key: cord-0969278-h4sm7oib authors: Bibi, Nousheen; Gul, Sana; Ali, Johar; Kamal, Mohammad Amjad title: Viroinformatics approach to explore the inhibitory mechanism of existing drugs repurposed to fight against COVID-19. date: 2020-08-22 journal: Eur J Pharmacol DOI: 10.1016/j.ejphar.2020.173496 sha: c564c48fecb0b26a57b11e4c06702fc12c3f38c4 doc_id: 969278 cord_uid: h4sm7oib The rapid breakout of the coronavirus disease of 2019 (COVID-19) has been declared pandemic with serious global concern due to high morbidity and mortality. As we enter the phase beyond limitations there is an urgent need for explicit treatment against COVID-19. To face this immediate global challenge, drug development from scratch is a lengthy process and unrealistic to conquer this battle. Drug repurposing is an emerging and practical approach where existing drugs, safe for humans, are redeployed to fight this harder to treat disease. A number of multi clinical studies have repurposed combined cocktail (remdesivir + chloroquine and favipiravir + chloroquine) to be effective against COVID-19. However, the exact mechanistic aspect has not yet been revealed. In the present study, we have tried to decipher the mechanistic aspects of existing medicines at the viral entry and replication stage via the structural viroinformatics approach. Here we implied the molecular docking and dynamic simulations with emphasis on the unique structural properties of host receptor angiotensin-converting enzyme 2 (ACE2), SARS-CoV2 spike protein and RNA dependent RNA polymerase enzyme (RdRp) of the SARS-CoV2. Deep structural analysis of target molecules exposed key binding residues and structural twists involved in binding with important pharmacophore features of existing drugs [(7-chloro-N-[5-(diethylamino)pentan-2-yl]quinolin-4-amine (chloroquine),N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide) (SSAA09E2), 2-ethylbutyl (2S)-2-{[(S)-{[(2R,3S,4R,5R)-5-{4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl}-5-cyano-3 (remdesivir) and 6-Fluor-3-oxo-3,4-dihydro-2-pyrazincarboxamid (favipiravir)]. It is evident from this structural informatics study that combo of chloroquine + SSAA09E2 with remdesivir or favipiravir could significantly restrain the virus at the entry and replication stage. Thus, drug repurposition is an attractive approach with reduced time and cost to treat COVID-19, we don't have enough time as the whole world is lockdown and we are in urgent need of an obvious therapeutics' measures. chloroquine) to be effective against COVID-19. However, the exact mechanistic aspect has not yet been revealed. In the present study, we have tried to decipher the mechanistic aspects of existing medicines at the viral entry and replication stage via the structural viroinformatics approach. Here we implied the molecular docking and dynamic simulations with emphasis on the unique structural properties of host receptor angiotensin-converting enzyme 2 (ACE2), SARS-CoV2 spike protein and RNA dependent RNA polymerase enzyme (RdRp) of the SARS-CoV2. Deep structural analysis of target molecules exposed key binding residues and structural twists involved in binding with important pharmacophore features of existing drugs [(7-chloro-N-[5-(diethylamino) In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) was identified in Wuhan, China and named COVID-19 by WHO. COVID-19 (coronavirus disease of 2019) has surprisingly caught the global community as of August 10, 2020, the number of COVID-19 cases going to reach twenty million (20, 055, 196) with over seven hundred thousands (734,536) deaths worldwide (https://www.worldometers.info/coronavirus/). . The virus swiftly blowout from China to over >200 countries worldwide already exceeding the 2003 SARS CoV1 epidemic (Fischer et al, 2020) . Currently, there are no effective medications against COVID-19/SARS-CoV2 although several research groups throughout the world are working to develop the vaccine. There is an urgent need for the development of effective prevention and treatment strategies for SARS-CoV2 outbreak. Drug repositioning or reprofiling is a promising strategy to conquer the battle against COVID-19 in a time-critical manner. The newly identified SARS-CoV2 belongs to β-coronavirus. It has more than 96% sequence similarity with SARS-CoV1 (Fischer et al, 2020) . SARS-CoV is a complex of structural and non-structural proteins (NSPs) (NSP-2-NSP-16) which are produced as cleavage products of the ORF1a and ORF1ab viral polyproteins (Ziebuhr, 2005) . Among these NSP-12 along with cofactors, NSP-7 and NSP-8 possess RNA-dependent RNA polymerase activity (Ahn et al, 2012 , Subissi et al, 2014 . Thus, Nsp12, a conserved protein in coronavirus, is an RNA-dependent RNA polymerase (RdRp) and the vital enzyme of coronavirus replication/transcription complex. All viruses (including SARS-CoV2) need host receptor proteins on the cell surface for successful entry and infection. Angiotensin-converting enzyme 2 (ACE2), located in the human lower respiratory tract, is recognized as a cell receptor for SARS-CoV (Jia et al, 2005) and self-assembly (structural proteins). Broad-spectrum antiviral agents (BSAAs) estimated 'safe-in-man' tested on early phase clinical trials have been advertised as good drug repurposing candidates (Andersen et al, 2020) . Chloroquine, an anti-malarial drug has been shown to have antiviral activity at entry and postentry stages of the COVID-19 infection (Gao et al, 2020) . It can boost the antiviral activity of remdesivir and possibly aid as a synergizer of BSAAs (Adedeji et al, 2013) . In the present study, we have used the integrated structural informatics approach to explore the pharmacological interactions and inhibitory mechanism of chloroquine, remdesivir, favipiravir and SSAA09E2at the entry and virus replication stage of SARS-CoV2. Using molecular J o u r n a l P r e -p r o o f 5 docking, dynamic simulations and structural proteomics techniques important structural crunch of ACE2 (Human receptor) enzyme, SARS-CoV2 spike protein and RNA-dependent RNA polymerase (RdRp) enzyme of SARS-CoV2 were identified and the inhibitory mechanism of existing drugs was uncovered through drug-target interactions. So, the existing drugs against other viruses are an attractive and credible approach to treat unexplained viruses, as rather than conventional one bug, one drug method one drug more than one viruses are a debate of the day. J o u r n a l P r e -p r o o f The 3-dimensional structure of the ACE2 (PDB ID:1R4L), SARS-CoV2 spike ectodomain structure (PDB ID:6vyB) and RdRp (PDB ID:6M71) were retrieved from Protein Data Bank (PDB) (Berman et al, 2000) . Because of unavailability of the three-dimensional structure of RNA-dependent RNA polymerase (RdRp) (at the time when the study was designed) the I-Tasser server (Zhang, 2008) was used to build the model for downward structure analysis. 6nur was used as a template by I-Tasser with a TM score of 0.63 and having a sequence identity of 96.36%. Ramachandran plot was used to assess the stereochemistry and validity of the built three dimensional protein model (Laskowski et al, 2018) . The structure of the ligand; remdesivir (CID: 121304016), favipiravir (CID: 492405), chloroquine (CID: 83818 ) and SSAA09E2 (CID: 2738575 ) were retrieved from the PubChem database (https://pusbchem.ncbi.nlm.nih.gov/). To find the appropriate binding orientations of the ligand dataset with the respective target automated dockings were performed using AutoDock 4.2 (Norgan et al, 2011) tool following standard protocol. Polar hydrogen atoms and Kollman charges were assigned to the receptor protein molecule. Energy minimization was performed using a Lamarckian genetic algorithm with default parameters. An energy-based clustering of docked complexes was performed with the RMSF tolerance of 1.0 Å. The resulting clusters were classified through the lowest energy representative of the individual cluster. Resulting complexes were evaluated for molecular interactions using UCSF Chimera (Pettersen et al, 2004) and Discovery Studio (https://accelrysdiscovery-studio-visualizer.software.informer.com/3.0/). (Zlenko, 2012) . The system was neutralized by the addition of Na + and Clˉ counter ions. Energy minimization (steepest descent algorithm for 500 steps) was executed by the tolerance of 1000 kJ/mol Å to eliminate initial steric clashes. After completing the minimization step systems were subjected to simulations for 50 ns time scale under constant temperature (300 K) and pressure (1 atm). To this end, electrostatic interactions were calculated using Particle Mesh Ewald (PME) algorithm. To investigate the stability behavior of spike protein, RdRp enzyme, and ACE2 receptor systems, visual molecular dynamics (VMD) (Humphrey et al 1996) , PyMol (http://www.pymol.org) and GROMACS tools were used. Because of the lack of tertiary structure of the RdRp, the homology modeling technique was used to build the three-dimensional structure of RdRp. Ramachandran plot of polymerase indicated the presence of more than 98% residues in sterically allowed regions ( Fig. 1 A, B and C). Three dimensional structure of RdRp protein predicted through the I-Tasser server with TM score of 0.632 indicates the correct topology of the model for further studies. C-score (confidence score J o u r n a l P r e -p r o o f 8 for predicting the quality of predicted model) of RdRp was 0.95 showed the fidelity of the model for further computational analysis. Pore analysis was also performed with the MOLE server (mole.upol.cz) (Fig. 1 D) . Viral spike protein, RdRp, and human receptor ACE2 were selected as the target for the present study to combat the COVID-19 disease at entry and replication stages. The effective therapeutic approach is to interfere with viral entry and replication for preventing kcal/mol) revealed an interaction with the residues at the binding interface with spike-RBD. Ser43 and Ser47 formed H-bonding with the phenyl moiety of entry inhibitor. Ser44 formed coordinate covalent interaction apart from hydrophobic interactions made by Phe40, Trp349, and Asp350 ( Fig. 2A, B, and D) . stacking interaction in addition to hydrophobic interactions formed by critical residues (Asp452, Arg555, Asp623, Thr680, Ser681, and Ser682) of catalytic site (Fig. 4 A, B and D) . The strong interaction of both remdesivir and favipiravir with RdRp of SARS-CoV2 suggests a worthy option to treat COVID-19 disease with these existing antiviral medicines. To measure the stability and structural behavior of ACE2 receptor, spike protein, and RdRp, Through comparative analysis, significant conformational changes were observed. Tyr546 to Arg555 loop region was tilted towards the center to make close contact with the drugs (remdesivir and favipiravir), Pro620 to Ala625 also showed fluctuation with the significant inward push towards the cavity. Ser681 to Gly683 region pf RdRp also tilted inwards to aid the Ser682 in the binding pose with drug molecule. In addition to these critical conformational changes, these a number of fluctuations was also detected in the loop regions and secondary structure lying far from the RdRp binding cavity (Fig. 5 E and F) . The current COVID-19 epidemic painfully realizes us that our existing treatment options for hard-to-treat-disease are limited. Despite the extensive research conducted after the breakout of SARS-2003 and MERS-CoV in 2012, at present, there is no explicit drug to treat these coronaviruses. With the evident cost-and time-consuming nature of de novo drug development, the fastest and correct option is to use existing medicines for other diseases to conquer this war against COVID-19. So, drug repurposition also called drug reprofiling is the most promising strategy to identify drugs against SAR-CoV2 in a time-dependent manner. J o u r n a l P r e -p r o o f number of studies were also confirmed that SARS-CoV2 efficiently spreads among humans with a high affinity for human ACE2 receptor protein (Walls et al, 2020) . The interaction of virus spike protein with host cell surface receptor, ACE2 is appealing since it recruits the infection process of the virus. RdRp synthesizes a full-length negative-strand RNA template that makes more viral genomic RNA . Thus, RdRp could be an attractive target to stop the COVID-19 illness caused by SARS-CoV2. The outbreak of this COVID-19 epidemic, triggered the researchers all over the world to explore drugs for potential therapeutics of this SARS-CoV2-induced respiratory disease. Scientists are struggling to obvious drug discovery against this virus. Several drugs such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical studies to test their efficacy and safety in the treatment of COVID-19 in China and all across the world; some promising results was achieved in March, 2020 and anti-malarial drug chloroquine was confirmed as an active drug in treating coronavirus and FDA has approved it on March 28, 2020 . an the On June 5, 2020 the investigators from United kingdom recovery trial announced that chloroquine and hydroxychloroquine do not prove promising to reduce the mortality rate of hospitalized COVID-19 patients and results were supported by another solidarity trial, the french discovery trial (https://www.sciencemag.org/news/2020). There is little hope that chloroquine could be beneficial at the early onset of disease before getting into the hospital. Many other trials are still paused to get-go signal from the safety committee to make sure that these trials will not harm the patients under study ( Kupferschmidt, June 9, 2020) . So, the controversy of chloroquine at present is the dispute of the day. remdesivir and ravipiravir, are just approved for a clinical trial as a drug to treat COVID-19 (Maxmen, 2020) . Although promising results were observed with remdesivir and favipiravir and many pharmaceutical companies are launching generic names for these drugs as COVID-19 treatment but to date, we are lacking sufficient pieces of evidence to call any drug as "game-changer". The present study was performed to identify the exact mechanistic actionof repurposed drugs at a structural level using viroinformatics approach. Through molecular docking and dynamic simulation studies, we can speculate that chloroquine and SAA09E2 (Viral entry inhibitors) can block the viral entry into the human cell by blocking the binding interface of spike-RBD (virus) and human ACE2 receptor protein. So, the binding of these inhibitors with the spike-RBD domain may affect the key conformational switches required for the infection process. Similarly, binding of these inhibitors with the ACE2 receptor protein is of significant interest and it is speculated based on these results that virus entry could be stopped by blocking the viral host interaction to cure this COVID-19 illness ( Fig. 6 A and B) . So, these can be proposed as dualaction inhibitors instead of one bug one drug notion. Our in-silico targeting of RdRp, via drug repurposition revealed important structural insights and important structural features of remdesivir and favipiravir to block the catalytic site so these drugs can block the synthesis of viral RNA. Docking analysis of remdesivir and favipiravir with RdRp showed strong binding with high binding free energy of -12.67 kcal/mol and -10.34 kcal/mol, respectively. Based on our computational analysis, we propose these antiviral drugs in combination with viral entry inhibitors as a good option to treat SARS-CoV2 associated respiratory disease. Likewise, our results support the computational (Wuet al, 2020) and muticlinical studies (Maxmen, 2020) conducted to find potential therapeutics measures in the war against COVID-19. The limitation of the present study is as it was designed in march 2020, FDA approved the use of chloroquine as a treatment option for COVID-19. But there is such a rush to find the specific treatment for this rapidly spreading virus that is creating problems for the scientist to make decisions. As the controversial use of chloroquine and other drugs in more than 200 solidarity trials running in the world, so the limitation of the study is the immense pressure and controversy in the scientific community about the use of chloroquine. Secondly, at the time of methodology design, crystal structure of the RdRp enzyme of SARS-CoV2 was not available, so we predicted computationally and now the crystal structure is available in Protein Data Bank None. The data supporting the findings of the article are available in the Protein Data Bank (PDB) at http://www.rcsb.org and PubChem database at https://pubchem.ncbi.nlm.nih.gov/. The authors declare no conflict of interest, financial or otherwise. . 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