Polyvalent Glycomimetic-Gold\nNanoparticles Revealing\nCritical Roles of Glycan Display on Multivalent Lectin–Glycan\nInteraction Biophysics and Antiviral Properties

Multivalent lectin–glycan interactions (MLGIs)\nare widespread\nand vital for biology, making them attractive therapeutic targets.\nUnfortunately, the structural and biophysical mechanisms of several\nkey MLGIs remain poorly understood, limiting our ability to design\nspatially matched glycoconjugates as potential therapeutics against\nspecific MLGIs. We have recently demonstrated that natural oligomannose-coated\nnanoparticles are powerful probes for MLGIs. They can provide not\nonly quantitative affinity and binding thermodynamic data but also\nkey structural information (<i>e.g</i>, binding site orientation\nand mode) useful for designing glycoconjugate therapeutics against\nspecific MLGIs. Despite success, how designing parameters (<i>e.g</i>., glycan type, density, and scaffold size) control their\nMLGI biophysical and antiviral properties remains to be elucidated.\nA synthetic pseudodimannose (psDiMan) ligand has been shown to selectively\nbind to a dendritic cell surface tetrameric lectin, DC-SIGN, over\nsome other multimeric lectins sharing monovalent mannose specificity\nbut having distinct cellular functions. Herein, we display psDiMan\npolyvalently onto gold nanoparticles (GNPs) of varying sizes (<i>e.g</i>., ∼5 and ∼13 nm, denoted as G5- and G13\npsDiMan hereafter) to probe how the scaffold size and glycan display\ncontrol their MLGI properties with DC-SIGN and the closely related\nlectin DC-SIGNR. We show that G5/13 psDiMan binds strongly to DC-SIGN,\nwith sub-nM <i>K</i><sub>d</sub>s, with affinity being enhanced\nwith increasing scaffold size, whereas they show apparently no or\nonly weak binding to DC-SIGNR. Interestingly, there is a minimal,\nGNP-size-dependent, glycan density threshold for forming strong binding\nwith DC-SIGN. By combining temperature-dependent affinity and Van’t\nHoff analyses, we have developed a new GNP fluorescence quenching\nassay for MLGI thermodynamics, revealing that DC-SIGN-G<i>x</i>-psDiMan binding is enthalpy-driven, with a standard binding Δ<i>H</i><sup>0</sup> of ∼ −95 kJ mol<sup>–1</sup>, which is ∼4-fold that of the monovalent binding and is comparable\nto that measured by isothermal titration calorimetry. We further reveal\nthat the enhanced DC-SIGN affinity with G<i>x</i>-psDiMan\nwith increasing GNP scaffold size is due to reduced binding entropy\npenalty and not due to enhanced favorable binding enthalpy. We further\nshow that DC-SIGN binds tetravalently to a single G<i>x</i>-psDiMan, irrespective of the GNP size, whereas DC-SIGNR binding\nis dependent on GNP size, with no apparent binding with G5, and weak\ncross-linking with G13. Finally, we show that G<i>x</i>-psDiMans\npotently inhibit DC-SIGN-dependent augmentation of cellular entry\nof Ebola pseudoviruses with sub-nM EC<sub>50</sub> values, whereas\nthey exhibit no significant (for G5) or weak (for G13) inhibition\nagainst DC-SIGNR-augmented viral entry, consistent to their MLGI properties\nwith DC-SIGNR in solution. These results have established G<i>x</i>-psDiMan as a versatile new tool for probing MLGI affinity,\nselectivity, and thermodynamics, as well as GNP–glycan antiviral\nproperties.