Structure, Mechanism, and Regulation of PACAP/VIP GPCR Subtypes

在药理上重要的B类G蛋白偶联受体中，PAC1/VPAC 垂体腺苷环化酶激活多肽受体(ADCYAP1R1/VIPR1/VIPR2)， ADCYAP1)和血管活性肠肽(VIP)与多种疾病有关，包括慢性 疼痛和应激相关的行为异常，是全球最常见的神经学疾病之一 今天的挑战。然而，针对这些受体的试剂的开发具有潜在的治疗作用 由于缺乏全长PAC1/VPAC受体的结构信息和机制 了解配体结合如何以不同方式驱动受体激活的构象变化 和有偏见的信号。因此，这项提议的总体目标是采用最先进的 模拟PAC1/VPAC受体结构和动力学的计算方法，目的是开发 小分子化合物来调节它们的功能。对于每个受体亚型，唯一的识别 允许特定配体相互作用和转导蛋白关联的结构特征将有助于 小分子配体的合理设计和优化。为了实现这一目标，将有三个具体目标 追求：(1)模拟和比较决定神经肽选择性和 生理相关的PAC1Null、PAC1Hop1、VPAC1和VPAC2受体亚型之间的功能；(2) 描述PAC1/VPAC受体-转导蛋白的相互作用和特异性；以及(3)建立和 优化小分子以选择性调节PAC1和VPAC受体。我们的初步数据显示 建立了PAC1Null受体的建模方法，展示了不同的信号行为 并鉴定出两种不同的PAC1Null拮抗剂。在目标1和目标2下，我们将 使用同源建模、蛋白质结构优化和分子动力学模拟来研究 无配体、与神经肽(PACAP/VIP)或转导蛋白复合的受体 (GS/GQ/β--逮捕)。特别是，我们将阐明配体特异性的关键结合部位以及 促进长期信号传递的构象状态。受体突变和结构将告知 和/或证实受体模型。在目标3下，我们将整合分子对接和模拟， 有机合成，以及分子和细胞分析，以开发选择性小分子调节剂。 特别是，将测试以正构和变构为靶点的策略。我们的多学科 方法是创新的，因为它提供了一种无与伦比的全面手段来调查 PAC1/VPAC受体处于不同的状态和功能状态。此外，拟议的研究是 意义重大，因为它将弥合在理解分子结构和动力学如何 可以决定PAC1/VPAC受体的机制。最后，考虑到目前治疗学的局限性，这些研究 可能为治疗具有挑战性的神经疾病提供新的机会和方法。

Among the pharmacologically important Class B G protein-coupled receptors (GPCRs), the PAC1/VPAC receptors (ADCYAP1R1/VIPR1/VIPR2) for pituitary adenylate cyclase activated polypeptide (PACAP, ADCYAP1) and vasoactive intestinal peptide (VIP) have been implicated in several disorders, including chronic pain and stress-related behavioral abnormalities which are among the most prevalent global neurological challenges today. However, the development of reagents targeting these receptors for potential therapeutics has been hampered by the lack of full-length PAC1/VPAC receptor structural information, and mechanistic understandings of how ligand binding can differentially drive conformational changes for receptor activation and biased signaling. Accordingly, the overarching aim of this proposal is to employ state-of-the-art computational methods to model PAC1/VPAC receptor structure and dynamics, with the goal of developing small-molecule compounds to modulate their functions. For each receptor subtype, the identification of unique structural features that allow for specific ligand interactions and transducer protein associations will facilitate the rational design and optimization of small-molecule ligands. Towards that goal, three specific aims will be pursued: (1) to model and compare structures and mechanisms that determine neuropeptide selectivity and function among the physiologically relevant PAC1Null, PAC1Hop1, VPAC1, and VPAC2 receptor subtypes; (2) to delineate PAC1/VPAC receptor-transducer protein interactions and specificity; and (3) to develop and optimize small molecules for selective PAC1 and VPAC receptor regulation. Our preliminary data have established the modeling methodology with the PAC1Null receptor, demonstrated distinct signaling behaviors of the PAC1/VPAC receptors, and identified two different PAC1Null antagonists. Under Aims 1 and 2, we will use homology modeling, protein structure refinement, and molecular dynamics simulations to study the receptors that are ligand-free and complexed with a neuropeptide (PACAP/VIP) or a transducer protein (Gs/Gq/β-arrestins). In particular, we will elucidate the binding sites that are key for ligand specificity as well as conformational states that facilitate long-term signaling. Receptor mutagenesis and constructs will inform and/or substantiate the receptor models. Under Aim 3, we will integrate molecular docking and simulations, organic synthesis, and molecular and cellular assays to develop selective small-molecule modulators. Especially, the strategies to target the orthosteric and allosteric sites will be tested. Our multidisciplinary approach is innovative as it provides an unparalleled and comprehensive means to investigate the PAC1/VPAC receptors in various conditions and functional states. Further, the proposed research is significant, because it will close fundamental gaps in understanding how molecular structures and dynamics can dictate PAC1/VPAC receptor mechanisms. Finally, given current limitations in therapeutics, these studies may offer new opportunities and approaches to treat challenging neurological disorders.