School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Infection Biology Unit, German Primate Centre - Leibniz Institute for Primate Research, 37077 Göttingen, Germany.
Nanoscale. 2024 Jul 25;16(29):13962-13978. doi: 10.1039/d4nr00484a.
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, , ∼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 (, 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 (, 5, 13, and 27 nm) and glycan density (, 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 (Δ°) 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 EC 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.
多价凝集素-聚糖相互作用（MLGIs）对于病毒感染和免疫调节至关重要。因此，它们的结构和生物物理数据不仅对于理解其基本机制非常有价值，而且对于设计针对靶标 MLGIs 的有效糖缀合物治疗药物也非常有价值。然而，对于一些重要的 MGLIs，这些信息仍然了解甚少，这极大地限制了研究进展。我们最近开发了高度糖基化的纳米颗粒，约 4nm 的量子点（QDs）或约 5nm 的金纳米颗粒（GNPs），作为 MLGIs 的机制探针。使用两种重要的模型凝集素病毒受体 DC-SIGN 和 DC-SIGNR，我们表明这些探针不仅可以提供用于定量 MLGI 亲和力的灵敏荧光测定法，而且还可以揭示有用的关键结构信息（结合位点取向和结合模式），用于 MLGI 靶向。然而，以前支架的小尺寸可能不是最大化 MLGI 亲和力和靶向特异性的最佳选择。在此，我们使用 α-甘露-α-1,2-双糖（DiMan）功能化的 GNP（GNP-DiMan）探针，系统地研究了 GNP 支架尺寸（5、13 和 27nm）和聚糖密度（100、75、50 和 25%）如何决定它们的 MLGI 亲和力、热力学和抗病毒特性。我们开发了一种新的 GNP 荧光猝灭测定格式，以最大程度地减少 GNP 强内滤效应在 MLGI 亲和力定量中的可能干扰，结果表明，增加 GNP 尺寸非常有利于提高 MLGI 亲和力。我们通过结合温度依赖性亲和力和范特霍夫分析进一步确定了 MLGI 的热力学，结果表明 GNP-DiMan-DC-SIGN/R 结合是焓驱动的，随着 GNP 尺寸的增加，有利的结合吉布斯自由能变化（Δ°）得到增强。最后，我们表明增加 GNP 尺寸可显著提高其抗病毒效力。值得注意的是，DiMan 涂层的 27nm GNP 可有效且稳健地阻断 DC-SIGN 和 DC-SIGNR 介导的假埃博拉病毒细胞进入，其 EC 分别约为 23 和 49pM，使其成为针对 DC-SIGN/R 介导的埃博拉病毒细胞感染的最有效糖缀合物抑制剂。我们的结果确立了 GNP-聚糖作为定量 MLGI 生物物理参数的新工具，并表明增加 GNP 支架尺寸可显著提高其 MLGI 亲和力和抗病毒效力。
