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

Exploring the binding efficacy of ivermectin against the key proteins of SARS-CoV-2 pathogenesis: an in silico approach


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: an in silico approach

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: an in silico approach

Discussion

Ivermectin is a popular choice of drug for treating various parasitic infections till today. Since 1987, this drug has been used to treat more than 3.7 billion onchocerciasis patients through the Mectizan Donation Programme sponsored by Merck for eliminating of onchocerciasis [20]. Furthermore, lymphatic filariasis was included in this program in 1998 [21]. Ivermectin is also a member of the three-drug combinatorial therapy alongside the albendazole and diethylcarbamazine [22]. Ivermectin is also efficacious against the Strongyloides, scabies and soil-transmitted helminths [23]. Moreover, ivermectin has also been explored as an endectocide for reducing malarial vectors to reduce the disease transmission [24]. Ivermectin exerts its parasiticidal action through binding and blocking the anion/Cl- ion channels located on the cell membrane, thereby causing disruption of the neuromuscular system leading to paralysis and death [23]. A major advantage of using this FDA-approved drug is its relatively benign nature at treatment doses in humans [25]. Recently, ivermectin has been reported for antiviral activity toward SARS-CoV-2 in vitro [13]. The study depicts that a low dose of ivermectin (5 micromolar) can induce 93% reduction in viral RNA from released virion and 99.8% reduction in cell-associated/unreleased virion after 24 h of incubation [13]. Interestingly, reduction of viral RNA was found to be increased up to 5000-times after 48 h of treatment [13]. Researchers have hypothesized that ivermectin binds and impairs Impα/β1 heterodimer, which plays a key role in binding the cargo protein of coronavirus and facilitates its translocation toward the nucleus [13]. Moreover, researchers have also claimed that ivermectin molecules may act as ionophores and be capable of producing osmotic lysis of the viral membrane [26]. Considering the high and rapid viricidal activity of ivermectin, involvement of a specific target is a question. Therefore, the present study was conducted in silico to explore the possible molecular targets of ivermectin in SARS-CoV-2 and the possible mechanism of interactions between ivermectin and the proteins involved in the viral pathogenesis. Such molecular interactions between ivermectin and the target proteins are most likely mediating the rapid and intense antiviral efficacy of ivermectin.


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