Influenza is an acute upper respiratory tract infectious disease caused by influenza viruses that seriously endangers human health. Influenza viruses are negative-sense RNA viruses belonging to the Orthomyxoviridae family. Based on the differences in viral nucleoprotein and membrane protein, influenza viruses are classified into types A, B, and C. Type A is pathogenic to humans and various animals, type B is only pathogenic to humans, and type C poses relatively minor risks to humans. The high antigenic variability of influenza viruses prevents humans from achieving permanent immunity. Antigenic variation is the most important biological characteristic of influenza viruses and is closely related to the periodic outbreaks and pandemics of influenza, as well as its prevention and treatment. Currently, the main measures for preventing and treating influenza include vaccination and medication. Drug treatments mainly consist of ion channel m2 blockers such as amantadine, beta blockers, antisense oligonucleotides, and neuraminidase inhibitors (NIs).

In the field of drug treatment research, the neuraminidase (NA) has emerged as a highly successful target for the development of anti-influenza drugs. The surface of influenza viruses is adorned with three crucial proteins: hemagglutinin (HA), neuraminidase (NA), and matrix protein (M2), each playing a pivotal role in the viral transmission cycle. Firstly, the HA of influenza viruses recognizes and binds to sialic acid receptors on the surface of host cells, allowing the virus to attach to the cell membrane. Subsequently, the virus enters the cell through receptor-mediated endocytosis to replicate. The matrix protein is responsible for forming ion channels in the viral envelope, facilitating the release and expression of viral genetic material. The replicated viral proteins and other components are then transported to the cell surface, embedded in the cell membrane, and assembled into new viral particles. Finally, mature influenza viruses detach from the host cells to proceed with the next stage of transmission.

During this stage, neuraminidase plays a vital role. Before detaching from the host cell, the mature virus maintains its final connection with the cell through the HA and sialic acid receptors. Neuraminidase's function is to hydrolyze these sialic acid receptors, severing the link between the virus and the host cell, enabling new infections. Additionally, the surface of newly formed viral particles is adorned with sialic acid receptors, allowing viruses to aggregate through mutual recognition via HA. Aggregated viral particles can be eliminated by the human immune system. However, neuraminidase prevents this aggregation by hydrolyzing sialic acid residues. Therefore, neuraminidase inhibitors (NIs) can block the viral lifecycle, effectively controlling the further spread of the virus in the respiratory tract.

To date, two major classes of NA have been identified, encompassing ten subtypes (N1-N10). Class I includes N1, N4, N5, N8, and N10, while Class II comprises N2, N3, N6, N7, and N9. The distinction lies in the presence of a "150-cavity" near the active center of Class I NA, which is absent in Class II. Despite low homology among different NA subtypes, the 19 amino acids in their active centers are highly conserved, providing a foundation for subsequent research efforts.

In recent years, neuraminidase inhibitors that have achieved milestones include sialic acid analogs, cyclohexenes, and five-membered ring compounds.

1. Sialic Acid-based NAIs

The currently marketed sialic acid-based drug is zanamivir, with the trade name Relenza. Shortly after its launch, zanamivir exhibited issues such as low oral bioavailability and the development of resistance, prompting researchers to shift their focus towards structural optimization. By etherifying the glycerol side chain of zanamivir, enhancing its hydrophobic interaction with neuraminidase, laninamivir was developed. Currently, laninamivir, as a long-acting drug, has been launched in countries like Japan and South Korea. Its structure is illustrated in the figure below.

In recent years, modifying the guanidino group of zanamivir has emerged as one of the strategies for drug improvement. Studies have found that modifying the guanidino group to reduce its alkalinity results in new compounds that exhibit varying degrees of inhibition against both H1N1 and H3N2 viruses. Comparative research has revealed that altering the guanidino group of zanamivir by reducing its alkalinity can enhance the bioavailability of the inhibitor, overcoming the limitations of zanamivir such as its inability to be administered orally. Additionally, this modification can strengthen the interaction with NA, thereby improving its inhibitory activity against NA.

Apart from modifications to the guanidino group, structural modifications to the C-1 carboxyl group of zanamivir also represent another approach for drug improvement. Furthermore, the polymers of zanamivir have garnered attention, with research reporting that the tetramer of zanamivir (TZ) exhibits stronger binding affinity with neuraminidase than the zanamivir monomer. The four pharmacophore groups of TZ can simultaneously engage with the four active sites on the NA tetramer. Notably, TZ is not only effective against seasonal H3N2 and H7N9 viruses but also inhibits their mutant strains. This research finding provides novel insights into the design of multivalent inhibitors.

2.Cyclohexene-based NAIs

The currently marketed cyclohexene-based drug is oseltamivir, known commercially as Tamiflu. As a prodrug of GS-4071, oseltamivir was the first orally administered neuraminidase inhibitor to be introduced into the market. Its structure is illustrated in the figure below. However, with the widespread use of the drug, the emergence of resistant strains has limited the application of oseltamivir.

In recent years, research on the modification and improvement of GS-4071 has primarily focused on optimizing the amino group structure, modifying the C-5 position, transforming guanidino derivatives, and leveraging the principle of bioisosterism to replace the carboxyl group with a phosphate group. Studies have found that structural optimization of the amino group in GS-4071 can enhance its affinity with the "150-cavity," thereby increasing its inhibitory effect. Structural modifications to the C-5 position of GS-4071 have led to the development of a series of 5-amino derivatives that exhibit inhibitory effects against various mutant virus strains. Furthermore, compounds obtained through the modification of the guanidino group have demonstrated potent inhibitory activity against both wild-type and drug-resistant neuraminidases.

3.Five-membered Ring NAIs

The primary five-membered ring drug currently available on the market is peramivir. Late-stage clinical studies on peramivir have revealed that, upon intravenous or intramuscular administration, it effectively inhibits highly pathogenic H5N1 influenza viruses. Its structure is illustrated in the figure below.

The presence of the guanidino group on the peramivir ring reduces its oral bioavailability. Some scholars have investigated removing the guanidino group from peramivir, but this significantly decreases its antiviral activity. Drawing on the successful design of guanidino derivatives of zanamivir and oseltamivir, structural modifications to the guanidino group to enhance oral bioavailability hold great promise. Additionally, the successful modification of the carboxyl groups in zanamivir and oseltamivir can also be applied to the modification of peramivir. Research has found that transforming the carboxyl group of peramivir into a phosphate group and synthesizing a series of mono- and diester derivatives not only strongly inhibits wild-type influenza viruses in humans and birds but also exerts potent inhibition against the oseltamivir-resistant strain H275Y. Furthermore, the monoester derivatives generally exhibit higher inhibitory activity than their corresponding phosphate derivatives.

4.Other Classes of NAIs

As research into synthetic neuraminidase inhibitors progresses, natural products with neuraminidase inhibitory activity have gradually become a research focus. Among them, flavonoid compounds exhibit excellent neuraminidase inhibitory activity. For instance, seven dihydroflavones with moderate inhibitory activity have been extracted from Sophora flavescens, with IC50 values ranging from 12 to 20 μM. Additionally, a xanthone isolated from Cudrania tricuspidata demonstrates neuraminidase inhibitory activity at the nanomolar level. Furthermore, three chalcones with anti-influenza virus activity have been obtained from Glycyrrhiza inflate, exhibiting varying degrees of inhibition against H1N1, H1N1(H274Y), and H9N2.

In addition to flavonoid natural products, other natural substances also exhibit neuraminidase activity. For example, Katsumadain A, extracted from Alpinia katsumadai, demonstrates strong inhibitory activity against H1N1(A/PR/8/34) with an IC50 value of 1.05 μM. Shikometabolin E and Shikometabolin F, isolated from Lithospermum erythrorhizon, also exhibit potent neuraminidase inhibitory activity, with IC50 values of 1.91 and 2.79 μM, respectively. Furthermore, through molecular docking and pharmacological activity testing with neuraminidase (NA), various active ingredients in Huanglian Jiedu Decoction, including coptisine, palmatine, epiberberine, oroxylin A, berberine, baicalin, and baicalein, have been found to possess neuraminidase inhibitory activity to varying degrees.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.