Bueno Ana B, Showalter Aaron D, Wainscott David B, Stutsman Cynthia, Marín Aranzazu, Ficorilli James, Cabrera Over, Willard Francis S, Sloop Kyle W
From the Centro de Investigación Lilly, Eli Lilly and Co., Alcobendas 28108, Spain and.
Endocrine Discovery and.
J Biol Chem. 2016 May 13;291(20):10700-15. doi: 10.1074/jbc.M115.696039. Epub 2016 Mar 14.
Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and characterization of small molecule GLP-1R ligands as possible therapeutics.
激活胰高血糖素样肽-1受体（GLP-1R）的治疗性干预可增强2型糖尿病患者的葡萄糖依赖性胰岛素分泌并改善能量平衡。研究肽配体激活GLP-1R的机制时，已利用诱变、受体嵌合体、光亲和标记、氢-氘交换以及配体结合胞外域的晶体学来建立受体同源模型。然而，这尚未实现设计或发现类药物非肽GLP-1R激活剂。最近，对GLP-1R阳性变构调节剂4-(3-苄氧基苯基)-2-乙基亚磺酰基-6-(三氟甲基)嘧啶（BETP）的研究确定，GLP-1R中的半胱氨酸-347通过共价修饰对阳性变构调节剂活性是必需的。为了推进GLP-1R的小分子激活，我们表征了BETP的促胰岛素分泌机制。在鸟苷5'-3-O-(硫代)三磷酸结合和INS1 832-3胰岛素瘤细胞cAMP测定中，BETP增强了GLP-1(9-36)-NH2刺激的cAMP信号传导。使用分离的胰岛，BETP以葡萄糖依赖性方式增强胰岛素分泌，这需要肽配体和GLP-1R两者。在共价机制研究中，PAGE荧光成像显示在用[(3)H]BETP孵育的表达GLP-1R的细胞的免疫沉淀实验中GLP-1R被标记。此外，我们研究了其他报道的GLP-1R激活剂和从筛选活动中鉴定出的化合物是否通过共价修饰调节GLP-1R。与BETP类似，发现几种分子以半胱氨酸-347依赖性方式增强GLP-1R信号传导。这些化学类型是与谷胱甘肽反应的亲电试剂，液相色谱/质谱法确定了缀合后形成的半胱氨酸加合物。总之，我们的结果表明共价修饰可用于将GLP-1R稳定在活性构象中。此外，这些发现为发现和表征作为可能治疗剂的小分子GLP-1R配体提供了药理学指导。
