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

Crystallization. The recombinant production and purification of SARS-CoV M pro with authentic N and C termini was described in detail previously. 48, 49 Using an Amicon YM10 membrane (EMD Millipore), the purified SARS-CoV M pro was concentrated to 21 mg·mL -1 in buffer A (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.5). Crystallization was performed by equilibrating 1 µL protein (mixed with 1 µL precipitant solution) against 500 µl reservoir containing 6 -8% polyethylene glycol (PEG) 6,000, 0.1 M MES (pH 6.0), at 20℃ using the vapor diffusion sitting-drop method. Compounds 11a and 11s were dissolved in 100% DMSO at 50 mM and 200 mM stock concentrations, respectively. A crystal of the free enzyme was soaked in cryo-protectant buffer containing 20% MPD, 6% PEG 6,000, 0.1 M MES, 7.5 mM 11a, pH 6.0, for 2 h at 20℃. Another set of free enzyme crystals was soaked in another cryoprotectant buffer with 6% PEG 6,000, 5% MPD, 0.1 M MES, 15% glycerol, 10 mM 11s, pH 6.0, for 2 h. Subsequently, crystals were fished and flash-cooled in liquid nitrogen prior to data collection.