The study targets the major proteases of SARS-CoV-2 involved in viral replication

Severe Acute Respiratory Syndrome The major protease of coronavirus 2 (SARS-CoV-2) is an enzyme involved in coronavirus transcription and replication. A new study led by S. Samar Hasnain of the University of Liverpool, UK, used the selenium-containing drug ebselen to use the SARS-CoV-2 main protease (M)._Professional).

They found that the selenium atom inactivates cysteine ​​in the catalytic pocket of the major protease — preventing SARS-CoV-2 growth and viral replication. Research results may help develop treatments to block the major proteases of the virus.

The researcher wrote:

“Our study is clearly an urgent concern for SARS-CoV-2, but broader of organoselenium compounds by a new chemical mechanism of protease cysteine ​​seleniumization in other current zoonotic betas. It has therapeutic applications. Coronavirus And what may appear in the future. “

The study of “a novel inhibitory mechanism of SARS-CoV-2 main protease by ebselen and its derivatives” bioRxiv* Servers and articles have been peer reviewed.

The rationale for current research

Previous studies by researchers used ebselen and five other derivatives to inhibit the major protease of SARS-CoV-2 and block its replication. They found that selenium-based ebselen compounds were most effective in inhibiting enzyme and viral replication.

Inhibition occurred because ebselen provided the selenium atom and formed a covalent bond at the major protease catalytic site. This blocked the histidine-cysteine ​​catalytic diad.

Based on the results, the team assumed in this current study that ebselen scaffolding could be used to improve selenium-based ebselen to further enhance antiviral activity.

result

Researchers investigated the reactivity of ebselen with the derivative Cys145 and whether it could inhibit the proteolytic activity of the SAR-CoV-2 main protease.

They found that ebselen and other compounds are potent inhibitors with sub-micromolar levels of IC.₅₀.. Two derivatives of ebselen, called MR6-7-2 and MR6-18-2, were twice as effective in inhibiting major proteases.

The team also tested ebselen and derivatives, and their ability to stop viral replication in SARS-CoV-2 infected human vero cells. Mr6-31-2 was three times more effective than the parent ebselen in promoting antiviral activity.

“These results show a clear on-target interaction between these compounds and M._Professional It has significant inhibitory power against SARS-CoV-2 and has the potential to develop treatments for COVID-19 patients. “

To determine the inhibition mechanism, researchers used an electron density map of selenium based on diffraction data using X-rays with wavelengths near the selenium absorption edge.

No selenium density was found in the ligand-free enzyme, but there was a strong density in the cysteine-histidine-catalyzed diad of the structure of the major protease treated with the ligand.

When using M, about 60% of selenium occupies space^Professional-80% when using Ebselen and M^Professional-Consistent with previous data found a bond in the catalytic pocket that causes the formation of a serenyl-sulfide bond with the MR6-31-2 crystal, Cys145.

The researchers observed that the inhibitory activity from ebuserene and its derivatives is induced by the selenium atom attached to the major protease catalytic site that inactivates cysteine.

Hydrolysis causes seleniumization of the catalyst site

Based on the results, the team suggested that this mechanism occurs due to hydrolysis at the major protease active site, which releases the phenolic by-product salicylanilide when interacting with ebselen.

The team used standard salicylanilide to optimize the LC / MRM-MS method and obtained a chromatogram to confirm this hypothesis. The ebselenium sample during incubation showed a peak of 6.37 minutes corresponding to standard salicylanilide.

The measured concentration of salicylanilide in the ebselenium sample was 1.28 ng / ml after 4 hours. This indicates that the formation of salicylanilide in the incubation of the main protease and ebselen occurs in a time-dependent manner.

The results confirm that the mechanism of seleniumization at the major protease active site occurred for the hydrolysis product.

“His41 assists in water-mediated attacks on intermediate adduct 2 in SNAr-type hydrolysis reactions, with intermediate 3 in a manner similar to the peptide hydrolyzed tetrahedral intermediate in the oxyanion hole of the active site. I suggest that it may be stabilized. “

*Important Notices

bioRxiv Publish preliminary scientific reports that should not be considered definitive as they are not peer-reviewed, guide clinical practice / health-related behaviors, and should not be treated as established information.