Identification of Influenza Endonuclease Inhibitors Using a Novel Fluorescence Polarization Assay

Abstract

Influenza viruses have been responsible for the largest pandemics in the previous century. Although vaccination and prophylactic antiviral therapeutics are the primary defense against influenza virus, there is a pressing need to develop new antiviral agents to circumvent the limitations of current therapies. The endonuclease activity of the influenza virus PAN protein is essential for virus replication and is a promising target for novel anti-influenza drugs. To facilitate the discovery of endonuclease inhibitors, we have developed a high-throughput fluorescence polarization (FP) assay, utilizing a novel fluorescein-labeled compound (Kd = 0.378 μM) and a PAN construct, to identify small molecules that bind to the PAN endonuclease active site. Several known 4-substituted 2,4-dioxobutanoic acid inhibitors with high and low affinities have been evaluated in this FP-based competitive binding assay, and there was a general correlation between binding and the reported inhibition of endonuclease activity. Additionally, we have demonstrated the utility of this assay for identifying endonuclease inhibitors in a small diverse targeted fragment library. These fragment hits were used to build a follow up library that that led to new active compounds which demonstrate FP binding and anti-influenza activities in plaque inhibition assays. The assay offers significant advantages over previously reported assays, and is suitable for high-throughput and fragment-based screening studies. Additionally the demonstration of the applicability of a mechanism-based ‘targeted fragment’ library supports the general potential of this novel approach for other enzyme targets. These results serve as a sound foundation for the development of new therapeutic leads targeting influenza endonuclease.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

“…Structural studies have now revealed new opportunities for drug discovery that target the essential endonuclease activity of the virus (24)(25)(26). In the current study, we had two primary goals; to confirm that the known endonuclease inhibitor L-742,001 targets the enzyme within the intact virus by generating resistant mutants, and to survey the resistance potential of the endonuclease by characterizing the sites of mutation.…”

“…S5A shows the full kinetic analysis from which V max and K M values of 3764190 s −1 and 1566 nM, respectively, were derived. We then measured the binding of our fluorescent probe (25) to PA N ΔLoop by fluorescent polarization, and the K d was found to be 44 nM (Fig. 3B).…”

“…For the FRET-based activity assay, the substrate is 5′-Cy5-GAATACTCAAGCTATGCATC-3IAbRQSp (20,32). Details of our fluorescent binding probe and the fluorescent polarization assay have been described (25).…”

Mutational Analysis of the Binding Pockets of the Diketo Acid Inhibitor L-742,001 in the Influenza Virus PA Endonuclease

“…Another endonuclease inhibitor identified by these investigators is flutimide, a fully substituted 1-hydroxy-3H-pyrazine-2,6-dione isolated from a fungus (21), which served as a lead compound for developing a series of more potent aromatic analogues (22). These discoveries were soon followed by others, resulting in a chemical variety of reported influenza virus endonuclease inhibitors, e.g., N-hydroxamic acid and N-hydroxyimide compounds (23), tetramic acid series and diketobutanoates (24), polyphenolic catechins (25), phenethylphenylphthalimide analogs derived from thalidomide (26), macrocyclic bisbibenzyls (27), a group of compounds bearing distinct pharmacophoric fragments (28), and 3-hydroxyquinolin-2(1H)-ones (29). An effort was also made to define the essential pharmacophore from the available structure-activity relationship (24).…”

Inhibition of PA endonuclease activity of influenza virus RNA polymerase by Kampo medicines

“…The endonuclease activity of the N-terminus of PA, which has a highly conserved sequence and has an essential function for influenza virus transcription and multiplication, has been identified as a potential target for developing novel anti-influenza virus drug (19) with low susceptibility to viral resistance. We demonstrate here that several Kampo medicines including Kakkonto, Shosaikoto, Saikokeishito, Keishito, Maobushisaishinto, and Maoto are able to inhibit recombinant PA endonuclease activity, whereas Kampo medicine Chikujountanto at a highest tested concentration of 100 μg/mL is unable to inhibit the enzyme activity.…”