Introduction to coronavirus: SARS, MERS and 2019-nCov, the prevention and treatment (IV)

 

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Introduction to coronavirus: SARS, MERS and 2019-nCov, the prevention and treatment (IV)


 

An overview of 3CLpro and PLpro proteins

3CLpro and PLpro are two proteases that play a role in the replication and packaging of next-generation viruses, and both process peptide translation from genomic RNA to structural or non-structural proteins. PLpro can also act as a deubiquitinase, its function is to deubiquitinate host cell proteins such as interferon factor 3 (IRF3), and inactivate the pathway of nuclear factor-activated B cell k light chain enhancer vated B cells (NF-kB). This can cause immune-infected host cells to be immunosuppressed. It is precisely because these two proteases are related to virus replication and control of host cells that they are vital to the virus, making them viable targets for antiviral drugs. Similar to the RdRp protein, 2019-nCoV and SARS-CoV have 96% significant sequence identity on the 3CLpro coding. 3CLpro will automatically form dimers, each of which contains two regions, the N-terminal catalytic region and the C-terminal region. Most of the amino acid residues on the catalytic region that show differences between the two viruses are on the protein surface. Although S46 (2019-nCoV) / A (SARS-CoV) may interact with substrates or inhibitors that bind to the active site, its small structural change from A (alanine) to S (serine) The binding of the small molecule inhibitor to the active site should not be significantly altered. Small molecule inhibitors that can effectively inhibit SARS-nCoV 3CLpro are expected to have similar effects on 2019-nCoV 3CLpro.

Unlike 3CLpro, PLpro from both viruses shared only 83% sequence. The different amino acid residues between the two viruses cover almost all surfaces of PLpro. Significant changes in amino acid composition can affect the interaction of two PLpro enzymes with their ligands. However, the three secondary structural components that form the active site did not change in the two PLpro proteins. Therefore, inhibitors developed for SARS-CoV PLpro may also be applicable for 2019-nCoV PLpro.

Most of the research over the past two decades for treatment of SARS-CoV has focused on the development of small molecules, peptides and peptidomimetic inhibitors of 3CLpro and PLpro. In terms of binding and inhibiting both proteases, many inhibitors are at μM levels. However, there are currently some highly effective low nanomolar inhibitors that can be used in combination with other protease inhibitor therapies to help fight the virus. We will classify inhibitors based on the proteases that are inhibited to prevent the virus from controlling the host cell. Each compound was tested using SARS-CoV, MERS-CoV or deubiquitinated cell models. Hundreds of small molecule compounds that inhibit 3CLpro and PLpro have been developed, and we have selected several of the most effective inhibitors since the early 2000s. These compounds are in the low micromolar range at inhibitory concentrations, and there is room for further development. However, extensive structure-activity relationship studies are available to guide which substituents need to be modified in targeted 2019-nCoV inhibitor studies. In addition, the summary can guide researchers and health professionals in the use of combination therapies with two or more of these compounds, and there are precedents for combination therapies in treating patients with coronavirus infection. One compound, 3CLpro-1  has an IC50 value of 200 nM for SARS-CoV. This strong inhibitory effect may be sufficient against the 2019-nCoV virus.

3CLpro and PLpro are both cysteine ​​proteases. Covalent inhibitors with high titers may be developed for them. Recently, Zhou and colleagues developed a class of potential covalent cysteine ​​protease inhibitors that specifically target coronavirus entry. Although not directly related to 3CLpro and PLpro, such small vinyl sulfone molecules are capable of inhibiting virus replication in the nM range. The team found that the combination of serine protease inhibitor camostat with carmustastat and its vinylsulfone cysteine ​​protease inhibitor was able to fight SARS-CoV. Compared with the control group, the survival rate of mice with SARS-CoV treated with the combination therapy was significantly improved. They studied several variants of small vinyl sulfone molecules. These small vinyl sulfone molecules provide a new structural backbone for the study of the structure-activity relationship of drugs. In addition, these small molecules can be tested for specific inhibitory effects on 3CLpro and PLpro to further elucidate their mechanism of action. Considering their highly effective inhibition of SARS-CoV, they may be equally effective for 2019-nCoV.

Other coronavirus subfamily

Further understanding of the Coronavirus subfamily could also provide inspiration for new treatment options, such as the study of feline coronavirus (FCoV) and its mutant form of feline infectious peritonitis virus (FIPV). It was found that the tripeptide bisulfite adducts GC376 and NPI64 have a significant inhibitory effect on FIPV replication at a concentration of 0.04 μM. FIPV and 3CLpro of SARS-CoV share approximately 50% sequence identity, but the overall structure is conservative. In FRT-based activity analysis, it was found that the IC50 value of GC376 for 3CLpro in SARS-CoV was 4.9 times that of FIPV. Combined with higher cytotoxicity of compounds (GC376 and NPI64 in CRFK cells are CC50> 150 μM and CC50 = 61.91 μM, respectively), these masked-aldehyde warheads inhibitors on 2019-nCoV 3CLpro should be studied as soon as possible.

Summary

2019-nCoV and SARS-CoV have very high sequence homology on their RdRp and 3CLpro proteins. Previous studies have identified small molecule inhibitors that target these two proteins in SARS-CoV. We assume that Remdesivir and 3CLpro-1 could be directly used to treat 2019-nCoV. Because Remdesivir is a drug in clinical trials, relevant Chinese authorities may negotiate with Gilead to decide whether it is possible to use the drug in 2019-nCoV patients. The 2019-nCoV spike protein's RBD-ACE2 binding region is significantly different from the SARS-CoV spike protein, especially in the two regions that directly interact with the ACE2 protein receptor. This difference ruled out the possibility of previously developing antibodies and therapeutic peptides against the SARS-CoV spike protein RBD for the treatment of 2019-nCoV. However, the development of homologous peptides of RBD and ACE2 and their cocktail therapy for inhibiting RBD-ACE2 interaction to prevent infection may be a rapid treatment option.

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