ITRACONAZOLE INHIBITS ENTEROVIRUS REPLICATION BY TARGETING THE OXYSTEROL-BINDING PROTEIN

Strating, Jeroen R.P.M.; van der Linden, Lonneke; Albulescu, Lucian; Bigay, Joëlle; Arita, Minetaro; Delang, Leen; Leyssen, Pieter; van der Schaar, Hilde M.; Lanke, Kjerstin H.W.; Thibaut, Hendrik Jan; Ulferts, Rachel; Drin, Guillaume; Schlinck, Nina; Wubbolts, Richard W.; Sever, Navdar; Head, Sarah A.; Liu, Jun O.; Beachy, Philip A.; De Matteis, Maria A.; Shair, Matthew D.; Olkkonen, Vesa M.; Neyts, Johan; van Kuppeveld, Frank J.M.

The interaction between ITZ and GFP-hOSBP-SII was investigated by MicroScale Thermophoresis (MST) [see e.g. (Jerabek-Willemsen et al., 2011; Seidel et al., 2013) ]. pEGFP-hOSBP-SII was transfected into HEK293T cells using polyethelyneimine (PEI) (Polysciences) and after ~5hrs the medium was replaced by expression medium (293SerumFree medium [Gibco] supplemented with GlutaMax [Gibco], 3 g/l Primatone-RL-UF, 2 g/l D-glucose monohydrate, 3.7 g/l NaHCO 3 , 1.5% DMSO, 100 U/ml penicillin and 100 μg/ml streptomycin). After two days, cells were harvested by centrifugation, washed with PBS and pelleted again. The cell pellet was snap-frozen in liquid nitrogen and stored at -80°C, or used directly for a purification. Cells were lysed in lysis buffer (50 mM Tris-HCl pH7.4, 250 mM NaCl, 1 mM EDTA, 0.5% nonidet-P40 with protease inhibitor complex [Roche]) for 15 min on ice, centrifuged for 20 min at ~20Kxg, and bound to StrepTactin beads (IBA). Beads were loaded in a column (Bio-Rad), unbound material was drained and beads were washed with washing buffer (50mM Tris-HCl pH7.4, 250 mM NaCl, 1 mM EDTA). Proteins were eluted in elution buffer (washing buffer with 10% glycerol, 2.5mM biotin and protease inhibitor complex), aliquoted, snapfrozen in liquid nitrogen and stored at -80°C. Purity of the protein preparations was checked by SDS-PAGE stained with GelCode Blue (Pierce) and protein concentrations were determined using Bradford reagent (Bio-Rad). For MST measurements, proteins were thawed on ice, centrifuged for 5 min at full speed in a table top centrifuge to remove any protein aggregates, and diluted in MST buffer (50 mM Tris-HCl pH7.6, 150 mM NaCl, 10 mM MgCl 2 ) supplemented with 0.05% Tween-20. ITZ was serially diluted in 16 two-fold dilution steps in MST buffer with Tween-20. Protein and ITZ dilutions were mixed, loaded in standard treated capillaries (NanoTemper Technologies) and measured using a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies) equipped with a blue filter set, which is compatible with GFP fluorescence. Measurements were performed at 22-24°C, 20% MST power, 20% LED power. Individual experiments were analyzed using the NTAnalysis software (NanoTemper Technologies), normalized fluorescence values of individual experiments (Fnorm[1/1000]) were exported to Excel, base-line corrected by substracting the average of the first three values (lowest ITZ concentrations, plateau for OSBP with no ITZ bound), and normalized to maximal binding using the amplitude calculated for each measurement by the NTAnalysis software. Data of three individual experiments were averaged. Data were plotted and a curve was fitted with the fit function from the law of mass action. Figure 2 , and Renilla luciferase levels were measured after 7 hr. Acute toxicity of the drug treatments was analyzed in parallel using an MTS assay as in Figure 1B (C). The toxicity of treatment with 100 μM ketoconazole prevents drawing a conclusion about any antiviral effect of this drug concentration on CVB3. (D) HeLa R19 cells were infected with RLuc-EMCV, treated with Hedgehog pathway antagonists as in Figure 2 , and Renilla luciferase levels were measured after 6 hr. (E) HeLa R19 cells were treated with 10 μM antifungal azoles for 6 hr, fixed and cholesterol was stained with filipin. Cholesterol was redistributed only by ITZ, posaconazole and ketoconazole, but not by fluconazole or voriconazole. (F) HeLa R19 cells were infected with RLuc-EMCV, treated with βest