The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic growth factors FGF-1, FGF-2, and VEGF were studied by surface plasmon resonance (SPR) on a BIAcore 3000 biosensor. Compared with heparin, PI-88 has at least 11-fold higher affinity for FGF-1 and at least 3-fold higher affinity for VEGF, but at least 13-fold lower affinity for FGF-2. To define the structural features of PI-88 that are important for growth factor binding, several analogues, such as dephosphorylated PI-88 and a sulfated pentasaccharide, were prepared. The binding interactions of these analogues with FGF-1, FGF-2, and VEGF were similarly studied by SPR, and structure-activity relationships were determined.

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as its anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

A surface plasmon resonance-based solution affinity assay is described for measuring the K(d) of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin-streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar K(d) values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.

A predictive relationship between calculated and observed binding affinities for the complexation of ligands to the fibroblast growth factors FGF-1 and FGF-2 based on molecular docking calculations is described. The majority of the ligands examined in this study have high conformational flexibility, and to account for this, multiple conformers were generated for each and subsequently used in flexible docking calculations. Two scoring functions, Gscore and Emodel, were used to quantify the protein:ligand interaction of which the Emodel score showed the best correlation with experimental binding energies. Both scoring functions, however, predicted similar locations for the ligand sulfate groups in the binding site. The van der Waals radii of nonpolar atoms of both the protein and ligand, which modify the effective sizes of both the protein binding site and the ligand, were also systematically altered by factors of 1.0, 0.9, and 0.8 in order to optimize the conditions for predictive docking. Least squares analyses of the Emodel scores against experimental binding energies yielded best r(2) values of 0.91 and 0.83 for FGF-1 and FGF-2, respectively, with slightly lower q(2) values. Optimized scale factor combinations in conjunction with the least squares lines of best fit based on the Emodel function were used to define a predictive model that was tested against ligands not included in the original set. Acceptable predictions of binding affinity were obtained for use in the initial screening of potential leadlike molecules for both FGF-1 and FGF-2.

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