Pefloxacin and its derivative, novel inhibitors of the SARS-CoV-2 Main protease (3CLpro) and their pharmacokinetics prediction: An in silico analysis

For over two years, COVID-19 pandemic has been a major global health concern and threat to human life. In the SARS-CoV2 macromolecules, the 3-chymotrypsin like protease (3CLpro or main protease) has been identified to be crucial and essential for viral survival, processing of the viral polyproteins and has been explored as a target in COVID-19 drug discovery.

Although vaccines and other various inhibitors have been designed and launched, the emergence of the variant of this virus has put an unrelenting effort of researchers to this end. Also, the high cost of manufacturing these molecules coupled with the occurrence of drug resistance is a concern.

Herein, Pefloxacin and its derivative for the first time were screened for their inhibitory activity against the SARS-CoV2 main protease through in silico analysis and their pharmacokinetic properties were evaluated. Interestingly, from the docking results, they both bind with high affinity at the active site of the protein. Moreover, they showed excellent pharmacokinetic and drug - likeness properties. Derivatization of Pefloxacin at the C7 position prevents its blood-brain barrier permeability.

Overall, the dual antibacterial and potential antiviral activities of these two molecules make them promising drug candidates for COVID-19 management.

Before now, fluoroquinolones and other synthetic analogs have been approved for their antibacterial activities and efficacy against respiratory tract infection [1,2]. Their mechanism of action is based on the ability to inhibit DNA Gyrase-topoisomerase II and topoisomerase IV - the enzyme crucial for DNA replication and synthesis [1]. A previous study has reported the biological activities of fluoroquinolones against varicella-zoster virus, and cytomegalovirus [3]. Additionally, clinical data have exposed the activities of fluoroquinolone and quinolone-based drugs against DNA and RNA viral infections [4].

The fluoroquinolone skeletal structure consists of a carboxylic group at C-3 and a bulky substituent at the C-7 position and the N1 position of the quinolone moiety. These key structural features and modifications have been identified and reported to enhance their antiviral activities and pharmacokinetic properties [5]. In line with this background, ciprofloxacin and his chalcone derivatives have been reported to inhibit the SARS-CoV2 main protease via molecular docking studies and in vitro studies [6]. Also, Moxifloxacin was reported to interact with the SARS-CoV2 protease via the preliminary in silico analysis [7]. In the same perspective, a Library of Quinolones-based agents were used to probe the different macromolecular targets of the SARS-CoV2 and several inhibitors were reported [8].

The coronavirus disease caused by the several acute syndrome coronavirus 2(SARS-CoV2) originated from Wuhan in china and displays symptoms ranging from fever, and dry cough to shortness of breath [9]. In the SARS-CoV2 macromolecules, 3-chymotrypsin like protease (3CLpro or main protease) is essential for the viral life cycle [10]. This enzyme plays a crucial role in the processing of the viral polyproteins and has been explored as a target in COVID-19 drug discovery [11]. According to crystallographic data, amino acid HIS 41, HIS 164, MET 49, MET 165, THR 190 and GLY 143 play a very crucial role in the stability of the ligand - Mpro complexes and are key amino acid residues in the active site [12,13].

Although vaccines and other various inhibitors have been designed as shown in Table 1, the emergence of the variant of this virus has put an unrelenting effort of researchers to this end. Also, the high cost of manufacturing these molecules is a disadvantage to developing countries. Unfortunately, the presence of comorbid conditions of mixed viral and bacterial infections coupled with the emergence of drug resistance demands more effort in drug discovery and repurposing.

Herein, for the first time, we probe and investigate the interaction of the SARS-CoV2 main protease with Pefloxacin and its derivate and studied the ligand-protein binding using in silico molecular docking techniques. We hypothesize that a structural modification at the C-7 position could modulate the antiviral activity of Pefloxacin and improve its pharmacokinetic properties.

Ligand preparation

Several compounds have shown their various antiviral activities from docking studies [29]. In this study, The PDB format of Pefloxacin and Hydroxychloroquine are retrieved from the PubChem database.Derivativeofpefloxacin-(1-ethyl-6-fluro-7-(4-(Nsubstitutedcarbamoylmethylphenyl)piperazyn-1-yl)-4-oxoquinoline-3-carboxylic acid was drawn and optimized with MolView and finally saved as a PDB format.

Following energy minimization and optimization of the ligands [30], they were all converted to PDBQT format using the graphical user interface version of PyRx virtual screening tool-python prescription 0.8.

Protein preparation

The 3D co-crystallized ligand of SARS-CoV-2 3CLpro (PDB ID: 6LU7) with the resolution of 2.16 Å was retrieved from the RCSB PDB database (http://www.rcsb.org/pdb). The protein was extracted to expose the active site and water of crystallization was removed [31]. Polar hydrogens were added and the protein was further optimized by the Autodock tool and Biovia Discovery Studio Visualizer before the docking process [32].

Molecular docking

The virtual screening of the compounds was done with hydroxychloroquine as the control using Pyrx software by autodock wizard as the engine for docking [33,34]. The configuration file for the grid parameters was generated using Auto Grid engine in Pyrex. Predefined XYZ Center Coordinate of -13.7042, 14.7095,72.0248 respectively having amino acid residues in the active site of the protein were set using the Vina wizard. The ligands with the highest binding energy, preferred orientation and lowest RMSD were considered to be ligands with very high affinity. Then the ligand-protein interactions were visualized by Biovia Discovery Studio Visualizer and the plausible binding modes are predicted [35]. Furthermore, the compounds were compared to hydroxychloroquine as the control.

Drug-likeliness properties

The Swiss ADME Predictor (http://www.swissadme.ch/) was used to calculate the Lipinski rule parameters and drug-likeliness properties [36-39].The canonical smiles of ligands were retrieved and entry was made into the server. These properties include molecular weight, number of hydrogen bond acceptor, number of hydrogen bond donor, topological polar surface area, and lipophilicity level. Human intestinal absorption, Blood brain barrier permeability were also investigated. Above mentioned parameters help to predict aqueous solubility and the ability of molecules to travel across the biological lipophilic membrane.

Virtual screening of the inhibitors of the SARS-CoV2 3CLpro (Main protease)

Table 2 molecular docking result reveals that pefloxacin and its analog for the first time have a very high binding affinity compared to hydroxychloroquine which is the control. Pefloxacin, Pefloxacin ananalognd Hydroxychloroquine have the binding energy of -7.9 kcal/mole, -7.6 kcal/mole and -6.0 kcal/mole respectively. The types of interactions and the amino acid residues in the active site that are involved in the interaction are shown in Figures 1-4. Pefloxacin interacts with the amino acid residues, THR 26, ARG 188 CYS 145, HIS 41, MET 49, MET 49. Pefloxacin derivative interact with the amino acid residues, THR 190, GLN 189, CYS 145, SER 144. Furthermore, Hydroxychloroquine interacts with amino acid residues- MET 49, GLN 189, ARG 188, MET 165, ASP 187, HIS 164, HIS 41, CYS 145, ASN 142, PHE 140, HIS 172.

Drug likeliness properties and in silico ADME prediction

Properties such as molecular weight, number of hydrogen bond acceptor, number of hydrogen bond donor, topological polar surface area, lipophilicity level, Human intestinal absorption, Blood brain barrier permeability were calculated for Pefloxacin, its analogue and hydroxychloroquine and the result is as shown in Table 3.

The 3CLpro (main protease) of the SARS-CoV2 has been identified as one of the invaluable macromolecular targets in COVID-19 drug discovery. The protease plays a vital role in the viral replication, processing of the polyproteins that are translated from the RNA molecules. Moreover, the key amino acid residues in the active site that have been mapped out include, HIS 41, HIS 164, MET 49, MET 165, THR 190 and GLY 143. Also, several classes of drugs including fluoroquinolones have been screened for their ability to inhibit the main protease of the SARS-CoV2 [6]. Although vaccines and other various inhibitors have been designed and launched, the emergence of the variant of this virus has also put an unrelenting effort of researchers to this end. Also, the high cost of manufacturing these molecules coupled to the occurrence of drug resistance is a disadvantage to developing countries.

We screened pefloxacin and a structural modification of the position C-7 analogue for antiviral activity and compared them with hydroxychloroquine.

Our result revealed that for the first time that pefloxacin and its analog bind with a very high affinity and have a binding energy of -7.9 kcal/mole and -7.6 kcal/mole respectively compared to the control. Although Ciprofloxacin had earlier been reported to have a binding energy of -7.0 kcal/mole [7] Pefloxacin has a higher affinity when comparing the scores. The analysis of the ligand-protein interaction shows that pefloxacin and its analog show a very vital interaction with the key amino acid residue at the active site as shown in Table 2. Pefloxacin interact with the amino acid residues THR 26, ARG 188 CYS 145, HIS 41, MET 49, MET 49. Pefloxacin derivatives interact with the amino acid residues THR 190, GLN 189, CYS 145, SER 144. Furthermore, Hydroxychloroquine interacts with amino acid residues- MET 49, GLN 189, ARG 188, MET 165, ASP 187, HIS 164, HIS 41, CYS 145, ASN 142, PHE 140, HIS 172.

As shown in Table 3, Pefloxacin and its analog both has an hydrogen bond donor atoms and hydrogen bond acceptor atom less than 5 and 10 respectively. Pefloxacin and its structurally modified form both have a low TPSA (65.78 and 94.88) respectively coupled with a bioavailability scores greater than zero. These cumulatively suggest a higher possibility of antiviral activities. Interestingly, a structural modification at the C7 position impedes the blood brain barrier permeability of pefloxacin. iLog p - values less than 5 has presented in the table authenticate an ideal lipophilicity nature of the molecules. The two molecules have a high intestinal absorption ability. To our advantage, they are not classified as Pan-Assay Interference Compounds (PAINS) reflecting that they bind to specific biological sites instead of interacting with random targets.

Favorably, the obtained calculations by the Swiss ADME predictor indicated that the two molecules meet the bioavailability requirement and are good drug candidates. However, the analog proposed is not commercially available but can be synthesized. It is also important to note that Pefloxacin as an example of fluoroquinolones are used as an antibiotic in the management of upper respiratory tract infections in cases of beta-lactam antibiotics resistance. Therefore, in this context, a dual mode of antiviral and anti-bacterial activity is possible to the great advantage of the COVID-19 patients.

For the first time, pefloxacin and its structural analog was screened for their inhibitory activity against the SARS-CoV2 main protease, and they both bind with high affinity compared to hydroxychloroquine via molecular docking approach. Docking pose are suggestive of the structural alteration and modification of antiviral activities. Providentially, these two drug molecules are potential drug candidate for COVID-19 management.

However, the derivatized compound needs to be synthesized. Also, in vitro, in vivo testing and further clinical trials need to be done on these molecules in order to elucidate their molecular mechanism and validate this novel finding.

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