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Dissecting Multivalent Viral Receptor-carbohydrate Interactions Using Polyvalent Multifunctional Glycan-Quantum Dot

Periodic Reporting for period 1 - DC-SIGN-MFN (Dissecting Multivalent Viral Receptor-carbohydrate Interactions Using Polyvalent Multifunctional Glycan-Quantum Dot)

Reporting period: 2018-07-13 to 2020-07-12

Multivalent lectin-glycan interactions (MLGIs) play a key role in facilitating viral infections, affecting hundreds of millions people worldwide. Understanding the underlying structural mechanisms is key to be able to design glycoconjugates that can potently and specifically block such interactions, thereby preventing infection. Compared to other anti-viral strategies, developing glycoconjugates to block virus entry to host is advantageous: it can prevent virus mutating and developing resistance. This is especially important for unstable RNA viruses, e.g. SARS COV-2 responsible for the Covid-19 pandemic, whose rapid mutation can severely hamper the effectiveness most anti-viral strategies. However, research advances have been hampered by inability of current methods to reveal key structural information (e.g. binding site orientation, distance, & binding mode) of some key cell membrane lectins. This is important because, (1) glycoconjugates's antiviral potency depends on critically the ability to match the spatial arrangements of MLGI partners; (2) many lectins have overlapping glycan specificity, this can prevent glycoconjugates from blocking other MGLIs non-specifically to induce severer side effects. For example, despite 20 years of research, the detailed structures of two closely related and vitally important tetrameric lectins, DC-SIGN and DC-SIGNR, remain unknown. They can bind to virus surface glycans to enhance the infections of many viruses (e.g. HIV, HCV and Ebola). Unfortunately, conventional biophysical techniques, e.g. SPR and ITC, although powerful in providing quantitative binding thermodynamics and kinetics, they cannot provide structural information.

This fellowship aims to develop a novel multimodal readout strategy (combining FRET, TEM and particle size analysis) using compact glycan-quantum dots (QDs) by exploiting multivalency and QD’s strong fluorescence and high contrast in TEM imaging. By tuning glycan structure, valency, inter-glycan spacing, it aims to create a perfect spatial & orientation match to those of glycan-binding-domains (CRDs) in DC-SIGN/R, leading to greatly enhanced binding affinity. By studying QD-glycans and their assemblies binding with DC-SIGN/R, we will reveal key structural data (e.g. CRD orientation, distance, binding mode) in DC-SIGN/R. It also aims to verify the binding data with native receptors on cell surfaces, correlate receptor binding affinity with virus inhibition potency, and study their immune cell activation.
We have developed a glycan-nanoparticle (NP, e.g. QD or gold nanoparticle, GNP) based multimodal readout strategy which dissected the different binding mode and affinity enhancing mechanism for DC-SIGN/R successfully. We find that DC-SIGN bind simultaneously with one glycan-NP and give impressive affinity enhancement (5-6 orders of magnitude over monovalent binding); whereas DC-SIGNR inter-crosslink with glycan-NPs and give much lower affinity enhancement. This result agrees with our proposed CRD orientation difference in DC-SIGN/R. We find that the glycan-NPs’ viral inhibition potency depends on not only the binding affinity but also binding mode with target lectins: only those showing simultaneous-, but not cross-link-, binding mode can to produce robust virus inhibition (Figure 1). Finally, we find that linking glycan-NPs together via rigid DNA linker can significantly enhance their binding affinity with DC-SIGNR, but not with DC-SIGN, suggesting that the glycan-NP assemblies may bind simultaneous with DC-SIGNR and can act as potent inhibitors against DC-SIGNR mediate viral infection. However, the planned dendritic cell immune response and virus inhibition studies were not possible due to the Covid-19 lockdown.

The fellow has received extensive trainings in ligand synthesis, protein production, labelling and FRET assays, as well as project management and grant writing skills. The fellow has also participated in supervising MChem and MSc students and received significant support from technical staff and other lab members. The fellow has collaborated with other group members to produce two research papers as co-author. The fellow prepared drafts, figures for publications, and participated weekly group meetings and monthly discussions.

Research Publications
(1) Wang, W., Kong, Y., Jiang, J., Tian, X., Li, S., Akshath, U.S. Tiede, C., Hondow, N., Yu, A., Guo, Y. and Zhou, D. Nanoscale, 2020,12(16), pp.8647-8655.
(2) Budhadev, D., Poole, E., Nehlmeier, I., Liu, Y., Hooper, J., Kalverda, E., Akshath, U.S. Hondow, N., Turnbull, W.B. Pöhlmann, S., Guo, Y., and Zhou, D. J. Am. Chem. Soc. 2020, 142(42), 18022-18034.

Conference presentation:
(1) U.S. Akshath, D. Budhadev, E. Kalverda, N. Hondow, W. B. Turnbull, Y. Guo and D. Zhou. Probing multivalent lectin-glycan interactions using multifunctional glycan-nanoparticles, Poster-Nanolithography of Biointerfaces Faraday Discussion, London, UK, 3-5/7/2019
(2) D. Budhadev, E. Poole, I. Nehlmeier, Y. Liu, E. Kalverda, J. Hooper, U. S. Akshath, W. B. Turnbull, S. Pöhlmann, Y. Guo and D. Zhou. Polyvalent Glycan-Nanoparticles as Multifunctional Structural Probes for Viral Receptors, DC-SIGN and DC-SIGNR. Poster- European Symposium on Biological and Organic Chemistry, 53rd ESBOC Symposium, Gregynog, UK, 17-19/5/2019.
(3) D. Budhadev, E. Poole, I. Nehlmeier, Y. Liu, E. Kalverda, J. Hooper, U. S. Akshath, W. B. Turnbull, S. Pöhlmann, Y. Guo and D. Zhou. Probing and Blocking DC-SIGN/R-glycan Interaction Mediated Virus Infection Using Polyvalent Multifunctional Glycan Gold Nanoparticles. Oral presentation- RSC Carbohydrate Early Career Researcher Webinar Series, Oral, 2/7/2020.
This project has exploit a new strategy to prepare glycan-QDs and their dimer assemblies via a rigid double-stranded DNA linker. We have found that the assembly size and inter-particle distance can be tuned via the linker DNA length. We find that DC-SIGN bind simultaneously with one glycan-QD to form small individual assemblies, while DC-SIGNR inter-crosslink with multiple QDs to produce extended assemblies. As a result, QD-glycans bind much more strongly to DC-SIGN than that to DC-SIGNR. However, the DNA-linked glycan-QD dimer bind to DC-SIGNR with the same affinity as to DC-SIGN, suggesting DNA mediated glycan-QD assemblies can be harnessed for specific DC-SIGNR targeting and robust viral infection, which has not been feasible for individual glycan-NPs. These findings will provide importance guidance toward the design novel glycan-NPs and their assemblies for potent, specific blocking of lectin receptor mediate viral infections. The robust surface and conjugation chemistries and NP assembly strategies can harnessed for novel nanomedicine development to potential compact against viral, bacterial infection, cancer and other human diseases.

It should be noted that this project has been focused on elucidating fundamental mechanisms underlying MLGIs, and establish new approaches and design rules for potent, specific lectin targeting. It is not focused on developing new products or medicines directly. However, the new tools and findings obtained from this study will likely to benefit human healthcare, the UK/EU pharmaceutical industries and provide socio-economic impact over the medium to long term (e.g. 5-15 years).

The new findings have been highlighted on the group research website and also the group tweeter account @ZhouDejian.
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