key: cord-0724733-3vyt2dko
authors: Sacco, Michael Dominic; Hu, Yanmei; Gongora, Maura Verenice; Meilleur, Flora; Kemp, Michael Trent; Zhang, Xiujun; Wang, Jun; Chen, Yu
title: The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition
date: 2022-03-15
journal: Cell Res
DOI: 10.1038/s41422-022-00640-y
sha: e265562aa8fd9abd4b46e282c3475a804aade0ce
doc_id: 724733
cord_uid: 3vyt2dko

nan

Using QuikChange site directed mutagenesis, Pro132 was mutated to His in a pET29a(+) vector with the cloned gene for the BetaCoV/Wuhan/WIV04/2019 SARS CoV-2 main protease, producing a gene that is equivalent to that found in the Omicron SARS-CoV-2. Protein was then expressed and purified as previously described 1 . Crystals of OM pro with GC-376 were grown by incubating 15 mg/mL OM pro overnight at 4 °C with two-fold molar excess GC-376. Protein was diluted to 5 mg/mL in gel filtration column buffer (20 mM Tris pH 8.0, 150 mM NaCl) and DMSO was added for a final concentration of 4 %. OM Pro was then mixed with crystallization buffer (20% PEG 3350, 0.2 M KNO 3 ) and allowed to grow in a hanging drop apparatus at 20 °C for approximately two weeks.

A plate-like crystal with sides of ~ 200 x 150 µm was transferred to a cryo-protectant solution of 27.5 % PEG 3350, 0.2 M KNO 3 , and 15% glycerol, and transferred to an Oxford Cryosystems nitrogen cryostream for data collection. An X-ray data set was collected at 100 K on a Rigaku MicroMax-007 HF microfocus rotating anode X-ray generator equipped with a Dectris EIGER R 4M detector. A total of 180 frames were collected with an exposure time of 120 s per frame and Δφ of 1°. The data were indexed and integrated using CrysAlis Pro (Rigaku, The Woodlands, Texas, USA) and scaled and merged with AIMLESS 2 in the CCP4 suite. The final model was solved via molecular replacement using PDB ID 7C6U as the reference model for Molrep 3 . This structure was subsequently refined using Refmac5 4 and Coot 5 .

Sequence data for SARS-CoV-2 isolates was gathered using the CoVsurver of the Global Initiative on Sharing Avian Influenza Data, Developed by A*STAR Bioinformatics Institute (BII), Singapore. All numbers shown are accurate as of Jan 15, 2022.

For determination of K m and V max , WT or P132H M pro was diluted in the reaction buffer (20 mM HEPES (pH 6.5), 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT, and 20% glycerol) to the final Time-dependent proteolysis was performed by diluting WT or P132H M pro to 100 nM in the reaction buffer and incubating at 37 °C for 0, 1, 3, 6, 12, 24, or 48 hrs. At each time point, 10 µM FRET substrate was added to initiate the reaction, which was monitored every 90 seconds for 1 h at 30 °C. Initial velocity was determined by linear regression for the first 15 min.

The binding of M pro inhibitors to WT or P132H M pro was determined by differential scanning fluorimetry (DSF) using QuantStudio TM 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA), as previously described 7, 8 . DSF was carried out in a 96-well PCR plate by mixing 

Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M(pro) and cathepsin L

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