title: Non-peptidic thrombospondin-1 mimics as fibroblast growth factor-2 inhibitors: an integrated strategy for the development of new antiangiogenic compounds.
abstract: Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic factor fibroblast growth factor-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the 15-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milan 20131, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Colombo
authors.forename: Barbara
authors.initials: B
authors.lastname: Margosio
authors.forename: Laura
authors.initials: L
authors.lastname: Ragona
authors.forename: Marco
authors.initials: M
authors.lastname: Neves
authors.forename: Silvia
authors.initials: S
authors.lastname: Bonifacio
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Annis
authors.forename: Matteo
authors.initials: M
authors.lastname: Stravalaci
authors.forename: Simona
authors.initials: S
authors.lastname: Tomaselli
authors.forename: Raffaella
authors.initials: R
authors.lastname: Giavazzi
authors.forename: Marco
authors.initials: M
authors.lastname: Rusnati
authors.forename: Marco
authors.initials: M
authors.lastname: Presta
authors.forename: Lucia
authors.initials: L
authors.lastname: Zetta
authors.forename: Deane F
authors.initials: DF
authors.lastname: Mosher
authors.forename: Domenico
authors.initials: D
authors.lastname: Ribatti
authors.forename: Marco
authors.initials: M
authors.lastname: Gobbi
authors.forename: Giulia
authors.initials: G
authors.lastname: Taraboletti
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000378:metabolism; D002823:Chorion / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D019700:Thrombospondin 1 / Q000737:chemistry / Q000502:physiology*
chemical_list: D020533:Angiogenesis Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D016222:Fibroblast Growth Factor 2