key: cord-0777694-6h10egwm
authors: Vardhan, Seshu; Sahoo, Suban K.
title: Exploring the therapeutic nature of limonoids and triterpenoids against SARS-CoV-2 by targeting nsp13, nsp14, and nsp15 through molecular docking and dynamic simulations
date: 2021-12-13
journal: J Tradit Complement Med
DOI: 10.1016/j.jtcme.2021.12.002
sha: 59f57a02da586a336f643f52e155b60ee5941826
doc_id: 777694
cord_uid: 6h10egwm

BACKGROUND AND AIM: The ongoing global pandemic due to SARS-CoV-2 caused a medical emergency. Since December 2019, the COVID-19 disease is spread across the globe through physical contact and respiratory droplets. Coronavirus caused a severe effect on the human immune system where some of the non-structural proteins (nsp) are involved in virus-mediated immune response and pathogenesis. To suppress the viral RNA replication mechanism and immune-mediated responses, we aimed to identify limonoids and triterpenoids as antagonists by targeting helicases (nsp13), exonuclease (nsp14), and endoribonuclease (nsp15) of SARS-CoV-2 as therapeutic proteins. EXPERIMENTAL PROCEDURE: In silico molecular docking and drug-likeness of a library of 369 phytochemicals from limonoids and triterpenoids were performed to screen the potential hits that binds effectively at the active site of the proteins target. In addition, the molecular dynamics simulations of the proteins and their complexes with the potential hits were performed for 100 ns by using GROMACS. RESULTS AND CONCLUSION: The potential compounds 26-deoxyactein and 25-O-anhydrocimigenol 3-O-beta-d-xylopyranoside posing strong interactions with a minimum binding energy of −10.1 and −9.5 kcal/mol, respectively and sustained close contact with nsp13 for 100 ns. The nsp14 replication fork activity was hindered by the tomentosolic acid, timosaponin A-I, and shizukaol A with the binding affinity score of −9.2, −9.2, and −9.0 kcal/mol, respectively. The nsp15 endoribonuclease catalytic residues were inhibited potentially by limonin, 25-O-anhydrocimigenol 3-O-alpha-l-arabinopyranoside, and asperagenin posing strong binding affinity scores of −9.0, −8.8, and −8.7 kcal/mol, respectively. Computationally predicted potential phytochemicals for SARS-CoV-2 are known to possess various medicinal properties.

After the emergence of severe acute respiratory syndrome coronavirus 2 (SARS- in December 2019, the fight against COVID-19 pandemic is still underway globally due to the continuous increase in infected cases and deaths [1, 2] . Structurally, SARS-CoV-2 has a positive sense single-stranded viral RNA genome of length ~30 kb with 16 non-structural proteins (nsp1 to nsp16), 3 structural proteins, and 8 accessory proteins [3] . Therapeutic targets such as papainlike protease (PLpro), 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), and spike glycoprotein (SGp) are targeted primarily for repositioning approved drugs and developing adjuvant therapies with phytochemicals and vaccines [4] [5] [6] [7] .

The three less focused targets, i.e., nsp13, nsp14, and nsp15 are also the critical components of the replication-transcription process to promote the life cycle of SARS-CoV-2.

The nsp13 belongs to the helicase superfamily-1 driven by the nucleoside triphosphate hydrolase (NTPase), and the RNA helicase activity is pivotal in SARS-CoV-2 RNA replication [8] [9] . In nsp13, the zinc-binding sites that function with the involvement of divalent metallic ions, i.e., Mg 2+ , Mn 2+ , Ca 2+ , or Zn 2+ critical in NTPase and ATPase activity [10] . Some studies have highlighted the importance of nsp13 for its inhibitory role in regulating type I interferon production, as well as the suppression of interferons (IFNs) levels due to nsp13 overexpression via the host USP13 [11] . Some recent in silico studies have proposed cepharanthine, idarubicin, and nilotinib as potential inhibitory agents against nsp13 [12] . The uridine-specific endoribonuclease nsp15 is highly conserved in the coronavirus family at the C-terminal catalytic domain to recognize uridine moiety, and it is also responsible for the interference of the host immune system [13] . There is a scope for experimenting and validating the key functions of nsp15 endoribonuclease with the host system, the uridine vanadate interaction with the catalytic residues including (HIS235, HIS230, LYS290, THR341, and GLY247) proposed to be the therapeutic target site to screen potential inhibitors. Along with nsp13, nsp15 also requires Mn 2+ metal ions for various key functions [14] . Tiparacil and uridine 2′,3′-vanadate have been reported to inhibit the putative active and catalytic sites of nsp15 [13] . The nsp14 Nterminal domain possesses exoribonucleolytic activity that belongs to the DEDDh exonucleases super family. An exonucleolytic activity is crucial for the RNA proofreading activity found in coronavirus replication is an exceptional feature not found in other viruses, which is critical for nidoviral evolution and genome expansion. It involves catalysis for the excision of endonucleoside monophosphates from nucleic acid [15] . The nsp14 requires two divalent metal ions and reactive water molecules. The nsp14 exonuclease requires divalent cations like Mg 2+ , Mn 2+ , Ca 2+ , Ni 2+ , Cu 2+ , Co 2+ , or Zn 2+ for inducing structural changes and reaction activity [16- J o u r n a l P r e -p r o o f 17] . The proofreading activity of nsp14 was associated with nsp10, and this activity could be hindered by sofosbuvir [18] .

Phytochemicals have a profound role in inhibiting the viral targets of SARS-CoV-2. The validated virtual screening of natural compounds was found to deliver antagonist activity against the therapeutic target in vitro and in vivo experiments. Some examples justify the antagonism, i.e., betulonic acid, savinin, bavachinin, neobavaisoflavone, kaempferol, quercetin, catechin and curcumin could potentially inhibit SARS-CoV-2 3CLpro. Some triterpenoids like limonin, ursolic acid, glycyrrhizin, and 7-deacetyl-7-benzoylepoxyazadiradione possess activity against Mpro, RdRp, and SGp-RBD [19] [20] [21] [22] . The active ingredient glycyrrhizin from Glycyrrhiza radix can inhibit the replication of SARS-CoV related viruses with minimal toxic effects [19] . The natural products such as berberine, baicalin, resveratrol, catechin, procyanidins are reported to interfere with SARS-CoV-2 cellular infection and replication through their action on autophagy [22] . The molecular docking studies of raddeanine, tomentodiplacone B, osajin, sesquiterpene glycoside, rhamnetin, silydianin, and eryvarinM showed higher binding affinity at the active site of nsp13 [23] . Similarly, guanosine-P3-adenosine-5',5'-triphosphate (G3A), and TCM 57025 pose strong binding affinity to guanine-N7 methyltransferase of nsp14 [24] . Sarsasapogenin, ursolic acid, ajmalicine, silymarin, curcumin, gingerol and rosmarinic acid were found to be effective inhibitors against nsp15 [25] . In a recent experimental study, glycyrrhizic acid nanoparticles were developed and applied against SARS-CoV-2 [26] .

Betulonic acid derivatives interfere with hCoV229E nsp15 mutant active site replication with an inhibitory concentration EC50 of 0.6 uM [27] .

Considering the role of nsp13, nsp14, and nsp15 in the SARS-CoV-2 life cycle including the importance of phytochemicals in formulating adjuvant therapies against COVID-19, as a part of our ongoing research [20, 28] , we are interested in finding promising lead compounds from limonoids and triterpenoids. We performed a computational screening for 369 limonoids and triterpenoids using molecular docking and pharmacokinetic properties, and proposed potential lead phytochemicals that could inhibit therapeutic targets and suppress the SARS-CoV-2 life cycle. In addition, the molecular dynamics simulations of docked complexes were performed for 100 to study the stability and flexibility of ligands at active site of target protein.

The FASTA sequences of helicases (nsp13), endonucleases (nsp14), and exonucleases (nsp15) were retrieved from NCBI databases with gene ID: QHD43415. The secondary structures were modelled using Modeller 10.0 protein structure modelling tool [29] . For nsp13 J o u r n a l P r e -p r o o f modelling, we chose X-ray crystallography structures, i.e., 5RL6, 6ZSL, 6XEZ, 6JYT retrieved from the RCSB protein data bank. The protein templates 5C8T, 7MC5, 7MC6 and 7D1Y were used for nsp14, whereas for nsp15 6WLC, 6VWW, 7KEG, and 7MED. The modelled protein structures were validated through Ramachandran plot, ERRAT plot, and Verify 3D structure validation by online tool (https://saves.mbi.ucla.edu/) [30] .

The 3D structure of 369 phytochemicals (limonoids and triterpenoids) were collected from the Chemical Entities of Biological Interest (ChEBI) database ( Table S1 ). The structure data files in '.sdf' were converted to '.pdb' format using the Open Babel 2.3.2 tool. Screening of 369 phytochemicals for ADME properties was performed by using the Swiss ADME online tool (http://www.swissadme.ch/index.php) [31] .

The Autodock4 tools was used to generate the 'pdbqt' file of the proteins and ligands [32] . The grid coordinates for the site-specific and random docking are given in Table S2 . The molecular docking is performed using the AutoDock Vina 1.1.2 scoring function, whereas the strawberry-pearl 5.32.1.1 script was used to perform virtual screening [33] . The Lamarckian genetic algorithm (LGA) was used for the molecular docking studies. The docked structures were analysed by using Biovia Discovery studio 2021.

The free energy of the protein-ligand interaction was computed by AutoDock 4.2. The free energy of binding is calculated as ΔG (bind) = ΔH -TΔS, where the ΔH represents the enthalpic, and TΔS the entropic contribution (only a negative ΔG value is energetically favourable). The energy of ligand and protein in the unbound state was estimated first, and then the energy of the protein-ligand complex was computed. The difference was used to estimate ΔG by following equation [34, 35] .

ΔG= (Vbound L-L -Vunbound L-L ) + (Vbound P-P -Vunbound P-P ) + (Vbound P-L -Vunbound P-L +ΔSconf)

The molecular dynamics simulation was performed by using the Groningen machine chemical simulations (Gromacs 2019.2) [36] . The best docked confirmation was chosen for molecular dynamics simulations. The ligand topologies were built by using the PRODRG server. Protein parameters were generated by using the GROMOS96 43a1 force fields. For the solvation system, we have generated a triclinic simulation box fitting the complex provided by the SPC water model. In addition, 0.15 M sodium chloride has been added to the system. The total system is set up for energy minimization and neutralisation of 5000 steps for structure optimizations. The system had been equilibrated for 300 K temperature and 1.0 bar pressure, J o u r n a l P r e -p r o o f and these parameters were set for 150000 steps to perform simulations. The equilibrated system is set for MD simulations run up to 50000000 steps per 100 ns on an Intel (R) Xeon (R) CPU E5-2680v4@2.40GHz computer. We analysed the molecular dynamic simulation data and position restraints of the docking complex at 100 ns trajectories by using Graphpad Prism 6.0 software. The simulation data was analysed for RMSD (root mean square deviation) of the given structure over time, RMSF (root mean square fluctuation) of each residue in the given structure, radius of gyration, solvent-accessible surface area, and average number of H-bonds in each frame over time. Thousands of frames of simulation structures were interpreted using the VMD and Pymol visualization tools.

The ion-dependent enzymes helicases (nsp13), exonucleases (nsp14), and endoribonuclease (nsp15) are involved in viral replication and proliferation activities of SARS-CoV-2. The protein structure was first prepared and validated. The DOPE scores of the best models of nsp13, nsp14, and nsp15 are given in Table S3 . Protein structure validations of nsp13, nsp14, and nsp15 by Ramachandran plot, Errat plot, and Verify 3D are shown in (Table S4 ) and molecular docking studies with nsp13, nsp14 and nsp15 ( Table S1) Ursonic and ursolic acid potentially interact with SARS-CoV-2 endoribonuclease, which is essential for the lifecycle of coronaviruses [69] . Experimental evidence highlighted limonin is useful against HIV-I and HTLV-I, whereas the theoretical studies supported inhibitory properties against SARS-CoV-2 RdRp [20] . Limonin and limonin glycoside pose strong affinity against TMPRSS2 and furin by blocking SARS-CoV-2 spike glycoprotein interacting with proteolytic cleavage sites [28, 70] . Some of the phytochemicals reported for anti-cancerous properties, like lucidenic acid A, are a tetracyclic triterpenoid found in ganoderma lucidum showed anti-cancerous and antioxidant activities [38] . Also, lucidenic A acid possesses J o u r n a l P r e -p r o o f cytotoxicity against P388 cancer cells at 17 nM. It is also considered as an active promoter of MMP-2 (matrix metallo proteases-2) [55, 56] . These research outcomes encouraged us to develop a library of limonoids and triterpenoids for screening potential phytochemicals against the three key therapeutic proteins targets nsp13, nsp14 and nsp15 of SARS-CoV-2.

Helicases, the NTP-dependent enzymes are widely spread in various kingdoms including the (+)RNA viruses with a genome greater than 7kb [48] . They play a vital role in unwinding the dsDNA/dsRNA substrates, displacing the proteins that are bound to nucleic acid, remodeling the DNA or RNA secondary structures, and translocating along with the doublestranded nucleic acid without unwinding [49] . The nsp13 of SARS-CoV-2 has 596 amino acids.

These amino acids are organised in a triangular pyramid shape with two RecA-like domains IA and IIA, and the domain IB forms a triangular base. The N-terminal zinc-binding domain is connected to the IB domain, and the stalk domain forms the apex of the pyramid and belongs to the SF1 super family of helicases ( Fig. 1a ) [50] . Nsp13 also shares similar structural features with eukaryotic Upf1 helicases. This also exhibits various enzymatic processes such as the hydrolysis of NTP during the capping mechanism, unwinding of RNA duplexes with 50-30 directionality, and the RNA triphosphatase activity [51] . Additionally, RNA unwinding activity stimulated by the interaction with the RdRp, nsp12 and nsp13 is highly conserved in all coronaviruses and acts as a key enzyme in viral replication [52] . In this context, a potent noncompetitive inhibitor (SSYA10-001) blocks viral replication by inhibiting the unwinding activity of the helicases nsp13 in SARS-CoV, MHV and MERS [53, 54] .

The study of ADME properties of 369 phytochemicals and their dock score at the active site of nsp13 showing more than SSYA10 resulted several lead compounds are tabulated in Table S5 . The dock score and inhibition constant of the five best compounds (26-deoxyactein, have been found to be cytotoxic against MCF-7 and HepG2 cancer cells [39, 40] . Timosaponin A-I is found in the rhizomes of Anemarrhena asphodeloides Bunge is reported for the treatment of diabetes mellitus, inhibiting dipeptidyl peptidase-4 DPP-4 activity at a concentration of 5-50 μM in a dose-dependent manner [41] .

J o u r n a l P r e -p r o o f Fig 2b) . The helicases protein's zinc-binding sites, nucleic acid binding sites, and ATP binding sites have maximum RMSD peaks between 0.4 nm and 0.8 nm (Fig 2a) . 

Nsp14 is an N-terminal exonuclease, a C-terminal N7-methyltransferase and a bifunctional enzyme for mRNA capping that is involved in replication [58] . During the virus life cycle, nsp14 exhibits specific processes including pathogenicity, genome recombination, and innate immune responses. From the 3' end of the growing RNA strand, the mismatched nucleotides were removed by the exoN functional domain, which corrects the strand for errors created by RdRp [8] . ExoN has a distance relationship with the N-terminal DeDD protein superfamily. The zinc finger 1 is present and involved in nsp10 binding with nsp14, which reduces the catalytic activity of the exonuclease. The zinc finger 2 is adjacent to the catalytic J o u r n a l P r e -p r o o f sites i.e. ASP90, GLU92, GLU191, HIS268, and ASP273 [59] . The five catalytic residues are predicted to coordinate with Mg 2+ ion and that is adjacent to the removal of incorporated nucleotide errors. ExoN activity is inhibited by blocking the amino acids DeDD, ASP and GLU [60] .

The potential lead compounds were identified through molecular docking where they bound at the active site of nsp14 and also obeyed the Lipinski rule for ADME properties ( Table   S6 ). The top 6 compounds and their interactions with the residues of nsp14 at the active site are summarized in Table 2 . Molecular docking of the exonuclease enzyme with tomentosolic acid pose at the active site through multiple non-covalent interactions with a dock score of -9.2 kcal/mol (Fig. 3) . The residues like VAL91, GLU191, HIS268, PHE146, GLU92, LEU149, GLY93, PRO141, ASN104, PHE190, ASP90, ASP273, MET276 and ASN252 of nsp14 are interacted with the tomentosolic acid ( [44, 45] . In addition, the triterpene lactone phytochemical absinthin listed in Table 2 found in artemisia absinthium showed antiinflammatory activity [42] . The proposed lead compounds bind at the active site of nsp14, and their various medicinal properties were also reported. showed that the ligands, i.e. tomentosolic acid, timosaponin A-I, and shizukaol A bound to the nsp13 protein were more stable. The residual fluctuations during the ligand bound to protein dynamics simulation were calculated as RMSF (Fig. 4b) . The fluctuations were noticed in the ligand interactions with protein active site residues. The peaks in the Fig. 4b showing the nsp14 behavior concerning ligands (tomentosolic acid, timosaponin A-I, and shizukaol A) bound to residues, i.e., 10-70, 100-120, 210-215, and 450-475. The maximum peaks between 0.5-1. Fig. S12, Fig. S13 and Fig. S14 , respectively. J o u r n a l P r e -p r o o f 

Nsp15 is an endoribonuclease (endoU) found in all coronavirus families. It cleaves RNA substrates 3' of uredines [61] . Nsp15 cleavage target regulates the RNA activation and accumulation of viral RNA. This eludes the active viral RNA from getting recognised by the host's antibodies and defenders. Initially, it is thought that nsp15 is involved directly in the replication of the viral genome [62] . Later, it was found that the coronaviruses with inadequate nsp15 were more operational and replicating. Nsp15 is known for implying its cleavage specificity. It is vital for the life cycle of the coronaviridae. The endonuclease function of nsp15 J o u r n a l P r e -p r o o f plays an important role in the viral life cycle. Nsp15 preferentially cleaves uredine-containing RNA substrates [63] . So, this is generally known as endoU for its particularity in partitioning.

Rather than its function in RNA replication and synthesis, this nuclease is quite important for dodging host immune responses. The crystal structure of nsp15 is composed of a common hexameric assembly that consists of dimers made up of nsp15 trimers. The nsp15 protomer consists of three domains which include the N-terminal domain, the middle domain, and the Cterminal endonuclease domain (Fig. 5a) [64] . Each domain performs their specific functions.

The N-terminal domain performs a vital function in the process of oligomerization, whereas the middle domain and the C-terminal endonuclease are profoundly homologous with the other endoU enzymes. Moreover, the nsp15 is said to be only active in its hexamer form. Recent studies stated that the molecular requirements of nsp15 to participate in the oligomerization are still unknown [65] . The active site of nsp15 consists of two histidine residues. These residues facilitate the catalysis and also resemble the RNase A active site. The nsp15 consists of conserved histidine residues at the active site that are involved in catalysis and reminiscent of the postulated RNase A. Catalysis of RNase A followed by a two-step reaction in which, initially a 2′-3′-cyclic phosphate is hydrolyzed to form a 3′-phosphate. HIS235, HIS250, LYS290, SER294 and TYR343 are 5′-UMP-bound nsp15 endoU active residues [13] .

The best compounds with drug-like properties and strong binding affinity to endoribonuclease catalytic site were screened ( Table S7) Limonin is a potent ligand having versatile medicinal properties posing at the nsp15 active site with strong interaction with the residues LYS290, HIS250, THR341, HIS235 and HIS250 (Fig. 5b) . It exhibits strong binding affinity with a minimum binding energy of -8.8 kcal/mol and a predicted inhibitory concentration (Ki) of 1.98 uM ( Table 3) that the ligands bound to nsp15 protein were more stable. The residual fluctuations during the ligand bound to protein dynamics simulation were calculated as RMSF (Fig. 6b) 

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The authors declare no conflict of interest.