key: cord-0976169-p8j6vosg
authors: Sixto‐López, Yudibeth; Martínez‐Archundia, Marlet
title: Drug repositioning to target NSP15 protein on SARS‐CoV‐2 as possible COVID‐19 treatment
date: 2021-03-13
journal: J Comput Chem
DOI: 10.1002/jcc.26512
sha: 1cf88bba35f3fd10c570b16010f8a66507e6b2fd
doc_id: 976169
cord_uid: p8j6vosg

SARS‐CoV and SARS‐CoV‐2 belong to the subfamily Coronaviridae and infect humans, they are constituted by four structural proteins: Spike glycoprotein (S), membrane (M), envelope (E) and nucleocapsid (N), and nonstructural proteins, such as Nsp15 protein which is exclusively present on nidoviruses and is absent in other RNA viruses, making it an ideal target in the field of drug design. A virtual screening strategy to search for potential drugs was proposed, using molecular docking to explore a library of approved drugs available in the DrugBank database in order to identify possible NSP15 inhibitors to treat Covid19 disease. We found from the docking analysis that the antiviral drugs: Paritaprevir and Elbasvir, currently both approved for hepatitis C treatment which showed some of the lowest free binding energy values were considered as repositioning drugs to combat SARS‐CoV‐2. Furthermore, molecular dynamics simulations of the Apo and Holo‐Nsp15 systems were performed in order to get insights about the stability of these protein‐ligand complexes.

SARS-CoV-2 belongs to lineage B β-CoVs, subgenus Sarbecovirus. 3 Structurally, SARS-CoV and SARS-CoV-2, are formed by four main proteins: Spike glycoprotein (S), membrane (M), envelope (E), and nucleocapsid (N). 1 Particularly, the S protein mediates the binding of the receptor to the Host through receptor binding domains (RBD). 1, 4 The genome of SARS-CoV-2 and also codifies for non-structural proteins (Nsps) contained in a replicase gene that, due to ribosomal frameshifting, it encodes for two ORFs: rep1a and rep1b. These are then translated into two polyproteins named: Pp1a and Pp1ab, which are processed into 3C-like proteases, encoded by Nsp5 (3CLpro), as well as a papain-like protease encoded by Nsp3 (PLP). After being processed, it yields into 16 viral Nsps, one of which is the Nonstructural protein 15 (Nsp15). Nsp15 encodes a nidoviral RNA uridylate-specific endoribonuclease (NendoU), that is highly conserved among vertebrate nidoviruses (coronaviruses and arteriviruses). It plays a critical role in the viral replication and transcription. 5, 6 Nsps in other coronaviruses, assemble into a membrane-associated replicasetranscriptase complex, with many other functions that are yet to be elucidated. 7 SARS-CoV and SARS-CoV-2 are two (out of seven) beta coronaviruses that are able to infect humans. These two CoV share a higher sequence identity in their constitutive proteins, such as the subunit 2 of the S protein (89.8%), 8 as well as the Nsp15 protein (88% sequence identity and 95% similarity). Structurally, they are also very similar, showing RMSD values of 0.47 Å (SARS-COV chain A; PDB id:

2H85 and SARS-CoV-2 chain A; PDB: 6VWW). 6 Particularly, the SARS-CoV Nsp15 protein, produced in Escherichia coli, has endoribonuclease (endoU) activity that preferentially cleaved the 5 0 of uridylates of RNAs. 9 Similar EndoU activities have been described in other CoV Nsp15s, including human CoV 229E, mouse hepatitis virus (MHV), avian infectious bronchitis virus (IBV), turkey CoV (TcoV). 10, 11 Some years ago, it also was proposed that the EndoU activity of Nsp15, could interfere with the innate immune response. 11, 12 The Nsp15 proteins from SARS-COV and SARS-COV-s are structurally very similar. Nsp15 is an hexameric endoribonuclease with a catalytic site formed by two Histidine residues and a Lysine residue, which is a similar characteristic to RNase A. 6, 7 Nsp15 forms a hexamer made of dimers of trimers. The 39 kDa monomeric unit, composed of 345 residues, folds into three domains: N-terminal, middle domain, and the C-terminal catalytic NendoU domain, which is the domain responsible for the catalytic activity. Additionally, Nsp15 is exclusively present on nidoviruses and is absent in other RNA viruses, making it as an ideal target for drug design, to combat SARS-CoV-2. 13, 14 Therefore, in this work, we aim to identify Nsp15 inhibitors with a virtual screening strategy through molecular docking analysis and by employing some of the approved drugs from the library of DrugBank database. Then, we performed molecular dynamics simulations, in order to study the stability and structural properties of the apo-Nsp15 as well as the ligand-Nsp15 complexes, which could provide insights about their potential use in the treatment for the Covid-19 disease.

In total, we retrieved 2467 ligands from the Drugbank database (https://www.drugbank.ca/), specifically considering only the approved for human. The ligands were further processed using Raccon, 15 a graphical user interface for AutoDock virtual screening, in order to transform them to a suitable format (pdbqt), for further molecular docking studies. In this procedure, only polar hydrogens were taken into account and Gasteiger charges were also added.

Focused molecular docking studies were carried out using Autodock vina. 16 Nsp15 protein structure of SARS-CoV-2 (PDB: 6VWW) and the Nsp15 protein for the SARS-CoV (PDB: 5H85) were retrieved from Protein Data Bank (PDB).

In total, 2467 ligands were tested against Nsp15. The grid box of was centered on catalytic residues: H235, H250, K290, T341, Y343, and S294 with grid box dimensions of 30 Å × 30 Å × 30 Å. 6 Compounds were prepared using Racoon, 15 while protein was prepared with AutoDock Tools 1.6.5. 17 Polar hydrogen atoms and Kollman charges were encompassed. The molecular docking process was carried out using Autodock Vina. 16 Virtual screening was carried out only on SARS-CoV-2 Nsp15, from which only the compounds with the lowest free energy of binding were retrieved. Then, those compounds were also tested against SARS-COV Nsp15, under the same molecular docking conditions stated above. Benzopurpurin B, the experimental SARS-CoV in vitro Nsp15 inhibitor, was employed as a positive control. Additionally, we could observe that our molecular docking approach reproduced the interactions previously reported, validating our docking procedure.

Molecular dynamic simulations of the apo (Nsp15) and holo (Nsp15-Elbasvir and Nsp15-Paritaprevir and Nsp15-Citrate), were carried out using the GROMACS package. This procedure was done by employing the OPLS force field for GROMOS (GROMACS 96). 18 The box dimension was settled at least 1.0 nm away from the wall of the dodecahedral box with periodic boundary conditions, solvated with SPC water molecules. Systems were neutralized with 12 atoms of Na. Energy minimization was carried out using the steepest descent method. Berendsen temperature coupling and isotropic pressure coupling were established in order to reach a stable environment (300 K, 1 bar). The particle mesh Ewald (PME) algorithm was applied to treat electrostatic and Van der Waals interactions, using the following values: cutoff for the short-range VdW (rvdw) was set to 1.0 nm and Coulomb cut-off (r coulomb) at 1.0 nm. All the bond lengths were constrained using the LINCS algorithm 19 and the time step was set to 0.002 ps. The complex was equilibrated for 10 ns, and the MD simulations were run for about 30 ns. MD simulations were further processed, by the removal of translational and rotational movements from the systems, before the calculation of the root-mean-square deviation (RMSD) and root-mean-square fluctuations (RMSF).

Conformational sampling of the protein was considered at 0, 10, 20, and 30 ns. Additionally, representative ensembles were obtained by RMSD conformational clustering algorithm using the GROMOS method. 20 We then performed a principal component analysis (PCA), also known as essential dynamic (ED). The PCA analysis is a statistical technique that allows to extract the large-scale collective motions of the atoms from the simulations, that are often correlated to its biological function and biophysical properties. 21 The performed method is described in detail elsewhere. 22 Citrate molecule was included in the MD simulation studied; due to the fact, it was reported to be bound to the Nsp15 catalytic site of SARS-CoV-2. 6 3 | RESULTS AND DISCUSSION

A virtual screening strategy, by means of Autodock Vina was carried out while employing only the approved compounds for human use, retrieved from the Drugbank database. A total of 2467 ligands were docked against Nsp15 SARS-CoV-2, from which 16 ligands were selected due to their lowest free binding energy, that spans from −10.9 to −9.0 kcal/mol (Table 1) .

These drugs are currently approved by the market with different indications. Difenoxine and Levocanbastine depicted free binding energy values lesser than −10.5 kcal/mol. However, Difenoxine is a controlled drug since it belongs to the opioids agents, while Levocabastine is for ophthalmic use. Other types of compounds that showed good free binding energy were Digoxin, Deslanoside, Acetyldigitoxin. Digitoxins are cardiac glycosides that show a sterol group as part of their chemical scaffold (Table 1 ). In early 2020, Ciclesonide, an inhaled steroid, was suggested to target Nsp15 by in vitro studies, specifically for coronavirus. 23 Thus, our findings might explain that the presence of sterol moiety, can be considered as a scaffold to target NSP15, and this group is needed and constitutes the first approach for further chemical modifications, with the aim to achieve better selectivity as well as the improvement of their ADMET properties. The use of Deslanoside, Digitoxin, and Acetylditgitorin, which are glycoside cardiotonics were not contemplated for further studies, since they usually have a narrow therapeutic margin, hindering the administration or patient adherence.

Ivosidenib, Entrectinib, and Irinotecan are drugs approved for cancer treatments, like leukemia, non-small cancer cell lung, and colorectal cancer, respectively, however, due to their several side effects, they were not considered for this study. In the case of sibutramine, this drug also depicted a good free binding energy (−9.8 kcal/mol) but it was recently withdrawn due to safety effects (cardiovascular events and strokes), and it was the reason it was not considered neither in this study. Atovaquone and Bedaquiline (−9.7 and − 9.6 kcal/mol, respectively) showed also favorable free binding energies, and they are used as antimalarial and antimycobacterial drugs, respectively.

Atovaquone was also used in an independent study as Mpro ligand for SARS-CoV-2 treatment. Thus, our approach proposes Atovaquone for COVID-19 treatment, due to its possible binding to Nsp15. 24 Additionally, Ergot derivatives, such as Dihydroergotamine and Ergotamine (−10 and − 9.6 kcal/mol, respectively) were also pointed out as possible ligands that target Nsp15, since they both are used for migraine treatment. Independently, ergotamine was proposed as a Mpro ligand, identified by combining molecular docking and MD simulation studies, 25 and it also was proposed to target several SARS-CoV-2 proteins such as: Nsp1, 2, 4, 5, 7, 8, 9, 10, and 12, papain-like proteinase, proteinase 3CL-PRO, RdRp, spike protein, Mpro, and others, making it a potential drug to be used for COVID-19 treatment. 25 Finally, Paritaprevir and Elbasvir, two antiviral drugs currently approved for hepatitis C treatment, depicted a binding free energy of −9.6 kcal/mol each. Thus, they were considered for further MD simulation studies, aside from the fact that independent studies have suggested the use of both compounds for COVID-19 treatment. 24, 25 (Scheme 1).

From the molecular docking studies, we could observe that the 15 compounds showed similar binding modes, due to their common chemical moieties, as discussed below.

Difenoxin and levocabastine complexes with Nsp15-SARS-CoV-2

show mainly hydrophobic interactions, by making π-π interactions with phenyl rings of residues H235 and Y343, and also showing π-cation interactions with K290; while on the other extreme, the phenyl groups interact with W333 through π-π interactions. Thus, the aromatic interactions seem to be crucial for both compounds, which are complemented with hydrogen bonding interactions at G248, H250, H235, and K290, with carboxylic group of both molecules ( Figures 1A and 2E,N) .

Deslanoside, Acetyldigitoxin, Digitoxin, on the other hand, share a similar binding mode, since they accommodated their glycoside portion into the catalytic site, by interacting through π-π or hydrophobic interactions with W333, Y343, K290, as well as hydrogen bonds with H235, H250, T341, S294, V292. The sterol portion mainly interacts with M219 and F241 ( Figures 1B and 2A,F,D) .

Digoxin, as the other molecules, accommodates the glycoside portion into the catalytic site but the sterol portion interacts with different residues, in comparison to other ligands: Deslanoside, Acetyldigitoxin, and Digitoxin. Docking studies suggested that Digoxin interacts with H235 and V339, by hydrophobic interactions, and forming hydrogen bonds with G230, E340, A232, and E234 ( Figure 2G ).

Therefore, for this group of molecules, it seems that the glycoside por- 2L ). Both compounds were able to form hydrogen bonds, π-π interactions, and halogen interactions, features that can further be taken into account for potential drug design strategies.

Both Bedaquiline and Atavaquone, in both cases accommodate the quinoline and naphthoquinone moieties respectively, on the catalytic site. This could be possible due to π-π interactions, established between Y343 and the aromatic systems. Meanwhile, the other aromatic extreme of both molecules (naphthalene and biphenyl, respectively) interacts with W333, via π-π, as well as the formation of a hydrogen bond with H235 ( Figures 1G and 2B,C) . As far as we know,

S C H E M E 1 Elbasvir and Paritaprevir, drugs proposed with possible activity against Nsp15 of SARS-CoV-2, obtained by virtual screening approach this is the first time that Bedaquiline has been suggested for its use in the Nsp15 inhibition. In the case of Atovaquone, it has been suggested as a Mpro ligand with antiviral efficacy, 24 which also could be due to Nsp15 inhibition.

Sibutramine accommodates its phenyl ring into the catalytic site, similarly to Atovaquone and Bedaquiline. Its anchorage is mainly due to π-π interaction with Y343, and is supported by hydrophobic interactions with the following residues: H25 and V292, K290, C291, Figure 1D ).

Dihydroergotamine showed better binding free energy than Ergotamine. The former ligand accommodates the ergoline ring in the catalytic site, interacting with Y343 via π-π, and with H235 and W333, via π-alkyl, as well as with residues surrounding the catalytic site via hydrophobic interactions (E234 and E340, D240) ( Figure 2H ). On the other hand, Ergotamine protruded the ergoline ring to the opposite direction, while interacting with residues such as L346 and V292. Its phenyl ring interacts with W333, and forms a hydrogen bond with the oxygen of the amide portion ( Figure 2J ). This binding mode is energetically less favorable and this coincides with the decreased free binding energy value. The same tendency in binding affinity was predicted by another in silico study. 31 Paritaprevir reaches the catalytic site and accommodates its macrocyclic portion and protruded toward the exterior, with the substituents that stabilize the structure ( Figure 1F ). Hydrogen bonds are formed between Y343, and oxygen of the sulphonamide, with the residue: G247 and G248 with the oxygen of the amide portion and oxygen of the macrocyclic hydroxyl group; as well as hydrophobic interactions with phenanthridine portion (V29, S294, and L346) ( Figure 2O ).

Elbasvir is accommodated along with the catalytic site, mainly by π-π interactions with W333 and H235. Hydrogen bonds are also observed with the residues Y343, S294, and E340 ( Figures 1C and 2I ). Molecular docking of these compounds, showed that molecules with good binding energy values against Nsp15, are those that are capable of establishing π-π interactions, as well as hydrogen bonds, and other non-bonded interactions, albeit to a less grade.

Thirty Nano seconds MD simulations of the Apo-Nsp15 system, as well as the complexes: Nsp15-Elbasvir, Nsp15-Paritaprevir, and Nsp15-Citrate were performed (Figure 3 ). From these RMSD results, it could be observed that the compounds: Elbasvir (RMSD: 

The Clustering analysis allow us to obtain the most representative ensembles from the equilibrium phase of the MD simulation, by considering the last 30 ns. The most populated conformations were concentrated in the first three clusters, from which more than 99% are represented for apoprotein, and in complex with Elbasvir and Patiraprevir, whereas 96% was represented by Citrate ( Table 2) . Figure 4C ). While, the comparison of the native structure versus the most populated cluster conformation of Nsp15-citrate shows a RMSD of 1.246 Å, with structural differences at the following segments: S2-I31, V36-T48, G170-K174, S198-E203, Y238-F269, localized along with the protein structure ( Figure 4D ). As can be seen, the ligands stabilize the Nsp15 protein structure and the tendency is in agreement with geometrical parameters (RMSF and RMSD).

In the detailed analysis of the interactions, we found that in the most populated cluster conformations of the complex Nsp15-Elbasvir, Elbasvir forms a double hydrogen bond with H235, as well as hydrophobic interactions with the following residues: M216, Y238, D240, G247, and V292. It was also observed that it remains within the catalytic site, forming a stable complex ( Figure 4B ). In the case of Paritaprevir, it forms a hydrogen bond with Y343, as well as hydrophobic interactions with Q45 and C291 ( Figure 4C ). Finally, Citrate only binds to K345 ( Figure 4C ). Our results are consistent with other studies, where was citrate was reported as Nsp-15 structure stabilizer. 31 

We gratefully acknowledge to CONACYT, to Instituto Politécnico Nacional, to COFAA-SIP/IPN and YSL thanks to CONACYT by PhD scholarship.

Yudibeth Sixto-López https://orcid.org/0000-0001-8903-3834

Marlet Martínez-Archundia https://orcid.org/0000-0002-6140-8953

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Epub 2020, 1. How to cite this article: Sixto-López Y, Martínez-Archundia M. Drug repositioning to target NSP15 protein on SARS-CoV-2 as possible COVID-19 treatment