A novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol 4-kinase IIIβ

Abstract

Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model.

Fig 1

Molecular structures of compounds 1 and 2.

Fig 2

Antiviral activities of compounds 1 and 2 on a single virus replication cycle. Cells were infected at low MOI with various HEV and HRV species, the cardiovirus EMCV, or the aphthovirus ERAV. After infection, 1 μM compound 1 or 3 μM compound 2 was added to the cells. After 8 h, cells were lysed by freeze-thawing to release intracellular virus particles, and the total virus titer was determined by endpoint titration. Bars represent means of three samples ± the SD.

Fig 3

Compound 1 is a potent inhibitor of enterovirus RNA replication. (A) BGM cells were infected with CVB3-Rluc at an MOI of 1. Following infection, the virus was removed and compound 1 or compound 2 was added to the cells. The values obtained with the replication inhibitor GuaHCl, used at 2 mM, are shown as a dashed line. (B) BGM cells were transfected with RNA transcripts of EV71 or CVB3 replicons. Immediately after transfection, the medium was replaced by fresh (compound-containing) medium. For the experiments in both panels A and B, at 8 h postinfection or posttransfection, cells were lysed to quantify the intracellular amount of luciferase as a measure of viral RNA replication. Data points or bars represent means of three samples ± the SD.

Fig 4

Resistance of CVB3 mutants to compound 1. (A and B) BGM cells were infected with CVB3 wild type, CVB3 3A-V45A, or CVB3 3A-H57Y (A) or transfected with the corresponding RNA transcripts of full-length infectious CVB3 clones (B). Immediately after infection or transfection, various concentrations of compound 1 were added to the cells. After 8 h, cells were lysed by freeze-thawing to release intracellular virus particles, and the total virus titer was determined by endpoint titration. (B) Bars represent means of three samples ± the SD. Significant differences compared to wild-type virus are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5

Compounds 1 and 2 specifically inhibit PI4KIIIβ in vitro. Recombinant PI4KIIIβ or PI4KIIIα was incubated in the presence of compound 1 (upper panel) or compound 2 (lower panel) with their substrate, phosphatidylinositol, in the form of Triton micelles and radioactively labeled ATP. After termination of the enzyme reaction with phosphoric acid, the amount of radioactive ATP that was incorporated into the micelles was quantified as a measure of PI4K activity. Data were converted to the percent inhibition relative to controls. Data points represent the means of three samples ± the SD.

Fig 6

Compound 1 binds in the ATP-binding pocket of PI4KIIIβ. (A) HeLa R19 cells were transfected with FAPP1-PH-GFP and treated 1 day later with 1 μM compound 1 for 1 h, after which endogenous PI4KIIIβ was stained. (B and C) HeLa R19 cells were cotransfected with plasmids carrying FAPP1-PH-GFP and either HA-tagged PI4KIIIβ wt (B) or PI4KIIIβ-Y583M (C). The next day, cells were treated for 1 h with 1 μM compound 1, after which the overexpressed PI4KIIIβ was stained with an antibody against HA. Nuclei were stained with Hoechst stain.

Fig 7

CVB3 replication in the presence of compound 1 or 2 is rescued by expression of a PI4KIIIβ mutant. BGM cells were transfected with HA-tagged PI4KIIIβ wt, PI4KIIIβ-Y583M or, as negative controls, the kinase-dead PI4KIIIβ-D656A or EGFP. Two days posttransfection, cells were infected with CVB3-Rluc in the presence of 1 μM compound 1 or 3 μM compound 2. After lysis of the cells at 8 h p.i., the amount of luciferase activity was quantified in the samples. Bars represent means of three samples ± the SD.

Fig 8

Plasma drug concentration levels of compound 2 in NMRI mice. Mice were treated with a single dose of compound 2 as either 1 mg/kg i.v. or 5 mg/kg orally. Terminal blood samples were collected from three animals per time point, up to 8 h postdosing. Samples were centrifuged, and the supernatant was subjected to mass spectrometry analysis. Data points represent the means of three samples ± the SD.

Fig 9

Antiviral activity of compound 2 in a CVB4-induced pancreatitis mouse model. (A) Pancreas histopathology (H&E stain) in CVB4-induced pancreatitis. Mice were infected with CVB4, treated with compound 2 (right panel) or left untreated (left panel), and sacrificed 3 days p.i. (B) Histopathological severity scoring for CVB4-induced pancreatitis. H&E-stained tissue sections were scored blindly for inflammation, necrosis, and edema by using a standardized scoring system. A score of 0 indicates complete absence of pathology, whereas a score of 3 refers to diffuse lesions throughout the tissue section. (C) Effect of compound 2 on serum markers for pancreatitis. At day 3 p.i., serum was collected and lipase/amylase were quantified in enzymatic colorimetric assays. (D) Effect of compound 2 on infectious virus production, which was quantified by titration of tissue homogenates from cell cultures.