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Discovery of Influenza Polymerase PA-PB1 Interaction Inhibitors Using an In Vitro Split-Luciferase Complementation-Based Assay.

ABSTRACT: The limited therapeutic options and increasing drug-resistance call for next-generation influenza antivirals. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. Here, we developed an in vitro split luciferase complementation-based assay to speed up screening of PA-PB1 interaction inhibitors. By screening 10,000 compounds, we identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Further mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. Overall, we developed a robust high throughput-screening assay for screening broad-spectrum influenza antivirals targeting PA-PB1 interaction and identified R151785 as a promising antiviral drug candidate.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7028398 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundIP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.ObjectivesBased on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1-604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and methodsIn the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.ResultsAs expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.ConclusionsIt seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

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Discovery of Influenza Polymerase PA-PB1 Interaction Inhibitors Using an In Vitro Split-Luciferase Complementation-Based Assay.

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