Controlled multivalent interactions in the inhibition of toxins via well-designed glycopolypeptides

Many critical recognition events in biology are mediated via multivalent interactions between multiple saccharide ligands and their protein receptors. These proteincarbohydrate interactions are therefore important and being extensively investigated as they play a crucial role in several processes including pathogen recognition, inflammation, cell signaling, differentiation, and adhesion of various bacterial toxins. Multiple research groups have investigated these interactions by developing multivalent polymeric antagonists for carbohydrate binding proteins. In our work, we have selected cholera toxin (CT) as a model example to study these multivalent bindings by developing multivalent inhibitors. Various investigations have employed diverse guidelines that are believed to govern multivalency in the design of inhibitors for CT-GM1 interactions. Although successful in many respects, they are limited by certain architectural features such as a lack of synthetic versatility, significant polydispersity, and uncontrolled density and arrangement of saccharide ligands. Thus the mechanism by which multivalency is functioning in these systems is impractical to analyze and control. A more detailed understanding of multivalent binding by polymeric materials therefore requires the development of well-designed glycopolymers in which architectural features are well defined and controlled. Our approach aims to develop polymers via protein engineering methods and to equip these polypeptides with multivalent sugar ligands via chemical methods, to competitively bind with such toxins and neutralize them. This method allows control over architectural features such as number and spacing of saccharide ligands on the polymer, precise placement of charges and conformation of the polymer backbone. Such control over the architectural features allows for more purposeful design of polymers for inhibition of the multivalent binding event. Polypeptides with chemically reactive natural or non-natural amino acid residues were synthesized and were coupled with saccharides to produce glycopolypeptides that were used to evaluate the inhibitory potencies against CT via the use of immunochemical assays. The data from our experiments confirms the relevance of appropriate saccharide spacing, polypeptide chain extension, saccharide linker conformation, and the systematic placement of charges on the polypeptide backbone in improving the inhibition of CT.