Drug Repurposing to Identify Therapeutics Against COVID 19 with SARS-Cov- 2 Spike Glycoprotein and Main Protease As Targets: an in Silico Study

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Drug repurposing to identify therapeutics against COVID 19 with SARS-Cov- 2 spike glycoprotein and main protease as targets: an in silico study.

Arun K.G, Sharanya C.S, Abhithaj J and Sadasivan C*

Department of Biotechnology and Microbiology, Kannur University, Thalassery Campus,

Kannur, Kerala-670661, India.

*Corresponding Author; e-mail: [email protected] Abstract

The total cases of novel corona virus (SARS-CoV-2) infections is more than one million and total deaths recorded is more than fifty thousand. The research for developing vaccines and drugs against SARS-CoV-2 is going on in different parts of the world. Aim of the present study was to identify potential drug candidates against SARS-CoV-2 from existing drugs using in silico molecular modeling and docking. The targets for the present study was the spike protein and the main protease of SARS-CoV-2. The study was able to identify some drugs that can either bind to the spike protein receptor binding domain or the main protease of SARS-CoV-2. These include some of the antiviral drugs. These drugs might have the potential to inhibit the infection and viral replication.

Introduction

During the end of 2019, outbreak of a chronic respiratory disease was reported in Wuhan province of China which took about 4000 lives and very large number of people infected. The disease was transmitted to many countries around the world in one or two months. As per the latest data, the total number of infections crossed one million around the globe and death toll reached more than 50,000. The deadly pathogen behind these pandemic was found to be a new member of betacorona virus in the Coronaviridae family. Phylogenetic analysis revealed its similarity to the SARS -CoV (Severe Acute Respiratory Syndrome corona virus) reported in China during 2002 and MERS-CoV (Middle East respiratory syndrome coronavirus) reported in Middle East during the year 2013 (1). This novel corona virus was named as SARS-CoV-2 or nCov-19. These are single stranded positive sense RNA virus enclosed within the nuclear envelop surrounded by a membrane and spike glycoprotein which aid in the host cell attachment. The RNA genome of this nCov-19 is about 30 kb in size (2). COVID 19 experience in human as a mild to moderate respiratory problems and can be recovered without any special treatment but elder peoples with diabetes, chronic respiratory diseases, cancer and cardiovascular disease are prone to high risk during this infection. According to the clinical practitioners report, patients with COVID-19 showed symptom of sore throat, cough, fever, muscle pain, tiredness and viral pneumonia. This virus spread from diseased person to other through coughing and sneezing and can be prevented by maintaining a proper distance with others and sanitising hands with alcohol frequently. So practicing personal hygiene and social distancing is the only way to prevent from this deadly pandemic (3, 4). Currently no drugs or vaccines are available to prevent this disease so the drugs for targeting specific protein in virus structure may be a milestone in the drug discovery process.

The genome analysis of Corona virus revealed the presence of structural and non-structural proteins. These include spike glycoprotein responsible for host cell attachment, RNA dependant RNA polymerase and papain like proteases. The receptor binding domain of the spike protein interacts with human Angiotensin- converting enzyme 2 (ACE-2) receptor for the invasion to host cell. The invasion is also activated by priming reaction by host serine protease TMPRSS211 (5). Once the virus invasion occur in the host cell, it release their single stranded RNA into host cell and multiply with the help of host cell protein synthesizing machinery and produce large amount of viral genome.

The spike glycoprotein present in the virus surface facilitates the infection via binding with ACE- 2 receptor (6, 7). The first Cryo EM structure of Novel corona virus spike protein was determined by Wrapp et al., in 2020 (8). The spike glycoprotein is is a homo-trimer and cleaved by furin-like proteases of host cell into SI and S2 subunits. The S1 subunit is the N-terminal domain which is responsible for the host cell attachment by binding to the cell membrane receptors through its receptor binding domain (RBD) and the S2 is the C-terminal domain (9, 10). The RBD of the S1 subunit is therefore responsible for the zoonotic transmission, recognition of host cell and invasion (Fig 1). The S2 subunit comprises two heptad regions, HR1 and HR2 and a lipophilic fusion protein which make the coiled helix structure of the subunit. During the time of infection the RBD in the spike protein binds to the host cell surface receptor leading to conformational changes in both the subunits (S1 and S2) and the fusion loop get exposed and fuse to membrane together with the heptad region, form a bundle fusion core and facilitate the fusion of viral and host cell membranes. A potential therapeutic approach against SARS-CoV-2 infection is to prevent the binding of the viral spike protein to the host ACE-2 receptor. Strong binding of small molecules to the RBD will be an efficient strategy to prevent the binding of RBD to ACE-2 receptor.

Proteases are one of the important and best studied proteins in corona viruses. It is very essential for the processing poly proteins that are translated from viral RNA (11). Inhibition of this viral protease enzyme will results into the blocking of viral replication. In this scenario, the development of inhibitors to the SARS-CoV-2 main protease gains importance in the drug discovery process against COVID-19. Recently Zhang et al solved the crystal structure of inhibitor bound SARS-CoV-2 main protease (12) (Fig 1).

The development of new therapeutics is an expensive and time consuming process. Normally it will take years to get the newly developed drug for the treatment. Drug repurposing is an efficient strategy in medicinal chemistry to bring faster and effective solutions to the unmet medical needs. Repurposing of existing drugs is considered as crucial therapeutic approach for the treatment of COVID-19, considering the fast pace of its spread around the world. The treatment with anti-viral drugs, Favilavir, is an example of such drug repurposing initiatives. Since the number of infections increasing drastically day by day, identifying potent candidates from the existing drugs has great importance. The current study aim to identify potent candidates that can bind with SARS-CoV-2 spike protein and main protease from approved drugs using molecular docking methods. Fig 1: (a) The crystal structure of SARS-CoV-2 spike protein. The human ACE-2 receptor binding domain of spike protein is shown in red colour (b) 3D structure of SARS-CoV-2 main protease. Residues in the active site (His-41, Gly-143, Phe-140 and Glu-166) are shown in red colour.

Methods

In silico screening studies were performed to identify potent drugs that bind to SARS-CoV-2 spike glycoprotein and main protease using Schrodinger suit (Maestro 11.5). The coordinate files of drugs (a total of 4531) were obtained from Drug central data base (http://drugcentral.org/) (13). The ligand structure was prepared and optimized by using lig prep module. The 3D structures of viral proteins, SARS-CoV-2 spike glycoprotein (PDB ID: 6VSB) and main protease (PDB ID: 6LU7), were downloaded from protein data bank. The energy minimization of the protein structures were carried out by protein preparation wizard of Schrodinger suit. Receptor grid was generated around the RBD of CoV-2 spike glycoprotein and around the active site of main protease. Virtual screening studies were performed using three types of docking option available in Schrodinger suit. Initial screening was conducted by High-Throughput Virtual Screening (HTVS) method and the high scoring compounds identified from the screening were further subjected to standard precision docking (SP Docking). Finally extra precision docking (XP docking) was carried out using the high scoring compounds predicted by SP Docking. Results and discussion

The in silico binding analysis showed that some of the drugs screened have the potential to bind either to SARS-CoV-2 spike protein RBD or to the SARS-CoV-2 main protease. The spike protein binds with human ACE-2 receptor through the RBD. The key amino acid residues of RBD involved in the interaction are, Tyr-449, Tyr-453, Tyr-489, Tyr-495, Tyr-505, Asn-487, Gly- 496, Thr-500, and Gly-502 (14). The docking study revealed that the drugs Cytarabin, Raltitrexed, Tenofovir, Cidofovir, Lamivudine and Fludarabine are potent binders to the RBD (Fig 2). The binding affinity expressed in terms of glide score and the RBD residues involved in the binding are given in Table 1. These compounds interacts with key amino acid residues such as Tyr-453, Tyr-495, Gly-496 and Tyr-505 that play important role in ACE-2 receptor binding. Also, the drugs like Azanidazole, Lamivudine, Rosoxacin and Fosfosal showed significant binding with the spike RBD (Table2).

The docking study revealed that the drugs Ribostamycin, Petrichloral, Valganciclovir, Framycetin, Tafenoquine and Vandetanib can bind to Cov-2 main protease (Fig 3). High binding scores were observed for these compounds (Table 3). The crystal structure of CoV-2 main protease (PDB ID: 6LU7) contains a potent ketoamide inhibitor in its active site. The binding is stabilized by hydrogen bonds with His-41, Gly-143, Phe-140 and Glu-166 (12). The docking study revealed that the drugs mentioned above can interact with these key residues on binding (Fig 3). Table 4 shows some other drugs that also can bind to the protease active site. Drug Glide score Residues involved in the hydrogen bonding with the drug

Cytarabine. -7.329 Tyr-453, Tyr-495, Gly-496, Tyr-505

Raltitrexed -6.599 Lys-417, Ile-418, Ser-494, Tyr-495, Gly-496

Tenofovir -6.799 Tyr-495, Gly-496, Tyr-505

Cidofovir -6.627 Tyr-495, Gly-496, Tyr-505

Lamivudine -6.846 Tyr-495, Gly-496, Tyr-505

Fludarabine -7.613 Ile-418, Gly-496, Tyr-505

Table 1: The Glide score and details of interaction of drugs, showed high binding affinity with SARS-Cov-2 spike protein receptor binding domain. Residues in RBD that are crucial for binding with human ACE-2 receptor is shown in red. Fig 2: Schematic diagrams of the drugs cytarabine (a), raltitrexed (b), tenofovir (c), cidofovir (d), lamivudine (e) and fludarabine (f) bound to RBD of SARS-Cov-2 spike protein. Drug Glide score kcal/mol Azanidazole -6.57 Lamivudine -6.568 Rosoxacin -6.58 Fosfosal -6.487 Mezlocillin -6.261 Chlorazanil -6.058 Levomefolic acid -6.174 Vidarabine -6.140 Tisopurine -6.085 Minoxidil -6.13 Succinylsulfathiazole -6.167 Zalcitabine -6.481 Thioguanine -6.28

Table 2: Binding affinity of some other drugs with SARS Cov-2 spike protein receptor binding domain.

Drug Glide score Protease active site residues ( kcal/mol) involved in the hydrogen bonding with the drug Ribostamycin -9.737 Asn 142, Cys 145, His 164, Glu 166, Gln 189 Petrichloral -8.82 Leu 141, His 164, Cys 145 Valganciclovir -8.56 Cys 145, His 163, Glu 166 Framycetin -8.55 Met 49, Asn 142, Cys 145, His 164, Glu 166 Tafenoquine -8.46 Phe 140, Leu 141, Gly 143, Glu 166 Vandetanib -7.84 His 164, Glu 166

Table 3: The Glide score and details of interaction of drugs showed high binding affinity with SARS-Cov-2 main protease active site. Fig 3: Schematic diagrams of the drugs ribostamycin (a), petrichloral (b), valganciclovir (c), framycetin (d), tafenoquine (e) and vandetanib (f) bound in the active site of SARS-Cov-2 main protease. Drug Glide score (kcal/mol) Ertugliflozin -7.548 Canagliflozin -7.318 Dapagliflozin -7.262 Cobimetinib -7.23 Clopenthixol -7.117 Dexlansoprazole -7.066 Diperodon -7.034 Loflucarban -7.228 Benzpiperylone -7.18 Benexate -7.1 Sisomicin -7.78 Talniflumate -7.199 Tobramycin -7.03 Oxyphencyclimine -7.12

Table 4: Binding affinity of some other drugs with SARS-CoV-2 main protease.

Summary

On the backdrop of the rapid spreading of SARS‐CoV-2 infections, drug repurposing has a significant importance to bring out effective medication to treat the disease without delay. In this work, we aimed to screen the drugs to test whether any one of these can be used to treat COVID 19. The computational docking studies revealed that the drugs like Cytarabine, Raltitrexed, Tenofovir, Cidofovir, Lamivudine and Fludarabine have high binding affinity towords the SARS‐ CoV-2 spike protein RBD. These drugs might have the potential to block the binding of SARS‐ CoV-2 spike protein to human ACE-2 receptor and thereby the infection. Similarly the drugs like Ribostamycin, Petrichloral, Valganciclovir, Framycetin, Tafenoquine and Vandetanib can bind to the active site of SARS-CoV-2 main proteases. Hence, these drugs might have the ability to inhibit the protease activity of the viral enzyme and thereby the viral replication. Since the study is based on in silco methods, further in vitro validation is needed to confirm the binding efficiency of these drugs with the target proteins. References

1. Wei-jie Guan, Zheng-yi Ni, Yu Hu, Wen-hua Liang, Chun-quan Ou, Jian-xing He, Lei Liu, Hong Shan, Chun-lia, Lei, David SC Hui, Bin Du, et al. Clinical Characteristics of Coronavirus Disease 2019 in China. The New Eng J Med. 2020; doi.10.1056/NEJMoa2002032 2. Swatantra K, Vimal KM, Anil KP, Madan LBB, Shailendra KS. Structural, glycosylation and antigenic variation between 2019novel coronavirus (2019-nCoV) and SARS coronavirus (SARS-CoV). Virus Dis. 2020;31(1):13–21. doi.10.1007/s13337-020-00571- 5 3. World Health Organization. Coronavirus disease (COVID-19) pandemic.https://www.who.int/emergencies/diseases/novel-coronavirus-2019 Date of accessed: March 31, 2020. 4. Robert V, Lucy CO, Ilaria D, Peter W, Charles W, Natsuko I, et al. Estimates of the severity of coronavirus disease 2019: a model-based analysis. www.thelancet.com/infection Published online March 30, 2020 https://doi.org/10.1016/S1473-3099(20)30243-7 5. Cynthia L, Qiongqiong Z, Yingzhu L, Linda VG, Steve PW, Linda JC, et al. Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Cent. Sci. 2020; 6 (3): 315-331. doi.10.1021/acscentsci.0c00272 6. Prabakaran P, Xiao X, Dimitrov DS. A model of the ACE2 structure and function as a SARS- CoV receptor. Biochem Bioph Res Co. 2004;314 (1): 235-241. 7. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, Luo S, et al. Receptor and viral determinants of SARS‐coronavirus adaptation to human ACE2. The EMBO journal 2005;24 (8): 1634- 1643 8. Daniel W, Nianshuang W, Kizzmekia SC, Jory AG, Ching LH, Olubukola A, et al. Cryo- EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;36: 1260–1263. 9. Fan W, Su Z, Bin Y, Yan-MC, Wen W, Zhi GS, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579: 265–269. doi:10.1038/s41586- 020-2008-3. 10. Yuan Y, Duanfang C, Yanfang Z, Jun M, Jianxun Q, Qihui W, et al. Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. Nat Commun. 2017;8(15092) doi: 10.1038/ncomms15092. 11. Hilgenfeld R. From SARS to MERS: Crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 2014;281:4085–4096. doi:10.1111/febs.12936 Medline 12. Linlin Z, Daizong L, Xinyuanyuan S, Ute C, Christian Dr, Lucie S, Stephan B, Katharina R, Rolf H. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020; doi. 10.1126/science.abb3405. 13. Oleg U, Jayme H, Jeffrey K, Cristian GB, Jeremy JY, Stephen LM, Stuart JN. Tudor I. Oprea, DrugCentral: online drug compendium. Nucleic Acids Research. 2017; 45 (D1): D932–D939. doi. 10.1093/nar/gkw993.

14. Jun L, Jiwan G, Jinfang Y, Sisi S, Huan Z, Shilong F, et al. Crystal structure of the 2019- nCoV spike receptor-binding domain bound with the ACE2 receptor. Biorxiv. doi.10.1101/2020.02.19.956235