User:Dora Bonadio/Sandbox 1: Difference between revisions

<Structure load='' size='350' side='right' caption='Main Protease' scene='84/845941/Biological_assembly/2'>

The M^pro protease (also known as 3CL^pro), 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^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 <ref name="Crystal_structure" />. As a [[protease]], M^pro 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^pro 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^pro 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.

The M^pro 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^pro 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.  

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 <ref name="Crystal_structure" />. 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 <ref name="ofmpro" />. 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 <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" />.  

As have been shown, because of its importance for viral replication, inhibiting SARS-CoV-2 M^pro 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^pro inhibitors toxic side-effects may be reduced <ref name="sars_mers" />. Therefore, CoV M^pro 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^pro <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^pro <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^pro and the α-ketoamide inhibitor <ref name="Crystal_structure" />.

*[https://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6Y2E  rcsb.org] - To view the primary and secondary structure of SARS-CoV-2 M^pro.