R&D Status of Drugs Based on Influenza Pathogenesis (Part One)

Influenza is an acute respiratory infectious disease caused by influenza virus. Influenza virus replicate by attacking epithelial cells of host and infects more cells, which triggers the immune system to attack and destroy infected tissues of respiratory system and induces overreaction of the immune system. Some patients develop common respiratory symptoms and systemic symptoms, such as muscular stiffness, fatigue and loss of appetite. A few people also have digestive tract symptoms, such as abdominal pain and diarrhea. In severe cases, such complications as pneumonia, meningitis, acute respiratory distress syndrome and shock may appear, which may lead to multiple organ failure and even death. 

Anti-influenza not only refers to inhibit virus replication, but also block the transmission of related inflammatory factors between immune cells, thus relieving excessive immune response and reducing the damage of the body caused by repairing immune response. In addition to the commonly used neuraminidase inhibitors and M2 blockers, new drugs are also being developed in other targets of virus replication and body immunity. Traditional Chinese medicine has always been known for its multi-target treatment. According to research, it was found that many active components in traditional Chinese medicine, such as quinones, flavonoids and alkaloids, are effective against influenza. In addition, the research found that the combination of Chinese and Western medicines has better anti-influenza therapeutic effect than single one of them. 

Drugs inhibiting neuraminidase, a surface protein of influenza virus 

The monomer of mushroom tetramer glycoprotein neuraminidase consists of stem structural domain immobilized on virus membrane and spherical head of catalytic active center. Neuraminidase cannot only cleave the glycosidic bond between hemagglutinin and sialic acid to catalyze sialic acid hydrolysis, but also expose the receptors on the cell surface by reducing the viscosity of respiratory mucus layer to enhance virus adsorption. It can also promote the spread of virus-containing tissue fluid to the lower respiratory tract to damage it in a short time. Neuraminidase is a widely developed and applied target at present. 

1. Chemical drugs and small molecule compounds 

Neuraminidase inhibitors (NAIs), as antiviral drugs, are widely used at present. They compete with neuraminidase (substrate) by reversibly binding with neuraminidase active sites to inhibit neuraminidase enzyme function and cut off the virus diffusion chain, thus preventing virus offspring from shedding from the host cell surface. The combination of zanamivir and neuraminidase active site E-119 forms hydrogen bond, which is designed as dry powder inhalation for local concentration in respiratory tract due to its poor bioavailability. Oseltamivir phosphate, the first oral anti-influenza western medicine, can bind to neuraminidase active site H275Y, among which R292K, E119V and H274Y are common drug-resistant mutation sites. Peramivir inhibits neuraminidase activity through the strong intermolecular interaction between its carboxyl and guanidino groups and neuraminidase active sites (including Asp151, Glu119, Glu227, etc.). R378Q, R378K and R378L are drug-resistant mutation points. Because of its poor bioavailability, it is currently the only anti-influenza drug approved by FDA for intravenous administration. Laninamivir octanoate can well inhibit the oseltamivir phosphate resistant strains, and its advantage is that it can stay in the lungs for a long time to maintain long-term inhibition. Using it once a week can effectively fight off influenza 

2. Active ingredients and compound prescriptions of traditional Chinese medicine 

Germacrone, a monocyclic sesquiterpene extracted from plant essential oil, can inhibit the replication and transcription of influenza virus and activity of neuraminidase in a dose-dependent manner. Combined with oseltamivir, it shows cumulative inhibition on virus infection in vivo and in vitro, representing that it has the potential to be developed alone or in combination with other drugs for the treatment of influenza. Cyanidin-3-sambubioside (C3S), a flavonoid extracted from black elderberry, can be used as a potential inhibitor of mutation of virus H274Y. Combined with H274Y mutant influenza virus, it forms a hydrogen bond-like force and shows good binding affinity. The combination of C3S with oseltamivir resistant to H274Y can represent strong antiviral activity. In addition, coptisine , berberine and geniposide extracted from coptidis decoction for detoxification can effectively inhibit neuraminidase-1 in a competitive way.

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Drugs inhibiting hemagglutinin, a surface protein of influenza virus 

Hemagglutinin is a spicate trimer composed of spherical head and stem region, which mainly mediates the introduction of influenza virus into host cells, including virus-cell binding and virus-host membrane fusion. The receptor binding site at the head of hemagglutinin binds to sialic acid on the cell surface, which allows viruses to enter through endocytosis. After entering into cells, the virus induces the conformational changes in the stem region of hemagglutinin in acidic environment, which leads to the fusion of virus membrane and host inner membrane and releases virus R neuraminidase genome into cytoplasm. The key step of hemagglutinin-mediated virus entry has become the potential target of anti-influenza drug development. 

1. Chemical drugs and small molecule compounds 

Arbidol, a broad-spectrum antiviral drug that has been approved for the treatment of influenza A and B in Russia and China, exerts the molecular glue function and interacts with the hemagglutinin protein of influenza virus to stabilize it during the transition from low pH to fusion state, thus inhibiting the hemagglutinin-mediated membrane fusion. That is the low pH-induced hemagglutinin refolding process. Arbidol can effectively inhibit the oxidative stress caused by the virus. In addition, it is also found that adding meta-hydroxyl to the thiophenol part of arbidol to replace the structured water molecules in the binding pocket can significantly increase the affinity for H3 and H1 subtypes. Compound CBS1116 can also interfere with hemagglutinin-mediated membrane fusion by binding to hemagglutinin stem region and interfering with hemagglutinin conformational changes triggered by low pH. In addition, the compound CR6261 also exerts antiviral effect by binding to the stem region of hemagglutinin in the similar way as CBS1116. 

Drugs inhibiting M2 ion channel of influenza virus 

M2 ion channel is the basis of RNA release in the process of influenza virus genome unwrapping and virus unwrapping. It can also be recognized by virus to enter and exit host cells under the proton action. In addition, it can inhibit autophagy degradation by blocking the fusion of autophagosome and lysosome. Although the current related clinical drugs are almost completely resistant, there is still potential to develop new drugs in promoting autophagy of infected cells. 

As the first generation of anti-influenza A drugs, M2 ion channel inhibitors, amantadine and rimantadine, can specifically inhibit M2 ion channel activity at low concentration, and can nonspecifically increase pH in host cells at high concentration, thus inhibiting or delaying acid-induced conformational changes of viral hemagglutinin. While most of the spreading human influenza viruses carry drug-resistant mutations (such as S31N, V27A and L26F) in the transmembrane structural domain of M2, which has led to nearly 100% drug resistance of the two drugs. The Centers for Disease Control and Prevention no longer recommend them for anti-influenza, because they can penetrate blood-brain barrier with potential central nervous system toxicity. Amantadine bromine thiophene, an amantadine derivative, inhibits virus from entering and existing host cells by targeting S31-M2 and N31-M2 channels. Compound J10688 is a host (cdc2 like kinase 1 (CLK1) inhibitor. CLK1 is responsible for the alternative splicing of M2 gene in the process of influenza virus infection and replication, while J10688 significantly down-regulates the phosphorylation of splicing factors SF2/ASF and SC35, thus regulating the alternative splicing of virus M2 gene. It shows anti-influenza virus activity in vivo and in vitro. Compound TCN-032 relieves influenza symptoms by specifically binding to M2 conserved extracellular domain, which prevents virus from sprouting and detaching. 

Drugs inhibiting the nucleoprotein of influenza virus 

Nucleoprotein, structural protein of influenza virus, plays a central role in virus replication. Nucleoprotein is the skeleton structure of vRNP, and its functions involve RNA accumulation, nuclear transport, viral RNA transcription and replication, etc. The folding of nucleoprotein in influenza virus strains is basically conservative, which means that the virus is not easily resistant to nucleoprotein inhibitors. Therefore, there is a good development prospect. 

F66 is the first reported RNA binding groove inhibitor targeting nucleoprotein, which may bind to RNA binding groove in R174-K184 epitope region and inhibit virus replication and transcription. Naproxen is also an RNA binding groove inhibitor targeting nucleoprotein, which cannot only directly inhibit the activity of influenza virus, but also inhibit the inflammatory reaction triggered by virus. Mini-genome analysis showed that RK424 could inhibit the activity of vR nucleoprotein of influenza virus, and then destroy the oligomerization of nucleoprotein induced by viral RNA. It can also combine with single nucleoprotein to destroy the interaction between nucleoprotein -RNA and nucleoprotein-nucleoprotein. Nucleozin, a small molecule compound, bridges two nucleoprotein molecules from bottom to bottom through Y289/N309 and Y52/Y313 pockets, preventing nucleoprotein from being introduced into nucleus and causing abnormal aggregation in cytoplasm, so as to inhibit replication of influenza virus. Compound S119-8 inhibits influenza virus replication by changing the oligomerization state of nucleoprotein, and has synergistic effect with oseltamivir and reduces drug resistance. 

Reference 

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

About the author: 

Xiao Michong, a drug quality researcher, has been committed to drug quality research and drug analysis method verification for a long time, and now works in a large Chinese drug R&D company, engaging in drug inspection and analysis and verification of analysis methods. 

Continue to Read Part Two: R&D Status of Drugs Based on Influenza Pathogenesis (Part Two)