Neuraminidase

Neuraminidase, also known as sialidase, is a group of glycoside hydrolase enzymes that cleave the glycosidic linkages of neuraminic acids. Neuraminidase enzymes are a large family, found in a range of organisms. The best-known neuraminidase is the viral neuraminidase, a drug target for the prevention of the spread of influenza infection. In the 1940s American scientist George Hirst identified in samples of influenza virus mixed with red blood cells (erythrocytes) a substance that broke down receptors on the surfaces of red cells. Shortly thereafter, German-born British biochemist Alfred Gottschalk discovered that these receptor-destroying enzymes were neuraminidases.

Classification

According to the catalytic mechanism, neuraminidase can be classified into two groups that respectively cleave exo or endo poly-sialic acids:

• Exo hydrolysis of α-(2→3)-, α-(2→6)-, α-(2→8)-glycosidic linkages of terminal sialic acid residues
• Endo hydrolysis of (2→8)-α-sialosyl linkages in oligo- or poly(sialic) acids

According to the sources, neuraminidase can be classified into three groups:

• Viral neuraminidase
• Bacterial neuraminidase
• Mammalian neuraminidase

There are nine known subtypes of influenza neuraminidase, many of which are found only in duck and chicken breeds. N1 and N2 subtypes were positively correlated with human epidemics, and N3 or N7 subtypes were found in some isolated death cases.

Viral Neuraminidase

Viral neuraminidase is a type of neuraminidase found on the surface of influenza viruses that enables the virus to be released from the host cell. Nine subtypes of neuraminidase are described for influenza A, whereas only one subtype was revealed for the influenza viruses B. Neuraminidases are required for influenza virus replication. When influenza virus replicates, it attaches to the interior cell surface using hemagglutinin. Sialic acids are found on various glycoproteins at the host cell surface, and the virus exploits these groups to bind the host cell. In order for the virus to be released from the cell, neuraminidase must enzymatically cleave the sialic acid groups from host glycoproteins. Since the cleavage of the sialic groups is an integral part of influenza replication, blocking the function of neuraminidase with neuraminidase inhibitors is an effective way to treat influenza.

Figure 1. Structure of Influenza, showing neuraminidase marked as NA and hemagglutinin as HA.

Structure

Influenza neuraminidase is a mushroom-shaped projection on the surface of the influenza virus, which comprises 470 amino acid residues. The three-dimensional structure of NA consists of four co-planar and roughly spherical subunits, and a hydrophobic region that is embedded within the interior of the virus' membrane. The NA's head region consists of one big domain, which is formed by six identical antiparallel β-sheets (motifs) organized in the form of a propeller-like structure. Influenza neuraminidase comprises a single polypeptide chain that is oriented in the opposite direction to the hemagglutinin antigen. The composition of the polypeptide is a single chain of six conserved polar amino acids, followed by hydrophilic, variable amino acids. β-Sheets predominate as the secondary level of protein conformation.

Mechanism

The enzymatic process of neuraminidase is divided into four steps. The first step is that when α-sialoside binds to sialidase, the ²C₅ chair conformation is twisted into a pseudo boat conformation. The second step leads to an oxocarbocation intermediate, the sialosyl cation. The third step is to form Neu5Ac initially as the α-anomer, and then mutarotation and release as the more thermodynamically-stable β-Neu5Ac.

Figure 2. Proposed mechanism of catalysis of influenza virus sialidase 4.

Inhibitors

All influenza viruses bear two surface glycoproteins, a hemagglutinin and a neuraminidase, which are the antigens that define the particular strain of influenza. The variation of these molecules over time permits the virus to evade human immune responses and therefore necessitates the formulation of a new vaccine each year. The hemagglutinin is a sialic acid receptor–binding molecule and mediates entry of the virus into the target cell. The neuraminidase cleaves the cellular-receptor sialic acid residues to which the newly formed particles are attached. This cleavage releases the viruses, which can now invade new cells. Without neuraminidase, infection would be limited to one round of replication, rarely enough to cause disease.

The ability of transition-state analogues of sialic acid to inhibit the influenza neuraminidase was first recognized in the 1970s, but the design of highly effective inhibitors became feasible when analysis of the three-dimensional structure of influenza neuraminidase disclosed the location and structure of the catalytic site. Potent inhibitors such as zanamivir closely mimic the natural substrate, fitting into the active site pocket and engaging the protein in the most energetically favorable interaction. Zanamivir is administered by oral inhalation, which delivers the drug directly to the respiratory tract. Oseltamivir was developed through modifications to the sialic acid analogue framework (including the addition of a lipophilic side chain) that allow the drug to be used orally.

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