Discovery of a Novel Specific Inhibitor Targeting Influenza A Virus Nucleoprotein with Pleiotropic Inhibitory Effects on Various Steps of the Viral Life Cycle

Abstract

Influenza viruses (IAV) continue to pose an imminent threat to human due to annual influenza epidemics outbreak and episodic pandemics with high mortality. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for new additional prophylactic and therapeutic options, including new antiviral targets and drugs with new mechanisms of action to prevent and treat influenza infection. Here we characterized a novel influenza A nucleoprotein (NP) inhibitor FA-6005 that inhibited a broad spectrum of human pandemic, seasonal influenza A and B viruses in vitro and protects mice against lethal influenza A virus challenge. The small molecule FA-6005 targeted a conserved NP I41 domain and acted as a potential broad, multi-mechanistic anti-influenza virus therapeutic since FA-6005 suppressed influenza virus replication and perturbed intracellular trafficking of viral ribonucleoproteins (vRNP) from early to late stage. Cocrystal structures of the NP/FA-6005 complex reconciled well with concurrent mutational studies. This study provides the first line of direct evidence suggesting that the newly-identified NP I41 pocket as an attractive target for drug development that inhibit the multiple functions of NP. Our results also highlighted FA-6005 as a promising candidate for further development as an antiviral drug for the treatment of IAV infection and provide chemical-level details for inhibitor optimization. Importance Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Therefore, there is an urgent need for broad-spectrum inhibitors to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and the emergence of antiviral drug resistance. Herein we identified a novel influenza A virus NP antagonist FA-6005 with broad-spectrum efficacy against influenza viruses and our study presented a comprehensive study of mode of action of FA-6005 with the crystal structure of the compound in complex with NP. The influenza inhibitor holds promise as an urgently sought-after therapeutic option offering a complementary mechanism of action to existing antiviral drugs for the treatment of influenza virus infection, and that should further aid development of universal therapeutics.

Discovery of ligustrazine and chalcone derivatives as novel viral nucleoprotein nuclear export inhibitors against influenza viruses

“…To further confirm the role of A9 in blocking viral infection, an IFA was performed to visualize the expression and translocation of the IAV NP. The NP is the most abundantly expressed protein during the influenza virus infection and plays important roles during the replication and transcription stage 28,32 . MDCK (Figure 5A) or A549 cells (Figure 5B) were challenged with A/Puerto Rico/8/1934 at an MOI of 1 and treated with DMSO (0.05%) or A9 (10 μM).…”

“…The recombinant NP‐N397S variant was rescued through an eight‐plasmid DNA transfection system. In this system, eight plasmids of DNA were transfected into cocultured 293T and MDCK cells 27,28 . Viral supernatants were harvested 72 h posttransfection, and propagated using 9 day old embryonated chicken eggs 29 .…”

“…The NP is the most abundantly expressed protein during the influenza virus infection and plays important roles during the replication and transcription stage. 28,32 MDCK (Figure 5A) or A549 cells (Figure 5B) were challenged with A/Puerto Rico/8/1934 at an MOI of 1 and treated with DMSO (0.05%) or A9 (10 μM). As shown in Figure 5A,B, the NP (stained green) in the cells treated with DMSO were distributed in the nucleus (stained blue) and cytoplasm at 10 h postinfection, while almost all of the NP treated with A9 was retained in the nucleus.…”

“…In this system, eight plasmids of DNA were transfected into cocultured 293T and MDCK cells. 27,28 Viral supernatants were harvested 72 h posttransfection, and propagated using 9 day old embryonated chicken eggs. 29 The inhibitory effects of A9 on the recombinant NP-N397S mutant virus and parent A/Puerto Rico/8/1934 virus were further tested through a viral yield reduction assay.…”

Antiviral Drugs in Influenza

“…The molecular target of FA-6005 is amino acid residue 41, thereby inhibiting the activity of the vRNP complex. The interaction of FA-6005 has been shown to reduce NP/vRNP export, impair the trafficking of circulating RNPs in the cytoplasm, perturb the virus uncoating process and vRNP import, and lead to disruption of the budding of daughter virions [ 150 ].…”