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Introduction

The M^pro protease (also known as 3CL^pro), is a viral non-structural protein from the virus SARS-CoV-2 ^[1], responsible for a major outbreak of the disease called Coronavirus Disease 2019 (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].

Function

Cleavage sites of SARS-Cov-2 proteases.

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

Structure

The M^pro is a protein of approximately 30 kDa ^[5][6] consisting of two containing 306 amino acid residues each ^[1]. This monomers dimerize forming a ^[1]. Each chain consists of : I (; residues 10-99), II (; residues 100-182), and III (; residues 198-303). Domains I and II comprise six-stranded antiparallel and domain III comprises ^[1][6]. The substrate-binding site is located between domains I and II with the containing the amino acid residues ^[1]. Domain III, in turn, has been shown to be involved in the regulation of M^pro dimerization, what is necessary for the catalytic activity of this enzyme once it helps to shape the ^[1][7]. This dimerization regulation is mainly through a between Glu290 of one monomer and Arg4 of the other monomer.^[1]. Moreover, the dimer has a that is predominantly between domain II of one monomer and the N-terminal residues of other monomer. Indeed, the N-terminal residue of each monomer interacts with Glu166 of the other monomer, helping shape the (notice how Glu166 is a key residue to shape the binding site).^[1] Therefore, The N-terminal of one monomer interacts with the other monomer by the to help dimerization.

Structural comparison with SARS-CoV M^pro

As mentioned above, SARS-CoV-2 M^pro has 96% sequence identity with SARS-CoV M^pro 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 M^pro among an important number of CoV M^{pro [6]}. However, an interesting difference found between SARS-CoV M^pro and SARS-CoV-2 M^pro is that in the first one there is a polar interaction between the domains III of each monomer, involving the residues Thr285, what is not found in the COVID-19 virus M^{pro [1]}. In fact, in SARS-CoV-2, the threonine is replaced by , an amino acid with hydrophobic side chain, leading to a higher proximity between the two domains III of the dimer ^[1].

An attractive drug target

As have been shown, because of its importance for viral replication, inhibiting SARS-CoV-2 M^pro activity could lead to viral replication blockage ^[1][6]. Moreover, no human proteases has been reported to have a similar cleavage specificity and so, in this aspect, M^pro inhibitors toxic side-effects may be reduced ^[3]. Therefore, CoV M^pro has been an attractive drug target among coronaviruses ^[3] and so it is for COVID-19 ^[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 M^{pro [6][8]}. Furthermore, a study carrying the pharmacokinetic characterization of an optimized M^pro provided useful framework for development of this kind of inhibitors toward coronaviruses ^[1]. It was showed that the interacts with the catalytic residue His41 and with residues Gly143 and Ser144 through hydrogen bonds, and that there is a nucleophilic attack of the catalytic Cys145 onto the α-keto group of the inhibitor. This can be seen in the formed between SARS-CoV-2 M^pro and the α-ketoamide inhibitor ^[1].

External Resources

• rcsb.org - To view the primary and secondary structure of SARS-CoV-2 M^pro.

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 1.16 1.17} 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].