2025-08-06 作者：lkr 来源：文献：Xenopus-derived glucagon-like peptide-1 and polyethylene-glycosylated glucagon-like
peptide-1 receptor agonists: long-acting hypoglycaemic and insulinotropic activities with potential therapeutic utilities
文献链接：https://pubmed.ncbi.nlm.nih.gov/29171021/
作者：Jing Han, Yingying Fei , Feng Zhou, Xinyu Chen, Ying Zhang, Lin Liu,Junjie Fu
原文摘要：
BACKGROUND AND PURPOSE
Incretin-based therapies based on glucagon-like peptide-1 (GLP-1) receptor agonists are effective treatments of type 2 diabetes. Abundant research has focused on the development of long-acting GLP-1 receptor agonists. However, all GLP-1 receptor agonists in clinical use or development are based on human or Gila GLP-1. We have identified a potent GLP-1 receptor agonist, xGLP-1B, based on Xenopus GLP-1.
EXPERIMENTAL APPROACH
To further modify the structure of xGLP-1B, alanine scanning was performed to study the structure -activity relationship of xGLP- 1B. Two strategies were then employed to improve bioactivity. First, the C-terminal tail of lixisenatide was appended to cysteinealtered xGLP-1B analogues. Second, polyethylene glycol (PEG) chains with different molecular weights were conjugated with the peptides, giving a series of PEGylated conjugates. Comprehensive bioactivity studies of these conjugates were performed in vitro and in vivo.
RESULTS
From the in vitro receptor activation potency and in vivo acute hypoglycaemic activities of conjugates 25 -36, 33 was identified as the best candidate for further biological assessments. Conjugate 33 exhibited prominent hypoglycaemic and insulinotropic activities, as well as improved pharmacokinetic profiles in vivo. The prolonged antidiabetic duration of 33 was further confirmed by pre-oral glucose tolerance tests (OGTT) and multiple OGTT. Furthermore, chronic treatment of db/db mice with 33 ameliorated non-fasting blood glucose and insulin levels, reduced HbA1c values and normalized their impaired glucose tolerance. Importantly, no in vivo toxicity was observed in mice treated with 33.
CONCLUSIONS AND IMPLICATIONS
Peptide 33 is a promising long-acting type 2 diabetes therapeutic deserving further investigation.
Abbreviations ALT, alanine aminotransferase; AST, aspartate aminotransferase; GLP-1, glucagon-like peptide-1; IPGTT, i.p. glucose tolerance test; mPEG-MAL, methoxy-PEG-maleimides; OGTT, oral glucose tolerance tests; RP-HPLC, reversed phase HPLC; SAR, structure–activity relationship; TC, serum total cholesterol; TG, triglycerides; T2DM, type 2 diabetes mellitus
mPEG-MAL（甲氧基聚乙二醇-马来酰亚胺）是一种重要的化学偶联剂，在生物医学领域具有应用，特别是在多肽和聚乙二醇化偶联物的制备中。马来酰亚胺官能团与多肽中的巯基在温和的条件下可以发生偶联反应，形成稳定的硫醚键。多肽和聚乙二醇化偶联物是生物医学领域中一种重要的化合物类型，它们通过特定的化学方法将多肽与聚乙二醇（PEG）连接起来，形成具有特殊性质和功能的偶联物。mPEG-MAL作为一种重要的化学偶联剂，在多肽和聚乙二醇化偶联物的制备中具有应用前景。
图为：mPEG-MAL结构式
mPEG-MAL 在多肽和聚乙二醇化偶联物合成中的应用：
将多肽和 mPEG-MAL 按照预定的浓度和摩尔比溶解在选定的反应溶剂中。在反应过程中，mPEG-MAL 中的马来酰亚胺基团（MAL）会与多肽中的巯基（-SH）发生特异性的点击化学反应，形成稳定的硫醚键。这种反应具有较高的选择性和效率，能够在温和条件下进行。如果多肽中没有天然的巯基，可以通过化学修饰方法在多肽特定位置引入巯基。在反应过程中，可适当搅拌或振荡反应溶液，以确保反应物充分混合，但搅拌速度不宜过快，以免产生过多泡沫或对多肽结构造成破坏。反应完成后，使用透析的方法去除未反应的 mPEG-MAL、多肽以及反应溶剂中的小分子杂质。选择合适截留分子量的透析袋，将反应混合物置于透析袋中，在大量的缓冲液或去离子水中透析，定期更换透析液，直至透析外液中检测不到小分子杂质为止。进一步的纯化可以使用色谱技术，如尺寸排阻色谱（SEC）或反相高效液相色谱（RP-HPLC）。SEC 可以根据产物和杂质分子大小的差异进行分离，而 RP-HPLC 则是利用产物和杂质在固定相和流动相之间分配系数的不同来实现分离，从而获得高纯度的多肽和聚乙二醇化偶联物。
图为：聚乙二醇结构式
结论：mPEG-MAL 中的马来酰亚胺（MAL）基团是一个高活性的官能团。在合成多肽和聚乙二醇化偶联物的过程中，它主要用于与多肽中的巯基（-SH）发生特异性的点击化学反应。这种反应具有高度的选择性，因为在生理条件或温和的反应条件下，马来酰亚胺基团能够准确地与巯基反应，形成稳定的硫醚键。例如，对于含有半胱氨酸残基的多肽，半胱氨酸的巯基就可以与 mPEG-MAL 的马来酰亚胺基团反应。这为将聚乙二醇（PEG）链连接到多肽上提供了可靠的化学连接方式，而且反应条件相对温和，通常在中性 pH 和室温到体温的温度范围内即可进行，减少了对多肽结构和活性可能造成的破坏。
