Design, synthesis, and characterization of Gs- and Gq-biased agonists of the Glucagon-like Peptide-1 Receptor (GLP-1R)

项目摘要/摘要 心血管疾病(CVD)在美国是主要的死亡原因，尤其是 在2型糖尿病(T2 DM)患者中流行。最近，胰升糖素样肽-1的激动剂 受体(GLP-1R)是一种G蛋白偶联受体，也是治疗T2 DM的常用靶点。 显示出良好的心血管益处，包括显著降低心血管疾病相关发病率和 死亡率。然而，批准的GLP-1R激动剂的更广泛的临床试验，其中许多是合成肽， 都产生了好坏参半的结果。因此，迫切需要了解推动 针对同一类试剂的不同作用模式，设计出更具针对性和有效性的治疗方法。 值得注意的是，GLP-1R的许多合成肽激动剂都表现出有偏向的激动剂，即配体 相对于内源性激动剂GLP-1，驱动对某些信号通路的偏好。这一现象 在许多其他GPCR中都可以看到。例如，盖尔曼实验室将N-上的α残基替换为β残基。 当处理表达PTH受体的细胞时，观察到G蛋白偏向。 我建议在这一策略的基础上开发新的GLP-1R合成肽激动剂，这些药物是有偏见的 将Gs和GQ通路作为更好地理解GLP-1R信号的工具。我将设计和合成α/β- GLP-1的多肽类似物，将N-末端的前八个氨基酸残基分别替换为 β残留物。将进行基于细胞的分析来测量cAMP生成(Gs)、钙动员(GQ)、 和β-arrestin-1，2募集，这代表了GLP-1R的主要信号通路，以表征 并确定其相对于GLP-1的偏向。 其次，我假设GLP-1R的偏向激活是独特的翻译后翻译的结果 多肽结合后及其下游的修饰(PTM)和蛋白质-蛋白质相互作用 结果，细胞内蛋白的表达和磷酸化发生了变化。为了验证这一假设，我将使用 基于MS的蛋白质组学研究纯化的GLP-1R的PTMS 先前使用Gs和GQ偏向多肽以及β-arrestin-1和-2偏向多肽进行治疗 由盖尔曼小组开发。第二次MS检测将在免疫共沉淀后进行 研究多肽结合后出现的独特的受体-蛋白质相互作用。最后，第三个MS 化验将获得下游蛋白质表达和磷酸化的变化，以深入了解 有偏向的激动剂结合产生的信号转导。这项建议的结果将有助于澄清 推动GPCRs信号转导的机制和帮助开发更安全、更有效的 治疗学。

Project Summary/Abstract Cardiovascular disease (CVD) is the major cause of death in the United Stand and is particularly prevalent in patients with type-2 diabetes mellitus (T2DM). Recently, agonists of the glucagon-like peptide-1 receptor (GLP-1R), a G protein-coupled receptor (GPCR) and common target for the treatment of T2DM, have shown promising cardiovascular benefits including a significant reduction in CVD-associated morbidity and mortality. However, broader clinical trials of approved GLP-1R agonists, many of which are synthetic peptides, have yielded mixed results. Thus, there is a critical need to understand the underlying mechanism driving the different modes of action for the same class of reagents to design more targeted and effective therapeutics. Notably, many synthetic peptide agonists of GLP-1R have demonstrated biased agonism, i.e., a ligand drives preference for certain signaling pathways relative to the endogenous agonist, GLP-1. This phenomenon is seen in many other GPCRs. For example, the Gellman lab replaced α residues with β residues on the N- terminus of the PTH peptide and observed a G-protein bias when treating cells expressing the PTH receptor. I propose to build on this strategy to develop novel synthetic peptide agonists of GLP-1R that are biased for the Gs and Gq pathways as tools to better understand GLP-1R signaling. I will design and synthesize α/β- peptides analogies of GLP-1 by replacing the first eight amino acid residues at the N-terminus, individually, with β residues. Cell-based assays will be performed to measure cAMP production (Gs), calcium mobilization (Gq), and β-arrestin-1, 2 recruitment, which represent the major signaling pathways of GLP-1R, to characterize the signaling profile and determine their biased relative to GLP-1. Next, I hypothesize that the biased activation of GLP-1R is the result of unique post-translational modifications (PTMs) and protein-protein interactions that arise after peptide binding and that downstream expression and phosphorylation of intracellular proteins is altered as a result. To test this hypothesis, I will use mass spectrometry (MS)-based proteomics to characterize the PTMs of purified GLP-1R that arise after treatment with the Gs and Gq biased peptides as well as β-arrestin-1 and -2 biased peptides previously developed in the Gellman group. A second MS assay will be performed after co-immunoprecipitation of the receptor to investigate unique receptor-protein interactions that arise after peptide binding. Finally, a third MS assay will access downstream changes in protein expression and phosphorylation for an in-depth understanding of the signal transduction produced by biased agonist binding. The results of this proposal will help elucidate the mechanisms driving the signal transduction of GPCRs and aid in the development of safer, more effective therapeutics.