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

胰高血糖素样肽 1 受体 (GLP-1R) 的 Gs 和 Gq 偏向激动剂的设计、合成和表征

• 批准号: 10388640
• 负责人: Kyle A Brown
• 金额: $ 2.61万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388640
• 关键词: Aftercare; Agonist; Alanine; Amides; Amino Acids; Automobile Driving; Basic Science; Binding; Biological Assay; Bioluminescence; Calcium; Cardiovascular Diseases; Cardiovascular system; Cause of Death; Cells; Characteristics; Clinical; Clinical Trials; Co-Immunoprecipitations; Complex; Cyclic AMP; Development; Diabetes Mellitus; Disease; Dose; Drug Targeting; Energy Transfer; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GCG gene; GLP-I receptor; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Homologous Gene; Hormone use; Individual; Libraries; Ligands; Mass Spectrum Analysis; Measures; Mentors; Morbidity - disease rate; Nature; Non-Insulin-Dependent Diabetes Mellitus; Obesity; PTH gene; Parathyroid Hormone Receptor; Pathway interactions; Patients; Pattern; Peptide Library; Peptide Synthesis; Peptides; Pharmacology; Phosphorylation; Plant Resins; Post-Translational Protein Processing; Postdoctoral Fellow; Prevalence; Production; Proteins; Proteomics; Reaction; Reagent; Receptor Signaling; Regulation; Signal Pathway; Signal Transduction; Spectrum Analysis; Testing; Therapeutic; Training; Translating; Woman; arrestin 1; arrestin 2; base; beta-arrestin; cardioprotection; design; effective therapy; experimental study; glucagon-like peptide 1; individualized medicine; insight; men; microwave electromagnetic radiation; mortality; novel; peptide B; peptide hormone; preference; protein expression; protein protein interaction; receptor; recruit; release of sequestered calcium ion into cytoplasm; response; success; synthetic peptide; therapeutically effective; therapy outcome; tool

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.

项目摘要/摘要 心血管疾病（CVD）是曼联摊位的主要死亡原因，尤其是 2型糖尿病（T2DM）患者普遍存在。最近，胰高血糖素样肽-1的激动剂 受体（GLP-1R），G蛋白偶联受体（GPCR）和治疗T2DM的共同靶标的 显示出有希望的心血管益处，包括大幅降低CVD相关的发病率和 死亡。但是，经认可的GLP-1R激动剂的更广泛的临床试验，其中许多是合成肽， 产生的结果混合了。那是迫切需要了解推动驱动的基本机制 同一类试剂的不同作用模式，以设计更有针对性和有效的治疗。 值得注意的是，GLP-1R的许多合成肽激动剂表现出偏见的激动剂，即配体 相对于内源性激动剂GLP-1，推动对某些信号通路的偏好。这个现象 在许多其他GPCR中都可以看到。例如，Gellman Lab取代了α保留在N-上保留的β 治疗表达PTH受体的细胞时，PTH肽的末端并观察到G蛋白偏置。 我建议以这种策略建立这种策略，以开发出有偏见的GLP-1R的新型合成肽激动剂 对于GS和GQ途径，作为更好地了解GLP-1R信号传导的工具。我将设计和合成α/β- 通过替换前八个氨基酸在N末端保留的GLP-1的肽类比，单独保留 β抗性。将进行基于细胞的测定，以测量营地产量（GS），钙动员（GQ）， 和β-arrestin-1，2募集，代表GLP-1R的主要信号通路，以表征 信号传导曲线并确定其相对于GLP-1的偏见。 接下来，我假设GLP-1R的偏置激活是独特的翻译后的结果 修饰（PTM）和肽结合后出现的蛋白质蛋白质相互作用，然后下游 结果改变了细胞内蛋白的表达和磷酸化。为了检验这个假设，我将使用 基于质谱（MS）的蛋白质组学以表征纯化的GLP-1R的PTM 以前用GS和GQ偏置肽以及β-甲蛋白-1和-2偏置肽治疗 在盖尔曼集团中开发。在共免疫沉淀后，将进行第二种MS分析 受体研究肽结合后出现的独特受体蛋白相互作用。最后，第三个MS 测定将访问蛋白质表达和磷酸化的下游变化，以深入了解 由偏置激动剂结合产生的信号转导的。该提案的结果将有助于阐明 驱动GPCR的信号传输的机制并有助于开发安全，更有效 疗法。