Non-peptidic Thrombospondin-1 Mimics as Fibroblast Growth Factor-2 Inhibitors AN INTEGRATED STRATEGY FOR THE DEVELOPMENT OF NEW ANTIANGIOGENIC COMPOUNDS

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 DDDDDNDKIPD-DRDN. 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.