Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

Linlin Zhang; Daizong Lin; Yuri Kusov; Yong Nian; Qingjun Ma; Jiang Wang; Albrecht von Brunn; Pieter Leyssen; Kristina Lanko; Johan Neyts; Adriaan de Wilde; Eric J. Snijder; Hong Liu; Rolf Hilgenfeld

The antiviral activity of the compounds was evaluated in a cytopathic effect (CPE) readout assay using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, innersalt]-based assay. Briefly, 24 h prior to infection, cells were seeded in 96-well plates at a density of 2.5 x 10 4 (RD cells) or of 1.7 x 10 4 (HeLa Rh) per well in medium supplemented with 2% FCS. For HRV2 and HRV14 infection, the medium contained 30 mM MgCl2. The next day, serial dilutions of the compounds and virus inoculum were added. The read-out was performed 3 days post infection as follows: The medium was removed and 100 μl of 5% MTS in Phenol Red-free MEM was added to each well. Plates were incubated for 1 h at 37°C, then the optical density at 498 nm (OD498) of each well was measured by microtiter plate reader (Saffire 2 , Tecan). The OD values were converted to percentage of controls and the EC50 was calculated by logarithmic interpolation as the concentration of compound that results in a 50% protective effect against virus-induced CPE. For each condition, cell morphology was also evaluated microscopically.