1 Scientific RepoRts | 6:31295 | DOI: 10.1038/srep31295 www.nature.com/scientificreports Miniaturizing VeGF: p eptides mimicking the discontinuous VeGF receptor-binding site modulate the angiogenic response Lucia De Rosa 1,* , Federica Finetti 2,* , Donatella Diana 1 , Rossella Di stasi 1 , sara Auriemma 1 , Alessandra Romanelli 3 , Roberto Fattorusso 4 , Marina Ziche 2 , Lucia Morbidelli 2,* & Luca Domenico D’Andrea 1,* The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. the VeGF/VeGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides difering in length and lexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor. All biological processes are inely regulated by a network of interactions between proteins whose characterization at molecular level can promote the design of novel protein binder drugs with therapeutic and diagnostic appli- cations. Peptides and peptidomimetics have been widely explored as protein-protein interaction modulators as they are advantageous with respect to protein-based (including antibodies) molecules and small organic com- pounds in spite of pharmacokinetic limitations 1 . In the last years, several examples of protein-binder peptides have been reported, achieved using either structure-based design, combinatorial or computational approaches. In particular, considering that molecular recognition between proteins is oten mediated by surface exposed loop or secondary structure motifs such as β-hairpins and α-helices, many eforts have been devoted to the development of peptides reproducing these protein interface elements 1,2 . Less explored and more challenging is the design of protein interface mimetic peptides reproducing multiple and discontinuous binding sites. A discontinuous bind- ing site is constituted by peptide segments which are close in the protein three-dimensional structure but distant in the primary sequence. Examples of discontinuous protein binding site/epitopes mimicry using peptides have been reported. hey have been assembled by fusing into a single molecular entity two or more linear amino acid segments using molecular scafolds or linkers 3–13 or selected by phage display from libraries of mimotopes 14 . We explored the design of peptides mimicking two interacting regions of the Vascular Endothelial Growth Factor (VEGF) for its receptor. VEGF is the main regulator of angiogenesis, a fundamental process for healing, reproduction and embryonic development 15 . he design of novel and efective angiogenic modulators is eliciting a considerable interest for therapeutic 16,17 and diagnostic applications 18 . VEGF is a homodimeric glycoprotein, covalently bound by two disulide bonds, characterized by a cystine knot motif 19 . It binds to two receptors on the surface of endothe- lial cells (ECs) (VEGFR1 and VEGFR2). he binding of VEGF to its receptors induces receptor dimerization and phosphorylation of the intracellular kinase domain which activate the intracellular pathways ending in EC proliferation and migration 20 . he analysis of the x-ray crystal structure of the complex between VEGF and the 1 Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Napoli, Italy. 2 Dipartimento di Scienze della Vita, Università di Siena, Via A. Moro 2, 53100 Siena, Italy. 3 Dipartimento di Farmacia, Università di Napoli “Federico II”, via Mezzocannone 16, 80134, Napoli, Italy. 4 Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, via A. Vivaldi 43, 81100, Caserta, Italy. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.D.D. (email: luca.dandrea@cnr.it) received: 29 April 2016 Accepted: 15 July 2016 Published: 08 August 2016 opeN