Isolation of novel cell-penetrating peptides from a random peptide library using in vitro virus and their modifications.
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.
Kamide
Kaeko
K
Advanced Medical Research Laboratory, Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kanagawa 227-0033, Japan. kamide.kaeko@me.mt-pharma.co.jp
Nakakubo
Hiroshi
H
Uno
Shusei
S
Fukamizu
Akiyoshi
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D019151:Peptide Library; D010455:Peptides; D054322:tat Gene Products, Human Immunodeficiency Virus