key: cord-0846425-v0h0ec96
authors: Kumar, Raj; Kumar, Vikas; Lee, Keun Woo
title: A computational drug repurposing approach in identifying the cephalosporin antibiotic and anti-hepatitis C drug derivatives for COVID-19 treatment
date: 2020-12-19
journal: Comput Biol Med
DOI: 10.1016/j.compbiomed.2020.104186
sha: 24c854186ed27ffe4f5786fe1534ff9d31fd3c0d
doc_id: 846425
cord_uid: v0h0ec96

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 1.4 million deaths worldwide. Repurposing existing drugs offers the fastest opportunity to identify new indications for existing drugs as a stable solution against coronavirus disease 2019 (COVID-19). The SARS-CoV-2 main protease (M(pro)) is a critical target for designing potent antiviral agents against COVID-19. In this study, we identify potential inhibitors against COVID-19, using an amalgam of virtual screening, molecular dynamics (MD) simulations, and binding-free energy approaches from the Korea Chemical Bank drug repurposing (KCB-DR) database. The database screening of KCB-DR resulted in 149 binders. The dynamics of protein-drug complex formation for the seven top scoring drugs were investigated through MD simulations. Six drugs showed stable binding with active site of SARS-CoV-2 M(pro) indicated by steady RMSD of protein backbone atoms and potential energy profiles. Furthermore, binding free energy calculations suggested the community-acquired bacterial pneumonia drug ceftaroline fosamil and the hepatitis C virus (HCV) protease inhibitor telaprevir are potent inhibitors against M(pro). Molecular dynamics and interaction analysis revealed that ceftaroline fosamil and telaprevir form hydrogen bonds with important active site residues such as Thr24, Thr25, His41, Thr45, Gly143, Ser144, Cys145, and Glu166 that is supported by crystallographic information of known inhibitors. Telaprevir has potential side effects, but its derivatives have good pharmacokinetic properties and are suggested to bind M(pro). We suggest the telaprevir derivatives and ceftaroline fosamil bind tightly with SARS-CoV-2 M(pro) and should be validated through preclinical testing.

Coronavirus disease 2019 (COVID-19) is a pandemic viral pneumonia and a threat to global public health. COVID-19 has resulted in over 1.4 million deaths from more than 61.8 million cases worldwide as of Dec 1, 2020 [1] . COVID-19 is caused by a novel coronavirus (nCoV) termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a member of the larger coronavirus family including severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS CoV) [2] [3] [4] . The main protease (M pro ) of SARS-CoV-2 is a non-structural protein that is responsible for processing the polyprotein translated from viral RNA and is a promising therapeutic target for COVID-19 [5] [6] [7] . Early evidence in SARS-CoV suggested that M pro inhibitors may abrogate the viral replication [8] . The atomic-level resolution of the SARS-CoV-2 protease structure must allow rational drug design against COVID-19 [4, 9, 10] . Drug repurposing (DR) provides the fastest possibilities to find existing drugs that may help treat COVID-19 [11] .

Currently, the anti-HIV-1 (human immunodeficiency virus) drugs lopinavir and ritonavir have been repurposed for SARS-CoV-2 M pro and tested in clinical trials in China [12] .

Lopinavir and ritonavir act in combination to abrogate the function of an important enzyme 'protease' which is essential for the viral replication. The efficacy and safety of a combination of an anti-HIV-1 drug darunavir and cobicistat are under phase 3 trials for COVID-19 (NCT04252274). Another example of drug repurposing is the Ebola drug remdesivir that has been approved by the U.S. Food and Drug Administration (FDA) for treating COVID-19 patients [13] [14] [15] . Chloroquine and hydroxychloroquine have been used to treat malaria and certain inflammatory conditions showed encouraging results against COVID-19 in vitro [16] [17] [18] . Corticosteroid methylprednisolone (NCT04244591, -19 may include nucleoside analogues, neuraminidase inhibitors, tenofovir disoproxil, lamivudine, and umifenovir [19, 20] . These studies reveal the potential of DR drugs in combating COVID-19. Here, we recruited an amalgam of dockingbased virtual screening, molecular dynamics (MD) simulations, and binding-free energy approaches to identify suitable existing drugs for the treatment of COVID-19.

The experimentally determined X-ray crystal structure of SARS-CoV-2 M pro (PDB:

6LU7) at a resolution of 2. 16 

MD simulations were performed to further understand the mechanism of protein-drug binding and to get dynamic information about the complex. The filtered hits from molecular descent algorithm by applying a maximum force of 1000 KJ/mol to avoid steric clashes. The   minimized system was then equilibrated under NVT (constant number of particles, volume,   and temperature) and NPT (constant number of particles, pressure, and temperature) independently. The NVT ensemble was applied for 500 ps at 300K using a V-rescale thermostat [26] . In the NPT ensemble, each system was equilibrated for 1 ns at 1 bar pressure controlled by Parrinnello-Rahman barostat [27] . The LINCS algorithm [28] and Particle mesh Ewald (PME) were used to restrain the bonds of heavy atoms and electrostatic interactions, respectively [29] . All simulations were carried out under periodic boundary conditions to avoid edge effects. Finally, a production run of 50 ns for each equilibrated system was performed under NPT conditions. The results were analyzed with DS, GROMACS, and visual molecular dynamics (VMD) software. GROMACS program 'gmx rms' was used for

calculating RMSD values of both protein and ligands. This program calculates RMSD values of atoms in a molecule with respect to a reference structure by least-square fitting the structure to the reference structure (t 2 = 0) as given below

Here, = ∑ , and ri( ) is the position of atom at time t. The protein backbone atoms were selected for calculating protein RMSD values while all ligand atoms were selected for calculating ligand RMSD [30] . The root mean square fluctuation (RMSF) and radius of gyration (Rg) calculations were performed by 'gmx rmsf' and 'gmx gyrate' utilities of GROMACS program, respectively.

Prediction of the binding affinities of small-molecule inhibitors to their biological targets plays an important role in structure-based drug design [31] . In this study, we exploited J o u r n a l P r e -p r o o f a GROMACS compatible program 'g_mmpbsa' to predict the protein-ligand binding free energies [32] . For calculating binding free energy, 50 snapshots of protein-ligand complexes were selected evenly from 0 to 50ns of MD trajectories [33] . The protein-ligand binding free energy is calculated as:

G solvation = G polar + G non-polar (5)

where x may indicate separated ligand or protein or a ligand-protein complex; E MM is the molecular mechanics potential energy in the vacuum; G solvation is the free energy of solvation;

SASA is the solvent-accessible surface area; γ = 0.02267 kJ mol -1 Å -2 or 0.0054 kcal mol -1 Å -2 is a coefficient related to surface tension of the solvent; b = 3.849 kJ mol -1 or 0.916 kcal mol -1 is a fitting parameter. The binding interaction between protein and ligand was calculated in three terms such as solvation (ΔE sol ), Van der Waals (ΔE vdw ), and the electrostatic (ΔE ele )

contribution. The final ΔG bind values for protein-ligand complexes were the average values from 0 to 50 ns of MD simulation trajectories.

An exhaustive computational approach including high-throughput virtual screening, molecular docking, MD simulations, and free energy calculations was used to identify potential drugs against the SARS-CoV-2 main protease enzyme. A workflow of the current study is represented in Figure 1 . Anti-HIV protease inhibitors lopinavir and ritonavir are J o u r n a l P r e -p r o o f recommended for the treatment of SARS-CoV and SARS-CoV-2 [4, 34, 35] . Therefore, we selected the bound cocrystal N3, lopinavir, and ritonavir as the reference inhibitors; these were used as standards for comparing the results obtained from our in silico study.

A workflow of the computational drug repurposing process for identifying potential drugs ceftaroline fosamil and telaprevir against the main protease of COVID-19.

SARS-CoV and SARS-CoV-2 main proteases are structurally similar and have a RMSD of 0.68 Å and highly conserved sequences with a striking sequence identity of 96.1%

( Figure 2 ) [36] . The close proximity of the inhibitor binding site is highly conserved and the only notable difference is an Ala46Ser mutation whose role is yet to be elucidated. The 

The experimentally determined three-dimensional structure of SARS-CoV-2 M pro Table   1 ).

The RMSD of protein backbone atoms and potential energy profiles obtained from the simulations indicate the overall stability of the simulated systems (Supplementary Figure S1 ) [39] . The RMSD values were between 0.15 nm to 0.36 nm for all the protein-drug complexes.

Varying RMSD values at the initial few nanoseconds of the simulations indicate the initial adjustment of ligands at the active site of SARS-CoV-2 M pro [39, 40, 41] . The analysis indicated that each system reached steady-state after convergence, and RMSD values remained within the acceptable value of <0.3 nm except the M pro -Atorvastatin complex (0.36 nm) during the 50 ns simulation run [42] .

The stability parameter potential energy (PE) was also analyzed (Supplementary Figure S1 ). Our analysis found that each simulation system obtained constant average potential energy profiles and remained stable during the simulation period. The RMSF calculations showed comparatively high fluctuations in Glu47 and Tyr154 versus the other residues. These residues are located away from the active site at the periphery of the protein (Supplementary Figure S2) . The radius of gyration calculations also showed steady profiles for all complexes, which makes the system compact without any aberrant behavior during the simulations (Supplementary Figure S3) . These results suggest that all protein-drug complex systems are stable and reliable for further analysis of binding modes, important molecular interactions, and free energy calculations.

J o u r n a l P r e -p r o o f

The substrate or inhibitor binding site of SARS-CoV-2 M pro can be divided into several subsites such as S1, S1', S2, and S3 ( Figure 2C ) [43] . The binding events of reference inhibitors and drugs were assessed at these subsites. The reference inhibitor N3 occupied the S1, S1', S2, and S3 subsites while lopinavir and ritonavir filled the S1', S2, and S3 subsites during the simulations (Figure 3 ). Telaprevir showed similar binding with SARS-CoV-2 M pro as N3 inhibitor occupying the S1, S1', S2, and S3 subsites ( Figure 4A ). Ceftaroline fosamil, atorvastatin, and clofazimine filled the S1', S2, and S3 subsites ( Figure 4B-D) . Sildenafil showed slightly different binding occupying S1, S1', and S2, subsites while everolimus filled only the S1' and S2 subsites ( Figure 4E&4F ). Interestingly, remikiren lost its binding with the SARS-CoV-2 M pro active site and moved out of the binding pocket which is also evident through a high binding energy value discussed in the next section ( Figure 4G ). hence, it has little value as a potential drug against COVID-19. We inferred that ceftaroline fosamil and telaprevir can bind with SARS-CoV-2 M pro more tightly than reference inhibitors.

J o u r n a l P r e -p r o o f 

Binding modes of ceftaroline fosamil and telaprevir were assessed using average structures calculated from MD simulation trajectories. Superimposition of complexes of SARS-CoV-2 M pro with reference inhibitors and drugs indicated that they bind at the same substrate/inhibitor binding cavity of the protein (Supplementary Figure S4) . Ceftaroline fosamil formed four hydrogen bond interactions with Thr24, Thr25, His41, and Thr45 Thr190, and Gln192 were also identified.

We further calculated the distance between hydrogen bond acceptor and donor atoms throughout the 50 ns simulations. The distances are represented as graphs which indicate that the average distance always remained less than the acceptable limit of 3.5 Å (Supplementary Figure S5 ). The reference inhibitor N3 has extensive hydrogen bonding interactions with eight bonds with SARS-CoV-2 M pro active site ( Table 4 ). Finally, 11 compounds having better docking scores when compared to the parent compound telaprevir and reference inhibitors, N3, lopinavir, and ritonavir were retained. Besides the telaprevir forming hydrogen bonds with residues Gly143, Ser144, Cys145, and Glu166, its derivatives also showed hydrogen bonds with other important active site residues such as Asn142, His164, and Gln189 (2D interactions, Supplementary Table 4 ). These results indicate that telaprevir derivatives have better binding properties and safer pharmacokinetic profiles than telaprevir.

The details of compounds with their 2D structures are shown in Table 3 . Sildenafil has been under Phase 3 clinical trials in China (NCT04304313). Remikiren has been reported as a potential drug against COVID-19 by Nguyen et al. [47] . Telaprevir is predicted to bind effectively to SARS-COV-2 papain-like protease (PL pro ) [48] . However, other potential drugs in our study have yet to be evaluated against COVID-19. The potential candidates were screened through exhaustive in silico approaches employing molecular docking, MD simulations, and free energy calculations. Our results suggested that ceftaroline fosamil and telaprevir have better binding energies than reference inhibitors. Ceftaroline fosamil occupies the S1', S2, and S3 subsites while telaprevir filled the S1, S1', S2, and S3

subsites of SARS-CoV-2 M pro active site. Moreover, ceftaroline fosamil formed a close contact with Ser46 residue which is mutated (Ala46Ser) in SARS-CoV-2 M pro versus SARS-CoV ( Figure 2D ). SARS-CoV M pro inhibitors may provide critical information about the molecular interactions necessary for targeting SARS-CoV-2 M pro due to their high sequence and structure similarities ( [53] , and feline (PDB: 6WTJ) [54] may bind to S1', S1, and S2 subsites. Some large inhibitors may bind to S1', S1, S2, and may extend J o u r n a l P r e -p r o o f through S3 subsites, for example N3 (PDB: 6LU7) [21] , alpha-ketoamide (PDB: 6Y2F) [43] , and X77 (PDB: 6W63) [55] . These compounds have dissimilar structures and sizes, yet they bind with and inhibit the activity of SARS-CoV-2 M pro . Therefore, a potential future perspective lies in the structure-activity relationship (SAR) studies which should provide greater insights into the selectivity of inhibitors of SARS-CoV-2 M pro .

Ceftaroline fosamil formed four hydrogen bond interactions including residues Thr24, Thr25, and His41 as observed in recently determined crystal structures of SARS-CoV-2 M pro (Table 4 ). Interestingly, telaprevir formed hydrogen bond interactions with important residues such as Gly143, Ser144, Cys145, and Glu166 suggesting its relevance as a potential SARS-CoV-2 M pro inhibitor. Telaprevir was previously used as a combination therapy against Hepatitis C virus (HCV) infection that was paired with ribavirin, peginterferon alfa-2a, and peginterferon alfa-2b [56] . Some side effects of telaprevir combination therapy were skin rashes and anemia in adverse events of treatment [57] . To avoid the side effects of telaprevir, analogs with better pharmacokinetic properties can be further synthesized and explored as M pro inhibitors.

Thus, we performed a substructure search in the PubChem database to identify telaprevir derivative compounds. The 11 resulting compounds had desirable pharmacokinetic properties such as good aqueous solubility, non CYP2D6 binding, low hepatotoxicity, high intestinal absorption, and inability to cross the BBB besides good binding properties with SARS-CoV-2 M pro active site. Therefore, we demonstrate that telaprevir derivative compounds identified by our in silico study have potential against COVID-19 and may bind to the SARS-CoV-2 main protease.

Our computational drug repurposing study identified ceftaroline fosamil and telaprevir as putative inhibitors against a critical COVID-19 drug target SARS-CoV-2 main protease. Our inhibitors formed hydrogen bonds with important residues such as Thr24, Thr25, His41, Gly143, Ser144, Cys145, and Glu166; these are consistent with the crystal structures of complexes of inhibitors bound with SARS-CoV and SARS-CoV-2 main proteases. A previously unexplored hydrogen bond interaction was identified in the case of ceftaroline fosamil with Thr45 located near the S1' subsite of SARS-CoV-2 M pro .

Considering the side effects of the telaprevir, its derivative compounds with better pharmacokinetic properties are suggested as potential antiviral candidates against COVID-19.

We recommend telaprevir analogs and ceftaroline fosamil identified in our study for in vitro testing and further validation through randomized clinical trials before being used in COVID-19 patients.

J o u r n a l P r e -p r o o f Table 4 . Hydrogen bond formation between SARS-CoV M pro active site residues and bound inhibitors of the selected complexes.

Thr24 Thr25 Thr26 His41 Phe140 Asn142 Gly143 Ser144 Cys145 His163 His164 Met165 Glu166 Gln189 Thr190 Gln192

J o u r n a l P r e -p r o o f

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The authors declare no conflicts of interest.J o u r n a l P r e -p r o o f