An array-based method to identify multivalent inhibitors

Abstract

Carbohydrate-protein interactions play a critical role in a variety of biological processes, and agonists/antagonists of these interactions are useful as biological probes and therapeutic agents. Most carbohydrate-binding proteins achieve tight binding through formation of a multivalent complex. Therefore, both ligand structure and presentation contribute to recognition. Since there are many potential combinations of structure, spacing, and orientation to consider and the optimal one cannot be predicted, high-throughput approaches for analyzing carbohydrate-protein interactions and designing inhibitors are appealing. In this report, we develop a strategy to vary neoglycoprotein density on a surface of a glycan array. This feature of presentation was combined with variations in glycan structure and glycan density to produce an array with approximately 600 combinations of glycan structure and presentation. The unique array platform allows one to distinguish between different types of multivalent complexes on the array surface. To illustrate the advantages of this format, it was used to rapidly identify multivalent probes for various lectins. The new array was first tested with several plant lectins, including concanavalin A (conA), Vicia villosa isolectin B4 (VVL-B(4)), and Ricinus communis agglutinin (RCA120). Next, it was used to rapidly identify potent multivalent inhibitors of Pseudomonas aeruginosa lectin I (PA-IL), a key protein involved in opportunistic infections of P. aeruginosa , and mouse macrophage galactose-type lectin (mMGL-2), a protein expressed on antigen presenting cells that may be useful as a vaccine targeting receptor. An advantage of the approach is that structural information about the lectin/receptor is not required to obtain a multivalent inhibitor/probe.

Figure 1

Different Multivalent Binding Modes and the Array Strategy.

Figure 2

Neoglycoproteins Used in the Model Studies

Figure 3

Comparison of ConA binding to Man6 and Man-α a) ConA binding at high neoglycoprotein density (1:0); shown are pairs of spots for Man-α and Man6 at a single ConA concentration (189 nM) and binding curves over a range of ConA concentrations. b) ConA binding at low neoglycoprotein density (1:7); shown are pairs of spots for Man-α and Man6 at a single ConA concentration (189 nM) and binding curves over a range of ConA concentrations. c) Proposed binding modes at high and low neoglycoprotein density. At high density, ConA binds Man-α via a bridging complex and Man6 via a 1:1 complex. At low neoglycoprotein density, a bridging complex with Man-α cannot be formed. d) Inhibition of ConA binding was evaluated by an ELISA-like assay at a range of neoglycoprotein concentrations and inhibition curves are shown for Man6 and Man-α. Man6 shows good inhibition while Man-α shows no inhibition.

Figure 4

Chemical Structures of Selected Glycans