Pickford Philip, Lucey Maria, Fang Zijian, Bitsi Stavroula, de la Serna Jorge Bernardino, Broichhagen Johannes, Hodson David J, Minnion James, Rutter Guy A, Bloom Stephen R, Tomas Alejandra, Jones Ben
Section of Endocrinology and Investigative Medicine, Imperial College London, London, UK.
Section of Cell Biology and Functional Genomics, Imperial College London, London, UK.
Br J Pharmacol. 2020 Sep;177(17):3905-3923. doi: 10.1111/bph.15134. Epub 2020 Jun 19.
Amino acid substitutions at the N-termini of glucagon-like peptide-1 (GLP-1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1 receptor agonists exendin-4 and lixisenatide lead to similar phenomena.
Exendin-4, lixisenatide and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand and their biased derivatives were assessed in mice.
Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was fivefold less potent for cAMP signalling. Both peptides induced extensive GLP-1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice with lixisenatide. N-terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose.
Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1 receptor trafficking via mechanisms unrelated to GLP-1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.
胰高血糖素样肽-1（GLP-1）受体激动剂肽N端的氨基酸取代会导致细胞内信号传导、亚细胞转运模式及体内疗效的差异。在此，我们旨在确定临床批准的GLP-1受体激动剂艾塞那肽-4和利司那肽在配体C端的序列差异是否会导致类似现象。
在不同细胞类型的一系列体外转运和信号传导试验中，对艾塞那肽-4、利司那肽以及具有偏向性信号特征的N端取代类似物进行了比较。开发了荧光配体和新的时间分辨荧光共振能量转移方法，以研究激动剂在细胞和亚细胞水平的行为。在小鼠中评估了每种母体配体及其偏向性衍生物的抗高血糖和厌食作用。
利司那肽和艾塞那肽-4显示出相同的结合亲和力，但利司那肽对cAMP信号传导的效力低五倍。两种肽均诱导血浆膜中广泛的GLP-1受体聚集，并迅速被内吞，但利司那肽处理后GLP-1受体再循环至细胞表面的速度较慢。这些综合缺陷导致利司那肽处理的小鼠最大持续胰岛素分泌减少，抗高血糖和厌食作用降低。两种配体的His1被Phe1 N端取代对其药理学有有利影响，导致胰岛素释放改善和血糖降低。
艾塞那肽-4 C端的变化通过与GLP-1受体占有率无关的机制影响信号效力和GLP-1受体转运。这些差异与它们控制血糖能力的变化有关，因此可能具有治疗相关性。
