Drug Discovery of Host CLK1 Inhibitors for Influenza Treatment

Abstract

The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.

Keywords: CLK1 inhibitor; CPE assay; baculovirus coinfection; influenza A virus; protein expression and purification; rational screening.

Figure 1

Construction of CLK1/pFastBac1 plasmid and the protein expression of CLK1 in insect cells. (A) A schematic of the steps to construct CLK1/pFastBac1 recombinant plasmid; (B) Identification of the constructed CLK1/pFastBac1 recombinant plasmid by both colony PCR assay and double digestion. C-1~5: clone 1 to 5. Cpos: positive clone. Cneg: negative clone; (C) Identification of recombinant bacmid CLK1/pFastBac1 by PCR analysis. NTC: non-template control; (D) Determination the optimal conditions to express the recombinant protein His₆-CLK1 by Coomassie Brilliant Blue staining.

Figure 2

Analysis of CLK1 recombinant protein purified by Ni-NTA column. (A) SDS-PAGE and Coomassie staining of purified His₆-CLK1 by Ni-NTA column after baculovirus-mediated expression in Sf9 insect cells. NPI50, NPI80 and NPI250 represent the increasing concentrations of imidazole in the elution buffer are 50, 80 and 250 mM, respectively. NPI250 shows purified His₆-CLK1 subsequently eluted from the Ni-NTA affinity column; (B) Western Blot analysis of CLK1 and His in crude insect cellular extract and purified His₆-CLK1 components.

Figure 3

Optimization of parameters in Kinase-Glo^® luminescent kinase assay. (A) Substrate concentration-dependent phosphorylation of CLK1; (B) Enzyme concentration-dependent phosphorylation of CLK1. ATP concentrations and MBP concentrations were 1 μM and 50 ng/μL, and incubation times were 90 min at 25 °C; (C) Time-dependent phosphorylation of CLK1. The incubation times of kinase reaction were set as 10, 30, 60, 90, 120, 150 and 180 min; (D) Mg²⁺ concentration-dependent phosphorylation of CLK1; (E) Mn²⁺ concentration-dependent kinase reaction of CLK1; (F) Temperature-dependent phosphorylation of CLK1; (G) ATP concentration-dependent phosphorylation of CLK1 with concentrations of 0.1, 1, 10 and 100 μM.

Figure 4

Evaluation of the screening model for CLK1 inhibitor. The calculated Z′ value was 0.70. RLU = relative light unit.

Figure 5

Virtual screening of lead compounds with CLK1 inhibitory effect. (A) Top five poses docking of ligand V25 in binding pocket of 2VAG, with RMSD value of 1.38 Å; (B) The interaction between ligand V25 and receptor is crucial for binding; (C) The proportion of different levels of scores according to virtual screening.

Figure 6

The structures and IC₅₀s of CLK1 inhibitors after screening based on Kinase-Glo^® Assay. (A) flavonoids; (B) tannins; (C) coumarins; (D) stilbenes.

Figure 6

The structures and IC₅₀s of CLK1 inhibitors after screening based on Kinase-Glo^® Assay. (A) flavonoids; (B) tannins; (C) coumarins; (D) stilbenes.