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== Introduction ==
== Introduction ==
The Mpro protease (also known as 3CLpro), is a viral non-structural protein from the virus SARS-CoV-2 <ref name="Crystal_structure">PMID:32198291</ref>, responsible for a major outbreak of the disease called [[Coronavirus Disease 2019 (COVID-19)]], declared pandemic by WHO in 11 march 2020 <ref name="WHO">WHO. COVID-19 situation reports [Internet]. [cited 2020 May 15]. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports </ref>. 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) <ref name="sars_mers">PMID:25039866</ref>.
The M<sup>pro</sup> protease (also known as 3CL<sup>pro</sup>), is a viral non-structural protein from the virus SARS-CoV-2 <ref name="Crystal_structure">PMID:32198291</ref>, responsible for a major outbreak of the disease called [[Coronavirus Disease 2019 (COVID-19)]], declared pandemic by WHO in 11 march 2020 <ref name="WHO">WHO. COVID-19 situation reports [Internet]. [cited 2020 May 15]. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports </ref>. 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) <ref name="sars_mers">PMID:25039866</ref>.
== Function ==
== 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 <ref name="Crystal_structure" />. As a [[protease]], Mpro is an enzyme that causes proteolysis, which means that it breaks protein peptide bonds by hydrolysis <ref name="fundamentals"> Sharma A, Gupta SP. Fundamentals of Viruses and Their Proteases. Viral Proteases and Their Inhibitors. 2017;1‐24. doi:[http://dx.doi.org/10.1016/B978-0-12-809712-0.00001-0]</ref>. Indeed, the Mpro processes the replicase polyprotein 1ab (pp1ab ~790 kDa) translated from the viral RNA ORF1ab <ref name="Crystal_structure" /><ref name="replication"> Enjuanes, Luis, ed. 2005. Coronavirus Replication and Reverse Genetics. Current Topics in Microbiology and Immunology. Berlin Heidelberg: Springer-Verlag. Doi:[https://doi.org/10.1007/b138038]</ref>. In fact, Mpro cleaves 11 sites of pp1ab and the recognition sequence at most sites is between Leu-Gln and (Ser, Ala, Gly) <ref name="Crystal_structure" /><ref name="sars_mers" />. Proteins resulting from this polyprotein cleavage are non-structural proteins (NSPs) and they seem to contribute with viral replication and transcription <ref name="replication" />. Thus, by processing an important number of non-structural proteins, this enzyme plays a critical role in SARS-CoV-2 replication.
[[Image:Clivagesites sarscov2.png |thumbnail|260px|alt=Cleavage sites of SARS-Cov-2 proteases.|Cleavage sites of SARS-Cov-2 proteases.]]
The M<sup>pro</sup> protein function is mainly deduced from the function of SARS-CoV virus M<sup>pro</sup>, which has a 96% amino acid identity and a highly similar three-dimensional structure with SARS-CoV-2 M<sup>pro</sup> <ref name="Crystal_structure" />. As a [[protease]], M<sup>pro</sup> is an enzyme that causes proteolysis, which means that it breaks protein peptide bonds by hydrolysis <ref name="fundamentals"> Sharma A, Gupta SP. Fundamentals of Viruses and Their Proteases. Viral Proteases and Their Inhibitors. 2017;1‐24. doi:[http://dx.doi.org/10.1016/B978-0-12-809712-0.00001-0]</ref>. Indeed, the M<sup>pro</sup> processes the replicase polyprotein 1ab (pp1ab ~790 kDa) translated from the viral RNA ORF1ab <ref name="Crystal_structure" /><ref name="replication"> Enjuanes, Luis, ed. 2005. Coronavirus Replication and Reverse Genetics. Current Topics in Microbiology and Immunology. Berlin Heidelberg: Springer-Verlag. Doi:[https://doi.org/10.1007/b138038]</ref>. In fact, M<sup>pro</sup> cleaves 11 sites of pp1ab and the recognition sequence at most sites is between Leu-Gln and (Ser, Ala, Gly) <ref name="Crystal_structure" /><ref name="sars_mers" />. Proteins resulting from this polyprotein cleavage are non-structural proteins (NSPs) and they seem to contribute with viral replication and transcription <ref name="replication" />. Thus, by processing an important number of non-structural proteins, this enzyme plays a critical role in SARS-CoV-2 replication.
== Structure ==
== Structure ==
The Mpro is a protein of approximately 30 kDa <ref name="replication" /><ref name="ofmpro">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. </ref> consisting of <scene name='84/845941/Assembly/3'>two protomers</scene> containing 306 amino acid residues each <ref name="Crystal_structure" />. This protomers dimerize forming a homodimer <ref name="Crystal_structure" />. Each protomer consists of <scene name='84/845941/Domains/1'>three domains</scene>: I (<scene name='84/845941/Domaini/1'>chymotrypsin-like</scene>; residues 10-99), II (<scene name='84/845941/Domains2/1'>picornavirus 3C protease-like</scene>; residues 100-182), and III (<scene name='84/845941/Domains3/1'>a globular cluster</scene>; residues 198-303). Domains I and II comprise six-stranded antiparallel β-barrels and domain III comprises five α-helices <ref name="Crystal_structure" /><ref name="ofmpro" />. The substrate-binding site is located between domains I and II with the <scene name='84/845941/Catalyticsite/1'>catalytic site</scene> containing the amino acid residues Cys145 and His41 <ref name="Crystal_structure" />. 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 <ref name="Crystal_structure" /><ref name="reveals"> PMID:12093723</ref>.
The M<sup>pro</sup> is a protein of approximately 30 kDa <ref name="replication" /><ref name="ofmpro">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. </ref> consisting of two <scene name='84/845941/Monomer/3'>monomers</scene> containing 306 amino acid residues each <ref name="Crystal_structure" />. This monomers dimerize forming a <scene name='84/845941/Assembly/5'>homodimer</scene> <ref name="Crystal_structure" />. Each chain consists of <scene name='84/845941/Domains/1'>three domains</scene>: I (<scene name='84/845941/Domaini/1'>chymotrypsin-like</scene>; residues 10-99), II (<scene name='84/845941/Domains2/1'>picornavirus 3C protease-like</scene>; residues 100-182), and III (<scene name='84/845941/Domains3/1'>a globular cluster</scene>; residues 198-303). Domains I and II comprise six-stranded antiparallel <scene name='84/845941/B_barrels/1'>β-barrels</scene> and domain III comprises <scene name='84/845941/A_helices/1'>five α-helices</scene> <ref name="Crystal_structure" /><ref name="ofmpro" />. The substrate-binding site is located between domains I and II with the <scene name='84/845941/Catalyticsite/1'>catalytic site</scene> containing the amino acid residues <scene name='84/845941/Cys145_his41/1'>Cys145 and His41</scene> <ref name="Crystal_structure" />. Domain III, in turn, has been shown to be involved in the regulation of M<sup>pro</sup> dimerization, what is necessary for the catalytic activity of this enzyme once it helps to shape the <scene name='84/845941/Substrate_binding_cleft/1'>substrate-binding site</scene> <ref name="Crystal_structure" /><ref name="reveals"> PMID:12093723</ref>. This dimerization regulation is mainly through a <scene name='84/845941/Glu290_arg4/2'>salt-bridge interaction</scene> between Glu290 of one monomer and Arg4 of the other monomer.<ref name="Crystal_structure" />. Moreover, the dimer has a <scene name='84/845941/N_terminal_interaction/1'>contact interface</scene> that is predominantly between domain II of one monomer and the N-terminal residues of other monomer. Indeed, the N-terminal residue <scene name='84/845941/Glu166_ser1/1'>Ser1</scene> of each monomer interacts with Glu166 of the other monomer, helping shape the <scene name='84/845941/Substrate_binding_cleft/1'>substrate-binding site</scene> (notice how Glu166 is a key residue to shape the binding site).<ref name="Crystal_structure" /> Therefore, The N-terminal of one monomer interacts with the other monomer by the <scene name='84/845941/Dimerization/1'>Glu166-Ser1 and Glu290-Arg1 interactions</scene> to help dimerization.
== Structural comparison with SARS-CoV M<sup>pro</sup> ==
== Structural comparison with SARS-CoV M<sup>pro</sup> ==
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 <ref name="Crystal_structure" />. Indeed, it has been shown that the substrate-binding pocket is a highly conserved region of Mpro among an important number of CoV Mpros <ref name="ofmpro" />. 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 <ref name="Crystal_structure" />. In fact, in SARS-CoV-2, the threonine is replaced by <scene name='84/845941/Ala285/1'>alanine</scene>, leading to a higher proximity between the two domains III of the dimer <ref name="Crystal_structure" />.
As mentioned above, SARS-CoV-2 M<sup>pro</sup> has 96% sequence identity with SARS-CoV M<sup>pro</sup> and as expected, also a highly similar three-dimensional structure <ref name="Crystal_structure" />. Indeed, it has been shown that the substrate-binding pocket is a highly conserved region of M<sup>pro</sup> among an important number of CoV M<sup>pro</sup> <ref name="ofmpro" />. However, an interesting difference found between SARS-CoV M<sup>pro</sup> and SARS-CoV-2 M<sup>pro</sup> 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<sup>pro</sup> <ref name="Crystal_structure" />. In fact, in SARS-CoV-2, the threonine is replaced by <scene name='84/845941/Ala285/1'>alanine</scene>, an amino acid with hydrophobic side chain, leading to a higher proximity between the two domains III of the dimer <ref name="Crystal_structure" />.
== An attractive drug target ==
== An attractive drug target ==
As have been shown, because of its importance for viral replication, inhibiting SARS-CoV-2 Mpro activity could lead to viral replication blockage <ref name="Crystal_structure" /><ref name="ofmpro" />. 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 <ref name="sars_mers" />. Therefore, CoV Mpro has been an attractive drug target among coronaviruses <ref name="sars_mers" /> and so it is for COVID-19 <ref name="Crystal_structure" /><ref name="ofmpro" />. 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 <ref name="ofmpro" /><ref name="elucidation"> 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:[https://doi.org/10.1016/j.jpha.2020.04.008].</ref>. Furthermore, a study carrying the pharmacokinetic characterization of an optimized Mpro <scene name='84/845941/13b/1'>α-ketoamide inhibitor</scene> provides useful framework for development of this kind of inhibitors toward coronaviruses <ref name="Crystal_structure" />. 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 <ref name="Crystal_structure" />.
As have been shown, because of its importance for viral replication, inhibiting SARS-CoV-2 M<sup>pro</sup> activity could lead to viral replication blockage <ref name="Crystal_structure" /><ref name="ofmpro" />. Moreover, no human proteases has been reported to have a similar cleavage specificity and so, in this aspect, M<sup>pro</sup> inhibitors toxic side-effects may be reduced <ref name="sars_mers" />. Therefore, CoV M<sup>pro</sup> has been an attractive drug target among coronaviruses <ref name="sars_mers" /> and so it is for COVID-19 <ref name="Crystal_structure" /><ref name="ofmpro" />. 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<sup>pro</sup> <ref name="ofmpro" /><ref name="elucidation"> 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:[https://doi.org/10.1016/j.jpha.2020.04.008].</ref>. Furthermore, a study carrying the pharmacokinetic characterization of an optimized M<sup>pro</sup> <scene name='84/845941/13b/1'>α-ketoamide inhibitor</scene> provided useful framework for development of this kind of inhibitors toward coronaviruses <ref name="Crystal_structure" />. It was showed that the <scene name='84/845941/13b2/1'>α-ketoamide inhibitor</scene> 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 <scene name='84/845941/Inhibitor_and_bindingsite_bond/1'>complex</scene> formed between SARS-CoV-2 M<sup>pro</sup> and the α-ketoamide inhibitor <ref name="Crystal_structure" />.
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.
== External Resources ==
== External Resources ==
*[https://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6Y2E rcsb.org] - To view the primary and secondary structure of SARS-CoV-2 Mpro.
*[https://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6Y2E rcsb.org] - To view the primary and secondary structure of SARS-CoV-2 M<sup>pro</sup>.
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 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].
FunctionFunction
Cleavage sites of SARS-Cov-2 proteases.
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]. Indeed, the Mpro processes the replicase polyprotein 1ab (pp1ab ~790 kDa) translated from the viral RNA ORF1ab [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]. 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.
StructureStructure
The Mpro 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 Mpro 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 MproStructural comparison with SARS-CoV Mpro
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 Mpro[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 monomer, 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 , an amino acid with hydrophobic side chain, leading to a higher proximity between the two domains III of the dimer [1].
An attractive drug targetAn attractive drug target
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]. 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]. Therefore, CoV Mpro 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 Mpro[6][8]. Furthermore, a study carrying the pharmacokinetic characterization of an optimized Mpro 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 Mpro and the α-ketoamide inhibitor [1].
External ResourcesExternal Resources
rcsb.org - To view the primary and secondary structure of SARS-CoV-2 Mpro.
↑ 3.03.13.23.3Hilgenfeld 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
↑ Sharma A, Gupta SP. Fundamentals of Viruses and Their Proteases. Viral Proteases and Their Inhibitors. 2017;1‐24. doi:[1]
↑ 5.05.15.2 Enjuanes, Luis, ed. 2005. Coronavirus Replication and Reverse Genetics. Current Topics in Microbiology and Immunology. Berlin Heidelberg: Springer-Verlag. Doi:[2]
↑ 6.06.16.26.36.46.5Jin, 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.
↑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
↑ 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].
