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

项目总结/摘要 心血管疾病（CVD）是美国的主要死亡原因， 在2型糖尿病（T2 DM）患者中普遍存在。最近，胰高血糖素样肽-1激动剂 GLP-1受体（GLP-1 R）是一种G蛋白偶联受体（GPCR），也是治疗T2 DM的常见靶点， 显示出有希望的心血管益处，包括显著降低CVD相关的发病率， mortality.然而，批准的GLP-1 R激动剂（其中许多是合成肽）的更广泛临床试验， 结果好坏参半。因此，迫切需要了解驱动这些变化的潜在机制。 同一类试剂的不同作用模式，以设计更有针对性和更有效的治疗方法。 值得注意的是，GLP-1 R的许多合成肽激动剂已显示出偏向性激动作用，即，配体 驱动相对于内源性激动剂GLP-1的某些信号传导途径的偏好。这种现象 在许多其他GPCR中也可以看到。例如，Gellman实验室将N-甲基-N-甲基- 在PTH肽的末端，并且当处理表达PTH受体的细胞时观察到G蛋白偏倚。 我建议在此基础上开发新的GLP-1 R合成肽激动剂， Gs和Gq通路作为更好地理解GLP-1 R信号传导的工具。我将设计并合成α/β- GLP-1的肽类似物，其通过将N-末端的前8个氨基酸残基分别替换为 β残基。将进行基于细胞的测定以测量cAMP产生（Gs）、钙动员（Gq）， 和β-抑制蛋白-1，2募集，代表GLP-1 R的主要信号传导途径，以表征 信号转导谱并确定其相对于GLP-1的偏倚。 接下来，我假设GLP-1 R的偏向性激活是独特的翻译后调节的结果。 修饰（PTM）和蛋白质-蛋白质相互作用后产生的肽结合和下游 结果细胞内蛋白质的表达和磷酸化被改变。为了验证这个假设，我将使用 基于质谱（MS）的蛋白质组学，以表征纯化GLP-1 R的PTM， 先前用Gs和Gq偏向肽以及β-抑制蛋白-1和-2偏向肽处理 Gellman集团开发的。在免疫共沉淀后进行第二次MS测定。 受体研究独特的受体蛋白质相互作用后出现的肽结合。第三个MS 分析将访问下游的蛋白质表达和磷酸化的变化，以深入了解 由偏向激动剂结合产生的信号转导。这项建议的结果将有助于阐明 驱动GPCR信号转导的机制，并有助于开发更安全、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.