Advanced Medical Research Laboratory, Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kanagawa 227-0033, Japan.
Int J Mol Med. 2010 Jan;25(1):41-51.
A number of cell-penetrating peptides (CPPs) have been reported, but their transduction efficiencies are too low to be used as intracellular carriers for therapeutic purposes. We conducted a comprehensive search to find novel CPPs using an in vitro virus (IVV) library, which presented random peptides consisting of 15 amino acids (diversity of the library was >10(12)). We found 9 kinds of novel CPPs with an intracellular translocation efficiency higher than that of the TAT peptide (YGRKKKRRQRRR). Interestingly, one of the novel CPPs, No. 14 (KLWMRWYSPTTRRYG), showed a dramatic improvement in translocation activity relative to the TAT peptide in CHO cells (>10-fold efficiency in 50 microM). As the intracellular translocation efficiency of No. 14 was increased by substitution Arg for Lys1 (14-1), we carried out alanine scanning on the basis of 14-1 to determine important amino acids for the intracellular translocation. The Ala substitution analysis showed that both Arg and Trp residues were important for the cell-penetrating activity and that their contribution was in the order Trp3<Arg12<Arg1<Arg5, Arg13<Trp6. Moreover, it was possible to substitute two Trp with other bulky amino acids such as Ile or Tyr. In this study, we showed that novel CPPs could be acquired by screening random peptides and modifying some amino acids could increase their cell-penetrating activity.
已报道了许多细胞穿透肽（CPPs），但它们的转导效率太低，无法用作治疗目的的细胞内载体。我们使用体外病毒（IVV）文库进行了全面搜索，以找到新的 CPP，该文库展示了由 15 个氨基酸组成的随机肽（文库的多样性>10^12）。我们发现了 9 种新型 CPP，其细胞内转位效率高于 TAT 肽（YGRKKKRRQRRR）。有趣的是，一种新型 CPP，即 No.14（KLWMRWYSPTTRRYG），在 CHO 细胞中的转位活性相对于 TAT 肽有了明显的提高（在 50 μM 时效率提高了 10 倍以上）。由于 No.14 的细胞内转位效率通过用精氨酸取代赖氨酸 1（14-1）而增加，因此我们在 14-1 的基础上进行了丙氨酸扫描，以确定对细胞内转位重要的氨基酸。Ala 取代分析表明，Arg 和 Trp 残基对细胞穿透活性都很重要，其贡献顺序为 Trp3<Arg12<Arg1<Arg5，Arg13<Trp6。此外，有可能用其他大体积氨基酸如异亮氨酸或色氨酸代替两个 Trp。在这项研究中，我们表明可以通过筛选随机肽获得新型 CPP，并且修饰某些氨基酸可以提高它们的细胞穿透活性。
