旁腺激素调节剂 1型受体（PTHR），一种调节血钙的医学上重要的G蛋白偶联受体（GPCR）， 磷酸盐稳态和骨重塑对PTH的反应。过量引起的有害性高钙血症 血液PTH水平，或PTHR多态性导致受体过度活跃，骨质疏松症和低钙- 由PTHR信号缺陷引起的mia是影响公众健康的许多骨和矿物质离子病变的基础。 健康先前通过R 01 DK-116780的支持解决了PTHR与Gs复合的近原子结构 并做出了一些发现，这些发现引导我们提出了一个假设， 研究，使用PTHR和药物化学方法的高分辨率结构，可以解决挑战， 发现和开发能够变构调节PTHR信号传导的小分子的挑战。 目标1将使用计算方法的组合，包括分子动力学（MD）模拟， 耦合到弹性网络模型（ENM）为基础的方法，预测PTHR网站，同时 可药用和变构的，并确定小分子，可以潜在地作为变构调节剂。这些 化合物将通过目标2和3中进行的实验的反馈进行反复改进和验证。 目标2将表征和优化（通过合成和/或使用先进的药物化学修饰）， 方法）这些命中化合物的药理学特征和细胞影响通过测定结合， 信号传导和PTHR在这些化合物存在下的运输特性。细胞研究将测试功能- 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）。 目的3将进行单粒子冷冻电镜研究，以解决PTHR与Gq复合物的结构， 通过Ca 2+和PKC信号通路介导PTH信号传导的G蛋白。新结构将 使我们能够确定不同的G蛋白-Gq和Gs-β偶联的机制基础， PTHR。结构数据将进一步用于模拟复合体的集体动力学， Gq蛋白与正构和变构配体结合之间的变构通讯机制 口袋 这项研究计划的意义在于它的第一种计算管道集成了 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，以鉴定具有潜在治疗效用的新型小分子， 骨和矿物质离子疾病。我们的研究可能最终证明对其他肽激素有用 GPCR将识别小分子作为未来治疗人类疾病的方法。

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.