Author: 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
Title: Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication Document date: 2020_2_10
ID: 7n8p9okf_68
Snippet: Assays with MERS-CoV and SARS-CoV were performed as previously described. 61, 63 In brief, Huh7, Vero, or Vero E6 cells were seeded in 96-well plates at a density of 1 × 10 4 (Huh7 and Vero E6) or 2 × 10 4 cells (Vero) per well. After overnight growth, cells were treated with the indicated compound concentrations or DMSO (solvent control) and infected with an MOI of 0.005 (final volume 150 µl/well in Eagle's minimal essential medium (EMEM) con.....
Document: Assays with MERS-CoV and SARS-CoV were performed as previously described. 61, 63 In brief, Huh7, Vero, or Vero E6 cells were seeded in 96-well plates at a density of 1 × 10 4 (Huh7 and Vero E6) or 2 × 10 4 cells (Vero) per well. After overnight growth, cells were treated with the indicated compound concentrations or DMSO (solvent control) and infected with an MOI of 0.005 (final volume 150 µl/well in Eagle's minimal essential medium (EMEM) containing 2% FCS, 2 mM L-glutamine, and antibiotics). Huh7 cells were incubated for two days and Vero/VeroE6 cells for three days, and differences in cell viability caused by virus-induced CPE or by compound-specific side effects were analyzed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer's instructions. Absorbance at 490 nm (A490) was measured using a Berthold Mithras LB 940 96-well plate reader (Berthold). Cytotoxic effects caused by compound treatment alone were monitored in parallel plates containing mock-infected cells. HCoV-229E-Rev: 5′-ggTCGTTTAGTTGAGAAAAGT -3′, and 229E-ZNA probe: 5'-6-Fam-AGA (pdC)TT(pdU)G(pdU)GT(pdC)TA(pdC)T-ZNA-3-BHQ-1 -3' (Metabion). Standard curves were prepared using serial dilutions of RNA isolated from virus stock. Data were analysed using GraphPad Prism 5.0; EC50 values were calculated based on a 4parameter logistic statistics equation. In parallel to the qPCR assays with inhibitors, cell viability assays were performed using Alamar-Blue™ Cell Viability Reagent (ThermoFisher) according to the manufacturer's instruction. CC50 values were calculated using inhibitor versus normalized response statistics equation by including proper controls (no inhibitor and 1% Triton-X-100-treated cells).
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