Mechanistic insights into the inhibitory activity of FDA approved ivermectin against SARS-CoV-2: old drug with new implications
The novel coronavirus (Covid-19) has become a great challenge worldwide since 2019, as no drug has been reported yet. Different clinical trials are still underway. Among them is Ivermectin (IVM), an FDA approved drug which was recently reported as a successful candidate to reduce SARS-CoV-2 viral load by inhibiting Importin-α1 (IMP-α1) protein which subsequently affects nuclear transport of viral proteins but its basic binding mode and inhibitory mechanism is unknown. Therefore, we aimed to explore the inhibitory mechanism and binding mode of IVM with IMP-α1 via different computational methods. Initially, comparative docking of IVM was performed against two different binding sites (Nuclear Localization Signal (NLS) major and minor sites) of IMP-α1 to predict the probable binding mode of IVM. Then, classical MD simulation was performed (IVM/NLS-Major site and IVM/NLS-Minor site), to predict its comparative stability dynamics and probable inhibitory mechanism. The stability dynamics and biophysical analysis of both sites highlighted the stable binding of IVM within NLS-Minor site by establishing and maintaining more hydrophobic contacts with crucial residues, required for IMP-α1 inhibition which were not observed in NLS-major site. Altogether, these results recommended the worth of IVM as a possible drug to limit the SARS-CoV-2 viral load and consequently reduces its progression. Communicated by Ramaswamy H. Sarma
1. Introduction In early December 2019, an outbreak of novel Coronavirus Disease-2019 (COVID-19) appeared in Wuhan, China(Huang et al., 2020; Zhu et al., 2020) that eventually becomes global ongoing pandemic owing to its contagious nature (Chen, Hu, et al., 2020). The Chinese center for disease control and prevention (CCDC) identified causative agent on January 7, 2020 from throat swab samples and named it Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a single-stranded RNA virus. The disease was named COVID-19 by the World Health Organization (WHO) (Sohrabi et al., 2020). The patients had developed symptoms of fever, dry cough, and sore throat (Chen, Mao, et al., 2020). Most of the cases were spontaneously recovered however; few had developed several fatal complications like organ failure, pulmonary edema, severe pneumonia, septic shock, and Acute Respiratory Distress Syndrome (ARDS). The SARS-CoV-2 outbreak was declared as a Public Health Emergency of International Concern (PHEIC) and a pandemic on January 30, 2020 and then on March 11, 2020 by the World Health Organization (WHO) (Vardhan). The total confirmed COVID-19 cases by 15 April 2020 reported to 1,914,916 across 210 countries with a total 123,0107 deaths (2020). Various studies have been conducted and some are still in process to develop COVID-19 vaccine. According to recent research conducted by WHO 21 vaccine candidates against COVID-19 are evaluated clinically (www.who.int) (Calina et al., 2020). So far remdesivir is the only drug approved for conditional marketing in European Union. Although no antiviral drug has been clinically approved for SARS-CoV-2 (Chen, Mao, et al., 2020), however clinical trials on various vaccine candidates are under way (Chen, Hu, et al., 2020; Gao et al., 2020; Gautret et al., 2020). In this regard, the antiviral activity of an Food and Drug Administration (FDA) authority approved antiparasitic drug Ivermectin (IVM) (Buonfrate et al., 2019; Canga et al., 2008), was studied for novel causative virus (SARS-COV-2) (Caly et al., 2020). In vitro studies of IVM had revealed antiviral activity against various viruses such as dengue virus, influenza virus, Human Immunodeficiency Virus (HIV-1) (Götz et al., 2016; Lundberg et al., 2013; Tay et al., 2013; Wagstaff et al., 2012). The investigation of its inhibition mechanism revealed that IVM inhibit the association of importin (IMP) α/β 1 heterodimer which is required for the transport of different viral proteins into the nucleus. Consequently, IVM serves to impede nuclear import and viral replication(Wagstaff et al., 2012). IVM has also expressed several other activities, nevertheless it is originally known as inhibitor of nuclear import of host (Kosyna et al., 2015; van der Watt et al., 2016) and viral proteins, for instance simian virus SV40 large tumor antigen (T-ag) (Tay et al., 2013), and the nonstructural protein 5 (NS5) of Dengue virus (Wagstaff et al., 2011; 2012) . It has markedly reduce the infection of RNA Viruses like influenza (Chen, Hu, et al., 2020; Götz et al., 2016).Venezuelan equine encephalitis virus (VEEV), West Nile Virus (Yang et al., 2020), and DENV 1–4 (Tay et al., 2013). This effective broad range activity of IVM for many distinct RNA viruses is due to the fact that these all viruses depend upon IMP α/β 1 for infection and replication (Caly et al., 2012; Jans et al., 2019). Additionally, various in vitro and in vivo studies has also reported the potency of IVM drug against the DNA viruses i.e. pseudo rabies virus (PRV) (Lv et al., 2018). Likewise, in year 2014–2017 clinical trials of IVM against DENV were also carried out, which revealed dengue inhibition and safety both, however no clinical benefits and alteration in viremia was detected. Considering all above information, Caly et al. (2020) investigated the therapeutic potential of IVM against SARS-COV-2. IVM showed a great inhibitory potential against IMPα/β protein and subsequently affect its nuclear transport. The IMPα/β is responsible for the transport of viral protein into and out of the nucleus and this movement is considered critical for several cellular processes such as differentiation and development which is also essential for disease states including oncogenesis and viral diseases. As mentioned earlier that the antiviral activity of IVM against both DENV and HIV-1 is strongly dependent upon importin α/β nuclear import of NS5 polymerase and HIV-1 integrase protein respectively. In case of COVID-19, causative agent SARS-CoV-2 a single stranded positive sense RNA virus displayed great resemblance to severe acute respiratory syndrome coronavirus (SAR-CoV). Investigation of SARS-CoV proteins indicated a vital role of IMPα/β1 during disease in signal-dependent nucleocytoplasmic shutting of the SARS-CoV nucleocapsid protein (Rowland et al., 2005; Timani et al., 2005; Wulan et al., 2015), that may affect host cell division (Hiscox et al., 2001; Wurm et al., 2001). Likewise, the accessory protein Open Reading Frames (ORF6) of SAR-CoV has shown to antagonize the antiviral activity of the STAT1 transcription factor by sequestering IMPα/β1 on the rough ER/Golgi membrane (Frieman et al., 2007; Vardhan). Above all, Caly et al. (2020) had also hypothesized that IVM dependent inhibition of nuclear transport could be a reasonable explanation of its inhibitory potential, but the basic inhibitory and binding mechanism of IVM with reference to IMP α/β1 is still unknown. Therefore, in this study, we aimed to explore the inhibitory mechanism and binding mode of IVM in association with importin-α1 protein by utilizing various computational approaches. In the first phase, comparative docking of IVM was performed against two different binding sites (Nuclear Localization Signals (NLS) major and minor sites) of importin-α1 to predict the probable binding mode of IVM. While in the second phase, we performed classical molecular dynamics simulation of both complexes, i.e. (I) IVM/NLS-Major site and (II) IVM/NLS-Minor site. 2. Materials and methods 2.1. Protein preparation The crystal structure of Importin-α1 in complex with a phosphomimetic peptide GM130 (PDB ID: 6K06) was accessed from RCSB Protein Data Bank with the resolution of 1.75 Å (Chang et al., 2019). As we need to explore the inhibitory mechanism of IVM within the binding cavity of target protein, the peptide and water molecules were removed and apo protein was selected and prepared. Furthermore, the protein structure was refined and optimized to relieve all the steric clashes via preparation wizard in MOE2019.01 (MOE, 2021). Afterwards, protein was charged and minimized by using AMBER99 forcefield to attain the lowest possible energy. 2.2. Molecule preparation The 2-D (.smi format) Ivermectin (IVM) was extracted from PUBCHEM (PubChem CID 6321424) (Pubchem.) and converted to 3-D format by MOE Builder Suite of MOE2019.01 (MOE, 2021). The molecule was prepared, charged and minimized by the MM94x forcefield. 2.3. Molecular docking protocol Since the binding mode of Ivermectin after binding with IMP-α1 has not been investigated yet. Therefore, first of all IVM molecule was docked into both NLS-major and minor site to investigate the comparative binding mode of Ivermectin within two different binding sites of IMP-α1 i.e. NLS-Major and NLS-Minor site. In this regard, the Dock module of MOE2019.01 (MOE, 2021) was utilized to analyze the suitable binding mode of Ivermectin. Before execution of docking, we benchmark different combination of scoring and placement methods of MOE Dock to find out the most suitable algorithm with respect to our target protein. In our case, induce fit docking protocol along with Triangle Matcher algorithm and LondonDG as initial scoring and re-scoring method gave the most reliable results with lowest binding energy, as compared to other combinations of algorithms. Afterwards, 30 conformations of IVM were generated and saved in .mdb file for each site. Finally, the best ligand conformation with highest scoring and lowest binding energy were visually inspected at molecular level to predict the probable binding mode of IVM within one cavity over other.
2.4. Molecular dynamic (MD) simulation protocol To investigate the comparative inhibitory mechanism and stability dynamics of Ivermectin within two different binding pockets of importin-α1, Molecular Dynamic Simulation of three systems were carried out including (i) Apo-system having importin-α1 protein, (ii) IVM/NLS-Major site of IMP-α1, and (iii) IVM/NLS-Minor site of IMP-α1. The topology of Ivermectin for all the system was generated using Automated Topology Builder (ATB) web server (Malde et al., 2011). While the topology of target protein was generated by using pdb2gmx module in explicit solvent under periodic boundary condition via application of an GROMOS56a force field (Berendsen et al., 1995). Then the systems were solvated in a cubic box of SPCE water molecules with a distance cutoff of 1.0 nm between the solute and edge of water box. The perturbed and unperturbed charges of the system were neutralized by adding appropriate number of counter ions. Energy minimization was performed at 10 KJ/mol with 50,000 steps of steepest descent algorithm by using Verlet cut off scheme (Páll & Hess, 2013) for each system to remove all the steric clashes and attain lowest possible energy. Then 1 ns equilibration was carried out under constant number of atoms, volume and temperature (NVT) at 300 K. The temperature was regulated by the velocity rescale algorithm. The LINCS holonomic constraints (Hess et al., 1997) were applied with the time step of 2 fs. The particle mesh Ewald (PME) method were used to treat the long-range electrostatic and van der Waals interactions (Hess et al., 1997). After that, during the second step, 1 ns equilibration following the, Parrinello–Rahman semi-isotropic pressure coupling (Parrinello & Rahman, 1981) was performed at constant pressure (1 atm) and temperature (NPT) at and 300 K. Finally, 80 ns long production MD was performed for each system to generate various sample snapshots for analyzing the possible inhibitory mechanism of IVM with respect to its conformational changes. All calculations were performed by GROMACS 5.1.2 (Abraham et al., 2015). The output trajectories of all systems were statistically analyzed to calculate various stability parameters involving root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (RoG) and hydrogen bonds. 2.5. Binding-free energy calculation Finally, total binding free energy of each simulated system were calculated by the Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) method. According to the stability of each system, last 20 ns of the MD trajectory were used to calculate △Gbind by using the following equation:
Ivermectin being an FDA approved drug, has well-established safety profile to be used in humans against various parasitic infections. Recent studies and reviews reported comparable safety of ivermectin at high doses as compared to its standard low dose except its safety profile in pregnancy is not evident yet. In recent study, Caly et al. reported antiviral activity of Ivermectin against SARS-CoV-2 (∼5000-fold reduction of viral RNA) with no toxicity. They hypothesized that this reduction in SARS-CoV-2 RNA is most likely through inhibition IMPα/β1 mediated nuclear import of viral proteins which needs further work up. Therefore, in our study, we explored the comparative binding mode and inhibitory mechanism of Ivermectin against two NLS-Major and NLS-Minor binding sites of IMP-α1. Our results discovered that the ivermectin reduces SARS-CoV-2 viral transport by inhibiting IMP-α1 protein after binding with its NLS-Minor site. The highest negative score in binding-free energy calculation brought forth the stable binding of IVM with NLS-Minor site as compared to NLS-Major site which was further confirmed during 80 ns simulation by investigating different stability parameters. During simulation, IVM stabilizes NLS-Minor site by establishing and maintaining more hydrophobic contacts than hydrogen bonds which is required for the inhibition of IMP-α1 protein. These hydrophobic contacts were not observed in case of NLS-Major site because of shifting from its stable conformation to unstable conformation with the pocket residues. Altogether, these results highlighted the worth of ivermectin along with its safety profile, to be considered as a possible drug to limit SARS-CoV-2 viral load and reduces the progression of this disease.
Credited to Urooj Qureshi