A focused sulfated glycoconjugate Ugi library for probing heparan sulfate-binding angiogenic growth factors.

A library of small molecule heparan sulfate (HS) mimetics was synthesized by employing the Ugi four-component condensation of d-mannopyranoside-derived isocyanides with formaldehyde as the carbonyl component and a selection of carboxylic acids and amines, followed by sulfonation. The library was used to probe the subtle differences surrounding the ionic binding sites of three HS-binding angiogenic growth factors (FGF-1, FGF-2 and VEGF). Each compound features 3 or 4 sulfo groups which serve to anchor the ligand to the HS-binding site of the protein, with a diverse array of functionality in place extending from C-1 or C-6 to probe for adjacent favorable binding interactions. Selectivity of binding to these proteins was clearly observed and supported by molecular docking calculations.

Synthesis of a heparan sulfate mimetic library targeting FGF and VEGF via click chemistry on a monosaccharide template.

A disulfated methyl 6-azido-6-deoxy-α-D-mannopyranoside template was used as a core structure for binding to the angiogenic growth factors FGF-1, FGF-2, and VEGF. The core structure was diversified in a rapid, parallel manner by employing the Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition ("click") reaction. The diversity was further extended by incorporating a Swern oxidation-Wittig reaction sequence on a click adduct of propargyl alcohol. Thus, the sulfated core was linked by various spacers to selected hydrophobic or polar motifs, which were designed to probe the protein surface surrounding the cationic heparan sulfate binding sites of the growth factors in order to improve affinity and selectivity. The affinities of the compounds for the growth factors were measured by surface plasmon resonance solution affinity assays. A lead compound was identified with micromolar binding affinity toward both FGF-1 and VEGF (K(d)=84 and 49 µM, respectively) and good selectivity over FGF-2 (29- and 51-fold, respectively).

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins.

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.

Design, synthesis, FGF-1 binding, and molecular modeling studies of conformationally flexible heparin mimetic disaccharides.

Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a D-galactose donor with a methyl beta-D-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solution in equilibrium between the (4)C1 and (1)C4 conformers, as confirmed by 1H NMR spectroscopy, thus mimicking the conformationally flexible L-iduronic acid found in heparin. Docking calculations showed that the predicted locations of disaccharide sulfo groups in the binding site of FGF-1 are consistent with the positions observed for co-crystallized heparin-derived oligosaccharides. Predicted binding affinities are in accord with experimental Kd values obtained from binding assays and are similar to the predicted values for a model heparin disaccharide.

Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88).

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.

An experimental and molecular-modeling study of the binding of linked sulfated tetracyclitols to FGF-1 and FGF-2.

The experimental binding affinities of a series of linked sulfated tetracyclitols [Cyc2N-R-NCyc2, where Cyc = C6H6(OSO3Na)3 and R = (CH2)n (n = 2-10), p-xylyl or (C2H4)2-Ncyc] for the fibroblast growth factors FGF-1 and FGF-2 have been measured by using a surface plasmon resonance assay. The KD values range from 7.0 nM to 1.1 microM for the alkyl-linked ligands. The binding affinity is independent of the flexibility of the linker, as replacement of the alkyl linker with a rigid p-xylyl group did not affect the KD. Calculations suggest that binding modes for the p-xylyl-linked ligand are similar to those calculated for the flexible alkyl-linked tetracyclitols. The possible formation of cross-linked FGF:cyclitol complexes was examined by determining KD values at increasing protein concentrations. No changes in KD were observed; this suggesting that only 1:1 complexes are formed under these assay conditions. Monte Carlo multiple-minima calculations of low-energy conformers of the FGF-bound ligands showed that all of the sulfated tetracyclitol ligands can bind effectively in the heparan sulfate-binding sites of FGF-1 and FGF-2. Binding affinities of these complexes were estimated by the Linear Interaction Energy (LIE) method to within a root-mean-square deviation of 1 kcal mol(-1) of the observed values. The effect of incorporating cations to balance the overall charge of the complexes during the LIE calculations was also explored.

Application of the four-component Ugi condensation for the preparation of sulfated glycoconjugate libraries.

A focused library of novel, sulfated glycoconjugates was synthesized by utilizing carbohydrate-derived blocks in the four-component Ugi condensation. Library members comprise a sulfated monosaccharide linked by various spacers to either an aromatic or monosulfated moiety, or a second sulfated monosaccharide. The affinities of these heparan sulfate (HS) mimetics for the HS-binding fibroblast growth factors FGF-1 and FGF-2 were measured via a surface plasmon resonance solution affinity assay.

Validation of molecular docking calculations involving FGF-1 and FGF-2.

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.

Probing the interactions of phosphosulfomannans with angiogenic growth factors by surface plasmon resonance.

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.