Repurposing of FDA approved drugs targeting Main protease MPro for SARS-CoV-2

SARS-CoV-2 is one of the greatest pandemics in the history. There is no medicine or vaccine yet discovered to control the outbreak. The paper deals with repurposing existing drugs to control the outbreak of SARS-CoV-2 virus. Ten FDA-approved drugs namely Indinavir, Nelnavir, Letermovir, Irinotecan, Elbasvir, Saquinavir, Darunavir, Raltegravir, Atazanavir and Amprenavir were studied. In silico methods for virtual screening of protein-ligand docking of these drugs against SARS-CoV-2 M Pro was performed. The binding eciency of the drugs against viral main protease M Pro was signicantly high to inhibit SARS-CoV-2. The results conrmed that Atazanavir, Nelnavir, and Letermovir not only occupied the active site of M pro but also showed increased binding anity (-10.36 kcal/mole, -9.47 kcal/mole and -9.43 kcal/mole) even more than of control drugs of Lopinavir (-8.71 kcal/mole) and Ritonavir (-8.08 kcal/mole). These repurposed drugs can be used in combination or individually as an alternative approach for rapid drug discovery against SARS-CoV-2


Introduction
The outbreak of SARS-CoV-2 started in December 2019 from the Wuhan, China has now created chaos by attacking over 200 countries jeopardize the global public health crisis. It has admonished the scienti c community to distil best science. According to the information shared by WHO in the public domain, as of May10, 2020 Covid19 has infected around 3,917,366 con rmed cases in more than 200 countries across the globe including a poignant 274, 361casualties, with a cumulative death date of >7% [1]. However, Patients infected with Covid19 having pre-existing co morbidities like cancer, diabetes, cardiovascular and respiratory disease are at risk of mortality [2] [3]. The scienti c community worldwide is still underway to gure out the outbreak of novel coronavirus 2019. At present, there are no vaccines and therapeutic interventions against this pandemic [4].
Drug repurposing strategies have developed as a competent tool for identi cation of existing potential drugs with well-known toxicity pro les [5]. Whereas these can be used in combination or individually as an alternative approach for rapid drug discovery against SARS-CoV-2 [6]. In the present study, we have employed an in silico methods for virtual screening of protein-ligand docking of FDA approved drugs against SARS-CoV-2 M Pro (PDB ID 7BQY) which have been shown recently in a co-crystallized form with its inhibitor N3occupying the active binding pocket of the enzyme. It was targeted and screened for selecting the number of FDA approved drugs in order to interact with M Pro (PDB ID 7BQY) and further blocking the active site [7]. The crystal structure of SARS-CoV-2 M Pro in apo form and (PDB ID: 6Y2E) and N3 bound structure (PDB ID: 7BQY) illustrates that protein is in crystallographic dimer form consisting of two identical monomer units. Every protomer consists of three domains. The interface of domain I and II domain consist of amino acid Cys145-His41 which collectively creates binding pocket of protein, where it's inhibitor N3 is bound in co-crystallised form [7] [8].
We have shortlisted the number of commercially available drugs that are already in use for treating humans countering by several viral infections and screened them for M pro active site binding. Our results have shown that Atazanavir, Nel navir, and Letermovir not only occupied the active site of M pro but also shows increased binding a nity even more than of our control drugs. While the rest of the compounds also hold appreciable binding a nity occupying the important active site determinants. We emphasize that all the repurposed drugs considered and screened in this study for disease COVID-19 should be validated and an in vitro inhibitory potential need to be analysed using various robust biophysical and biochemical procedures leading clinical trials.

Proteins/Macromolecules
The crystal structure of SARS-CoV-2 main protease (M pro ) (PDB ID: 7BQY) structures were retrieved from protein data bank (https://www.rcsb.org/), in .pdb format [7]. The structures of the target protein SARS-CoV-2 M pro with a covalent inhibitor, N3, was available in the PDB and it contains two chains, A and B, which form a homo dimer. Thereafter the chain A of PDB le was optimized for macromolecule preparation.
Protein structure alignment and homology analysis  [9]. The protein sequence were obtained in FASTA format and multiple sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) [10]. The DALI server was used for homology analysis and creating superimposed structure of both proteins [11].

Literature survey and drug selection
The list and structures of FDA approved drugs were retrieved from the drug bank (https://www.drugbank.ca/) [12]. The 10 FDA-approved drug compounds (Table 1) used in the present study were Indinavir, Nel navir, Letermovir, Irinotecan, Elbasvir, Saquinavir, Darunavir, Raltegravir, Atazanavir and Amprenavir against viral protease that could inhibit SARS-CoV-2 main protease M Pro .
Thereafter, the molecules which were optimized, were saved in the PDB format for further processing.

HCV infection
Determination of Active Sites CASTp 3.0 software (http://sts.bioe.uic.edu/castp/ server) was used for determining the active sites by using binding pocket analysis tool [13]. The active site residues were used to evaluate the Grid box and docking results. The binding pocket of the ligand in the catalytic site was manually obtained followed by veri cation by docking analysis.

Molecular Docking
Crystal structure of main protease M Pro and 3D structure of selected FDA was obtained from RCSB protein data bank (PDB ID: 7BQY) and drug bank (https://www.drugbank.ca/) respectively. The drug repurposing was done through molecular docking using the 3-D structure of protein PDB and drug compounds. The Molecular docking analysis was done using AutoDock 4.2, which works on the lamrackian genetic algorithm (GA) along with empirical free energy scoring function so as to identify the most favourable binding posture of the chosen FDA approved drugs [14]. The interaction of the ligand with the active site of the main protease M Pro was determined by energy score (S, Kcal/mol). Low energy score indicates good a nity. Pymol version 1.7.4.5 Edu was used for visualization of the docked results [15].

Results And Discussion
The SARS-COV-2 virus contains around ~30,000 nucleotide long single-stranded positive sense RNA molecule [(+)ssRNA], having5' capped region along with 3' poly-A tail, which immediately recognized by eukaryotic machinery and undergoes translation [16]. The Virus genome consists of six open reading frames (ORFs) out of which rst ORF encodes by the 5' end of the genome. This rst ORF further splits intoORF1a and ORF1b which encodes two polypeptides pp1a and pp1ab. These two candidates are responsible for the synthesis of 16 non-structural proteins (nsP1-16), rest ORFs are utilized in synthesizing remaining four important structural proteins: Spike protein (S), Envelope proteins (E), Membrane proteins (M) and Nucleocapsid protein (N). [17] The virus uses S protein which is heterotrimeric and interacts with the ACE2 (a homolog of angiotensin-converting enzyme 2) which act as a virion receptor. [18] This facilitates host-pathogen interaction enabling viral entry into host cell [19].
Thereafter, genomic RNA acts as a template recognized by host cell machinery to synthesize 2 polyproteins pp1a and pp1ab which undergoes proteolysis [20]. It also gives important non-structural proteins including two proteases; Papain like protease (P Pro ) and Chymotrypsin like protease (3CLpro) or main protease (M Pro ) [21] [22]. These important proteases further process the polyprotein in sequencespeci c manner synthesizing 16 different nsPs. These are directly associated with the further replication and transcription of the virus by forming replication transcription complex (RTC) [23]. Therefore, targeting the main protease M Pro represents the attractive drug target for restricting the further production of nonstructural viral proteins. This will inhibit replication events of the virus life cycle. Moreover, no evidences have been found of inhibiting human protease activity upon targeting viral proteases, thus, precluding the chances of cellular toxicity even though inhibiting the main protease [24].
As of now, there is no prominent treatment or vaccine that is reported or approved speci cally for treating Covid19 patients [25]. However scienti c community around the globe putting all efforts in research endeavors towards the advancement of remedial intercessions and researching viral drug targets [26]. Recently scientist has deduced some of the important X-ray crystal structure of viral proteins such as 3CL like protease, papain-like protease, and spike (S) protein [27] [28]. From the above nding, it was seen that virus gets attached with angiotensin-converting enzyme 2 (ACE2) receptors which are present in the lower respiratory tract providing entry into the lungs [29].
The understanding of SARS-CoV-2 virus with that of previously studied SARS-CoV virus, was performed through protein structure alignment and sequence similarity and the results are presented in Fig. 1. Nonstructural proteins (nsPs) of corona viruses are individual functional proteins formed by the proteolysis of its long polypeptide precursor which is processed by chymotrypsin like proteases accompanying papain proteases. [30] Protein sequence similarity analysis of M Pro on comparing SARS-CoV-2 with that of SARS-CoV shows remarkable protein sequence similarity with around 96% of identical protein sequence along with conserved amino acid sequence among both CoVs, while SARS-CoV-2 virus keeps mutating, suggesting M Pro as ideal drug target (Fig. 1).
The binding e ciency of the drugs was determined through molecular docking. The crystal structure of COVID-19 main protease in complex with an inhibitor N3 at resolution of 1.7 0 A was extracted from protein data bank (PDB ID-7BQY) and visualized using Pymol molecular visualization tool presented in Fig. 2. The amino acid residues interacting with ligand were retrieved by using CASTp 3.0 server by using binding pocket analysis tool and presented in table 2.
The molecular docking analysis of the drugs revealed strong interaction with higher energies and binding a nity against the SARS-CoV-2 Mpro. The potential antiviral drugs bind with the unrestrained conformation in the same active groove of protein, where it was co-crystallized with bound native ligand.
Intermolecular interaction and ranking based on the molecular docking binding a nity and binding energies of the potential drugs is shown in table 3. Re-docking of two reference compounds (lopinavir and ritonavir) were also done in order to check the credibility of the software Autodock 4.2. It was found to be almost same indicating the high delity of the docking method. In this study, our focus is on top drug candidates among selected 10 drugs for further analysis as these drugs showing a range of binding a nity from (-10.36 to -5.81). Despite of that among 10 drug compounds, the top 3 drug compounds atazanavir, nel navir and letermovir were showing binding a nity even higher than of our reference compounds (Fig. 2).
The global outbreak of novel coronavirus SARS-CoV-2rapidlyspreading disease named Covid-19, declared asa pandemic by the World Health Organization. This novel virus has affected nearly 206 countries globally after getting hike exponentially in the second week of March 2020.
In the recent study of crystal structure of SARS-CoV-2 main protease M Pro (PDB ID: 7BQY) in complex with an inhibitor N3 at 1.7 A, it is shown that targeting the main protease M Pro represents the best attractive drug target. It can restrict the production of non-structural viral proteins thus, inhibiting replication events of the virus life cycle. [7] However, testing the already approved drugs against the COVID-19 as drug repurposing strategy facilitates the discovery of potential drug candidates. Ritonavir and Lopinavir are thoroughly researched and established FDA approved protease inhibitors for HIV. [31] [32] Previously these drug candidates were also suggested for treatment of SARS and MERS [33]. Additionally, this combination has been utilized in COVID-19 patients to control the infection. [ Table  3). Interestingly, it was found that our top three drugs Atazanavir, Nel navir and, Leteromovir were interacting with a much more appreciable binding a nity even better than that of our reference drugs.
However, we emphasize that all the repurposed drugs considered and screened in this study for disease COVID-19 should be validated and in vitro inhibitory potential needs to be analyzed during various robust biophysical and biochemical procedures leading clinical trials.

Conclusion
Repurposing of FDA approved drugs could be one of the potential solutions to control the outbreak of SARS-CoV-2 virus in the present scenario. The crustal structure analysis showed that M Pro represents the best attractive drug target for the virus. Atazanavir, Nel navir, Letermovir, Indinavir, Irinotecan, Amprenavir, Raltegravir, Saquinavir, Darunavir, and Elbasvir FDA approved drugs were investigated as the potential inhibitor of SARS-CoV-2 main protease M Pro . Molecular docking retrieved that Atazanavir, Nel navir and, Leteromovir showed signi cant binding a nity of -10.36 kcal/mole, -9.47 kcal/mole, -9.43 kcal/mole respectively which is even better than the reference drug. The study can help in designing drugs for controlling and preventing the COVID-19 outbreak.
Declarations Figure 1 (A) Superimpose protein structure of main protease MPro. SARS-CoV-2 is shown as yellow while SARS-CoV as green and conserved amino acid residues highlighted with blue. (B) Multiple sequence alignment of amino acid sequence of SARS-CoV-2 and SARS CoV. Identical amino acid sequence of each protein is highlighted as green along with residues marked underneath with * show catalytic residues and amino acids marked with # shows ligand binding residues of different subsites.

Supplementary Files
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