Previous Review: "Current Status of Drug Research and Development Targeting the Pathogenesis of Influenza (Part 1)"

Drugs that Inhibit the RNA Polymerase of Influenza Virus

The RNA polymerase of influenza virus is a protein complex composed of PA, PB1, and PB2 subunits. PA possesses endonuclease activity and can induce proteolysis, PB1 exhibits RNA polymerase activity, and PB2 recognizes and binds to the 5'mRNA cap structure of the host, playing a significant role in the replication and transcription of the genome. Currently, RNA polymerase inhibitors have a low mutation frequency and mild adverse reactions in clinical practice, making them an important target for anti-influenza drug research.

1. Chemical Drugs and Small Molecule Compounds

The nucleoside analog ribavirin, primarily targeting inosine monophosphate dehydrogenase (IMPDH), inhibits viral RNA replication by reducing intracellular GTP concentration and blocking viral protein synthesis, which can also induce RNA replication mutations. When combined with glycyrrhizic acid, it can significantly reduce the expression levels of inflammatory factors IL-6, TNF-α, and IL-1β. Pimodivir, currently in phase III clinical trials, targets the PB2 subunit of the RNA polymerase and inhibits viral gene expression by preventing polymerase binding and the 7-methyl GTP cap structure on host precursor mRNA, showing activity against multiple strains of influenza A virus and strains with low sensitivity to NAIs.

Baloxavir marboxil, which is currently available in the US and Japan, targets the endonuclease activity of the viral PA subunit. Once it binds to PB2.8, it can prevent the polymerase from cleaving precursor mRNA, effective against both influenza A and B viruses, including those resistant to oseltamivir. The purine nucleoside analog favipiravir is active against almost all influenza viruses. After being ribosylated and phosphorylated in cells, it forms the drug's active form, favipiravir ribotriphosphate (RTP), which is misrecognized by the viral RNA polymerase as a purine and incorporated into newly synthesized RNA, leading to chain termination during RNA synthesis. Additionally, it exerts antiviral effects by inducing fatal mutations. Due to the COVID-19 pandemic and other reasons, this drug has been approved for use in China.

2.Active Ingredients of Traditional Chinese Medicine

Isorhamnetin can block the PB2 operation in RNA polymerase to prevent viral replication and also reduce the production of reactive methoxy groups caused by influenza A infection. The main components of silymarin extracted from milk thistle seeds, such as silibinin and silydianin, can prevent the virus from entering host cells, regulate autophagy to inhibit the formation of oxidative stress, and trigger the cascade of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor kappa-B kinase (IKK). The main active ingredient of Melia toosendan, toosendanin, has a stronger binding affinity to PA than known PA inhibitors. It disrupts nuclear transport by altering the nuclear localization of PA protein, leading to cytoplasmic accumulation of PA and inhibition of early replication of influenza virus mRNA. It also interferes with virus entry into host cells by inhibiting protein kinase B activity.

Drugs to Alleviate Excessive Immune Response

Anti-inflammatory immunity is an essential step for the body to fight against influenza virus invasion. Inflammatory factors, as signaling molecules of the immune system, are released through signaling pathways such as NF-κB, Nrf2, JAK-STAT, RLRs, and AMPK to defend the body. In addition to inhibiting the replication and spread of influenza virus, it is also necessary to block the transmission of inflammatory factors between immune cells to prevent excessive immune response in the human body.

1. Chemical Drugs and Small Molecule Compounds

Intravenous immunoglobulin (IVIG) contains IgG antibodies and has dual therapeutic effects of immune substitution and immune regulation. It can reduce mortality and morbidity associated with severe influenza and is often used as a first-stage intervention measure in intensive care environments for influenza patients. Peroxisome proliferators-activated receptors γ (PPARγ) have functions such as enhancing lipid and glucose metabolism, cell differentiation, and inhibiting inflammation. In cases of excessive immune response, it can reduce the number of neutrophils and macrophages and decrease the production of inflammatory factors. N-acetylcysteine can inhibit the activity of myeloperoxidase, reduce the number of neutrophils, macrophages, and levels of proinflammatory factors, thereby reducing lung inflammation and pulmonary edema.

2.Active Ingredients, Compound Formulas, and Traditional Chinese Medicine (TCM) Preparations

Cucurbitacin saponins can effectively reduce the levels of IL-1β, TNF-α, IL-4, IFN-γ, TXA2, and PGE2 in lung tissues, thereby enhancing the ability to resist influenza A virus infection by reducing lung inflammation. Andrographolide, known as a natural antibiotic, exerts its anti-influenza virus activity by participating in the activation of NF-κB, Nrf2, JAK-STAT, and RLRs signaling pathways, reducing virus load and the expression of inflammatory cytokines induced by infection, and ultimately reducing influenza virus-induced cell death. Additionally, Xiyanping injection, which contains andrographolide as its main component, combined with oseltamivir, generally shows good clinical efficacy.

The active ingredients of the TCM licorice include glycyrrhizin, glycyrrhetic acid, and isoliquiritigenin. Glycyrrhizin acts on 11-β-hydroxysteroid dehydrogenase type 1 in IL-1 signaling pathways within host cells, preventing monocyte recruitment and reducing the levels of proinflammatory molecules (CXCL10, IL-6, CCL2, and CCL5). It activates IFN-γ and reduces the expression of NF-κBp38 and JNK, possesses antioxidant activity, and inhibits the formation of reactive oxygen species (ROS) induced by influenza virus. Furthermore, it can be used as an adjuvant in the preparation of anti-influenza drugs. Isoliquiritigenin alleviates lung inflammation by inhibiting inflammatory factor gene expression and inflammatory cell accumulation under the activation of the PPAR pathway.

The classic TCM formula Gegen Tang (Pueraria Decoction) induces a Th1-type immune response by promoting the production of IL-12 and IFN-γ, reduces the expression of Toll-like receptor 7 (TLR-7) signaling pathway and TNF-α, and improves the Th1/Th2 immune balance, thereby reducing excessive immune response. Shenfu injection effectively prevents a decline in MAP and reduces lung tissue damage by inhibiting the transcription, expression, translocation, and secretion of high mobility group protein B1 (HMGB1), increasing the expression of TLR-4 signaling and the production of IκB-α, and reducing the expression levels of p65, p50, TNF-α, and IL-1β in the lungs. In other words, it prevents the occurrence of an inflammatory cytokine storm by inhibiting the HMGB1-NF-κB pathway.

Multi-target Anti-influenza Drugs

Some anti-influenza drugs achieve the dual effects of inhibiting influenza virus replication and anti-inflammatory immunity through the action of multiple components, targets, and pathways. Currently, such drugs are mostly concentrated in some undeveloped active ingredients of traditional Chinese medicine.

1. Chemical Drugs and Small Molecule Compounds

Salinomycin is an effective killer of influenza A and B viruses. It blocks the nuclear migration of NP, causing the cytoplasmic accumulation of NP during virus entry, especially in perinuclear endosomes. It also prevents endosome acidification and inactivates the M2 proton transport function. Furthermore, when combined with oseltamivir, it can significantly improve therapeutic efficacy and can be used as an adjuvant treatment for influenza with low drug resistance or sensitivity to existing drugs. Nitazoxanide is an oral anti-parasitic drug that also has antiviral and immunomodulatory effects. It targets influenza virus by inhibiting the maturation and translation of HA, upregulates IFN and various IFN-induced genes to exert anti-inflammatory effects, and also has a synergistic effect when combined with oseltamivir.

2. Active Ingredients of Traditional Chinese Medicine and TCM Preparations

Catechin compounds bind to NA near the conserved adjacent residue 430 cavity (secondary sialic acid binding site) of the influenza virus structure, inhibiting the virus from detaching. They also significantly reduce the content of autophagy-related protein LC3B in a dose-dependent manner and exert anti-influenza virus effects by affecting the conformation of HA. Natural catechins can even be used as inactivators to prepare inactivated viruses.

Curcumin, a polyphenolic 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 disrupting the stacking interaction between Y148 and RNA bases. Curcumin can significantly inhibit influenza virus-induced oxidative stress and inhibit the activation of TLR2/4, NF-κBp38/JNK, and MAPK signals induced by influenza virus through the activation of the Nrf2 signaling pathway.

The aloe anthraquinone component aloin inhibits the TGF-β-mediated immune response by inhibiting influenza virus NA and preventing NA-mediated TGF-β activation, enhancing the immunity of virus HA-specific T cells. This allows for more infiltration of HA-specific Th and Tc cells in the lungs and restores the antiviral response in NS1-inhibited STAT1-mediated transfected cells, enhancing the production of IFN-γ and TNF-α. Its flavonoid components such as quercetin, catechin, and kaempferol can significantly inhibit the synthesis of M2-related segments in viral mRNA and inhibit M2 protein expression by binding to key functional sites (PHE47A and LEU43A). Additionally, they exhibit antiviral activity against H1N1 or H3N2 by inhibiting influenza virus-induced autophagy. Aloe polysaccharide mediates immune regulation by activating macrophages to produce NO and secrete cytokines (TNF-α, IL-6, and IFN-γ), while also inhibiting M2.

The flavonoid extracts of Scutellaria baicalensis include baicalin, wogonin, oroxylin A, and glycosides. Baicalin can prevent autophagy induced by the mTOR signaling pathway inhibited by H3N2, induce IFN-γ production in Th, Tc, and NK cells, and activate the JAK-STAT1 signaling pathway. Wogonin can inhibit influenza-induced AMPK phosphorylation and activation, enhance antiviral signaling and reduce inflammatory responses by enhancing IRF3 phosphorylation and the activation of IL-stimulated genes (such as MxA and OAS) under the induction of type I and III IFNs. The flavonoid extracts of Scutellaria baicalensis not only inhibit HA and NA activity, reduce the expression of TNF-α, IL-6, and MCP-1, and increase the expression levels of IFN-γ and IL-10 in lung tissue to regulate inflammatory responses, but also indirectly help the body recover by affecting the intestinal flora. In the treatment of influenza A-induced lung injury, the extract has better therapeutic effects than the single baicalin component.

In vitro experiments have found that the aqueous extract of Isatis indigotica root, S-03, has the effect of inhibiting HA and increasing the activity of antioxidant enzymes such as SOD, CAT, GSH-Px, and total antioxidant capacity. Additionally, the polysaccharide extract of Isatis indigotica root weakens the upregulation of inflammatory factors induced by influenza virus by activating the TLR-3 signaling pathway, significantly reducing the expression of IL-6, IP-10, MIG, and CCL5, and exhibiting inhibitory effects against human influenza viruses (H1N1 and H3N2) as well as avian influenza viruses (H6N2 and H9N2).

Influenza poses a significant threat to human survival. Due to the priority use of NA inhibitors in treatment strategies, there is a continuous emergence of resistant strains, and some drugs have severe side effects, such as neurotoxicity caused by adamantanes. In the face of the ever-increasing number of resistant strains, many effective compounds are still in the screening test stage, and clinical trials and market availability of related drugs still require a long period of time. The multi-target mechanism of action of traditional Chinese medicine (TCM) is not yet clear. Many drugs are only effective in vitro, with unknown mechanisms of action in vivo, and rely solely on long-term medication experience for influenza treatment. Additionally, some TCM ingredients have issues such as poor solubility, stability, and low bioavailability. Currently, there are two main aspects in antiviral drug research: one is to optimize or design new drugs targeting existing targets; the other is to identify new targets and develop new antiviral drugs.

Reference

Chen Jinfeng, He Jun, Xu Tao. Research Progress of 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.