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

Testing for inhibitory activity of candidate compounds. Initially, we performed a quick assessment of the inhibitory activity of the candidate compounds towards the enteroviral and coronaviral replicons at a concentration of 40 µM in Huh-T7 cells. Compounds that were relatively powerful and non-toxic at this concentration, were assayed in a dose-dependent manner to estimate their half-maximal effective concentration (EC50) as well as their cytotoxicity (CC50), as described. 29 In brief, different concentrations of a-ketoamides (40 µM in screening experiments or increasing concentration (0, 1.25, 2.5, 5, 10, 20, 40 µM) when determining the EC50) were added to growth medium of replicon-transfected Huh-T7 cells. Twenty-four hours later, the cells were washed with 1 mL phosphate-buffered saline (PBS or OPTIMEM, Invitrogen) and lysed in 0.15 mL Passive lysis buffer (Promega) at room temperature (RT) for 10 min. After freezing (-80 o C) and thawing (RT), the cell debris was removed by centrifugation (16,000 x g, 1 min) and the supernatant (10 or 20 µl) was assayed for Firefly or Renilla luciferase activity (Promega or Biotrend Chemikalien) using an Anthos Lucy-3 luminescence plate reader (Anthos Microsystem).