Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo

Abstract

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ). A good correlation between PI4KIIIβ activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIβ inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIβ inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIβ were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIβ is deleterious.

Fig 1

Antiviral effects of aminothiazole series of compounds that appear to target PI4KIIIβ. (A) Structures of example compounds compared to PIK93 and T-00127-HEV1. (B) Titer of wild-type HRV-B14 and I42V HRV-B14 mutant after being in the presence of compounds at 10× EC₅₀ for 24 h (n = 1). (C) Inhibitory effects of compounds (10 μM) on in vitro activities of lipid kinases (averages of two experiments with standard deviations [error bars]). (D) Correlation of HRV-A16 antiviral and replicon activity and in vitro PI4KIIIβ activity.

Fig 2

Confirmation of mechanism of action of aminothiazole series through siRNA knockdown of PI4KIIIβ. (A) Expression of PI4KIIIβ determined by qRT-PCR and Western blot analysis. The qRT-PCR results are averages of four samples with standard deviations (error bars). Two passages of H1-HeLa cells were examined by Western blotting (passage 6 and 44). The positions of molecular weight in kDa are shown to the right of the Western blot. (B) Effect of knockdown of various lipid kinases or irrelevant gene (IRR) via siRNA transfection on HRV infection of H1-HeLa cells at two multiplicities of infection (MOIs). Data are averages of four samples with standard deviations. The data are representative of three experiments. (C) Expression of lipid kinases as determined by qRT-PCR after siRNA treatment for 6 days (averages of four samples with standard deviations). (D) Effect of compound 1 targeting PI4KIIIβ and the protease inhibitor rupintrivir on HRV infection in siRNA-transfected H1-HeLa cells (MOI of 0.05). Data are averages of four samples with standard deviations. The data are representative of three experiments.

Fig 3

Repeat dosing in SJL mice suggests that short-term inhibition of PI4KIIIβ is deleterious. Survival graphs after compound 2 and T-00127-HEV1 were dosed at 50 and 250 mg/kg over 7 days (n = 20 for each dose).