Beyond the Amino Acid Sequence: Glycosylation
How Simple Questions Open New Frontiers in Drug Discovery—The Battle Between Influenza Viruses and Sialic Acid
In the latter half of the previous article, I explained how human-adapted influenza viruses initiate infection. Hemagglutinin (HA), a protein on the viral surface, primarily recognizes terminal sialic acid residues on glycans displayed by airway epithelial cells, particularly the Siaα2-6Gal motif in which sialic acid is α2,6-linked to galactose. This recognition enables the virus to attach to the cell. A molecular battle over these glycans unfolds between the virus and our cells.
Oseltamivir (brand name: Tamiflu), one of the most commonly used treatments for influenza, inhibits the viral enzyme neuraminidase. Neuraminidase cleaves terminal sialic acid residues, allowing newly formed virus particles to detach from the surface of infected cells. By blocking this enzyme, oseltamivir suppresses the release and spread of progeny virus particles. In other words, it is a strategy that “blocks the virus’s exit.”
But could we take the opposite approach and eliminate the virus’s “point of entry”? Once we understand how infection occurs, a simple question naturally arises.
“If HA binds to glycans containing α2,6-linked sialic acid to initiate infection, could we create a drug that specifically disrupts this interaction and thereby develop a highly effective treatment for influenza?”
This is, in many ways, a classic drug-discovery strategy. Yet therapies that directly target the interaction between HA and sialic acid have not become established as standard influenza treatments. Why not? The answer lies in the physicochemical challenges posed by the protein-glycan interactions, as well as in the virus’s sophisticated survival strategies.
Question 1: Why Aren’t There Drugs That Block the Virus’s “Point of Entry”?
When considered as a one-to-one interaction, the binding between HA and a sialic acid-containing glycan is actually very weak. At first glance, this might make the interaction seem easy to disrupt.
However, the surface of an influenza virus particle is studded with numerous HA trimers, while the cell surface displays a dense array of sialic acid-containing glycans. The virus uses multiple HA molecules to bind simultaneously to multiple glycans, allowing it to adhere firmly to the cell surface. This works much like a hook-and-loop fastener: each individual interaction is weak, but when many interactions occur simultaneously, they collectively generate strong binding. This overall binding strength resulting from multiple simultaneous interactions is known as “avidity” (Figure 1).
For this reason, even if a small molecule blocks some of the available binding sites, many other contacts remain possible, making it difficult to prevent the virus as a whole from attaching to the cell surface. In addition, a drug that directly targets HA may lose effectiveness as HA mutates. Even if an inhibitor is successfully developed, changes in or around the binding site could reduce the drug’s activity.
Question 2: Then Why Not “Mask” the Host Receptors?
If directly targeting the virus is difficult, another possibility would be to bind preemptively to the sialic acid-containing glycans on our cell surfaces and “mask” them so that the virus can no longer attach. This reversal of perspective seems attractive, but here again, the complexity of biological systems presents a major challenge.
Of course, sialic acid is not present merely for influenza viruses to exploit. It is widely found at the termini of glycans on glycoproteins and glycolipids and is involved in a variety of physiological functions, including cell-cell recognition, cell signaling, and regulation of immune responses. For example, immune cells express receptors known as Siglecs, which recognize sialic acid-containing glycans and help regulate immune-cell activity. Broad or prolonged masking of these glycans could therefore interfere not only with viral attachment but also with normal glycan recognition and cellular functions.
Another possibility would be to use sialic acid-binding lectins, derived from plants, bacteria, or other organisms. However, lectins can bind multiple glycans simultaneously, cross-link cells, and alter intracellular signaling. Some lectins can also be cytotoxic and, as foreign proteins, may trigger immune responses. For these reasons, directly using naturally occurring lectins as safe therapeutic agents is far from straightforward.
Local delivery to the respiratory tract might reduce systemic effects. Even so, an important challenge remains: how can we safely mask only the relevant glycans, at the right location, and only for the required period of time.
A Reversal in Thinking: “Removing” Rather Than “Masking” Sialic Acid
One possible way around this dilemma is not to “mask” sialic acid, but to “remove” it temporarily. DAS181, also known as Fludase, is a drug candidate based on this concept (1). DAS181 is an inhaled recombinant fusion protein containing a bacterial sialidase. It is designed to enzymatically cleave terminal sialic acid residues in both α2,3- and α2,6-linkages from glycans on respiratory epithelial surfaces, thereby reducing the number of attachment sites available to the virus. Because this approach targets a host-cell feature exploited by the virus, it may be less susceptible to mutations in HA.
However, sialic acid also contributes to normal mucosal function and immune regulation. The extent and duration of its removal—and the safety of doing so—must therefore be carefully evaluated. DAS181 remains under development, and it is not yet clear whether it will ultimately become an anti-influenza therapy. Nevertheless, the idea of “temporarily modifying a host-cell feature used by the virus, rather than continually chasing a rapidly mutating virus” points toward a distinct direction in antiviral drug development.
Glycans attached to proteins are far more than decorative structures. They can act as molecular markers by which viruses recognize host cells, as gateways to infection, and as targets for new therapeutic strategies.
Glycan structures are not directly encoded by the genetic code. Yet they exert a major influence on both viral infection strategies and our own approaches to drug discovery. The battle between influenza viruses and sialic acid offers a striking example of the profound biological roles played by glycans.
1) Triana-Baltzer GB, et al. DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis. J Antimicrob Chemother. 2010;65:275-284. doi:10.1093/jac/dkp421.
Multiple hemagglutinin (HA) trimers on the surface of a human influenza virus recognize terminal Siaα2-6Gal motifs on glycans displayed by airway epithelial cells. In these motifs, sialic acid is α2,6-linked to galactose. Although each individual HA–glycan interaction is weak, simultaneous binding between multiple HA trimers and multiple glycans generates strong overall viral attachment, known as avidity. In living cells, these glycans are carried by glycoproteins and glycolipids. This illustration is schematic; the numbers, sizes, arrangement, and scale of the molecules are not shown in their actual proportions.
Author Profile
Toshifumi Takao
Professor Emeritus, Osaka University; Ph.D. in Science.
From his fourth year as an undergraduate until retirement, he was affiliated with the Institute for Protein Research, Osaka University, where he consistently engaged in research and education in protein chemistry and structural biology.
He currently serves as a Specially Appointed Professor (part-time) at the same institute and as a Life Science Fellow in the Product Division, Rigaku Corporation.
In 2022, he was awarded an honorary doctorate in biochemistry by the University of Havana, Cuba.
