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Development of orthogonal peptide bioconjugation methodology for application in targeting GLP-1R.

机译：正交肽生物共轭方法的开发用于靶向GLP-1R。

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摘要

Bioconjugation is a powerful tool for precise modification of biomolecules such as peptides and proteins. Peptide bioconjugation has been a topic of interest for a long time now. Here, we have explored the potential of some of the robust and popular peptide bioconjugation strategies such as maleimide-thiol chemistry, Cu-catalyzed azide-alkyne cycloaddition (CuAAC/click chemistry), native chemical ligation (NCL) and Suzuki-Miyaura cross coupling. The orthogonality of these bioconjugation reactions were utilized to develop an efficient methodology for labeling a peptide with multiple tags such as a cell penetrating K9 peptide, a biotin, and a fluorescein. Its application was demonstrated in a biological system by performing a cell penetration experiment. The peptide was visualized by fliorescence to confirm the cell penetration and it was pulled out from the cell lysate using streptavidin beads by virtue of biotin-streptavidin interaction. Having demonstrated the orthogonality, the bioconjugation tools were further employed to engineer glucagon-like peptide-1 (GLP-1) in order to target its receptor. Using native chemical ligation (NCL) and click chemistry, a facile, yet efficient strategy for the synthesis of dimeric constructs of full length GLP-1 and an N-terminus GLP-1 analog was developed. Also, maleimide-thiol chemistry was employed to modify a high affinity GLP-1 analog, EM2198. To minimize non-specific uptake of EM2198-based PET tracers, differently charged and uncharged amino acid-containing hydrophilic tails were conjugated to the C-terminus of EM2198. While positively charged and negatively charged tails did not minimize the non-specific uptake, peptide 37 with an uncharged penta-Ser tail inhibited non-specific uptake by liver, lungs, and spleen but not by the kidneys. Lastly, the site specific cleavage of GLP-1 peptide bonds by NEP 24.11 was shielded by the introduction of side-chain to side-chain lactam bridges at specific positions. Also, as emphasized by their lower potencies, the lactam bridge near the N-terminal random coil of GLP-1 appeared to be less tolerated than the one distant from the N-terminus. These peptides showed promise as PET tracers for beta cell mass (BCM) imaging as revealed by the in vivo and ex vivo PET/CT imaging experiments, however high renal uptake was also observed.
机译：生物缀合是用于精确修饰生物分子（如肽和蛋白质）的强大工具。肽生物缀合已经成为很长一段时间以来的关注话题。在这里，我们探索了一些健壮且流行的肽生物共轭策略的潜力，例如马来酰亚胺-硫醇化学，Cu催化的叠氮化物-炔烃环加成（CuAAC /点击化学），天然化学连接（NCL）和Suzuki-Miyaura交叉偶联。这些生物结合反应的正交性被用于开发一种有效的方法，以标记带有多个标签的肽，例如细胞穿透性K9肽，生物素和荧光素。通过进行细胞渗透实验，证明了其在生物系统中的应用。通过荧光使肽可视化，以确认细胞渗透，并利用链霉亲和素珠通过生物素-链霉亲和素相互作用将其从细胞裂解物中抽出。已经证明了正交性，生物缀合工具被进一步用于工程化胰高血糖素样肽-1（GLP-1），以靶向其受体。利用天然化学连接（NCL）和点击化学，开发了一种简便而有效的策略，用于合成全长GLP-1和N末端GLP-1类似物的二聚体构建体。同样，采用马来酰亚胺-硫醇化学来修饰高亲和力GLP-1类似物EM2198。为了最大程度地减少基于EM2198的PET示踪剂的非特异性摄取，将带不同电荷和不带电荷的含氨基酸的亲水性尾巴缀合至EM2198的C端。尽管带正电荷和带负电荷的尾部不能使非特异性摄取最小化，但是具有不带电荷的五-Ser尾巴的肽37抑制了肝，肺和脾脏的非特异性摄取，但抑制了肾脏。最后，通过在特定位置引入侧链至侧链内酰胺桥来屏蔽NEP 24.11对GLP-1肽键的位点特异性切割。同样，如其较低的效力所强调的，GLP-1 N端无规卷曲附近的内酰胺桥似乎比远离N端的内酰胺桥耐受性差。如体内和离体PET / CT成像实验所揭示的，这些肽有望作为PET示踪剂用于β细胞质量（BCM）成像，但是也观察到了高肾脏摄取。

著录项

  • 作者

    Manandhar, Bikash.;

  • 作者单位

    The University of Texas at Dallas.;

  • 授予单位 The University of Texas at Dallas.;
  • 学科 Polymer chemistry.;Organic chemistry.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 167 p.
  • 总页数 167
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 康复医学;
  • 关键词

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