Repurposing of anisomycin and oleandomycin as a potential anti-(SARS-CoV-2) virus targeting key enzymes using virtual computational approaches

Department of Public Health, College of Public Health and Health Informatics, University of Ha'il, Ha'il, Kingdom of Saudi Arabia Department of Biology, College of Science, Hail, P.O. 2440, University of Ha’il City 2440, Saudi Arabia Laboratory of Genetics, Biodiversity and Valorization of Bio-resources (LR11ES41), University of Monastir, Higher Institute of Biotechnology of Monastir, Avenue Tahar Haddad, BP74, 5000 Monastir, Tunisia Molecular Diagnostics and Personalised Therapeutics Unit, University of Ha'il, Ha'il, Kingdom of Saudi Arabia School of Electronic Science and Engineering, University of Electronic Science and Technology of China, Chengdu 610054, Sichuan, China Laboratory of Bioresources: Integrative Biology and Valorization, (LR14-ES06), University of Monastir, Monastir 5000, Tunisia Department of Pharmaceutics, College of Pharmacy, University of Hail, Kingdom of Saudi Arabia Department of Pharmaceutics, Faculty of Pharmacy, University of Khartoum, Sudan Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt Department of Chemistry, College of Science, Qassim University, Buraidah 51452, Saudi Arabia University of Monastir, Faculty of Science of Monastir, Avenue of the Environment, 5019 Monastir, Tunisia Department of Chemistry, Faculty of Science and Arts of Baljurashi, Albaha University, Saudi Arabia Faculty of Science of Sfax, Department of Chemistry, University of Sfax, B.P. 1171, 3000 Sfax, Tunisia


Introduction
A novel highly pathogenic viral infection from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was discovered over a year ago in Wuhan City, Hubei Province, China. On February 11, 2020, the virus was named SARS-CoV-2 by the International Virus Classification Commission (ICTV). Simultaneously, the disease was named by the World Health Organization (WHO) as coronavirus infectious disease 2019 (COVID-19) (1-3). As a human pathogen, SARS-CoV-2 has been declared as a global pandemic by the World WHO based on the rate of increasing spread and the fatality of the viral infection. In comparison to SARS-CoV and MERS-CoV, it was found to be faster transmissible from human-to-human (4)(5)(6). Released on March 19, 2021, the confirmed coronavirus cases accounted for more than 122 million and more than 2.7 million deaths. By the 23 th of September 2021, the total confirmed cases reached 231 million with more than (4,735,316) total death around the world. In Saudi Arabia, there are more than (546.792) confirmed cases with more than 8684 deaths (https://www.worldometers.info/coronavirus). According to the genome sequencing data of SARS-CoV-2, there is more than 82% sequence identity with SARS-CoV and MERS-CoV and >90% sequence identity for essential enzymes and structural proteins, containing genes encoding 3C-like proteinase, RNAdependent RNA polymerase (RdRp), 20-O-ribose methyltransferase, spike protein, envelope protein, nucleocapsid phosphoprotein, and several unknown proteins (7,8).To date, there are no approved therapeutic drugs to prevent the expansion of human SARS-CoV-2 and its wide-spreading despite some vaccines which is still in debate and unfavored option for a wide spectrum of people, underling the necessity of an immediate need for antivirals. One of the interesting strategies is to assess their ability to inhibit any SARS-CoV-2 proteins essential for the viral lifecycle. Thereby, four targets including viral ACE2, M pro ; PL pro , Receptor Binding Domain of Spike protein and Furin have been chosen. PL pro is crucial in the process of coronavirus replication and infection of the host inducing cleavages of N-terminus of the replicate poly-protein to release Nsp1, Nsp2 and Nsp3, which is essential for enabling virus replication (9,10). 3CLpro as Nsp5 is essential for the life cycle of the virus (11). RdRp is also known as Nsp12.RdRp is a crucial replicase that catalyzes the synthesis of a complementary viral RNA and thus plays a central role in the replication and transcription of COVID-19 virus genes, possibly with the aid of nsp7 and nsp8 as co-factors (12). Nsp 14 is a nonstructural protein 14 of coronaviruses important for the viral replication and transcription and works as S-adenosyl methionine (SAM)-dependent (guanine-N7) methyl transferase (N7-MTase) (13). The coronavirus main protease (M pro ) is considered as the most important target for SARS-CoV-2 drug design that permits the viral gene expression and replication by the proteolytic cleavage of replicase polyproteins, without which the virus replication is severely hampered (14). Currently, although modern medicine is leaning towards the use of phytocompounds produced by plants as secondary metabolites with broad-spectrum activity (15)(16)(17)(18)(19)(20), research have been focused also on the repositioning of existing molecules with therapeutic effect and good availability.
Based on the above facts, target selection and validation are the crucial steps in drug repurposing. Therefore, the main purpose of this work is to test two approved drugs named anisomycin and oleandomycinused as antibiotics for various therapeutic effects. Consequently, anisomycin and oleandomycin will be docket to investigate potential binding-conformation of the ligands of these antibiotics to the binding sites of the SARS-CoV-2 target proteins. Furthermore, as a key step toward unraveling their molecular mechanisms as well as predicting drug side-effects and drug repositioning opportunities, in silico target prediction along with their ADMET parameters have been assessed.

Materials and methods
Anisomycin ( Figure 1) as an antibiotic, is a translational inhibitor secreted by Streptomyces spp., strongly activates the stress-activated mitogenactivated protein (MAP) kinases JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) and p38/RK (also known CSBP for Cytokinin Specific Binding Protein) in mammalian cells, thereby preventing elongation and causing polysome stabilization. Oleandomycin ( Figure 1) is a macrolide antibiotic synthesized from strains of Streptomyces antibioticus, commercialized under three names and in two forms: as pure oleandomycin ("matromycin," Pfizer; "romicil," Hoffmann-La Roche) and as a mixture with twice its weight of tetracycline ("sigmamycin," Pfizer). The spectrum of activity on micro-organisms is, therefore, wider than that of penicillin and streptomycin.

Molecular docking
The three-dimensional structure of the target protein was retrieved from RCSB Protein Data Bank (21) with PDB ID:2AJF. Chain A, formed by 597 residues, and bound with ligand "2-acetamido-2deoxy-beta-D-glucopyranose" in the binding pocket, was considered for docking with selected ligands-Oleandomycin, and Anisomycin. The target protein structure was further analyzed for the presence of domain using NCBI CD Search to search the Conserved Domain Database (22). Docking is based on two major aspects-search algorithm and scoring function. Search algorithms are used to identify the orientation and conformations of the ligand-bound in the binding pocket of the receptor (23). Scoring functions are employed to differentiate between active and random compounds and to predict binding free energies in ligand-protein docking (24). Docking of protein ligands was carried out using AutoDock Vina (19), GOLD (20,21), and LibDock from Discovery Studio Client v20.1.0.19295. The docking softwares used to employ different algorithms to improve binding accuracy. AutoDock Vina uses Broyden-Fletcher-Goldfarb-Shanno algorithm (25). However, GOLD employs a degree of freedom in the binding site that corresponds to the reorientation of hydrogen bond donor and acceptor groups (26)(27)(28). The binding affinity and gold fitness scores were obtained from AutoDock Vina and GOLD, respectively for obtaining the best orientation and conformation of the ligands. These values were further correlated with the experimental values. In order to get accurate results, all the docking experiments were performed with the default parameters.

Docking using AutoDock Vina
PDBQT files of receptor protein and ligands were prepared using the Graphical User Interface program AutoDock Tools (ADT). The bound ligand was removed and the grid box was created with size 60 × 60 × 60 XYZ points with a grid spacing of 0.375 Å and grid center was designated at dimensions (x, y, and z): 8.098, 23.137, and 50.858, around the ligand bind site. Protein and ligands were set to rigid during the docking procedure and a configuration file consisting of protein and ligand information along with grid box properties was prepared for executing docking using AutoDock Vina. The ligand pose with the lowest binding energy/ binding affinity was selected for exploring close intra-molecular interactions with the receptor.

Docking using GOLD (Genetic Optimization for Ligand Docking)
In the GOLD suite, the wizard was used for docking protein and ligands with default parameters. The active site with a 06 Å radius sphere was defined by selecting the bound ligand with in the protein. 10 solutions for each ligand were obtained by applying default Genetic Algorithm settings. The best ligand was selected based on the highest GoldScore fitness function. The ligand and the protein docked complex was further analysed for close intra-molecular interactions.
The molecular docking and visualization studies were also carried out with the help of a commercially available site-features-directed docking (LibDock) program in Discovery Studio (29). The protein 2AJF_A was prepared by adding protein and the binding site was defined by selecting the ligand "2acetamido-2-deoxy-beta-D-glucopyranose" from the Current selection and defining the xyz coordinates as 7.877869, 23.220714, and 50.836553 with a radius of 10.32 Å. The docking preferences were set to "high quality", and the "Best" Confirmation method with maximum conformations of 255 was selected. The ligand pose with the highest LibDock Score was selected to form docked complex with the receptor for further analysis.

Intra-molecular Interactions in docked complex
Docked complexes of 2AJF_A with Oleandomycin and Anisomycin obtained using AutoDock Vina, GOLD, and LibDock were further analysed for intramolecular interactions using the View Interaction tool from Discovery Studio Client v20.1.0.19295. Interacting residues of protein and ligand were visualized in 3D and 2D view.

Domain Identification
CD-search revealed the presence of Peptidase_M2 domain, an Angiotensin-converting enzyme, starting from 1 -588 residues in 2AJF_A.

Molecular target predictions
Molecular target predictions are important to find the phenotypical side effects or potential crossreactivity caused by the action of small biomolecules were obtained by using the web tool (http://www.swisstargetprediction.ch/) and entering the smile formats of the desired drugs to obtain the targets. The prediction concerns the putative targets of the given molecule by utilizing 2D and 3D similarity index with known ligands.

Results and discussion Molecular docking
In order to found the potential treatment for COVID-19, in silico approach has been proposed to validate the repositioning of two drugs such as anisomycin and oleandomycin. Based on the least binding scores, interactions of the selected drugs at the active site of the different SARS-CoV-2-receptors are shown in Figures 2 and 3.
The results depicted in Tables 1 and 2 provided that anisomycin and oleandomycin fit well to the binding site of the selected targets, especially with M pro exhibiting the lowest binding score, -7.62 kcal/mol and -59.72 kcal/mol, respectively.

Docking studies of anisomycin and oleandomycin with the SARS-CoV-2 M pro
SARS-CoV-2 M pro (3C-like proteins) is a cysteine proteases enzyme that can hydrolyze proteins with the help of its Cys-amino acid residues present in the active site and has been proved as the potential target protein to prevent the spread of infection by inhibiting the cleavage of the viral polyprotein.

Docking studies of anisomycin and oleandomycin with the SARS-CoV-2 NSP15
The Nonstructural uridylate-specific endoribonuclease named Nsp-15 as an appropriate drug target against SARS-CoV2, essential for its lifecycle and virulence was located in the N terminal domain, leaves 2'-3'-cyclic phosphates 5' to the cleaved bond (https://swissmodel.expasy.org/repository/species/269 7049) in which the mechanism of action is independent of the endonuclease activity. Nsp15 affects viral replication by interfering with the host's innate immune response by suppressing the type I IFN (IFN-α/β, thus eluding detection of viral mRNA by double-stranded RNA sensors. NSP15 is reported to slow viral replication more than any other target (30).

Docking studies of anisomycin and oleandomycin with the SARS-CoV-2 Furin
Furin qualified as a target protein for screening of anti-viral compounds is a kind of proprotein convertases enzyme that cycles between the trans-Golgi network and the cell surface, where it recognizes the cleavage motif on protein precursors and converts them to functional proteins through cleavage (31). I was present on the plasma membrane of the host cell may also be playing a very crucial role in the entry of the SARS-CoV-2 virus within cells. The furin recognition site within the SARS-CoV-2 S protein is very similar to that of the highly virulent avian and human influenza viruses suggesting that it may act on S protein during viral production (32).

Pharmacokinetics
In order to ensure the drug-likeness properties during the time of preclinical analysis trial in drug discovery and development, assessment of absorption, distribution, metabolism, excretion and toxicity (ADMET) is very crucial for attractive molecules to possess the best chance to become effective drugs (33)(34)(35)(36)(37)(38). From the output of some ADMET properties shown in Table 3, it was shown that the liver and intestine cytochrome P450 enzymes (CYP1A2, CYP3A4, CYP2C19, CYP2D6, and CYP2C9) interact with drugs and are responsible for their metabolism. CYP2D6 is responsible for the metabolism of a wide range of compounds in the liver. Its, inhibition by a drug induces the problem of a drug interaction.
Results revealed no inhibition and therefore the lack of any interaction. Anisomycin was found to not act as a P-glycoprotein (P-gp) inhibitor or substrate, however, Oleandomycin was P-g substrate and P-g I inhibitor but not P-g II inhibitor. Their skin permeability was -3.04 and -2.735 units, respectively. They are non-mutagenic and none of them have shown hERG I and hERG II inhibition activity. Their LD 50 values were 2.681and 2.981mol/kg, respectively and their chronic oral rat toxicity (LOAEL) values were 1.109 and 1.722 (log mg/kg_bw/day) without any skin sensitivity. Hence, based on the ADMET analysis, both Anisomycin and Oleandomycin were confirmed to be permissible as available potent drugs with exceptional druglike properties and therefore they can be further assessed for their in vitro SARS-CoV-2 inhibitory activities (Table 3).

Target prediction
Molecular Target studies are important to understand the molecular mechanisms underlying a given phenotype or bioactivity, to rationalize possible side-effects and predict off-targets. An efficient drug will perform its mechanism of action by interacting with the proteins, enzymes and other bio-macromolecules. Based on their resemblance with known drugs, we can estimate the desired drug targets. The top 50 results of the closely associated receptors based on Target, Common Name, Uniprot ID, ChEMBL-ID, Target Class, Probability and Known actives in 2D/3D were depicted as a pie-chart ( Figure 4). As shown, Anisomycin has 13.3% enzyme and 6.7% protease whereas oleandomycin predicts 24% enzyme and 8% protease, as targets.   Considering the fatality of SARS-CoV-2 and its high infection rate, finding new therapeutic agents, especially drugs is actually a race against time. There is no approved treatment available to eliminate the virus despite the appearance of some vaccines. Docking and scoring software is used widely to enhance the drug design and predict the interaction between drugs and macromolecules in pharmaceutical products. Therefore, SARS-CoV-receptors were used for blinding the docking analysis of some known drugs. As regards toM pro , its structure comprises three domains, domain 1 (residues 10 to 99), domain 2 (residues 100 to 182) and domain 3 (residues 198 to 303). It is a cysteine protease formed by Cys-145 and His-41 catalytic dyad in its active center which is highly conserved among the coronavirus proteases and plays a major role in substrate binding and the activity of the enzyme. The amino acid residue Hisbehave as a common acid-base and Cys-is is very well known for its nucleophilic character, exactly responsible for Michael addition reactions to the α, βunsaturated ketones and nucleophilic attack to the ketones in biological reactions. The proteolytic process is believed to be dependent on the active site cysteine (Cys-145) side chain thiolate nucleophile attack on the amide bond of the substrate (39,40). The -SH group of Cys145 is ion-paired with His41 forming Cys145-His41 catalytic dyad, which differs from most serine proteases that have a catalytic Ser-His-Asp triad in their active sites (40,41). Therefore, this protein constitutes an essential opportunity to identify a potential drug candidate as SARS-CoV-2. Consequently, a good SARC-CoV-2 M pro inhibitor should contain either conjugated ketone (type-I) or active carbonyls (aldehydes or ketones; type-II) with sufficient hydrophobic parts for non-covalent interactions. PL pro is a multifunctional cysteine protease that processes viral polyproteins to a functional replicase complex leading to viral spread (41). The0 SARS-CoV-2 PL pro shares 83% sequence similarity with SARS-CoV and was involved in deubiquitination, de-ISGylation which obstruct the important signaling pathways causing viral invasion of the innate immune response by the expression of type I interferon (42,43).
It has been reported that furin may be involved in the proteolytic processing of S protein to make its conformation suitable for binding on ACE2 receptors (46). The priming of SARS-CoV-2 S protein by furin would hypothetically make many more cells susceptible to infection, as compared to S protein priming by TMPRSS-2 alone Furin protease has a binding site from residue 109 to 574, with the presence of catalytic triad Asp153-His194-Ser368 and an additional oxyanion hole at Asn295 (47,48). Also, it possesses allosteric sites, where inhibitors can bind and change the conformation of the active site.
As shown both anisomycin and oleandomycin confirm the presence of the triad Asp153-His194-Ser368 as well as Asn295 and therefore might be used for prevention and treatment of the COVID19. Pharmacokinetic studies will be beneficial for scientists to search out safe and effective drug candidates in the initial stage of drug discovery. Based on the aforementioned results, the drug-likeness of anisomycin and oleandomycin have been validated. Also, they were predicted to be good inhibitors of the SARS-CoV proteins and could be propitious as therapeutics for SARS-CoV-2 infection.

Funding
This research has been funded by the Scientific Research Deanship at the University of Ha'il -Saudi Arabia through project number RG-20 230.