Review of Part One: [R&D Status of Drugs Based on Influenza Pathogenesis (Part One)] 

Drugs inhibiting influenza virus RNA polymerase 

Influenza virus RNA polymerase is a protein complex composed of PA, PB1 and PB2 subunits. PA has endonuclease activity and can induce proteolysis. PB1 has RNA polymerase activity, and PB2 can recognize and bind to the host 5'mRNA cap structure, so as to play an important role in genome replication and transcription. At present, RNA polymerase inhibitors have become an important target for anti-influenza drug research because of their low mutation frequency and mild side effect. 

1. Chemical drugs and small molecule compounds 

Ribavirin, a guanosine ribonucleoside analogue, targets inosine 5' monophosphate dehydrogenase (IMPDH), which can inhibit viral RNA replication and induce RNA replication mutation by reducing the concentration of GTP in cells and preventing viral protein synthesis. Additionally, combined with glycyrrhizic acid, ribavirin can significantly reduce the expression levels of inflammatory factors IL-6, TNF-α and IL-1β. Pimodivir, which has entered Phase III clinical trials, targets PB2 subunit of RNA polymerase and inhibits the expression of virogene by preventing polymerase binding and cap structure of 7-methyl GTP in pre-mRNA host. And it also has activity against various kinds of influenza A viruses and strains with low sensitivity to NAIs. Baloxavir, which has been listed in the United States and Japan at present, targets the endonuclease activity of PA subunit virus. Once it is combined with PB2.8, it can prevent polymerase from cutting pre-mRNA, which is effective for influenza A, B viruses and even oseltamivir-resistant viruses. Favipiravir, a purine nucleoside analogue, is active against almost all influenza viruses. After ribosylation and phosphorylation in cells, it produces a drug-active form of nucleoside triphosphate fampiravir RTP. Viral RNA polymerase misrecognizes this form as purine and it is mistakenly incorporated into newborn RNA, which leads to chain termination in the process of RNA synthesis. In addition, it exerts anti-virus effects by inducing fatal mutation. Due to the outbreak of COVID-19 and other reasons, this drug has been listed in China. 

2. Active ingredients of traditional Chinese medicine 

Isoquercitrin can prevent virus replication and reduce the active methoxy group caused by influenza A infection through blocking the operation of PB2 in RNA polymerase. Silybin and silidianin, the main components of silymarin extracted from seeds of silybum marianum, can prevent virus from entering host cells, inhibit the formation of oxidative stress by regulating autophagy, and trigger extracellular signal regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor kappa-B kinase (IKK) cascade. The binding affinity of toosendanin, the main active component of toosendan fruit, binding to PA is stronger than that of known PA inhibitors. Toosendanin destroys nuclear transport by changing the nuclear localization of PA protein, which leads to cytoplasm accumulation of PA and inhibits early replication of influenza virus mRNA. It can also interfere with virus entering host cells by inhibiting the activity of protein kinase B. 

Drugs for relieving excessive body immunity 

Anti-inflammatory immunity is an indispensible step for the body to fight against influenza virus. Inflammatory factors, as immune signal molecules, are released through NF-κB, Nrf2, JAK-STAT, RLRs, AMPK and other signaling pathways for self-defense. In addition to inhibiting the replication and spread of influenza virus, the excessive body immunity triggered by the transmission of inflammatory factors between immune cells should be blocked.

1. Chemical drugs and small molecule compounds 

Intravenous immunoglobulin (IVIG) contains IgG antibody. It has dual therapeutic effects of immune substitution and immune regulation, which can reduce the mortality and morbidity related to severe influenza. IVIG is often used as the first-stage intervention measure in the influenza intensive care environment. Peroxisome proliferators-activated receptors γ (PPAR γ) can enhance lipid and glucose metabolism and cell differentiation and inhibit inflammation. It can reduce the number of neutrophilic granulocytes and macrophages, as well as the production of inflammatory factors, in the excessive body immune. N-acetylcysteine can inhibit the activity of myeloperoxidase, reduce the number of neutrophilic granulocytes and macrophages and the level of pro-inflammatory factors,so as to reduce lung inflammation and pulmonary edema. 

2. Active ingredients, compound prescriptions of traditional Chinese medicine and Chinese patent medicine 

Polygalasaponin can effectively reduce the levels of IL-1β, TNF-α, IL-4, IFN-γ, TXA2 and PGE2 in lung tissue, and enhance the anti-influenza A infection ability by alleviating lung inflammation reaction. Andrographolide is known as a natural antibiotic drug. Its anti-influenza activity is to reduce the viral load and the expression of inflammatory cytokines caused by infection and ultimately reduce the cell death induced by influenza virus by participating in the activation of NF-κB, Nrf2, JAK-STAT and RLRs signaling pathways. Additionally, the combination of Xiyanping injection with andrographolide as the main component and oseltamivir has a good clinical efficacy. The active components of traditional Chinese medicine licorice include glycyrrhizin, enoxolone and isoliquiritigenin, among which the glycyrrhizin acts on corticosteroid 11-β-dehydrogenase isozyme 1 in IL-1 signaling pathway to enter host cells. Hence, it hinders monocyte recruitment and reduces the levels of pro-inflammatory molecules (CXCL10, IL-6, CCL2 and CCL5), activates IFN-γ and reduces the expression of NF-κBp38 and JNK. Glycyrrhizin has antioxidant activity and can inhibit the formation of reactive oxygen species (ROS) induced by influenza virus. Therefore, it can be used as adjuvant for preparing anti-influenza vaccine. Isoliquiritigenin inhibits inflammatory factor gene expression and inflammatory cell aggregation by the activation of PPAR pathway, thus alleviating lung inflammation. Through promoting the production of IL-12 and IFN-γ, Gegen decoction, a classical prescription, can induce Th1 system immune response, reduce Toll like receptor 7 (TLR-7) signaling pathway and TNF-α expression, and improve Th1/Th2 immune balance, thus alleviating excessive immune response. Shenfu injection can increase the expression of TLR-4 signal and the production of IκB-α and reduce the expression levels of p65, p50, TNF-α and IL-1β in lung by inhibiting the transcription, expression, translocation and secretion of high mobility group protein B1 (HMGB1), thus effectively preventing the decrease of MAP and alleviating lung tissue injury. In other words, the whole process also refers to prevent inflammatory factor storm by inhibiting HMGB1-NF-κB pathway. 

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Multi-target anti-influenza drugs 

Some anti-influenza drugs have the dual effects of inhibiting influenza virus replication and anti-inflammatory immunity through multi-component, multi-target and multi-pathway action. At present, most of these drugs exist in some active ingredients of traditional Chinese medicine to be developed. 

1. Chemical drugs and small molecule compounds 

Salinomycin, as an effective killer of influenza A and B viruses, can cause the cytoplasm accumulation of NP in the process of viral entry, especially in the perinuclear endosome by blocking the nuclear migration of NP, and it can also prevent the endosome acidification and inactivate the proton transport function of M2. In addition, the combination of salinomycin and oseltamivir can significantly improve the curative effect, which can be used as an adjuvant therapy for influenza with low drug resistance or sensitivity to existing drugs. Nidazonide, as a kind of oral antiparasitic drug, has antiviral and immunomodulatory effects. It further targets influenza virus by inhibiting the maturation and translation of HA, and plays an anti-inflammatory role by up-regulating IFN and various IFN-inducible genes. It also has synergistic effect when combining with oseltamivir. 

2. Active ingredients of traditional Chinese medicine and Chinese patent medicine 

Catechinic acid compounds inhibit the escaping of virus by binding to NA near the cavity near residue 430 (sub-sialic acid binding site) conserved structure of influenza virus. It also significantly reduces the content of autophagy-related protein LC3B in a dose-dependent manner and exert anti-influenza effect by affecting the conformation of HA. Natural catechinic acid can even be used as inactivators for preparing inactivated virus vaccines. 

Curcumin, a polyphenol substance, interferes with the binding of HA to host cell receptors, and its derivatives have significant binding affinity with all subtypes of HA. It also reduces the RNA binding affinity of NP by interrupting the stacking between Y148 and RNA bases, which can obviously inhibit the oxidative stress induced by influenza virus and inhibit the activation of TLR2/4, NF-κBp38 /JNK and MAPK signals induced by influenza virus by activating Nrf2 signal. 

Anthraquinone-containing aloin inhibits the immune response mediated by TGF-β by inhibiting influenza virus NA and blocking the activation of TGF-β mediated by NA. It improves the immunity of virus HA specific T cells, which makes HA specific Th and Tc infiltrate more in lung, restores STAT1-mediated antiviral response in transfected cells inhibited by NS1, and enhances the production of IFN-γ and TNF-α. Its flavonoids, including quercetin, catechinic acid and kaempferol, can significantly inhibit the synthesis of M2 related segments in virus mRNA and inhibit the expression of M2 protein by binding with key functional sites (PHE47A and LEU43A). In addition, it can inhibit influenza virus-induced autophagy to produce antiviral activity against H1N1 or H3N2. Aloe polysaccharide mediates immunomodulation by activating macrophages to produce NO and secrete cytokines (TNF-α, IL-6 and IFN-γ), and at the same time, it can also inhibit M2. 

The flavonoid extracts of scutellaria baicalensis include baicalein, wogonin, oroxylin and glycosides. Baicalin can prevent autophagy induced by mTOR signaling pathway inhibited by H3N2, and it can also induce IFN-γ production in Th, Tc and NK cells and activate JAK-STAT1 signaling pathway. Wogonin can inhibit the phosphorylation and activation of AMPK induced by influenza. Under the induction of type I and type III IFN and through the phosphorylation of IRF3 and the activation of IL-stimulating genes (such as MxA and OAS), wogonin can enhance the anti-viral signal transduction and alleviate the inflammatory reaction. The flavonoid extracts of scutellaria baicalensis cannot only inhibit the activities of HA and NA, reduce the expression of TNF-α, IL-6 and MCP-1 and increase the expression level of IFN-γ and IL-10 in lung tissue to regulate the inflammatory reaction, but also indirectly promote body recover by affecting intestinal flora. In the treatment of lung injury induced by influenza A, the extract has better therapeutic effect than baicalin alone. 

In vitro experiments showed that S-03, the extracts from indigowoad root water, could inhibit HA and increase the activity of antioxidant enzymes, such as SOD, CAT, GSH-Px, and total antioxidant capacity. The extracts from isatis root polysaccharide inhibit human influenza viruses (H1N1 and H3N2) and avian influenza viruses (H6N2 and H9N2) by activating TLR-3 signaling pathway, which weakens the up-regulation of proinflammatory factors induced by influenza virus and significantly reduces the expression of IL-6, IP-10, MIG and CCL5. 

Influenza is a great threat to human survival. Due to the priority treatment strategy of using NA inhibitors, there are endless drug-resistant strains emerging. Some drugs even have serious toxic side effects, such as amantadine central nervous system toxicity. In the context that there are many drug-resistant strains emerging in endless, many effective compounds are still in the stage of screening test, and it will take a long time for clinical trials and listing of related drugs. The multi-target mechanism of traditional Chinese medicine is still unclear. Many drugs are only effective in vitro experiments, and their mechanism of action in vivo is unknown. The physicians use traditional Chinese medicine for the treatment of influenza treatment only rely on their years of experience. For some traditional Chinese medicine components, there are still such problems as poor solubility and stability and low bioavailability. At present, there are two main aspects in the research of antiviral drugs, the optimization or design of new drugs for existing targets; and the finding of new targets to develop new antiviral drugs.

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 

Review of Part One: [R&D Status of Drugs Based on Influenza Pathogenesis (Part One)]