Vipul Kumar1, Sudhakar Kancharla2, Manoj Kumar Jena3*
1Department of Biotechnology, School of Bioengineering and Biosciences,
Lovely Professional University, Punjab, India.
2Devansh Lab Werks, 234 Aquarius Drive, Homewood, Alabama, USA 35209.
3Department of Biotechnology, School of Bioengineering and Biosciences,
Lovely Professional University, Punjab, India.
Since the outbreak of severe acute respiratory syndrome corona Virus -2 (SARS-CoV-2) has happened in December 2019 in Wuhan, China, the cases of novel coronavirus disease (COVID-19) is rapidly increasing worldwide. In the absence of specific drugs against COVID-19, the fast and reliable choice would be repurposing of existing drugs. Here, we have chosen one of the crucial enzymes of the SARS-CoV-2, Papain like protease (PLpro) and its mutant C111S for the structure-based in-silico screening of the FDA approved drugs. Firstly, the alignment of the wild type and mutant PLpro was done, and no significant change in the global structure was observed. Then based on the docking study, we have reported the best 3 compounds against a mutant and wild type PLpro. These lead compounds include amikacin and mafenide, which are well-known antibiotics. The binding affinity, as well as number of polar and non-polar interactions, indicates their potential against the PLpro. This computational study strongly suggests the experimental validations of the predicted compounds for a confident claim.
The recent outbreak of severe acute respiratory syndrome corona Virus -2 (SARS-CoV-2) has taken millions of lives and has given a major challenge to the world medical system.1 It has been declared as pandemic worldwide by world health organization (WHO). As of 12th May, it has been spread in 216 countries, and 294190 confirmed deaths have been reported worldwide due to novel coronavirus disease (COVID-19) (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). This outbreak has given a major threat to the world economy and the health care system.
SARS-CoV-2 belongs to the virus family coronaviridae and possess large positive-sense RNA (~30kb) of genetic material.2,3 SARS-CoV-2, Middle East Respiratory Syndrome Virus (MERS) and SARS-CoV are included in the beta class of the coronavirus.3-5 And they all are known to infect both animals and humans. All the beta class coronaviruses attack the respiratory system of humans mainly and the major reason for pneumonia.6,7 The severity in the current outbreak and the number of infected cases is suggesting that SARS-CoV-2 is more contagious than previously known coronaviruses8,9
The complete genome sequencing of SARS-CoV-2 revealed that it encodes several structural and non-structural proteins, which helps in the entry and replication of the virus. Papain, like protease (PLpro) or chymotrypsin-like protease, is one of the crucial enzymes of the SARS-CoV-2, which helps in the virus replication inside the host cell.10,11 PLpro has been reported as part of the replicase polyprotein 1a of SARS-CoV-2, but it gets cleaved out from the protein due to its own catalytic activity and performs its major catalytic activity inside the host cytoplasm. It has been reported that after invading the host cell, the viral genome is released as a single-stranded positive RNA. Subsequently, it is translated into viral polyproteins using host cell protein translation machinery, which is then cleaved into effector proteins by viral proteinase, PLpro.12-14 The recent study has shown that PLpro shows significant homology with other coronaviruses and could be an effective therapeutic target for COVID-19.15
Many attempts have been made till now for developing new drugs from natural resorces, repurposing of existing drugs, vaccines and antibodies through in-silico and experimental methods16-29. Recently food and drug administration (FDA) has approved remdesivir, an antiviral drug for the treatment of COVID-19 due to this severe emergency (FDA news release 2020). The repurposing of the existing drug has always been a reliable and fast way to combat any new outbreak of diseases, taking this into consideration we have done a virtual screening of FDA approved drugs against PLpro. Two variant of the PLpro, a wild type (wt) and a C111S mutant (mt) has been chosen for the study, and 1657 FDA approved drugs were screened against both of the protease variants. Top 3 drugs have been reported according to the binding affinity towards each of the PLpro variants.
The structure of all the SARS-CoV-2 targets was retrieved from the RCSB protein data bank. The structure of the wildtype PLpro having PDB ID 6W9C (Osipiuk J et al., unpublished data) and mutant PLpro (C111S) having PDB ID 6WRH (Osipiuk J et al., unpublished data) were retrieved. Pre-processing including removal of water molecules, the addition of polar hydrogen atoms, and the deletion of non-essential heteroatoms were done using Discovery studio software 2020.30
A total of 1657 FDA approved drug library was retrieved from the ZINC15 database in SDF format.31 All these drugs were minimized using UFF forcefield with conjugate gradient protocol using PyRx software.32 Further docking was performed using Autodock vina33, built inside the PyRx Software.
From the docking study of the 1657 FDA drug compounds, based on the binding energy, the top 3 compounds have been reported against both wt PLpro and mt PLpro. Furthermore, the critical polar and non-polar interactive residues were analysed and have been shown using Discovery studio software.
Computational structure-based drug discovery has been known for fast and reliable screening of the lead molecules against any therapeutic target. Given the spread of the COVID-19, we tried to investigate the potential of already approved drugs against the replication of the virus. Till now two main proteases 3CLpro and PLpro are known of this virus which, cleaves different polyproteins, which further helps in the replication of the virus inside the host cells. Lots of attention so far has been given to the 3CLpro as its 3D structure came early and it becomes an easy target for structure based drug research34-36 ,hence in this study we studied an another curicial enzye PLpro. The PLpro plays a crucial role in SARS-CoV-2 replication by cleaving the polyproteins and making them functional which are involved in the replication of the virus inside the host cells, hence targeting its catalytic activity could hinder the viral replications. We have done a structure-based virtual screening of the FDA approved drugs against the PLpro. The virtual screening was performed with all the 1657 FDA approved compounds, by generating the grid at the substrate-binding site of the protease. The main catalytic residues, namely ASP164, VAL165, CYS270, LYS274, VAL30, were selected for generating the grid. The size of the grid was 25 Angstrom3 with centre of X= -30.29, Y = 31.21, Z= 31.43. The result of the screening showed that amikacin had the highest binding affinity with a binding energy of -6.35Kcal/mol, followed by mafenide (-5.83Kcal/mol) and eflornithine (-5.75 Kcal/mol). Amikacin is an approved aminoglycoside antibiotic that mainly works against gram-negative bacteria by hindering their replication activity.37,38 Also, mafenide is an another antibiotic agent recommended for the tissue burn, which works against both gram-positive and gram-negative Bactria.39,40 While eflornithine is used for sleeping sickness as in the treatment of facial hirsutism.41 Amikacin was making five hydrogen bonds namely, ASN109, GLY163, GLU167, GLY271, THR301, while two electrostatic interactions with ASP164 and ASP302. Further mafenide was making three hydrogen bonds with PLpro Gly163, Tyr264, and Thr301 and one electrostatic bond with Asp164. And, eflornithine was involve in making one hydrogen bond, Thr301 and three electrostatic interactions, Arg166, Asp164 and Asp302. The polar, as well as non-polar interactions between these drugs and the PLpro, have been shown in Figure1. As already reported in some studies previously that hydroxychloroquine works better when taken with antibiotic azithromycin42, indicating the potential of the antibiotics against this virus. Also, the high binding affinity of the anti-bacterial agents against PLpro indicating that these antibiotic agents could be the alternative option in the management of COVID-19 infection. This computational study warrants the careful and thorough investigation of these lead antibiotics through in-vitro and in-vivo assays against SARS-CoV-2.
Figure 1: The polar and non-polar interaction between wild type Papain like protease (wt PLpro) and drugs. (A) wt PLpro- amikacin (B) wt PLpro- mafenide (C) wt PLpro- eflornithine.
Recently it has been noticed that the major reason for the inefficacy of the trial of novel as well as existing drugs against SARS-CoV-2 is the mutations in the different strains. Recently the C111S mutant of the PLpro structure was submitted in the PDB (6WRH), gave an opportunity to test these drugs against this mutant. Firstly, the alignment of the mt PLpro was done with wt PLpro to investigate the global changes in the structure due to mutation. And it was found that the change in RMSD was insignificant with a value of 0.68 Angstrom. Further, to identify the drugs against mt PLpro, the screening with the same 1657 FDA drugs was done, making the grid as the exact same location as for wt PLpro. The result obtained was interesting, as, in spite of no significant change at the binding site as well as globally in the structure between wildtype and mutant, no drug was found to mutual against wt and mt PLpro in the top three lead compounds based on the binding energy. The result against mt PLpro showed that trimipramine had the highest binding affinity with binding energy -5.53Kcal/mol, followed by protriptyline (-5.33Kcal/mol) and lenalidomide (-5.32Kcal/mol). Currently, both trimipramine and protriptyline are used as an anti-depressant, and they both are also known to be immunomodulatory agents.43-46 While lenalidomide is a thalidomide analog, is also an immunomodulatory agent possessing immunomodulatory properties and is recommended for the treatment of multiple myeloma.47, 48 The trimipramine was involved in various pi-alkyl intercations, LEU162, PRO248, AS267, TYR273 and one electrostatic bond with ASP164. Similarly, protriptyline was also making many pi-alkyl interaction, LEU162, PRO248, ASN267and further it was making two elctrostic bond with ASP164 and ASP302 in the best docking pose. And, lenalidomide was one hydrogen bond with TYR273 and two pi-alkyl interactions with PRO247 and PRO248. Interactions between the predicted drugs and the mt PLpro has been shown in Figure 2. The results indicating that these FDA approved drugs could also inhibit the functional activity of the mt PLpro, although various other physiological factors, as well as experimental validations, will be required for any confident claim.
The scientist across the globe is still in the search of best novel or existing drug which could cure the COVID-19, in this pandemic situation the discovery of drugs could be accelerated with the help of computational power. This study could help the scientific community in the further investigation of the potential of these drugs by cutting the cost and time in the screening of the drugs.
Figure 2: The polar and non-polar interaction between mutant (C111S) Papain like protease (mt PLpro) and drugs. (A) mt PLpro- trimipramine (B) mt PLpro- protriptyline (C) mt PLpro- lenalidomide.
This computational study strongly suggests a need to investigate the predicted anti-bacterial agents and immunomodulatory drugs against SARS-CoV-2 through in-vitro and in-vivo assays. In this current pandemic situation and the absence of the specific drug against the virus, the repurposed approved drugs would be an effective, quick, and cheap option for the management of COVID-19.
5. CONFLICT OF INTEREST:
There is no conflict of interest exists among the authors.
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Received on 03.07.2020 Modified on 07.09.2020
Accepted on 13.10.2020 © RJPT All right reserved