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.

Multicycle CPE-reduction assay Subconfluent monolayers of the indicated cell lines seeded in 96-well plates were treated with serial dilutions of ITZ and infected with virus at the lowest MOI that resulted in full CPE within 3 days. The medium contained 2% fetal bovine serum. Subsequently, cells were incubated at 37°C for three days until complete CPE was observed in the infected and untreated virus controls. Cell viability was determined with an MTS assay by incubating the cells with AQueous One Solution Cell Proliferation Assay (Promega) and measuring the optical density of each well at 490 or 498 nm using a microplate reader. Raw optical density values were converted to percentage of untreated and uninfected cell controls after subtraction of background values obtained with virus controls. The concentration of compound that inhibits virus-induced cell death by 50% (50% effective concentration [EC 50 ]) was calculated by nonlinear regression analysis. Cytotoxicity of ITZ was assessed in a similar set-up, and 50% cytotoxic concentration (CC 50 ) values were derived from cell viability values determined with an MTS assay. Each experiment was performed at least in triplicate.