Isocorilagin, isolated from Canarium album (Lour.) Raeusch, as a potent neuraminidase inhibitor against influenza A virus.

Canarium album (Lour.) Raeusch (C. album) as a normally medicinal and edible plant has been used widely in Asian countries and is considered a source of phytochemicals that are beneficial to human health. Here, we showed at the first time isocorilagin, a polyphenolic compound isolated from C. album, displayed antiviral activity against diverse strains of influenza A virus (IAV), including A/Puerto Rico/8/34 (H1N1), A/Aichi/2/68 (H3N2) and NA-H274Y (H1N1) with IC50 value of 9.19 ±â€¯1.99, 23.72 ±â€¯2.51 and 4.64 ±â€¯3.01 µM, respectively. Further mechanistic studies revealed that it clearly inhibited neuraminidase activity of IAV and directly influenced the virus release. The molecular docking studies presented isocorilagin could bind to the highly conserved residues in the active sites of NA, implying that isocorilagin may be effective against various influenza strains and not susceptible to produce drug resistance. Taken together, the results strongly suggest that isocorilagin has potential to be an effective, safe and affordable neuraminidase inhibitor against a diverse panel of IAV strains. More importantly, our work expands the biological activities of C. album extracts and provide a new option for the development of anti-influenza drug.

A Small-Molecule Compound Has Anti-influenza A Virus Activity by Acting as a ''PB2 Inhibitor".

With regular influenza epidemics and the prevalence of drug-resistant influenza virus strains, it is extremely crucial to develop effective and low-toxicity anti-influenza A virus drugs that act on conserved sites of novel targets. Here, we found a new anti-influenza virus compound, 1,3-dihydroxy-6-benzo[ c]chromene (D715-2441), from a library of 8026 small-molecule compounds by cell-based MTT assay and explored the underlying mechanisms. Our results revealed that D715-2441 possessed antiviral activities against multiple subtypes of influenza A viruses (IAVs) strains, including H1N1, H5N1, H7N9, H3N2, the clinical isolate 690 (H3), and oseltamivir-resistant strains with the H274Y NA mutation, and suppressed the early steps in the virus replication cycle. Further mechanistic studies indicated that D715-2441 clearly inhibited viral polymerase activity and directly influenced the location of the PB2 protein. Moreover, binding affinity analyses confirmed that D715-2441 bound specifically to the PB2cap protein. Further, protein sequence alignment and a computer-aided molecular docking indicated that highly conserved amino acid residues in the cap-binding pocket of PB2cap were possible binding sites for D715-2441, which indicates that D715-2441 might be employed as a cap-binding competitor. Moreover, the combination of D715-2441 and zanamivir possessed a remarkable synergistic antiviral effect, with an FICI value of 0.40. In conclusion, these results strongly suggest that D715-2441 has potential as a promising candidate against IAV infection. More importantly, our work offers novel options for the strategic development of PB2cap inhibitors of IAV.

TRAF3 Positively Regulates Host Innate Immune Resistance to Influenza A Virus Infection.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is one of the intracellular adaptor proteins for the innate immune response, which is involved in signaling regulation in various cellular processes, including the immune responses defending against invading pathogens. However, the defense mechanism of TRAF3 against influenza virus infection remains elusive. In this study, we found that TRAF3 could positively regulate innate antiviral response. Overexpression of TRAF3 significantly enhanced virus-induced IRF3 activation, IFN-ß production, and antiviral response, while TRAF3 knockdown promoted influenza A virus replication. Moreover, we clarified that inhibiting ubiquitinated degradation of TRAF3 was associated with anti-influenza effect, thereby facilitating antiviral immunity upon influenza A virus infection. We further demonstrated the key domains of TRAF3 involved in anti-influenza effect. Taken together, these results suggested that TRAF3 performs a vital role in host defense against influenza A virus infection by the type-I IFN signaling pathway. Our findings provide insights into the development of drugs to prevent TRAF3 degradation, which could be a novel therapeutic approach for treatment of influenza A virus infection.

Methyl brevifolincarboxylate, a novel influenza virus PB2 inhibitor from Canarium Album (Lour.) Raeusch.

Methyl brevifolincarboxylate (MBC) was isolated from ethyl acetate extract of Canarium album (Lour.) Raeusch. The structure was identified, and the effect on influenza A virus infection was evaluated. MBC exhibited inhibitory activity against influenza virus A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) with IC50 values of 27.16 ± 1.39 µM and 33.41 ± 2.34 µM. Mechanism studies indicated that MBC inhibited the replication of influenza A virus by targeting PB2 cap-binding domain. Our results demonstrated MBC was a potent PB2 cap-binding inhibitor and represented as a new type of promising lead compound for the development of anti-influenza virus drugs from natural products.

Dihydromyricetin is a new inhibitor of influenza polymerase PB2 subunit and influenza-induced inflammation.

Development of new and effective anti-influenza drugs is critical for the treatment of influenza virus infection. The polymerase basic 2 (PB2) subunit as a core subunit of influenza A virus RNA polymerase complex is considered to be an attractive drug target for anti-influenza drug discovery. Dihydromyricetin, as a natural flavonoid, has a wide range of biological activities, but its anti-influenza A virus activity is ambiguous. Here, we found dihydromyricetin could inhibit the replication of a variety of influenza A virus strains. Mechanism studies demonstrated that dihydromyricetin reduced viral polymerase activity via selective inhibition of viral PB2 subunit, and decreased relative amounts of viral mRNA and genomic RNA during influenza A virus infection. The binding affinity and molecular docking analyses revealed that dihydromyricetin interacted with the PB2 cap-binding pocket, functioned as a cap-binding competitor. Interestingly, dihydromyricetin also reduced cellular immune injury by inhibiting TLR3 signaling pathway. Additionally, combination treatment of dihydromyricetin with zanamivir exerted a synergistic anti-influenza effect. Altogether, our experiments reveal the antiviral and anti-inflammatory activities of dihydromyricetin in vitro against influenza virus infection, which provides a new insight into the development of novel anti-influenza drugs.

Discovery of dihydropyrrolidones as novel inhibitors against influenza A virus.

More effective prophylactic and therapeutic strategies to combat influenza viruses are urgently required worldwide because the conventional anti-influenza drugs are facing drug resistance. Here, dihydropyrrolidones (DHPs), the products of an efficient multi-components reaction, were found to possess good activities against influenza A virus (IAV). Primary structure-activity relationship indicated that the activities of DHPs were greatly influenced by substituents and four of them had IC50 values lower than 10 µM (DHPs 5-2, 8, 14 and 19: IC50 = 3.11-9.23 µM). The activities against multiple IAV strains and mechanism of DHPs were further investigated by using 5-2 (IC50 = 3.11 µM). It was found that 5-2 possessed antiviral effects against all the investigated subtypes of IAVs with the IC50 values from 3.11 to 7.13 µM. Moreover, 5-2 showed very low cytotoxicity with CC50 > 400 µM. Results of mechanism study indicated that 5-2 could efficiently inhibit replication of IAV, up-regulate the expression of key antiviral cytokines IFN-ß and antiviral protein MxA, and suppress the production of the NDAPH oxidase NOX1 in MDCK cells. These results indicated that 5-2 could be used as a potential inhibitor against wide subtypes of IAVs.

Key amino acid residues of neuraminidase involved in influenza A virus entry.

Generally, influenza virus neuraminidase (NA) plays a critical role in the release stage of influenza virus. Recently, it has been found that NA may promote influenza virus to access the target cells. However, the mechanism remain unclear. Here, we reported that peramivir indeed possessed anti-influenza A virus (IAV) activity in the stage of viral entry. Importantly, we verified the critical residues of influenza NA involved in the viral entry. As a result, peramivir as an efficient NA inhibitor could suppress the initiation of IAV infection. Furthermore, mutational analysis showed NA might be associated with viral entry via amino acids residues R118, E119, D151, R152, W178, I222, E227, E276, R292 and R371. Our results demonstrated NA must contain the key amino acid residues can involve in IAV entry.

The role of oxidative stress in influenza virus infection.

Virus-induced oxidative stress plays an important role in the regulation of the host immune system. In this review, we provide backgrounds of the pathogenic mechanism of oxidative stress induced by influenza virus and the specific oxidant-sensitive pathways, and highlight that antioxidant is one of the effective strategies against influenza virus infection.