The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.

• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• Fluorometry methods   MeSH
• Humans MeSH
• Piperidines pharmacology   MeSH
• Pyridines MeSH
• Pyrimidines MeSH
• Pyrroles MeSH
• RNA Caps metabolism   MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   metabolism   MeSH
• High-Throughput Screening Assays * methods   MeSH
• Viral Proteins * antagonists & inhibitors   metabolism   MeSH
• Influenza A virus drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

• MeSH
• Crystallography, X-Ray MeSH
• Quantum Theory MeSH
• Models, Molecular MeSH
• Mutation genetics   MeSH
• Mutant Proteins metabolism   MeSH
• Protein Subunits antagonists & inhibitors   chemistry   metabolism   MeSH
• Protein Domains MeSH
• Pyridines chemistry   pharmacology   MeSH
• Pyrimidines chemistry   pharmacology   MeSH
• Pyrroles chemistry   pharmacology   MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   chemistry   metabolism   MeSH
• Thermodynamics MeSH
• Drug Resistance, Viral drug effects   MeSH
• Viral Proteins antagonists & inhibitors   chemistry   metabolism   MeSH
• Influenza A virus drug effects   enzymology   MeSH
• Publication type
• Journal Article MeSH