title: Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.
abstract: N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawakami
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Ogawa
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hatta
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.affiliation: Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST) , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Natsume
mesh_terms: D000596:Amino Acids / Q000494:pharmacology*; D001565:Benzoates / Q000737:chemistry; D001593:Benzyl Compounds / Q000737:chemistry; D002474:Cell-Free System / Q000378:metabolism; D003500:Cyclization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010456:Peptides, Cyclic / Q000737:chemistry / Q000494:pharmacology*; D057786:Peptidomimetics / Q000737:chemistry / Q000494:pharmacology*; D034444:Peptoids / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D051176:beta Catenin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C000622574:3,5-bis(chloromethyl)benzoic acid; D000596:Amino Acids; C000622575:BCP1 peptoid-peptide chimera; D001565:Benzoates; D001593:Benzyl Compounds; D010456:Peptides, Cyclic; D057786:Peptidomimetics; D034444:Peptoids; D012333:RNA, Messenger; D051176:beta Catenin; D012343:RNA, Transfer