key: cord-323908-8dgngwmw
authors: He, Zhesheng; Zhao, Wencong; Niu, Wenchao; Gao, Xuejiao; Gao, Xingfa; Gong, Yong; Gao, Xueyun
title: Molecules inhibit the enzyme activity of 3-chymotrypsin-like cysteine protease of SARS-CoV-2 virus: the experimental and theory studies
date: 2020-05-31
journal: bioRxiv
DOI: 10.1101/2020.05.28.120642
sha: 
doc_id: 323908
cord_uid: 8dgngwmw

SARS-CoV-2 has emerged as a world public health threat. Herein, we report that the clinical approved auranofin could perfectly inhibit the activity of 3-chymotrypsin-like cysteine protease (Mpro or 3CLpro) of SARS-CoV-2. Gold cluster could significantly inhibit 3CLpro of SARS-COV-2. Phenyl isothiocyanate and Vitamin K3 could well suppress the activity of 3CLpro. For Mpro inhibition, IC50 of auranofin, Vitamin K3, phenyl isothiocyanate, gold cluster are about 0.51μM, 7.96μM, 10.13μM, 1.61μM, respectively. These compounds may be with potentials for treatment SARS-CoV-2 virus replication. Especially for FDA approved auranofin, it is an anti-inflammation drug in clinic, thus it may with strong potential to inhibit virus replication and suppress the inflammation damage in COVID-19 patients. Gold cluster is with better safety index and well anti-inflammation in vitro/vivo, therefore it is with potential to inhibit virus replication and suppress the inflammation damage caused by COVID-19 virus. As Au(I) ion is active metabolism specie derived from gold compounds or gold clusters in vivo, further computational studies revealed Au ion could tightly bind thiol group of Cys145 residue of 3CLpro thus inhibit enzyme activity. Also, phenyl isothiocyanate and Vitamin K3 may interact with thiol group of Cys145 via Michael addition reaction, molecular dynamic (MD) theory studied are applied to confirmed these small molecules are stable in the pocket and inhibit Mpro activity.

A new coronavirus named as COVID-19 virus (also called SARS-CoV-2 virus) is responsible for the 2020 pandemic outbreak in the world. The viral 3-chymotrypsin-like cysteine protease (3CLpro, also called Mpro) enzyme controls this COVID-19 virus replication and is essential for its life cycle. Therefore, 3CLpro is drug target in the case of SARS-CoV-2. Our biochemistry studied revealed that the gold compounds such as auranofin, Isothiocyanate compounds such as phenyl isothiocyanate, vitamin K such as vitamin K3, and gold cluster such as glutathione coated gold cluster could well inhibit the activity of 3CLpro. Further DFT and MD computational studies also verified these small molecules could interact Cys145 and amino acid residues of Mpro, thus suppress the activity of Mpro. These compounds could serve as potential anti-SARS-CoV-2 lead molecules for further drug studies to combat COVID-19.

Recombinant COVID-19 virus 3CLpro with native N and C termini was expressed in Escherichia coli and subsequently purified following the recently reported work (1) . The full-length gene encoding COVID-19 virus Mpro was synthesized for Escherichia coli expression. Briefly, the expression plasmid was transformed into Escherichia coli cells and cultured in Luria Broth medium containing 100 μg/ml ampicillin at 37 °C. When the cells were grown to OD600 of 0.6-0.8, 0.5 mM IPTG was added to the cell culture to induce the expression at 16 °C. After 10 h, the cells were harvested by centrifugation at 3,000g. The cell pellets were resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl), lysed by high-pressure homogenization, and then centrifuged at 25,000g for 

In order to characterize 3CLpro enzymatic activity, we used a For Mpro inhibition, IC50 of phenyl isothiocyanate is about 10 .13µM, data showed as following Figure 3 . Phenethyl isothiocyanate is a constituent of cruciferous vegetables that has cancer preventive activity including lung, prostate, and breast cancer (7) . The potential of this compound to inhibit COUID-19 virus is an interesting topic as phenethyl isothiocyanate is a good candidate to be a dietary supplement. 

It is widely accepted that gold compounds metabolism in vivo and the produced Au(I) would interact with specific thiol group of protein to interfere their normal functions (12) . The interactions between the gold atom with the binding pockets of proteins were studied by density functional theory (DFT) calculations. For the calculations, only the main amino acids of the protein binding pockets were taken into account. All geometries were fully optimized using the B3LYP method in conjunction with the SDD basis set for Au and the 6-31G(d,p) for other nonmetals (13, 14, 15) .

The SDD pseudopotential was also applied for Au. During optimization, SMD4 solvation model was utilized to model the water environment. All the calculations were carried out using Gaussian 09 package (16) . The binding energy (Eb) between Au and the pocket ligands were calculated using the following equation: Eb = EAu+ + Eligands -EAu-ligands. where EAu+, Eligands, and EAu-ligands were the total energies of Au+ ion, the ligands, and the Au-ligand complex structures, respectively. Eligands were the single-point energy for the Au-ligand complex with the Au atom removed from the system. 

Density-functional thermochemistry. III. The role of exact exchange

Relativistic effects in gold chemistry. I. Diatomic gold compounds

A complete basis set model chemistry. II. Open-shell systems and the total energies of the first-row atoms

Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions

Scalable molecular dynamics with NAMD

OpenMM simulations using the CHARMM36 additive force field