In Silico Comparison of Main Proteinase Inhibitors for Different Coronaviruses

Coronaviruses are enclosed positive stranded RNA viruses with spike protein protrusions that permit the virus to penetrate and affect host cells. The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), as mortal human CoV illnesses, has sparked considerable interest in the medical community. The fast and global outbreak of a novel human coronavirus generated by a novel progeny of coronavirus 2 (CoV-2) has promoted an urgent need to identify an effective target for COVID-19 treatment. The main proteinase MPro has been prominented as an appealing therapeutic target for coronaviruses, which is responsible for the transcriptase and replicase of coronaviruses. The identification of prospective medications is an imperative and critical need for the medical community. Molecular docking was used to describe the protease and asses the capacity of various well-known and laboratory-tested natural MPro inhibitors. Seventy-sixth natural compounds with known inhibitory activity and four medicines reported against CoV-1 were chosen for molecular docking study. Our in-silico studies reveal that many of these molecules show high binding affinity for several CoV-2 proteases and compare favorably to CoV-1 and MERS proteases. Our research indicates that these molecules could be anti CoV-2 MPro. implying their possibility for reprofiling as antiviral leads with broad scope .


Introduction
Coronaviruses are related to the family Coronaviridae, subfamily Coronavirinae, order Nidovirales, and are enclosed RNA viruses with a positive strand containing a helical protein shell, including genomes of 27-31 kb.They are categorised via four genera α, β, γ and δ.These viruses possess prominent spikes on their surface that give them structure like a crown, allowing them to bind to the respiratory systems and digestive tracts of birds and mammals, and they are restricted to one host species 1,2 .
Three coronaviruses have been documented worldwide.In 2002-2003, Guangdong, China identified a zoonotic incident delivered by civet cats and bats, resulting in severe acute respiratory syndrome (SARS).The SARS-CoV infection caused over 700 deaths.The pandemic was ended by using proper cleanliness and quarantine measures 3,4 .Another zoonotic transition involving camels was noted in 2012 in Saudi Arabia, resulting in infections of the lower respiratory tract in humans.640 people died as a result of Middle East respiratory syndrome (MERS-CoV) 5, 6 .In 2019, a Published Online First: January, 2024 https://dx.doi.org/10.21123/bsj.2024.9091P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal novel pandemic called novel coronavirus illness (nCoV) with symptoms similar to SARS was identified in China.SARS coronavirus 2 (SARS-CoV-2) has been distinguished to be the root of COVID-19, an outbreak of respiratory diseases in humans that results in acute pneumonia 7 .Despite numerous attempts and programs to contain the disease spread, it has spread rapidly throughout the world, with the related fatality rate increasing.
According to the WHO, there have been over 1.6 million deaths.Because of the pathogen's novelty, no antiviral medicines or vaccines can lower the severity of the sickness or treat it.Furthermore, the acuteness of this virus has resulted in increased research on the sickness, resulting in a better perception of its aetiology, administration, and therapy 1 .
SARS-CoV-2 is the seventh familiar coronavirus in humans, following NL63, 229E, HKU1 and OC43, SARS and MERS-CoV.The majority of human coronaviruses originated from bat covids and were transferred to humans via a moderator host 8 .
The main protease (M Pro or NSP5), also called 3-Chymotrypsin-like protease (3CL Pro ), has a cleavage range similar to the 3C protease of picornavirus 13 .It is related to the cysteine protease group and cleaves polypeptides pp1ab at 11 locations with a sequence of Leu-Gln* (Ser, Ala, Gly) (*: cleavage site) 14,15 , a process started by the M Pro autocleavage from polyproteins pp1a/pp1ab.This cleavage process corresponds to M Pro in SARS-CoV 15-17   .In addition, the protease catalytic site includes a dyad of Cys145 and His41.The protease has three domains and the catalysis lies among domains I and II 15,18 .In the P2 domain of SARS-CoV polyproteins, there are three M Pro cleavage sites include Met, Phe, or Val.Other coronaviruses, otherwise, lack similar cleavage sites.The Zhang group published the X-ray structure of the COVID-19 M Pro complexed with a peptidomimetic α-ketoamide ligand at 1.95 Å.This research gave the first structural knowledge of the 3CL Pro complexed SARS-CoV-2, which is a viable target for drugs to limit and inhibit infection of SARS-CoV-2 in patients 15 .
Studies conducted recently have disclosed that chloroquine, hydroxychloroquine, ritonavir, lopinavir, remdesivir, azithromycin, dexamethansone, and ivermectin have the promise of inhibiting the severity of the disease in SARS-CoV-2 carriers [19][20][21][22] .Alternatively, numerous phytochemicals have been described in the literature to have potential antiviral action, which could be used as an alternative to limit coronavirus reproduction 23 .Natural molecules have a great chemical variety, a cheaper production cost than biotechnological compounds or outcomes synthesized by combinatorial chemistry, and have milder or no adverse effects than chemical medications 24 .
In our study, after determining the crystal structure of M Pro , we started searching for enzymes with a good identity ratio with this enzyme using FASTA alignment.We found two enzymes with a good identity ratio of 70 and 80%.These enzymes have been laboratory tested in vitro with some inhibitor classes that we chose to be studied theoretically with COVID-19 to find the best compounds and compared with other enzymes.In this context, the binding affinity of different inhibitor classes of phthalhydrazide-substituted ketoglutamine analogues, metal linked compounds, α, βunsaturated peptidomimetics, aescin, anilides, isatin, aryl boronic acids, and other compounds were studied on the viral proteases binding sites from CoV-2, CoV-1 and MERS using molecular docking investigations and testing their molecular interaction and binding energy.

Modelling of Ligands
A library of 80 anti-SARS-CoV active inhibitors was created from scratch or based on reported X-ray structures for their locations.TL-3 and the known α-ketoamide ligands of SARS-CoV-2 M Pro were obtained from their X-ray structures from protein data bank (https://www.rcsb.org)with PDB codes: 4K4P and 6Y2F.The remaining 78 inhibitors, were either sketched in 2D using ChemBioDraw Ultra 13 or retrieved from PubChem and saved in sdf format.MOE was also used to convert their 2D structures to 3D structures and to minimise them.The tested inhibitors and complexed ligands were then transferred to a certain database and stored as a mdp file in preparation for use in the coronavirus docking investigation.MMFF: Amber 10 force field was employed to optimize the designed structures.

Preparation of Viral Proteases
The crystal structures were created using the detailed technique outlined previously 25,26  A typical structure of PDB may contain metal ions, water molecules, co-crystallised ligands and cofactors.In addition, multimeric structures may require a reduction to one unit.All water molecules were removed except that existed in active site if existed, and all structures were preprepared for docking with the MOE module (Molecular Operating Environment) (http://www.chemcomp.com),which adds protons to structures where protons were absent and sets the force field at pH 7.0.Subsequently, the ligands complexed with these enzymes were chosen to create a radius sphere of 4.5 Å, which was dubbed the ligand binding site.

Molecular Docking Investigations of Anti-SARS-CoV Inhibitors with Main Protease Targets
Molecular docking investigations of prepared anti-SARS-CoV inhibitors on the active places of prepared SARS-CoV-2 M Pro , SARS-CoV M Pro and MERS-CoV CL Pro were performed using the MOE software (Molecular Operating Environment) (http://www.chemcomp.com).To find a potent inhibitor with potential enzyme-inhibitory properties for treating CoV-2, we selected M pro as a target enzyme.The chemical compositions of natural compounds and drugs employed in the docking study are shown in Fig. 1.To apply charges and parameters, the MMFF94x force field was used.After creating and isolating the active site with MOEs surface and mapping module, the ligands were docked on the inside surface of the target receptor employing the Dock module of MOE.To perform docking studies, triangle matcher and refinement approaches were used.For each trialed ligand, the rigid receptor was used as the refining protocol and the GBVI/WSA dG as the scoring protocol to choose the best pose from 100 varied poses.The active site was used as ligand atoms, and automatic rotational bonds were permitted.To their default rates, the scoring methodologies were adopted.After the docking processes were completed, the poses acquired were analysed, and the top ones with the best suited docking score values in the active site were picked.

Results and discussion
Molecular docking or other computer-aided techniques are effective tools for studying the molecular features of bindings between protein and ligand through drug development for a number of previously prominent and lethal diseases, involving the coronavirus (SARS) [27][28][29] .In this research, MOE software was used to conduct a computational analysis of many natural products against coronavirus target main proteases in order to discover the top hits from each family among 80 compounds, depending on their docking scores.The superior-ranked compounds had higher negative docking score rates and higher M Pro linking affinities.Consequently, in two stages, twelve hits that could potentially be CoV-2 M Pro inhibitors were identified.First, a doc was run on all enzymes with PDB codes: 6Y2F, 6LU7, 6WTT, 7C8U, 7CA8 and 7JQ2 for CoV-2, 1UK4 and 3C3N for CoV-1, 4RSP for MERS-CoV for compounds known as CoV-1 and MERS inhibitors, and 790 compounds emerged as the best pose for each compound with the enzymes already indicated Table 1.
In the second stage, we selected the best docking score related to each family of compounds with CoV-2 and compared it to CoV-1 and MERS 2D interaction graphs discovered by MOE Fig. 2.These findings clearly reveal that each of the molecules binds to the proteases active sites and hence may be predicted to decrease enzyme activity and thus limit viral multiplication.Furthermore, ADME-Toxicity investigated the physiochemical features of these ligands using the filter Lipinski's rule of 5 criteria for determining drug identity.The molecular features that are significant for a drug pharmacokinetics within a human body are established by Lipinski's rule.Lipinski's five criteria for a typical drug 1) a molecular mass of fewer than 500 g/mol, 2) no more than 5 hydrogen-bond donors, 3) no more than 10 hydrogen-bond acceptors, 4) a partition factor (log P) for octanol/water not larger than 5.
Violations of three or more of the criteria does not meet drug-likeness criteria when administered through the oral track 30 .Based on physiochemical features of the top twelve docked ligands and by matching to Lipinski's criteria, we concluded nine compounds fit totally and three others partially for containing violations Table 4.
As a result, computational investigations resulted in the identification of certain molecules as possible inhibitors of M Pro of CoV-2, which demonstrated the top binding scores and affinities.Furthermore, our computational studies reveal that these substances could prohibit other viral proteases as SARS-CoV-1 3CL Pro and MERS-CoV CL Pro , and by comparing the results, we can assume that molecules with higher docking scores than CoV-1 and/or MERS may show higher inhibitory activity than SARS-CoV-1 and MERS at lower concentrations when tested in the lab, and thus could be developed into potential pharmaceutical candidates for COVID-19.

Conclusion
Main proteinase is an intriguing target for inhibiting the viral reproduction cycle and treating infection with COVID-19.The goal of this work was to use in silico techniques to analyse the antiviral ability of a set of previously known inhibitors against 3CL Pro of coronavirus.The 3CL Pro of coronaviruses may be significantly inhibited by these inhibitors.Between the studied 80 compounds, compounds number 7, 9, 10, 12, 50, 62, 66, 79, and 80 showed binding interactions higher than those of 3CL Pro of CoV-1 and/or MERS-CoV and successfully avoided detection during drug-likeness tests.These findings imply that we have explored good hits nominee for the improvement of therapeutic medicines against COVID-19.Animal investigations and proper clinical trials will eventually be required to prove the possible preventative and therapeutic impact of these substances.

Figure 1 .
Figure 1.Chemical compositions of natural compounds and drugs tested against M Pro of SARS and MERS-CoV

Table 2
. The docking investigation of chosen compounds to M Pro of COVID19 yielded binding affinities ranging from -6.53 to -9.80 kcal/mol, which were compared to CoV-1 and MERS docking scores Table2.These compounds include the top scores of TL-3, reserpine, α-ketoamide, leupeptin, loxistatin, and other inhibitors from each class listed in Table1.Compounds 7, 46, 79 and 80 were found to have docking score values against CoV-2 lower than CoV-1 but higher than MERS, whereas compound 46 had the top linking affinity to those viral proteases.As a result, compound 46 was the tightest docked molecule to M Pro that embedded the coronavirus target protein.Compound 12 was in the second place in the list, docking at -9.57, with CoV-2 3CL Pro , a docking score surpassing CoV-1 and MERS.Compounds 9, 10, 50, 62, 66 and 78 followed a similar pattern.The TL-3 ranked third against CoV-2, with a docking score of -9.48 kcal/mol, compared to -8.24 kcal/mol for CoV-1 and -9.60 kcal/mol for MERS 3CL Pro .The interactions of the best inhibitors with amino acid units of 3CL Pro of CoV-2 Table3revealed that these molecules mostly engaged with the units via Hbonding and hydrophobic effects.The outcomes of the finest molecular docking inhibitors in the 3CL Pro active site of CoV-2 are shown by their individual