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The Mpro protease (also known as 3CLpro), is a viral non-structural protein from the virus SARS-CoV-2 ^[1], responsible for a major outbreak of the disease called COVID-19, declared pandemic by WHO in 11 march 2020 ^[2]. It has an important role in virus replication, as it’s responsible for cleavage of the polyproteins of the virus, alongside with papain-like protease(s) ^[3](3).

Function

The Mpro protein function is mainly deduced from the function of SARS-CoV virus Mpro, which has a 96% amino acid identity and a highly similar three-dimensional structure with SARS-CoV-2 Mpro ^[1]. As a protease, Mpro is an enzyme that causes proteolysis, which means that it breaks protein peptide bonds by hydrolysis ^[4] (4). Indeed, the Mpro processes the replicase polyprotein 1ab (pp1ab ~790 kDa) translated from the viral RNA ORF1ab ^[1][5](1, 5). In fact, Mpro cleaves 11 sites of pp1ab and the recognition sequence at most sites is between Leu-Gln and (Ser, Ala, Gly) ^[1][3](1, 3). Proteins resulting from this polyprotein cleavage are non-structural proteins (NSPs) and they seem to contribute with viral replication and transcription ^[5] (5). Thus, by processing an important number of non-structural proteins, this enzyme plays a critical role in SARS-CoV-2 replication.

Structure

The Mpro is a protein of approximately 30 kDa ^{[5] [6]} (5, 6) consisting of two protomers containing 306 amino acid residues each ^[1]. This protomers dimerize forming a homodimer ^[1]. (To view the primary and secondary structure of SARS-CoV-2 Mpro visit https://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6Y2E). Each protomer consists of three domains: I (chymotrypsin-like; residues 10-99), II (picornavirus 3C protease-like; residues 100-182), and III (a globular cluster; residues 198-303). Domains I and II comprise six-stranded antiparallel β-barrels and domain III comprises five α-helices ^[1][6] (1, 6). The substrate-binding site is located between domains I and II with the catalytic site containing the amino acid residues Cys145 and His41 ^[1]. Domain III, in turn, has been shown to be involved in the regulation of Mpro dimerization, what is necessary for the catalytic activity of this enzyme once it helps to shape the substrate-binding site ^{[1] [7]} (1, 7).

An attractive drug target

As mentioned above, SARS-CoV-2 Mpro has 96% sequence identity with SARS-CoV Mpro and as expected, also a highly similar three-dimensional structure ^[1]. Indeed, it has been shown that the substrate-binding pocket is a highly conserved region of Mpro among an important number of CoV Mpros ^[6](6). However, an interesting difference found between SARS-CoV Mpro and SARS-CoV-2 Mpro is that in the first one there is a polar interaction between the domains III of each protomer, involving the residues Thr285, what is not found in the COVID-19 virus Mpro ^[1]. In fact, in SARS-CoV-2, the threonine is replaced by alanine, leading to a higher proximity between the two domains III of the dimer ^[1].

Structural comparison with SARS-CoV M^pro

As have been shown, because of its importance for viral replication, inhibiting SARS-CoV-2 Mpro activity could lead to viral replication blockage ^[1][6] (1, 6). Moreover, no human proteases has been reported to have a similar cleavage specificity and so, in this aspect, Mpro inhibitors toxic side-effects may be reduced ^[3](3). Therefore, CoV Mpro has been an attractive drug target among coronaviruses ^[3](3) and so it is for COVID-19 ^[1][6](1, 6). Indeed, virtual drug screening, structure-assisted drug design, and high-throughput screening are been used to repurpose approved pharmaceutical drug and drug candidates targeting SARS-CoV-2 Mpro ^{[6] [8]} (6, 8). Furthermore, a study carrying the pharmacokinetic characterization of an optimized Mpro α-ketoamide inhibitor provides useful framework for development of this kind of inhibitors toward coronaviruses ^[1]. It was showed that the α-ketoamide inhibitor interacts with the catalytic site of the enzyme through two hydrogen bonding interactions, as can be seen in the complex formed between SARS-CoV-2 Mpro and an α-ketoamide inhibitor ^[1].

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References

1. ↑ ^{1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15} Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, Becker S, Rox K, Hilgenfeld R. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors. Science. 2020 Mar 20. pii: science.abb3405. doi: 10.1126/science.abb3405. PMID:32198291 doi:http://dx.doi.org/10.1126/science.abb3405
2. ↑ WHO. COVID-19 situation reports [Internet]. [cited 2020 May 15]. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports
3. ↑ ^{3.0 3.1 3.2 3.3} Hilgenfeld R. From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 2014 Sep;281(18):4085-96. doi: 10.1111/febs.12936. Epub 2014 Aug 11. PMID:25039866 doi:http://dx.doi.org/10.1111/febs.12936
4. ↑ Sharma A, Gupta SP. Fundamentals of Viruses and Their Proteases. Viral Proteases and Their Inhibitors. 2017;1‐24. doi:[1]
5. ↑ ^{5.0 5.1 5.2} Enjuanes, Luis, ed. 2005. Coronavirus Replication and Reverse Genetics. Current Topics in Microbiology and Immunology. Berlin Heidelberg: Springer-Verlag. Doi:[2]
6. ↑ ^{6.0 6.1 6.2 6.3 6.4 6.5} Jin, Zhenming, Xiaoyu Du, Yechun Xu, Yongqiang Deng, Meiqin Liu, Yao Zhao, Bing Zhang, et al. 2020. ‘Structure of M pro from SARS-CoV-2 and Discovery of Its Inhibitors’. Nature, April, 1–5. https://doi.org/10.1038/s41586-020-2223-y.
7. ↑ Anand K, Palm GJ, Mesters JR, Siddell SG, Ziebuhr J, Hilgenfeld R. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. EMBO J. 2002 Jul 1;21(13):3213-24. PMID:12093723 doi:10.1093/emboj/cdf327
8. ↑ Mirza, Muhammad Usman, and Matheus Froeyen. 2020. ‘Structural Elucidation of SARS-CoV-2 Vital Proteins: Computational Methods Reveal Potential Drug Candidates against Main Protease, Nsp12 Polymerase and Nsp13 Helicase’. Journal of Pharmaceutical Analysis, April. Doi:[3].