Based on the Current Status of Drug Research and Development Targeting the Pathogenesis of Influenza (Part 1)

Influenza is an acute respiratory infectious disease caused by the influenza virus. The influenza virus completes replication by attacking the host's epithelial cells, thereby infecting more cells, triggering the immune system to attack and destroy the infected tissues of the respiratory system, and leading to an overreaction of the immune system. Common symptoms include respiratory problems accompanied by systemic symptoms such as general aches and fatigue, loss of appetite, and in some cases, abdominal pain, diarrhea, and other gastrointestinal symptoms. In severe cases, complications such as pneumonia, meningitis, acute respiratory distress syndrome, shock, and other conditions may occur, leading to multiple organ failure and even death.

To fight influenza, it is not only necessary to inhibit viral replication, but also to block the transmission of related inflammatory factors between immune cells, alleviate excessive immune responses, and reduce the damage caused by the repair of immune responses. In addition to commonly used neuraminidase inhibitors and M2 blockers, new drugs are currently being developed targeting other aspects of viral replication and immune response. Traditional Chinese medicine has always been known for its multi-target therapy, and studies have found that many active ingredients in traditional Chinese medicine, such as quinones, flavonoids, and alkaloids, can effectively resist influenza. Furthermore, research has found that combining traditional Chinese medicine with Western medicine for the treatment of influenza produces better results than using either alone.

Inhibitors targeting the neuraminidase protein on the surface of influenza virus

The mushroom-shaped tetrameric glycoprotein neuraminidase is composed of a stem domain anchored on the viral membrane and a globular head serving as the catalytic active center. Neuraminidase not only catalyzes the hydrolysis of sialic acid by cleaving the glycosidic bond between hemagglutinin and sialic acid, but also enhances virus adsorption by reducing the viscosity of the respiratory tract mucus layer and exposing cell surface receptors. Additionally, it can promote the dissemination of virus-containing tissue fluid to the lower respiratory tract, causing short-term damage to the respiratory tract. Neuraminidase is currently the most targeted and widely developed enzyme for this purpose.

1. Chemical Drugs and Small-molecule Compounds

Neuraminidase inhibitors (NAIs) are currently the most widely used antiviral drugs. They competitively inhibit the enzymatic function of neuraminidase by reversibly binding to its active sites, thereby cutting off the virus diffusion chain and preventing the detachment of virus progeny from the host cell surface. Zanamivir forms hydrogen bonds with the neuraminidase active site E-119 and is designed as an inhaled powder formulation for local concentration in the respiratory tract due to its poor bioavailability. The first oral anti-influenza western medicine, oseltamivir phosphate, can bind to the neuraminidase active site H275Y, with R292K, E119V, and H274Y being common drug-resistant mutation sites. Peramivir inhibits neuraminidase activity through strong intermolecular interactions between its carboxyl and guanidino groups with the neuraminidase active sites (including Asp151, Glu119, and Glu227), with R378Q, R378K, and R378L being its drug-resistant mutation sites. Due to its poor bioavailability, it is currently the only FDA-approved anti-influenza drug administered by intravenous injection. Laninamivir octanoate has a good inhibitory effect on oseltamivir-resistant strains, with its greatest advantage being its ability to maintain long-term inhibition in the lungs for a prolonged period, requiring only once-weekly inhalation for effective anti-influenza treatment.

2.Active Ingredients and Compound Formulas in Traditional Chinese Medicine

The monocyclic sesquiterpene jimatone extracted from plant volatile oils can inhibit the replication, transcription, and neuraminidase activity of influenza virus in a dose-dependent manner. When combined with oseltamivir, it shows a cumulative effect on inhibiting viral infection both in vivo and in vitro, indicating its potential for development as a standalone or combined therapy for influenza. The flavonoid compound cyanidin-3-sambubioside (C3S) extracted from black elderberry can serve as a potential inhibitor for the H274Y mutation of the virus. It forms a hydrogen bond-like interaction with the H274Y mutant influenza virus and displays good binding affinity. When combined with oseltamivir, which is resistant to H274Y, it exhibits considerable antiviral activity. Additionally, berberine, berberine sulfate, geniposide, and other components in the water extract of Huanglian Jiedu Tang can effectively inhibit neuraminidase-1 in a competitive manner.

Drugs that Inhibit the Hemagglutinin Protein on the Surface of Influenza Virus

Hemagglutinin, a spike-like homotrimer composed of a globular head and a stalk region, primarily mediates the entry of influenza virus into host cells, including virus-cell binding and virus-host membrane fusion. The receptor-binding sites located on the head of hemagglutinin bind to sialic acid on the cell surface, enabling the virus to enter through endocytosis. After virus entry, the acidic environment induces a conformational change in the stalk region of hemagglutinin, leading to the fusion of the virus membrane with the host endosome membrane and the release of the virus's RNA genome into the cytoplasm. This crucial step mediated by hemagglutinin has become a potential target for anti-influenza drug development.

1. Chemical Drugs and Small-molecule Compounds

Arbidol, a broad-spectrum antiviral drug approved in Russia and China for the treatment of influenza A and B, interacts with the hemagglutinin protein of influenza virus through its molecular glue function, stabilizing it during the transition to the fusion state at low pH and inhibiting hemagglutinin-mediated membrane fusion, i.e., the low pH-induced hemagglutinin refolding process. It can also effectively inhibit virus-induced oxidative stress. Furthermore, it has been found that adding a meta-hydroxy group to the benzenethiol moiety of arbidol to replace the structured water molecules in the binding pocket can significantly increase its affinity for H3 and H1 subtypes. The compound CBS1116 similarly disrupts hemagglutinin-mediated membrane fusion by binding to the stem region of hemagglutinin and interfering with the low pH-triggered conformational changes of hemagglutinin. Additionally, the compound CR6261 also exerts its antiviral effect by binding to the stem region of hemagglutinin through a similar mechanism as CBS1116.

Drugs that Inhibit the M2 Ion Channel of Influenza Virus

The M2 ion channel serves as the basis for the release of RNA during the unpacking and uncoating of the influenza virus genome. It is also recognized by the virus to facilitate entry and exit from host cells under the action of protons. Additionally, it can block the fusion of autophagosomes and lysosomes to inhibit autophagic degradation in cells. Although current clinical drugs targeting this channel are almost completely resistant, there is still potential for developing new drugs in promoting autophagy in infected cells.

The first-generation anti-influenza drugs, M2 ion channel inhibitors such as amantadine and rimantadine, can specifically inhibit M2 ion channel activity at low concentrations, and at high concentrations, they can non-specifically increase the pH within host cells, thus inhibiting or delaying the acid-induced conformational change of the viral hemagglutinin. Most circulating human influenza viruses carry drug-resistant mutations (such as S31N, V27A, and L26F) in the M2 transmembrane pore domain, resulting in nearly 100% resistance to these two drugs. Due to their ability to cross the blood-brain barrier and potentially affect the central nervous system, the Centers for Disease Control and Prevention no longer recommend their use for influenza treatment.

The amantadine derivative, amantadine bromide thiophene, targets the S31-M2 and N31-M2 channel pores, inhibiting virus entry and exit from host cells to fight influenza. Compound J10688 is an inhibitor of host cell division cycle 2-like kinase 1 (CLK1), which is responsible for the alternative splicing of the M2 gene during influenza virus infection and replication. J10688 significantly downregulates the phosphorylation of splicing factors SF2/ASF and SC35, thereby regulating the alternative splicing of the viral M2 gene and exhibiting anti-influenza virus activity both in vitro and in vivo. Compound TCN-032 specifically binds to the conserved extracellular domain of M2, preventing viral budding and release to alleviate influenza symptoms.

Inhibitors of Influenza Virus Nucleoprotein

The nucleoprotein, a structural protein of influenza virus, plays a central role in viral replication. It serves as the backbone of the viral ribonucleoprotein (vRNP) complex and is involved in RNA packaging, nuclear transport, and viral RNA transcription and replication. The folding of the nucleoprotein in influenza virus strains is largely conserved, which means that the virus is less likely to develop resistance to nucleoprotein inhibitors, thus presenting promising prospects for drug development.

F66 is the first reported RNA binding groove inhibitor targeting the nucleoprotein, which likely binds to the RNA binding pocket in the epitope region of R174-K184 to inhibit viral replication and transcription. Naproxen is also an RNA binding groove inhibitor targeting the nucleoprotein, which not only directly inhibits influenza virus activity but also inhibits virus-triggered inflammatory responses. Microgenome assay revealed that RK424 disrupts viral RNA-induced nucleoprotein oligomerization by inhibiting influenza virus vRNP nucleoprotein activity, and also binds to monomeric nucleoprotein to disrupt nucleoprotein-RNA and nucleoprotein-nucleoprotein interactions. The small-molecule compound Nucleozin bridges two nucleoprotein molecules from bottom to top through the Y289/N309 and Y52/Y313 pockets, preventing nucleoprotein from entering the nucleus and causing abnormal aggregation in the cytoplasm to inhibit influenza virus replication. Compound S119-8 inhibits influenza virus replication by altering the oligomerization state of nucleoprotein, and has a synergistic effect when combined with oseltamivir, reducing drug resistance.

Next Article: "Current Status of Drug Research and Development Based on the Pathogenesis of Influenza (Part 2)"

References

Chen Jinfeng, He Jun, Xu Tao. Research Progress in Drugs Based on the Pathogenesis of Influenza [J]. Chinese Pharmacological Bulletin, 2021, 37(05):606-612.

About the Author

Xiaomichong, a researcher in pharmaceutical quality, has been dedicated to pharmaceutical quality research and verification of drug analysis methods for a long time. Currently, she works in a large domestic pharmaceutical research and development company, engaged in drug inspection analysis and verification of analytical methods.