title: Multimodal biopanning of T7 phage-displayed peptides reveals angiomotin as a potential receptor of the anti-angiogenic macrolide Roxithromycin.
abstract: Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Takakusagi
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address: tkksg@rs.noda.tus.ac.jp.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takakusagi
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Toizaki
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Yaichi
authors.initials: Y
authors.lastname: Kawakatsu
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Madoka
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Meiwafosis Co. Ltd., 1-14-2 Shinjuku, Tokyo 160-0022, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Meiwafosis Co. Ltd., 1-14-2 Shinjuku, Tokyo 160-0022, Japan.
authors.forename: Ryushi
authors.initials: R
authors.lastname: Fukuda
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Nakazaki
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Kobayashi
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address: sugawara@rs.noda.tus.ac.jp.
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
mesh_terms: D020533:Angiogenesis Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D000091082:Angiomotins; D017123:Bacteriophage T7 / Q000737:chemistry*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008840:Microfilament Proteins; D015394:Molecular Structure; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D058525:Quartz Crystal Microbalance Techniques; D015575:Roxithromycin / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: C423985:AMOT protein, human; D020533:Angiogenesis Inhibitors; D000091082:Angiomotins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D019151:Peptide Library; D010455:Peptides; D015575:Roxithromycin
keywords: Angiogenesis; Angiomotin; QCM-D; RELIC; Roxithromycin; T7 phage display