Multimodal biopanning of T7 phage-displayed peptides reveals angiomotin as a potential receptor of the anti-angiogenic macrolide Roxithromycin

• Published in: European journal of medicinal chemistry 2015-01, Vol.90, p.809-821
• Main Authors: Takakusagi, Kaori, Takakusagi, Yoichi, Suzuki, Takahiro, Toizaki, Aya, Suzuki, Aiko, Kawakatsu, Yaichi, Watanabe, Madoka, Saito, Yukihiro, Fukuda, Ryushi, Nakazaki, Atsuo, Kobayashi, Susumu, Sakaguchi, Kengo, Sugawara, Fumio
• Format: Article
• Language: English
• Subjects: Angiogenesis; Angiogenesis Inhibitors - chemical synthesis; Angiogenesis Inhibitors - chemistry; Angiogenesis Inhibitors - pharmacology; Angiomotin; Bacteriophage T7 - chemistry; Cell Proliferation - drug effects; Cell Survival - drug effects; Dose-Response Relationship, Drug; Humans; Intercellular Signaling Peptides and Proteins - metabolism; MCF-7 Cells; Membrane Proteins - antagonists & inhibitors; Membrane Proteins - metabolism; Molecular Structure; Peptide Library; Peptides - chemistry; QCM-D; Quartz Crystal Microbalance Techniques; RELIC; Roxithromycin; Roxithromycin - chemical synthesis; Roxithromycin - chemistry; Roxithromycin - pharmacology; Structure-Activity Relationship; T7 phage display; Tumor Cells, Cultured
• ISSN: 0223-5234; 1768-3254
• DOI: 10.1016/j.ejmech.2014.12.015

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. [Display omitted] •Roxithromycin (RXM) derivatives were synthesized for T7 phage display (T7PD) biopanning.•Biopanning using three different strategies independently identified RXM-recognizing (poly)peptides.•Bioinformatics analysis using the (poly)peptides indicated angiomotin (Amot) as a potential RXM-binding protein.•Binding assays confirmed the direct interaction between RXM and Amot via the extracellular domain.