Discovery and investigation of lead compounds as binders to the extra-domain B of the angiogenesis marker, fibronectin

Angiogenesis, the growth of new blood vessels from preexisting vessels, is a rare event in the adult (except in the case of the female reproductive cycle), but is a characteristic feature of diseases such as cancer, blinding ocular disorders (e.g., retinopathies), and rheumatoid arthritis. The ED-B domain of fibronectin, a domain of 91 amino acids, inserted by a mechanism of alternative splicing of the primary transcript into the fibronectin molecule, is a high-quality marker of angiogenesis and a target for molecular intervention, characterized by a high number of acidic residues on its surface, as well as some solvent-exposed hydrophobic residues. A library of 113 low molecular-weight organic compounds, containing both an aromatic moiety and at least one positive charge, was screened for binding to the ED-B domain, using two-dimensional heteronuclear NMR spectroscopy. One lead compound, 2,2-diphenylethylamine, was found that binds specifically to the ED-B domain, albeit with a dissociation constant in the millimolar range. Chemical modification of this scaffold revealed structural determinants required for binding, as well as amino acid residues in the ED-B domain responsible for the interaction. The results presented represent the basis for the development of high-affinity, low molecular-weight binders by using a linked-fragment approach or elongating the scaffold by means of combinatorial chemistry.