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

A buffer containing 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3, was used for all the enzymatic assays. Two substrates with the cleavage sites of M pro and 3C pro , respectively (indicated by the arrow, ↓), Dabcyl-KTSAVLQ¯SGFRKM-E(Edans)-NH2 and Dabcyl-KEALFQ¯GPPQF-E(Edans)-NH2 (95% purity; Biosyntan), were employed in the fluorescence resonance energy transfer (FRET)based cleavage assay, using a 96-well microtiter plate. The dequenching of the Edans fluorescence due to the cleavage of the substrate as catalyzed by the proteases was monitored at 460 nm with excitation at 360 nm, using a Flx800 fluorescence spectrophotometer (BioTek). Curves of relative fluorescence units (RFU) against substrate concentration were linear for all substrates up to beyond 50 µM, indicating a minimal influence of the inner-filter effect. Stock solutions of the compounds were prepared by dissolving them in 100% DMSO. The UV absorption of 11a was found to be negligible at l = 360 nm, so that no interference with the FRET signal through the inner-filter effect was to be expected. For the determination of the IC50, different proteases at a specified final concentration (0.5 µM SARS-CoV or HCoV-NL63 M pro , 2 µM CVB3 3C pro , 3 µM EV-A71 3C pro ) were separately incubated with the inhibitor at various concentrations (0 to 100 μM) in reaction buffer at 37℃ for 10 min. Afterwards, the reaction was initiated by adding FRET peptide substrate at 20 μM final concentration (final volume: 50 μl). The IC50 value was determined by using the GraphPad Prism 6.0 software (GraphPad). Measurements of enzymatic activity were performed in triplicate and are presented as the mean ± standard deviations (SD).