Spacing and Orientation of the Saccharide Epitopes Is Important for ConA Interaction

Low molecular weight divalent and trivalent ligands have also been generated to probe ConA ligand recognition. As had been shown with the hepatic lectin (see Section V.C) [96,97] and influenza virus hemagglutinin (see Sections VI.E-VI.F) [98], low molecular weight ligands can achieve high potencies, displaying inhibitory potencies 100- to 1000-fold higher than those of the corresponding monovalent saccharides. To investigate the effect of epitope spacing and orientation in ConA binding, Roy and coworkers constructed a series of low molecular weight bivalent ligands in which the saccharide epitopes were appended through linkers differing in length and composition (Fig. 15) [89,90]. The ability of these bivalent molecules to inhibit ConA binding to immobilized mannan (a yeast glycoprotein) was tested in an enzyme-linked lectin assay (ELLA) (Fig. 8). The structure of the linking group had subtle effects on the ConA inhibitory properties of the bivalent molecules. The bivalent ligand with a six-carbon linker, 17c, found to be superior in this study, was 5.3 times more effective per saccharide at inhibition than a-^-nitrophenyl-manno-pyranoside [89]. Roy and coworkers also examined the effects of higher order valency in a related series of related ligands (Fig. 16) [57]. Bi-, tri-, and tetravalent ligands, 18, 19, and 20, were 138, 210, and 231 times more active than a-Me-mannose, respectively, when analyzed on a molar scale. However this translates to 69-, 70-, and 57.8-fold increases in potency when determination is made on a per-saccharide-residue basis. On a multivalent display, additional ligands that do not contribute productively to binding can detract from the observed potency [86]. This has also been observed in the case of polyacrylamide displays targeting influenza virus, where maximal activity is observed at a low ligand density (see Sections VI.A-VI.B) [99-101].

The abilities of small bi-, tri-, and tetravalent molecules to inhibit ConA likely arise from their propensities to cross-link lectin multimers. Molecular modeling of Roy's ligands suggests that the spacers linking the saccharide recognition epitopes

Figure 15 Roy's bivalent mannose ligands directed against ConA. To investigate spacing effects, the ligands are designed with variable length linkers.

are not long enough to span the 65 A required to simultaneously occupy two binding sites within a single ConA tetramer (D. Mann, unpublished results, 1999). Another potential mode of action for these ligands is suggested by Brewer's finding that many oligosaccharides and glycopeptides can form cross-linked lectin complexes [102]. A turbidimetric assay monitoring the formation of cross-linked ConA precipitates supports this model for Roy's ligands (Fig. 7). Thus, the best small-molecule inhibitors of ConA may be those that can cross-link the lectin. Dendrimers that exhibit enhanced inhibitory activity toward ConA also appear to function by this mechanism [87,88]. Regardless of their mode of action, data generated with the bivalent ligands highlight the necessity for proper spacing between ligands to elicit optimal multi-valent interactions.

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