Institute of Radiopharmacy, Helmholtz Centre Dresden-Rossendorf, 01314 Dresden, Germany.
Nucl Med Biol. 2012 Nov;39(8):1202-12. doi: 10.1016/j.nucmedbio.2012.06.003. Epub 2012 Jul 10.
Phosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two (18)F-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7.
Radiolabeling was accomplished with the prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) using both, conventional and microfluidic-based bioconjugation of [(18)F]SFB to N-terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 °C and 37 °C and small animal PET in BALB/c mice were utilized for radiopharmacological characterization.
Isolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [(18)F]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone (<1% ID/mg protein). In vivo biodistribution of (18)F-labeled peptide dimers was determined with small animal PET revealing a superior biodistribution pattern of sC18-containing peptide dimer MQSpTPL-sC18 [(18)F]4.
([18)F]SFB labeling of the phosphopeptide-CPP dimers using a microfluidic system leads to an improved chemoselectivity towards the N-terminal NH(2) group compared to the conventional labeling approach. Cell-penetrating peptide sC18 can be considered as an ideal molecular shuttle for intracellular delivery of the Plk1-PBD-binding hexaphosphopeptide as demonstrated by its favourable radiopharmacological profile.
磷酸肽是研究细胞蛋白磷酸化/去磷酸化过程及其在各种信号转导途径中作用的有趣化合物。然而，由于磷酸肽的负电荷磷酸部分，磷酸肽在细胞中的作用研究受到严重限制，导致其穿过细胞膜的能力很差。本研究描述了两种（18）F 标记的磷酸肽-穿膜肽二聚体的合成和放射药理学评价。将 polo 样激酶-1 结合的六磷酸肽 H-Met-Gln-Ser-pThr-Pro-Leu-OH 与穿膜肽（CPP）偶联，CPP 为 cathelicidin 衍生肽 sC18 或人类降钙素衍生物 hCT(18-32)-k7。
使用 prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) 通过传统和基于微流控的生物偶联，将 [(18)F]SFB 连接到肽二聚体的磷酸肽部分的 N 末端，完成放射性标记。在 4°C 和 37°C 下，用人癌细胞系 HT-29 和 FaDu 细胞进行细胞摄取研究，并在 BALB/c 小鼠中进行小动物 PET，用于放射药理学特征分析。
使用 [(18)F]SFB 进行传统生物偶联时，分离的放射性化学产率范围为 2%至 4%。使用微流控技术，可获得高达 26%的显著提高的分离放射性化学产率。放射性标记的磷酸肽和磷酸肽-CPP 二聚体的细胞摄取研究表明，与单独的磷酸肽相比，两种磷酸肽二聚体在 2 小时后，细胞内摄取率均提高了 50% ID/mg 蛋白（<1% ID/mg 蛋白）。小动物 PET 测定了（18）F 标记的肽二聚体的体内分布，发现含有 sC18 的肽二聚体 MQSpTPL-sC18 [(18)F]4 的体内分布模式更好。
与传统标记方法相比，使用微流控系统对磷酸肽-CPP 二聚体进行 [(18)F]SFB 标记，可提高对 N 末端 NH2 基团的化学选择性。穿膜肽 sC18 可作为 Plk1-PBD 结合六磷酸肽的理想分子穿梭，如放射药理学特征所示，可实现其在细胞内的有效递药。
