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 PA(N) 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 (K(d) = 0.378 μM) and a PA(N) construct, to identify small molecules that bind to the PA(N) 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 that 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.

Figure 1

Three known RdRp influenza endonuclease activity inhibitors (1–3) and the structure of the fluorescent ligand (4) that was developed for our FP assay.

Figure 2

Direct binding experiment to determine the K_d and dynamic range of 4. (◊) 4 alone (0% binding) (●) PA_N protein + 4 after a 15-min incubation (K_d = 0.378 μM). (■) PA_N protein + 4 after a 24-h incubation (K_d = 0.353 μM).

Figure 3

Competitive binding experiment measuring the displacement of 4 from PA_N. (▼) DMSO negative control (0% inhibition) (◊) 250 μM compound 2 (100% inhibition) (●) Compound 2 (K_i = 0.09 μM) (■) DPBA (K_i = 0.48 μM) (▲) Compound 3 (K_i = 0.85 μM) (♦) Compound 19 (no inhibition).

Figure 4

Viral plaque inhibition of candidates identified by FP. (▼) DMSO negative control (0% inhibition), (♦) oseltamivir carboxylate positive control (100% inhibition), (○) Compound 16 (IC₅₀ >100 μM), (□) Compound 43 (IC₅₀ >100 μM), (∆) Compound 31 (IC₅₀ >50 μM), (◊) No infection, (●) Compound 42 (IC₅₀ = 14.28 ± 0.07 μM), (▽) Compound 44 (IC₅₀ = 18.61 ± 0.09 μM), (■) Compound 45 (IC₅₀ = 12.68 ± 0.08μM), (▲) Compound 2 (IC₅₀ = 3.13 ± 0.08 μM).