Bithiazole Inhibitors of Phosphatidylinositol 4‐Kinase (PI4KIIIβ) as Broad‐Spectrum Antivirals Blocking the Replication of SARS‐CoV‐2, Zika Virus, and Human Rhinoviruses

Abstract Over half a century since the description of the first antiviral drug, “old” re‐emerging viruses and “new” emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co‐infections, make the war against viruses quite challenging. Herein we report a host‐targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS‐CoV‐2 at low micromolar and sub‐micromolar concentrations. However, while the anti‐hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS‐CoV‐2 entry/replication is debated.


1) Chemistry
General. All commercially available chemicals were purchased from Merck or Fluorochem and, unless otherwise noted, used without any previous purification. Solvents used for work-up and purification procedures were of technical grade. TLC was carried out using Merck TLC plates (silica gel on Al foils, SUPELCO Analytical). Where indicated, products were purified by silica gel flash chromatography on columns packed with Merck Geduran Si 60 (40-63 µm). 1 H and 13 C NMR spectra were recorded on BRUKER AVANCE 300 MHz and BRUKER AVANCE 400 MHz spectrometers.
Chemical shifts (δ scale) are reported in parts per million relative to TMS. 1  Microwave Irradiation Experiments. Microwave reactions were conducted using a CEM Discover Synthesis Unit (CEM Corp., Matthews, NC). The machine consists of a continuous focused microwave power delivery system with an operator-selectable power output from 0 to 300 W. The temperature inside the reaction vessel was monitored using a calibrated infrared temperature control mounted under the reaction vessel. All experiments were performed using a stirring option whereby the reaction mixtures were stirred by means of a rotating magnetic plate located below the floor of the microwave cavity and a Teflon-coated magnetic stir bar in the vessel. ADME analysis. UV/LC-MS analyses of water solubility (for compounds 1, 4a, 4b and 4c), PAMPA, and Stability Tests (in human plasma and medium/serum solution) were performed by using Agilent 1100 LC/MSD VL system (G1946C) (Agilent Technologies, Palo Alto, CA) constituted by a vacuum solvent degassing unit, a binary high-pressure gradient pump, an 1100 series UV detector, and an 1100 MSD model VL benchtop mass spectrometer. MSD single-quadrupole instrument was equipped with the orthogonal spray API-ES (Agilent Technologies, Palo Alto, CA). The pressure of the nebulizing gas and the flow of the drying gas (nitrogen used for both) were set at 40 psi, 9 L/min, respectively. The capillary voltage, the fragmentor voltage, and the vaporization temperature were 3000 V, 70 V, and 350 °C, respectively. MSD was used in the positive and negative ion mode.
Spectra were acquired over the scan range m/z 50-1500 using a step size of 0.1. Chromatographic separation was obtained using a Phenomenex Kinetex C18-100Å column (150 x 4.6 mm, 5 μm S3 particle size) at room temperature and gradient elution with a binary solution (eluent A: H2O, eluent B: ACN; both eluents were acidified with formic acid 0.1% v/v). The analysis started with 5% of B (from t = 0 to t = 3 min), then B was increased to 95% (from t = 3 to t = 12 min), then kept at 95% (from t = 12 to t = 18 min) and finally return to 5% of eluent B in 2.0 min. The flow rate was 0.6 mL/min and injection volumes were 10 μL. UV detection was monitored at 254 nm. Quantification of the single compound was made by comparison with appropriate calibration curves.
The water solubility of compound 4d was determined using an LC-MS/MS system consisting of a Varian apparatus (Varian Inc) including a vacuum solvent degassing unit, two pumps (212-LC), an MSD triple quadrupole mass spectrometer (Mod. 320-LC) with ES interface and Varian MS Workstation System Control Vers. 6.9. Chromatographic separation was obtained using a Phenomenex Kinetex C18-100Å column (20 x 2.1 mm, 2.6 μm particle size) and gradient elution with a binary solution (eluent A: H 2 O, eluent B: ACN; both eluents were acidified with formic acid 0.1% v/v). The analysis started with 5% of B (from t = 0 to t = 1 min), then B was increased to 95% (from t = 1 to t = 8 min), then kept at 95% (from t = 8 to t = 10 min) and finally return to 5% of eluent     Apparent permeability (Papp) and membrane retention (%MR) were calculated according to the equations reported in reference [1].

Cells and viruses for ZIKV/SARS-CoV-2 testing
The H/PF/2013 ZIKV strain belonging to the Asian lineage was kindly provided by the Istituto and 1% Penicillin/Streptomycin (Pen/Strep, Euroclone). The same medium with a lower FBS concentration (1%) was used for the viral propagation and drug susceptibility testing. Cells were incubated at 37°C in a humidified incubator supplemented with 5% CO2. All the viral stocks were titrated by plaque reduction assay (PRA) as previously described. [5] Drugs and cytotoxicity assay Sofosbuvir (cat. HY-15005) and camostat mesylate (cat. HY-13512) were purchased from MedChem Express (https://www.medchemexpress.com) and resuspended in 100% DMSO and water, respectively. Once determined the CC50, for each compound was chosen a not-toxic dose used as starting drug concentration in the subsequent antiviral assays.

Antiviral assays (ZIKV, SARS-CoV-2)
To determine the antiviral activity of candidate compounds against ZIKV and SARS-CoV-2, a direct yield reduction assay (DYRA) based on infection of cells in presence of serial drug dilutions was performed as previously described. [6,7] Briefly, 7,000 Huh7 cells per well were infected with 50 TCID50 of ZIKV in 96-well plates for 1 h at 37°C with 5% CO2 and after virus removal, serial dilutions of each tested compound, starting from the not-toxic dose, were added to the cells and incubated at 37°C with 5% CO2. Similarly, 10,000 Calu-3 were infected with 100 TCID50 of SARS-CoV-2 in 96well plates for 2 h at 37°C with 5% CO2 and after virus removal, serial dilutions of each tested S8 compound were added to the cells and incubated at 37°C with 5% CO2. After 72 h of incubation, the antiviral activity was measured on cell monolayer by immunodetection assay (IA). [6,7] The IA protocol was adapted for the detection of SARS nucleocapsid protein in infected Calu-3 cells. Briefly, fixation and permeabilization were performed as previously described and cell lines were incubated The half-maximal inhibitory concentration (IC50) was calculated through a non-linear regression analysis of the dose-response curves generated with GraphPad PRISM software version 6.01. In each test, sofosbuvir and camostat mesylate were used as reference compounds against flaviviruses and SARS-CoV-2, respectively. Infected and uninfected cells without drugs were used to calculate the 100% and 0% of viral replication, respectively. Selectivity Index (SI) was calculated as ratio between CC50 and IC50.

Multicycle CPE reduction antiviral assay (HRV-02, HRV-14)
A multicycle CPE reduction assay to determine the antiviral activity of the compounds was performed as previously described in ref. [8]. In short, subconfluent HelaR19 (ATCC catalogue no. CCL2) were treated with serial dilutions of compounds and the cells were subsequently infected with virus at a MOI 0.01 for HRV-02 and HRV-14. Cells were incubated at 37°C for 3 days until full CPE was observed in virus infected untreated cell controls. In parallel, a cell viability assay to determine cytotoxicity was performed using the Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's protocol. The optical density at 490 nm was determined using a BioTek Gen5 microplate reader. The concentration of compound that inhibits virus-induced CPE by 50% (EC50) was calculated by nonlinear regression analysis with GraphPad Prism Version 6.

PI(4)P lipid staining
The staining methods was performed as previously described in (doi: 10.1042/BJ20090428) with slight modifications. HeLa R19 cells grown on coverslips in 24-well plates were treated with 10 μM of compound for 1 hour, fixed with 2% formaldehyde (FA) in PBS for 15 minutes and permeabilized with 100 μM digitonin in Buffer A (20 mM Pipes, pH 6.8, 137 mM NaCl, 2.7 mM KCl) for 10 minutes.
The cells were blocked with 5% Normal Goat Serum (NGS) and 50 mM NH4Cl in Buffer A for 45 minutes. Between each step, cells were washed three times PBS. The cells were incubated with the primary antibody mouse anti-PI4P IgM (Echelon Biosciences, Z-P004) diluted 1:100 in buffer A supplemented with 5% NGS for 1 hour on ice, and with the secondary antibody Alexa Fluor 594 Goat anti-Mouse IgM (Invitrogen) diluted 1:400 in buffer A supplemented with 5% NGS and 1:1000 DAPI solution for another hour also on ice. The cells were fixed with 2% FA in PBS for 10 minutes, washed with PBS + 50 mM NH4Cl and mounted with Fluorsave on coverslides. The slides were imaged with an Olympus BX60 fluorescence microscope.

S10
Supplementary Figure S1: Effect of compounds 4a-4d on intracellular PI4P levels. HeLa R19 cells were treated with 10 μM compound, stained for PI4P and imaged with fluorescence microscopy. As a positive control, the known PI4Kβ inhibitor BF738735 was used. [9] Examples are shown from one experiment representative of two independent experiments. Scale bars correspond to 5 μm.

Viability assays in human lymphocytes
Human peripheral blood mononuclear cells were isolated by means of Lympholyte density gradient centrifugation starting from buffy coat. Mononuclear cells were incubated for 4h with the compounds in study at 50M or with the vehicle (DMSO 0.5%). Following incubation with propidium iodide 10 µg/mL for 1 minute in the dark, at room temperature, mononuclear cells were immediately subjected to flow cytometry analysis: viable cells were identified as cells negative for the red fluorescence. The percentage of viability among lymphocytes, identified through the gating in the forward scatter (FSC)side scatter (SSC) (FSC low: SSC low) plot of mononuclear cells, was calculated with respect to lymphocytes exposed to vehicle (DMSO 0.5%).

In vitro kinase inhibition assays
Recombinant full length, HIS6-tagged PI4KIIIβ was purchased from ProQinase at 30 °C for 10 min. Reactions were stopped by adding 5μL of phosphoric acid 0.8%. Aliquots (10 μL) were then transferred into a P30 Filtermat (PerkinElmer), washed five times with 0.5% phosphoric acid and four times with water for 5 min. The filter was dried and transferred to a sealable plastic bag, and scintillation cocktail (4 mL) was added. Spotted reactions were read in a scintillation counter (Trilux, Perkinelmer). IC50 values were obtained according to Equation (1), where v is the measured reaction velocity, V is the apparent maximal velocity in the absence of inhibitor, I is the inhibitor concentration, and IC50 is the 50% inhibitory concentration. PI and PS were dissolved in chloroform/methanol 9:1 and mixed at a 1:3 ratio. After chloroform/methanol evaporation, water was added to 1:62.5 w/v and the mixture sonicated to clarity.