key: cord-0946930-i10e721u
authors: Tao, Xuan; Zhang, Lu; Du, Liubing; Lu, Kai; Zhao, Zhennan; Xie, Yanxuan; Li, Xiaobo; Huang, Shuxiang; Wang, Pei-Hui; Pan, Ji-An; Xia, Wei; Dai, Jun; Mao, Zong-Wan
title: Inhibition of SARS-CoV-2 replication by zinc gluconate in combination with hinokitiol
date: 2022-03-01
journal: J Inorg Biochem
DOI: 10.1016/j.jinorgbio.2022.111777
sha: 878d6afbccd768df972a98157f34ef1d88ffc79f
doc_id: 946930
cord_uid: i10e721u

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is currently the major challenge to global public health. Two proteases, papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro or Mpro), are indispensable for SARS-CoV-2 replication, making them attractive targets for antiviral therapy development. Here we screened a panel of essential metal ions using a proteolytic assay and identified that zinc gluconate, a widely-used zinc supplement, strongly inhibited the proteolytic activities of the two proteases in vitro. Biochemical and crystallographic data reveal that zinc gluconate exhibited the inhibitory function via binding to the protease catalytic site residues. We further show that treatment of zinc gluconate in combination with a small molecule ionophore hinokitiol, could lead to elevated intracellular Zn(2+) level and thereby significantly impaired the two protease activities in cellulo. Particularly, this approach could also be applied to rescue SARS-CoV-2 infected mammalian cells, indicative of potential application to combat coronavirus infections. Our studies provide the direct experimental evidence that elevated intracellular zinc concentration directly inhibits SARS-CoV-2 replication and suggest the potential benefits to use the zinc supplements for coronavirus disease 2019 (COVID-19) treatment.

listed in Table S1 . The expression and purification of PLpro C112S mutant was similar to WT-PLpro.

The expression plasmid of authentic SARS-CoV-2 3CLpro was prepared as previously described. 1 In brief, SARS-CoV-2 3CLpro gene was PCR-amplified using the nsp5 as template with primers listed in Table S1 . The 12 nucleotides encoding amino acids AVLQ were added before the Ser1 of 3CLpro to generate the autocleavage site of SARS-CoV-2 3CLpro. The 24 nucleotides encoding amino acids GPH 6 were added at the C-terminus to form a PreScission cleavable site. The PCR product was digested by BamHI and XhoI and ligated into pGEX-6p-1 vector, which was digested by the same restriction enzymes to generate pGEX-6p-1-3CLpro plasmid.

The pGEX-6p-1-3CLpro plasmid was transformed into BL21(DE3) cell for protein expression. 5 mL overnight culture at 37°C was transferred into 1 L fresh Luria-Bertani (LB) medium supplemented with 50 μg/mL carbenicillin with agitation (220 rpm) until OD 600 reaches 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM to induce protein expression. The purification procedures were the same with described in SARS-CoV-2 PLpro purification. Expression plasmid of 3CLpro D155A/C156S mutant was generated via site-directed mutagenesis using WT-3CLpro plasmid as a DNA template. The primers used for site-directed mutagenesis are listed in Table S1 . The expression and purification of 3CLpro D155A/C156S mutants were similar to WT-3CLpro.

The fluorogenic peptide substrate Dabcyl-FRLKGGAPIKG(Edans)V (greater than 95% purity) was synthesized by Genscript. The proteolytic assay buffer contains 20 mM Tris-HCl, pH 7.5, 300 mM NaCl and 1 mM TECP. Metal ions were added into protein and incubate for 20 minutes on ice before measurements. Enzymatic reactions were carried out at 30°C in black 96-well well plate. Proteolytic activities of SARS-CoV-2 PLpro were measured by monitoring APIKG(Edans)V production with the excitation and emission wavelengths of 329 nm and 520 nm respectively within 30 minutes on a Cytation 3 multi-mode plate reader. Fluorescence intensity was converted to the amount of hydrolyzed substrate using a standard curve drawn from the fluorescence measurements of well-defined concentrations of APIKG(Edans)V peptides. Initial reaction rates were calculated for each reaction within 10 minutes. The initial reaction rates and substrate concentrations were fitted to Michaelis-Menten curve using Graphpad software to obtain V max and K m values.

The fluorogenic peptide substrate MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 (greater than 95% purity) was synthesized by Genscript. The reaction buffer contains 20 mM Tris-HCl, pH 7.5, 300 mM NaCl and 1 mM TECP. Metal ions were added into proteins to pre-incubate 20 minutes on ice before measurements. Relative activity was calculated as ∆RFU mutant /∆RFU wild type ×100%..

The interactions between metal ion and SARS-CoV-2 PLpro/3CLpro were examined using the fluorescence-based protein thermal shift assay. The fluorescent dye Sypro orange (Sigma) was used to monitor the unfolding of SARS-CoV-2 PLpro/3CLpro. The reaction mixtures were added in a 96-well PCR plate containing 2 μL proteins (final concentration 1 mg/mL), 4 μL metallic compounds (final concentration 50 μM), 2.5 μL 8× Sypro orange dye and 11.5 μL reaction buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl and 1 mM TECP). The screening was carried out using a StepOnePlus thermal cycler (Life Technologies) with the procedures of heating from 25 °C to 99 °C at a rate of 1 °C/min. The fluorescence intensity was monitored with the excitation and emission wavelengths of 490 nm and 530 nm, respectively. Each compound was measured in triplicates.

To examine the deubiquitination activity of PLpro, the reaction mixture contains 100 nM SARS-CoV-2 PLpro, 1 μM ubiquitin-AMC (BioVision) and 10 μM zinc gluconate in 20 mM Tris-HCl buffer (pH 7.5). Zinc gluconate was added into proteins to pre-incubate 20 minutes on ice before measurements. The SARS-CoV-2 PLpro deubiquitination activity was determined by monitoring AMC released by the enzymes using Cytation 3 multi-mode reader at the excitation and emission wavelengths of 380 nm and 460 nm, respectively. 

Production of Authentic SARS-CoV Mpro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction