Synthesis and Nuclear Magnetic Resonance Structural Evaluation of Oxime-Linked Oligosialic Acid-Based Glycodendrimers.

A series of four oxime-linked octavalent sialic acid and oligosialic acid poly(ether amidoamine) glycodendrimers were synthesized. In the attachment of the sialic acids to the dendrimer core, chemoselective oxime bonds were formed between the unprotected sugars (sialic acid or α-2,8-linked di- through tetra-sialic acids) and the aminooxy-terminated dendrimer core in a microwave-mediated reaction, resulting in good to excellent yields (58-100%) of the fully functionalized octavalent glycodendrimers. Next, using a combination of 1D and 2D nuclear magnetic resonance and working from the inside outward, we employed a systematic method to assign the proton and carbon signals starting with the smallest linkers and dendrimer cores and moving gradually up to the completed octavalent glycodendrimers. Through this approach, the assignment of the protons and carbons was possible, including the E- and Z-isomers related to the oxime dendrimer to sugar connections and relative quantities of each. These glycodendrimers were designed as broad-spectrum inhibitors of viral pathogens.

Plasticity of the 340-Loop in Influenza Neuraminidase Offers New Insight for Antiviral Drug Development.

The recently discovered 340-cavity in influenza neuraminidase (NA) N6 and N7 subtypes has introduced new possibilities for rational structure-based drug design. However, the plasticity of the 340-loop (residues 342-347) and the role of the 340-loop in NA activity and substrate binding have not been deeply exploited. Here, we investigate the mechanism of 340-cavity formation and demonstrate for the first time that seven of nine NA subtypes are able to adopt an open 340-cavity over 1.8 µs total molecular dynamics simulation time. The finding that the 340-loop plays a role in the sialic acid binding pathway suggests that the 340-cavity can function as a druggable pocket. Comparing the open and closed conformations of the 340-loop, the side chain orientation of residue 344 was found to govern the formation of the 340-cavity. Additionally, the conserved calcium ion was found to substantially influence the stability of the 340-loop. Our study provides dynamical evidence supporting the 340-cavity as a druggable hotspot at the atomic level and offers new structural insight in designing antiviral drugs.

The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View.

The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (α2,3 and α2,6 sialyl N-acetyllactosamine) has been demonstrated by NMR. The NMR-based distinction between the signals of those sialic acids in the glycans covalently attached to the spike protein and those belonging to the exogenous α2,3 and α2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly 13 C-labelled trisaccharides at the sialic acid and galactose moieties. STD-1 H,13 C-HSQC NMR experiments elegantly demonstrate the direct interaction of the sialic acid residues of both trisaccharides with additional participation of the galactose moieties, especially for the α2,3-linked analogue. Additional experiments with the spike protein in the presence of a specific antibody for the N-terminal domain and with the isolated receptor binding and N-terminal domains of the spike protein unambiguously show that the sialic acid binding site is located at the N-terminal domain.

SARS-CoV-2 and other coronaviruses bind to phosphorylated glycans from the human lung.

SARS-CoV, MERS-CoV, and potentially SARS-CoV-2 emerged as novel human coronaviruses following cross-species transmission from animal hosts. Although the receptor binding characteristics of human coronaviruses are well documented, the role of carbohydrate binding in addition to recognition of proteinaceous receptors has not been fully explored. Using natural glycan microarray technology, we identified N-glycans in the human lung that are recognized by various human and animal coronaviruses. All viruses tested, including SARS-CoV-2, bound strongly to a range of phosphorylated, high mannose N-glycans and to a very specific set of sialylated structures. Examination of two linked strains, human CoV OC43 and bovine CoV Mebus, reveals shared binding to the sialic acid form Neu5Gc (not found in humans), supporting the evidence for cross-species transmission of the bovine strain. Our findings, revealing robust recognition of lung glycans, suggest that these receptors could play a role in the initial stages of coronavirus attachment and entry.

Synthetic O-acetylated sialosides facilitate functional receptor identification for human respiratory viruses.

The transmission of viruses from animal reservoirs to humans poses major threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding of how viruses of animal origin have adapted to binding to a cell surface component and/or receptor of the new host. Here we report on the specificities of human and animal viruses that engage with O-acetylated sialic acid, which include betacoronaviruses, toroviruses and influenza C and D viruses. Key to these studies was the development of a chemoenzymatic methodology that can provide almost any sialate-acetylation pattern. A collection of O-acetylated sialoglycans was printed as a microarray for the determination of receptor specificity. These studies showed host-specific patterns of receptor recognition and revealed that three distinct human respiratory viruses uniquely bind 9-O-acetylated α2,8-linked disialoside. Immunofluorescence and cell entry studies support that such a glycotope as part of a ganglioside is a functional receptor for human coronaviruses.

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein's glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry.

Bioinformatics studies on a function of the SARS-CoV-2 spike glycoprotein as the binding of host sialic acid glycans.

SARS-CoV and SARS-CoV-2 do not appear to have functions of a hemagglutinin and neuraminidase. This is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. The S1 N terminal Domains (S1-NTD) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of SARS-CoV and SARS-CoV-2, are here predicted to be "hiding" sites for recognizing and binding glycans containing sialic acid. This may be important for infection and the ability of the virus to locate ACE2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. It might even open up the possibility of an alternative receptor to ACE2. The prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. Nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between SARS-CoV-2 sequences and to consider the effects of viral mutations.

Adaptive Flexible Sialylated Nanogels as Highly Potent Influenza A Virus Inhibitors.

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand-receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC50 values in low picomolar concentrations and also blocks the virus entry into MDCK-II cells.