Polyvalent Glycomimetic-Gold Nanoparticle Conjugates reveal effects of glycan display on Multivalent Lectin-Glycan Interactions

Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for biology, making them attractive ther-apeutic targets. Unfortunately, the structural and biophysical mechanisms of several key MLGIs remain poorly understood, limiting our ability to design spatially matched glycoconjugates as potent therapeutics against targeting MLGIs. A synthetic pseudo-dimannose (psDiMan) ligand has been shown to selectively bind to a dendritic cell surface tetrameric lectin, DC-SIGN, over some other multimeric lectins sharing monovalent mannose specificity but having distinct cellular functions. Herein, we display psDiMan polyvalently onto gold nanoparticles (GNP-psDiMan) to probe how scaffold size and glycan valency control their multivalent binding properties with DC-SIGN. We reveal that GNP-psDiMan binds strongly, with sub-nM Kds, to DC-SIGN and their binding affinity is enhanced with the increasing GNP scaffold size. Interestingly, there is a minimal, GNP size-de-pendent, glycan density threshold for forming strong MLGIs with DC-SIGN. Moreover, we have developed a new fluorescence quenching method for probing MLGI thermodynamics by quantifying MLGI affinities under varying temperatures in combi-nation with the Van’t Hoff analysis. We reveal that DC-SIGN binding with GNP-psDiMans is enthalpy driven, with the standard binding enthalpy changes (H0s) of ~ -100 kJ/mol, matching well to those measured by isothermal titration calorimetry. Such H0 values are ~4 folds that of the corresponding monovalent psDiMan-DC-SIGN binding, implying that all 4 binding sites in DC-SIGN are engaged in binding to GNP-psDiMan. Dynamic light scattering studies further show that DC-SIGN binds glycans from the same GNP-psDiMan. Finally, using particles pseudotyped with the Ebola virus glycoprotein, we show that GNP-psDiMans potently inhibit DC-SIGN-dependent augmentation of Ebola virus cellular entry with sub-nM level EC50 values. In-terestingly, such EC50 values are comparable to their DC-SIGN binding affinities measured by fluorescence quenching in solu-tion. These results indicate that GNP based fluorescence quenching is a versatile method to reveal MLGI thermodynamics and binding determinants of high affinity MLGI interactions, and to predict GNP-glycan antiviral properties.