Institute for Molecular Bioscience , The University of Queensland , Brisbane 4072 , Queensland , Australia.
Whitehead Institute for Biomedical Research , 9 Cambridge Center , Cambridge , Massachusetts 02142 , United States.
ACS Chem Biol. 2018 Oct 19;13(10):2973-2980. doi: 10.1021/acschembio.8b00653. Epub 2018 Oct 5.
Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.
许多天然存在的肽类具有较差的蛋白水解稳定性，这限制了它们的治疗应用。环肽是源自植物的环状肽，由于其高度受限的结构，包括头到尾的环状骨架和形成半胱氨酸结核心的三个二硫键，而具有抗蛋白水解的作用。这种结构使它们成为可以将肽序列（表位）接枝到其上的有用支架。在这项研究中，针对鼠类 II 类 MHC 分子的羊驼衍生纳米抗体 VHH7 被用于将环肽靶向递送至抗原呈递细胞（APC）。测试了 MCoTI-I 和在环 6 中接枝有 HA 标签（YPYDVPDYA）的 MCoTI-I（MCoTI-HA）的免疫原性。为了产生所需的 VHH7-肽缀合物，使用了一种定点 sortase A 催化反应与无铜应变促进的环加成反应相结合的方法。单独的 MCoTI-I 没有显示出任何明显的抗体反应，因此表明环肽具有免疫沉默支架的能力。相比之下，与 VHH7 缀合的 MCoTI-I 引发了针对环状或线性 MCoTI-I 的抗体，因此表明了将环肽靶向 APC 并可能靶向其他细胞类型的简单而稳健的方法。当 MCoTI-HA 与 VHH7 缀合时观察到了类似的抗体反应，但对线性 HA 标签本身没有反应性，这表明环肽呈现的表位和线性表位之间的构象约束存在差异。应用于 MCoTI-HA 的市售 HA 抗体的研究证实，肽免疫原的构象会影响其反应性。因此，识别约束性表位的抗体的产生可能受益于接枝到环肽等支架上。更广泛地说，这项研究验证了一个原型环肽，它是一种肽家族的成员，在许多其他研究中已被证明可作为药物设计支架有用，可有效地在体内到达特定靶标。
