N8NeuraminidaseWithZanamivir

The influenza viruses contain two glycoproteins in their membrane which aid in function: hemagglutinin and neuraminidase. Different types of neuraminidases and hemagglutinins define specific strains of influenza which are identified with the abbreviation HxNy where x is the class of hemagglutinin and y is the class of neuraminidase in the virus' membrane. Neuraminidases are exosialidases which are categorized into groups labeled N1 through N9. Neuraminidase 8 (N8) is one of the nine serotypes and consists of 390 amino acid residues with a total weight of 43,490 Da. The isoelectric point of N8 is 5.71 and is classified as part of the group one family of neuraminidases which include N1, N4, N5, and N8 (4, 1). N8 neuraminidase in complex with Zanamivir (2HTQ) is important in the study of antiviral drugs and viral inhibition. Exosialidases work to cleave alpha-ketosidic linkages that connect sialic acid and carbohydrate residues on both cellular glycoproteins and viral glycoproteins. This allows newly formed viral particles to exit the infected cell and prevents their aggregation while promoting further infection. Furthermore, N8 cleaves neuraminic acid residues from respiratory tract mucins allowing the virus to penetrate the protective mucosa and infect the surrounding tissue (2).

The N8 structure is a homotetramer with globular subunits assembling to form a circular structure connected to viral particle membranes by a thin 60 to 100 Å long stalk (5, Figure 1).

Each of the four subunits is composed of six antiparallel beta strands forming a 'beta propeller' structure with the active site at its center able to bind one Zanamivir molecule (1, Figure 2). The entirety of the structure is mostly composed of beta strand and random loop secondary structure motifs. The random loops in the protein provide the most structural variety and contribute significantly to function especially as seen by the 150-loop discussed below. The active site is located at the N-terminus of the central parallel strands and is approximately 16 Å in diameter and 10 Å deep. The active site consists of functional and structural amino acids of which, the functional residues are in contact with sialic acid. In comparison to the active sites of group two neuraminidases, those of group one have slightly different structures as well as a small cavity proximal to the active site which allows for the development of inhibitors such as Zanamivir (2). This cavity seen in group one is a result of small differences not present in group two neuraminidases such as the nonpolar side chain of residue Val-149 pointing away from the active site and residue's Glu-119 carboxylate moiety pointing in the opposite direction as that of group two. The resulting cavity is accessible from the active site and increases the active site cavity width by 5 Å. Furthermore, the binding of inhibitors to N8 is a two-step process whereby Zanamivir or other inhibitors bind to the enzyme which then undergoes a slight conformational change.

In each subunit of the N8 tetramer a second neuraminic acid binding site (HB-site) exists and is formed by highly conserved amino acid residues. Residues 367, 370, and 372 are all serines involved in neuraminic acid binding as seen conserved between N8 in complex with Zanamivir [2HTQ] and Neuraminidase Subtype N9 Complexed with 30 MM Sialic Acid [2C4A] (3). Also conserved between the two enzymes from different groups are the Asn-400 and Trp-403 residues that interact with the substrate. The only variation between the two proteins at the HB-site is that of residue Lys-432 which is highly specific to N9 neuraminidases (12, Figure 3). Although the function of the HB-site is still uncertain, it is thought to act as an alternative neuraminic acid binding site. Interestingly, the changes described above can be found in unique strains such as H9N2, H2N2, and H3N2 which have caused human pandemics. This data suggests that some species of poultry can act as intermediates in the spread of influenza to humans (2).

The active site of neuraminidases contains eight highly conserved amino acid residues that interact directly with the substrate as well as ten other residues which are conserved in all strains of influenza virus and aid in molecular stability (7). The development of a functional inhibitor to these enzymes depended highly on the improvement of structural resolution technology. Before improvements in X-ray crystallography and other imaging techniques, inhibitors were used based on catalytic properties of the neuraminidases giving rise to drugs such as 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en). Higher resolution of active site residues and orientation fueled structure-based drug design giving rise to 4-deoxy-4-guanidino-Neu5Ac2en (Zanamivir). Zanamivir's large and basic guanidino group significantly increased affinity for the enzyme at the conserved residues Glu-119 and Glu-227 (7). Zanamivir's structure plays a role in its unique high affinity as a result of its similarity to neuraminidase's natural substrate Neu5Ac (figure 4a, 4b). The structural similarity confers high selectivity of the inhibitor to viral neuraminidases and lower selectivity for endogenous human sialidases which could create side effects. A 10,000-fold increase in affinity paired with high viral selectivity makes Zanamivir a highly effective and safe drug in comparison to other available antivirals such as oseltamivir (figure 4c). However, Zanamivir's high polarity and rapid excretion limits its distribution to an inhaled form of the drug.

The intense study of these sialidases and their inhibitors is especially relevant in recent decades as the prevalence of epidemics has risen and, furthermore, it is of dire importance to this day with the current COVID-19 pandemic testing public health infrastructure. Further research has brought up the question of viral mutation and resistance to synthesized drugs Zanamivir and oseltamivir; two of only four drugs approved by the FDA for influenza infection. Unfortunately, a viable influenza strain that is resistant to oseltamivir has been found due to mutations which block the rearrangement of residue Glu-276 at the active site (7). This strain exhibits reduced affinity for oseltamivir but is still sensitive to Zanamivir. No viable strains of influenza exhibit resistance to Zanamivir, however, resistant strains such as G70C4-G have been synthesized in vitro (8). This variant strain contains a mutation at the conserved residue Glu-119 resulting in a glycine residue taking its place. Furthermore, the strain also exhibits a Ser-186 to phenylalanine mutation in its haemagglutinin enzymes which works synergistically with the Glu-119 mutation to confer higher in vitro resistance. Such a strain mutating in vivo could be catastrophic and more research is being carried out to increase the variety of antiviral drugs available to combat the possibility of such mutation (8).

As mentioned previously, binding of substrates to group one sialidases is a two-step process characterized by binding and a slight conformational change. The neuraminidase protein contains a small cavity termed the 150-cavity which is capped by the 150-loop. This loop is composed of six amino acids from locations 147-152 which is relatively conserved (13). The conformation change occurs due to the rearrangement of the '150-loop' upon binding of the substrate or inhibitors. Before binding occurs, the conformation of group one neuraminidases is in an open conformation that is characterized by a larger active site cavity. Upon binding, the 150-loop rearrangement takes place and gives rise to the closed conformation where closing of the loop tightly coordinates the inhibitor or substrate.

Although both group one and group two neuraminidases contain the 150-loop structural motif, there can be significant differences between their amino acid composition and contribution to the protein's function. Bioinformatic software or databases, such as the Dali server of PSI-BLAST, can aid in the visualization and comparison of different proteins. The Dali server searches databases for protein tertiary structure and emphasizes similarity through comparison of intermolecular distance. The server computes a z-score which indicates greater level of similarity with greater magnitudes of the z-score (3). PSI-BLAST similarly searches databases for proteins but focuses on primary structure similarity. An E-value is produced that also indicates similarity of proteins, however, in this server lower E-value magnitudes equate to greater protein similarity (9). Using the N9 protein discussed before, Neuraminidase Subtype N9 Complexed with 30 MM Sialic Acid [2C4A], and the two servers, the similarity of the two neuraminidases can be interpreted as relatively close with an E-value of 6e-117 and a z-score of 62.6 (3, 9). When comparing the 150-loop, differing amino acid residues such as a Lys-150 residue in N8 neuraminidase and a His-150 residue in N9 neuraminidase can be noticed. Differing compositions of the proteins alter conformation and therefore the binding of substrates. However, conserved residues such as Asp-151, albeit positioned slightly differently in N8 and N9, reveal the relationship between the two proteins (6, 9, 12). The open conformation of group one neuraminidases as a result of the 150-loop is the subject of ongoing research and provides opportunities for the development of new generation anti-influenza drugs (7). New drugs such as a divalent Zanamivir could increase the bind time of the inhibitor and therefore make administration of the drug easier but the search for a universal neuraminidase influenza inhibitor is still ongoing. As the modern world struggles in its battle with COVID-19, a greater emphasis is being placed on the 'arms-race' against viral mutations. Further developments in anti-viral medications have the potential to save millions of lives once a novel viral mutation threatens human lives once again.