Exploring the binding efficacy of ivermectin against the key proteins of SARS-CoV-2 pathogenesis:

Aim: COVID-19 is currently the biggest threat to mankind. Recently, ivermectin (a US FDA-approved antiparasitic drug) has been explored as an anti-SARS-CoV-2 agent. Herein, we have studied the possible mechanism of action of ivermectin using in silico approaches. Materials & methods: Interaction of ivermectin against the key proteins involved in SARS-CoV-2 pathogenesis were investigated through molecular docking and molecular dynamic simulation. Results: Ivermectin was found as a blocker of viral replicase, protease and human TMPRSS2, which could be the biophysical basis behind its antiviral efficiency. The antiviral action and ADMET profile of ivermectin was on par with the currently used anticorona drugs such as hydroxychloroquine and remdesivir. Conclusion: Our study enlightens the candidature of ivermectin as an effective drug for treating COVID-19.


The Coronaviridae family of viruses has engraved its name in history by cursing humankind with three major blows – the severe acute respiratory syndrome (SARS) caused by SARS-CoV, middle east respiratory syndrome (MERS) caused by MERS-CoV, and the latest pandemic outbreak in the form of COVID-19 caused by SARS-CoV-2 ]. Hitherto, there are over 21.29 million confirmed cases of COVID-19 globally, which has already taken 0.76 million lives till the mid of August 2020 []. The SARS-CoV-2 belongs to the β-Coronavirus genus of a 2B group of the Coronaviridae family. This viral strain consists of four major structural proteins such as S protein, which encompasses the spike, E forming the envelope, M for the membrane and N for the nucleocapsid. This nucleocapsid contains a 29,903 base long, positive-sense single-stranded RNA genome []. The virus transmits from person to person mainly via close physical contact and by respiratory aerosols which are produced during coughing, sneezing, and even talking []. It is well assumed that the virus may also spread via fecal matters and by fomite transmission, which occurs when a person comes into contact with a contaminated surface []. Unrestricted domestic and international air travels from COVID hotspots are also considered significant contributors to the global spread of this viral infection [].

To date, several postulations are available regarding the mechanism of the pathogenesis of the virus in a human host. However, the actual mechanistic pathway is still undefined. In general, aerosol droplets containing the virus particle gains access into the human respiratory system, precisely the alveolar membranes []. After its entry into the respiratory system, the spike glycoprotein ectodomain present on the viral capsid binds to the angiotensin-converting enzyme-2 (ACE2) transmembrane receptor protein, consequently, the RNA genome enters the alveolar cells by receptor-mediated endocytosis []. The viral RNA-dependent RNA polymerase (RDRP; replicase) is eventually translated from its mRNA strand with the help of its main protease enzyme, and the replicase enzyme catalyzes the rapid replication of the viral genome alongside other structural proteins required for reconstructing new viral particles []. In addition to these, interactions between the viral antigens and host immune cells are considered as a crucial determinant factor of the immunopathological attributes of COVID-19 []. Proinflammatory responses induced from host–virus interactions trigger vasodilation, accumulation of humoral factors that ultimately result in fever, abnormal alveolar exchange and breathing difficulty, leading to death of patients [].

While the pandemic is spreading faster than wildfires, the unavailability of ratified drugs and or vaccine against the same has made the situation more alarming. In this context, recent studies on the use of hydroxychloroquine (an antimalarial drug) in combination with the antibiotic azithromycin [] and antiretroviral drugs like remdesivir, EIDD-2801 or favipiravir have shown effectiveness against SARS-CoV-2 []. Based on this, ivermectin has been recently reported as the most active agent against COVID-19 among the US FDA-approved drugs in vitro trial []. Ivermectin is a macrocyclic lactone natively used to treat a broad spectrum of parasitic infestations including lymphatic filariasis and onchocerciasis [14]. Interestingly, a recent study claims that the drug inhibits the replication of SARS-CoV-2 in in vitro condition and can reduce the spread of the virus by approximately 5000-times within 48 h while being tested in vitro using primate cell lines [13]. Considering the therapeutic promise of ivermectin against COVID-19 [15], the present study has been conducted to represent the efficacy of this drug against the four most crucial functional proteins of SARS-CoV-2 using advanced biocomputational approaches. Moreover, the efficacy of ivermectin has been compared with two of the recently used anticorona drugs, namely hydroxychloroquine and remdesivir.


Materials & methods


Data mining

The commercial ivermectin formulation is comprised of a racemic mixture of -O-dimethyl-22,23-dihydroavermectin B1a (ivermectin B1a) and 5-O-dimethyl-22,23-dihydroavermectin B1b (ivermectin B1b) and both structures were used in this study. 3D structures of ivermectin homologs, hydroxychloroquine and remdesivir were retrieved from PubChem compound library (https://pubchem.ncbi.nlm.nih.gov/). The structures were converted in .pdb format for further use. The structure of each ligand of ivermectin obtained from the Pubchem library was converted to 3D conformer (Supplementary Figure 1A) with minimal energy using Frog2 server. The 3D conformers of both remdesivir and hydroxychloroquine were downloaded from PubChem library. All these 3D conformers were used in protein–ligand docking study.

Full-length amino acid sequences of human ACE2 receptor protein (Accession ID: AAT45083.1), Human TMPRSS2 (Accession ID: AAH51839.1), SARS-CoV-2 Spike S1 receptor-binding domain (RBD; Accession ID: pdb|6M17|F) and SARS-CoV-2 NSP9 replicase enzyme (Accession ID: pdb|6W4B|A) were retrieved from NCBI protein database (www.ncbi.nlm.nih.gov). Furthermore, the crystal structure of SARS-CoV-2 protease (Protein Data Bank [PDB] ID: 6Y2E [DOI: 10.2210/pdb6Y2E/pdb]) was obtained from the RCSB PDB (www.rcsb.org). The crystal structure was generated ab initio by using x-ray diffraction techniques with a resolution of 1.75Å. A resolution below 3.0Å suggests good structural detailing which is desirable for molecular docking studies. This structure was introduced to PyMOL software application, whereby water molecules present in the original crystal structure were separated and removed from the native structure of the protein such to avoid undesirable interferences. On the other side, the structure of S2 subunit of spike protein was separately modeled by using the amino acid sequence of S2 and PDB ID 6VYB as a template. Crystal structure of the SARS-CoV-2 in native form, the RDRP was acquired from PDB (ID: 6M71). 3D structure of target proteins from SARS-CoV-2 and humans are represented in Supplementary Figure 1B–H.


Results


Molecular docking studies

In the present study, molecular docking was used to explore the targets of ivermectin in SARS-CoV-2 and to determine the comparative therapeutic efficacy with hydroxychloroquine and remdesivir, which are currently in use for treating COVID-19. While working with the molecular models, the quality of emulation of the molecular mechanics is known to depend on the feature of the models used for docking [17]. Therefore, we checked the stereochemical quality of each model. It was found that all the models had more than 92% of residues in favored regions, and it may indicate an optimal stereochemical quality that can be used for further studies (Supplementary Figure 2). Docking studies conducted using Hex provides E-value for every binding conformation, which is just inversely proportionate to the binding efficiency of the structure characterized by negative E-value. Suspiring confidence from the above assessment, protein–ligand docking studies were performed to gain insight into the most probable and efficient binding conformations of ivermectin with the proteins of interest. The results have been furnished in the subsequent subsections mentioned in the below.


Interaction of ivermectin with the spike glycoprotein of SARS-CoV-2

Our experimental data on the docking of ivermectin on SARS-CoV-2 spike protein (in native form) revealed a strong binding of the compound with an energy value of -261.74 and -287, respectively, for B1a and B1b homologs. Spike protein is a homotrimeric protein with two functional S1 subunits and one structural S2 subunit [18]. Therefore, we checked the actual binding site of ivermectin isomers in the spike protein through separate docking using S1 and S2 subunits. Results of molecular docking using the Hex software program are shown in Figure 1A and Table 1. It was observed that the ivermectin homologs can bind with both S1 (the receptor-binding domain of the spike protein) and S2 subunits of the SARS-CoV-2 spike protein. But, the strength of the binding of ivermectin isomers were more intense on the S2 subunit (Figure 1A & Table 1). Energy value (ETot- values) for the interaction of B1a and B1b were -372.99 and -393.29 for S1 protein while -395.9 and -411.6. Therefore, it may be inferred that binding of ivermectin at S2 subunit of spike protein may cause an allosteric effect, which in turn can induce a conformational change in the whole protein or receptor-binding S1 subunit. Ivermectin B1a has been found to be the better molecule in targeting spike protein or its subunits than B1b isomer. We also scrutinized the stability of ivermectin-SARS-CoV-2 spike protein complex through molecular docking analysis stated in the later part of the manuscript.



Exploring the binding efficacy of ivermectin against the key proteins of SARS-CoV-2 pathogenesis:

Interaction of ivermectin with SARS-CoV-2 replicase & RDRP

Ability of transcribing RNA using replicase and/or RDRP is one of the unique pathogenic hallmarks of SARS-CoV-2. In this connection, we have investigated whether the ivermectin could bind to RNA-synthesizing machinery, in other words, the viral replicase and/or RDRP enzyme or not. Our data revealed that the 5-O-dimethyl-22,23-dihydroavermectin B1a and ivermectin B1b homologs are able to bind with viral replicase (NSP9) with respective energy value of -327.47 and -352.2 (Table 1). Furthermore, we have also found that this strong interaction between replicase and ivermectin is due to intense binding of ivermectin at the RDRP domain (Figure 2B). Ivermectin B1b isomer was found to be the better molecule to form strong interaction with both replicase and which revealed very weak interaction with ivermectin though both of the ivermectin isomers were found to interact with the target protein (Figure 2A–B & Table 1). Major interacting residues of ivermectin forming noncovalent bonds with replicase and RDRP are presented in Figure 2A–B. Alike other protein targets, the binding affinity of ivermectin B1b to replicase and/or RDRP was higher than the binding of ivermectin B1a (Figure 2A & B).


Exploring the binding efficacy of ivermectin against the key proteins of SARS-CoV-2 pathogenesis:

Conclusion

Developing an effective therapeutic against COVID-19 is currently the utmost interest to the scientific communities. The present study depicts comparative binding efficacy of a promising FDA-approved drug, ivermectin, against major pathogenic proteins of SARS-CoV-2 and their human counterparts involved in host–pathogen interaction. Herein, our in silico data have indicated that ivermectin efficiently utilizes viral spike protein, main protease, replicase and human TMPRSS2 receptors as the most possible targets for executing its antiviral efficiency. Therefore, ivermectin exploits protein targets from both virus and human, which could be the reason behind its excellent in vitro efficacy against SARS-CoV-2 as reported by Caly et al. [13]. Ivermectin B1b isomers have been found to be the more efficacious molecule out of the two homologs. Intriguingly, comparison of the in silico efficiency of ivermectin with currently used anticorona drugs, such as hydroxychloroquine and remdesivir, indicated toward the potential of ivermectin to target the major pathogenic proteins of SARS-CoV-2. Ivermectin is a popular antiparasitic drug and is also safe in children, younger adults, pregnant and lactating ladies. Development of pulmonary delivery of ivermectin through synthesis of better ivermectin formulation has been reported recently and this is expected to shorten the treatment duration and lead to better outcomes [33]. It is noteworthy to mention that many anti-SARS-CoV-2s are now being tested for their efficacy in shaping the immune response of humans, through targeting the cell surface as well as intracellular toll-like receptors [34,35]. In this context, ivermectin could be an effective option as well. Considering all these facts, the present study explores the therapeutic targets of ivermectin against SARS-CoV-2 and enlightens the possibility of using this drug in COVID-19 clinical trials shortly.


Credited to Future medicine


 


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