Probing scaffold size effects on multivalent lectin–glycan binding affinity, thermodynamics and antiviral properties using polyvalent glycan-gold nanoparticles

Abstract

Multivalent lectin–glycan interactions (MLGIs) are pivotal for viral infections and immune regulation. Their structural and biophysical data are thus highly valuable, not only for understanding their basic mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information for some important MGLIs remains poorly understood, greatly limiting research progress. We have recently developed densely glycosylated nanoparticles, e.g., ∼4 nm quantum dots (QDs) or ∼5 nm gold nanoparticles (GNPs), as mechanistic probes for MLGIs. Using two important model lectin viral receptors, DC-SIGN and DC-SIGNR, we have shown that these probes can not only offer sensitive fluorescence assays for quantifying MLGI affinities, but also reveal key structural information (e.g., binding site orientation and binding mode) useful for MLGI targeting. However, the small sizes of the previous scaffolds may not be optimal for maximising MLGI affinity and targeting specificity. Herein, using α-manno-α-1,2-biose (DiMan) functionalised GNP (GNP-DiMan) probes, we have systematically studied how GNP scaffold size (e.g., 5, 13, and 27 nm) and glycan density (e.g., 100, 75, 50 and 25%) determine their MLGI affinities, thermodynamics, and antiviral properties. We have developed a new GNP fluorescence quenching assay format to minimise the possible interference of GNP's strong inner filter effect in MLGI affinity quantification, revealing that increasing the GNP size is highly beneficial for enhancing MLGI affinity. We have further determined the MLGI thermodynamics by combining temperature-dependent affinity and Van't Hoff analyses, revealing that GNP-DiMan–DC-SIGN/R binding is enthalpy driven with favourable binding Gibbs free energy changes (ΔG°) being enhanced with increasing GNP size. Finally, we show that increasing the GNP size significantly enhances their antiviral potency. Notably, the DiMan coated 27 nm GNP potently and robustly blocks both DC-SIGN and DC-SIGNR mediated pseudo-Ebola virus cellular entry with an EC₅₀ of ∼23 and ∼49 pM, respectively, making it the most potent glycoconjugate inhibitor against DC-SIGN/R-mediated Ebola cellular infections. Our results have established GNP-glycans as a new tool for quantifying MLGI biophysical parameters and revealed that increasing the GNP scaffold size significantly enhances their MLGI affinities and antiviral potencies.