title: Development of systemic in vitro evolution and its application to generation of peptide-aptamer-based inhibitors of cathepsin E.
abstract: Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC(50). This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.
authors.affiliation: Rational Evolutionary Design of Advanced Biomolecules, Saitama (REDS), Saitama Small Enterprise Promotion Corporation, #552, Saitama Industrial Technology Center, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kitamura
authors.forename: Chuya
authors.initials: C
authors.lastname: Yoshida
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kinoshita
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kadowaki
authors.forename: Yoko
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Tayama
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Kawakubo
authors.forename: Mohammed
authors.initials: M
authors.lastname: Naimuddin
authors.forename: Md
authors.initials: M
authors.lastname: Salimullah
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
authors.forename: Kazunori
authors.initials: K
authors.lastname: Hanada
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
mesh_terms: D000595:Amino Acid Sequence; D052158:Aptamers, Peptide / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D001483:Base Sequence; D020059:Cathepsin E / Q000037:antagonists & inhibitors*; D018076:DNA, Complementary / Q000235:genetics; D019020:Directed Molecular Evolution; D055808:Drug Discovery; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D052158:Aptamers, Peptide; D018076:DNA, Complementary; D019151:Peptide Library; D011480:Protease Inhibitors; D020059:Cathepsin E