Vitamin C Binds to SARS Coronavirus-2 Main Protease Essential for Viral Replication
Tek Narsingh Malla, Suraj Pandey, Luis Aldama, Dennisse Feliz, Moraima Noda, Ishwor Poudyal, George N Phillips Jr, Emina A Stojković, Marius Schmidt
doi:10.1101/2021.05.02.442358
There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS-Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the protein's active site at 2.5 Ångstrom resolution. The crystal structure of the Vitamin C 3CLpro complex may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but on related proteases of other infectious viruses.
Supplementary Material Expression. The CoV-2 3CLpro sequence was synthetized (GenScript) for optimized expression in E. coli according to sequence information published previously (Zhang, Lin, Sun, et al., 2020) . In short, the N-terminus of 3CLpro is fused to glutathione-S-transferase (GST). It further has a 6-His tag at the c-terminus. The N-terminal GST will be autocatalytically cleaved off after expression due to an engineered 3CLpro cleavage sequence. Although the His tag can be cleaved off by a PreScission protease, the tag did not interfere with crystallization and consequently was left on. Overexpression and protein purification protocols were modified from previous reports. E. coli were grown to 0.8 OD 600 at 37 o in terrific broth. Expression was induced by 1 mmol/L IPTG at 25 o C. After 3 h of expression, the culture was induced a second time (1 mmol/L IPTG), and shaken overnight at 20 o C. The yield is about 80 mg for a 6 L culture. Cells were resuspended in lysis buffer (20mM Tris Base, 150 mmol/L NaCl, pH 7.8.). After lysis of the bacterial cells, debris was centrifuged at 50,000 g for 1 hour. The lysate was let stand at room temperature for 3 h (overnight is also possible). After this, the lysate was pumped through a column containing 15 mL of Talon Cobalt resin (TAKARA). The resin was washed without using imidazole using a wash cycle consisting of low salt (20 mmol/L Tris Base, 50 mmol/L NaCl, pH 7.8), high salt (20 mmol/L Tris Base, 1 mol/L NaCl, pH 7.8) and..
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