Fuchigami Takeshi, Itagaki Kohnosuke, Ishikawa Natsumi, Yoshida Sakura, Nakayama Morio
Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Bioorg Med Chem Lett. 2019 Oct 1;29(19):126629. doi: 10.1016/j.bmcl.2019.126629. Epub 2019 Aug 19.
Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [I]I-NCP (11.2 ± 0.44% vs 1.75 ± 0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ± 0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.
天冬酰胺内肽酶或天冬酰胺基内肽酶是一种在某些癌症中过度表达的酶，参与癌症的迁移、侵袭和转移。我们开发了放射性碘标记的肽（[I]I-LCP）或荧光素标记的肽（FL-LCP），其具有可穿透细胞的d-精氨酸九聚体，通过天冬酰胺内肽酶可裂解的接头与阴离子d-谷氨酸九聚体融合，用作测量和监测天冬酰胺内肽酶活性的肽探针。通过改变其不可裂解序列，类似地制备并评估了FL-NCP和[I]I-NCP的不可裂解探针作为阴性对照探针。具有天冬酰胺内肽酶可裂解或不可裂解序列的模型肽（分别为LCP和NCP）与重组人天冬酰胺内肽酶反应，只有LCP被该酶消化。[I]I-LCP在天冬酰胺内肽酶阳性的HCT116细胞中的摄取显著高于[I]I-NCP（11.2±0.44% vs 1.75±0.06%剂量/毫克）。FL-LCP在HCT116细胞中的积累相当低（4.75±0.29%剂量/毫克蛋白质），但与FL-NCP的水平无显著差异。低浓度的[I]I-LCP（40 pM）在天冬酰胺内肽酶裂解后可能有效地内化。另一方面，更高浓度的FL-LCP衍生物（1 mM）的细胞摄取可能受到高浓度聚阴离子的限制。在荷瘤小鼠中的体内生物分布研究表明，[I]I-LCP在30分钟时的肿瘤摄取为每克注射剂量的1.34%（% ID/g）。30分钟时的肿瘤/血液和肿瘤/肌肉比率分别为0.63和1.77，表明[I]I-LCP在肿瘤中的积累不足以用于体内成像。尽管需要进一步的结构修饰来改善药代动力学性质，但[I]I-LCP已被证明是开发用于监测活体受试者中天冬酰胺内肽酶活性的核医学成像探针的有效支架。
