IN-SILICO STUDY: COX2 (CYCLO-OXYGENASE 2), AN UNKNOWN TARGET OF HYDROXYCHLOROQUINE REPOSITIONED FOR THE TREATMENT OF COVID 19 INFECTION AND CARDIOVASCULAR EFFECTS

IN-SILICO STUDY: COX2 (CYCLO-OXYGENASE 2), AN UNKNOWN TARGET OF HYDROXYCHLOROQUINE REPOSITIONED FOR THE TREATMENT OF COVID 19 INFECTION AND CARDIOVASCULAR EFFECTS

Hamza Nadjib Merad-Boudia ^*, Majda Dali-Sahi, Baya Guermouche and Nouria Dennouni-Medjati

Department of Biology, Analytical Chemistry and Electrochemistry Laboratory, Abou Bekr Belkaid University, Tlemcen, 13000, Algeria.

ABSTRACT: Hydroxychloroquine, widely used to treat certain autoimmune diseases and inflammatory conditions, has been repositioned as a new lead against the COVID-19 pandemic. Preliminary studies have suggested that hydroxychloroquine inhibits the entry of the SARS-CoV2 virus mediated by the ACE2 receptor and is a particularly interesting anti-inflammatory and immunomodulatory action in the cytokine storm during COVID-19 infection. Furthermore, cardiovascular effects have been reported in hypertensive patients. These last are thought to result from the inhibition of cyclooxygenase 2. Our results reveal for the first time in atomic detail how hydroxychloroquine, one of the most controversial drugs, has an affinity for a new molecular target (COX2) with a docking score: -7.6 (kcal/mol) and a root mean square deviation (RMSD = 2.542). Hydroxychloroquine binds to cyclo-oxygenase 2 via two hydrogen bonds (H-GLY136 and H-GLY45) and four hydrophobic bonds: Cl -CYS36, 0 -CYS36, 0 -CYS47 and 0 -PRO154). This mechanism has never been elucidated. Hydroxychloroquine proved to be promiscuous. Its structure, which differs from that of non-steroidal anti-inflammatory drugs , tends to show activity against a wide variety of biological targets : As an enzyme inhibitor (bioactivity = 0.15), GPCR receptor ligand (bioactivity = 0.35), ion channel modulator (bioactivity = 0.30), kinase inhibitor (bioactivity = 0.44) and protease inhibitor (bioactivity = 0.12). This approach improves the understanding of its mechanism of action and the side effects attributed to it.

Covid 19, Cardiovascular effects, Hydroxychloroquine, Cyclooxygenase 2, Molecular docking

INTRODUCTION: While the main scientific research has focused on chronic non-communicable diseases in recent decades. The year 2019-2020 was marked by the emergence of a new COVID-19 epidemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ^{1, 2}. This pandemic has challenged health systems around the world ³.

Hydroxychloroquine (4-amino-quinoline), widely used to treat some autoimmune diseases and inflammatory affections, has been repositioned as a new lead against COVID-19 infection ^{4, 5}.

Preliminary studies have suggested that hydroxy-chloroquine inhibits ACE2 receptor-mediated entry of the SARS-CoV2 virus ^{6, 7} and has an anti-inflammatory and immunomodulating action particularly interesting in the cytokine storm during COVID-19 infection ⁸. Thereafter, new research has highlighted the unfavourable evolution of this infection in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs) ⁹. Besides the gastro-digestive effects, serious cardiovascular complications have been previously reported when using cyclo-oxygenase. In the light of scientific debates led by researchers from all over the world on the benefit/risk ratio of hydroxychloroquine, the objective is to check if, by sharing anti-inflammatory properties with NSAIDs by binding to cyclooxygenase, hydroxychloroquine, causes fatal cardiovascular effects in hypertensive patients with Covid 19.

MATERIALS AND METHODS:

Preparation of Receptor: In the literature, several crystalline structures of COX-1 and COX-2 in complex with several inhibitors and substrates have been identified. With arachidonic acid (their physiological substrate) ¹⁰, classic NSAIDs such as Diclofenac classic NSAIDs such as Diclofenac ¹¹, Naproxen ¹², as well as coxibs such as celecoxib ¹³). COX-2 is induced by inflammation, while COX-1 is physiological. The X-ray crystallographic structure of ibuprofen bound to cyclooxygenase-2 (4PH9) (Mus musculus) ¹⁴, was obtained from the Protein Data Bank (PDB) (http://www.rcsb.org) with a resolution <3.00 Å (resolution: 1.81 Å, free R-value: 0.197, working R-value: 0.160, observed R-value: 0.162). This file is not directly used by the Autodock 1.5.6 tools. It was first visualized using Discovery Studio Visualizer version 2.5.5.

Discovery Studio is a software package of biological molecular design for chemists and computational biologists. (Discovery Studio 2.5 (CDOCKER Dock, Dassault Systemes BIOVIA, USA). Water molecules, ligands and other heteroatoms have been removed from the protein. This crystallographic structure has been preserved without any treatment for molecular docking. Co-crystallized inhibitors/substrates (ibuprofen) were used to define the corresponding active site, the flexible residues within this active site is a criterion for the validation of mooring calculations (re-docking). By a literature review, common residues involved in the binding site were identified ^{10, 15, 12, 16, 14}. The PDB file of the protein has been efficiently prepared using the AutoDock / Vina (Molecular Graphics Lab, Le plugin Scripps Research Institute, La Jolla, CA, USA) , and saved as pdbqt file. Polar hydrogens and Kollman chrages have been added.

Preparation of the Ligand: The necessary information about hydroxychloroquine was collected on the ChemSpider platform (http://www. chemspider.com/): Hydroxychloroquine (Molecular formula: C₁₈H₂₆ClN₃O, Average mass: 335.871 Da, Monoisotopic mass: 335.176453 Da, ChemSpider ID: 3526). - Chloroquine (Molecular formula C₁₈H₂₆ClN₃, Average mass 319,872 Da, Mono-isotopic mass 319.181519 Da, ChemSpider ID: 2618). Their chemical structures are almost similar to ¹⁷. These two anti-malarial and anti-rheumatic drugs have antiviral activity in-vitro. Chloroquine interferes with different stages of the viral life cycle ¹⁸. The 3D structure of the Ligand was downloaded from the Drugbak database (https://www.drugbank. ca/): Access number DB01611. Then the identification and verification were performed on Discovery Studio Visualizer version 2.5.5. The ligand was then exported to AutoDock tools 1.5.6. Torsional connections have been verified. The ligand was saved in the same docking folder in pdbqt format. Charges were then added.

Molecular Docking Protocol: Docking calculations were performed using the standard AutoDock Vina parameters. AutoDock Vina docking programme is generally in the top position among all the methods tested in CASF-2013. Vina is the best of all methods in terms of reception power. Ligand minimisation has a significant impact, reducing the performance difference between AutoDock and Vina ¹⁹. The active site was placed in a 40 × 50 × 40 Å grid box, in the geometrical center of the set of selected flexible residues at 0.375 Å as the grid spacing. Values of the root mean square deviation (RMSD) between the docking and the initial poses have been calculated. Results of the docking were initially examined in Discovery Studio. (Discovery Studio 2.5 (CDOCKER Dock, Dassault Systemes BIOVIA, USA). The best poses were ranked according to Vina scores (kcal/mol), evaluated by the free bond energies (score S, kcal/mol) and the binding interactions between the ligand atom and the residues of the active site. To predict the bioactivity of hydroxychloroquine for cox2 receptors, we used Molinspiration (https://www. molinspiration.com/). This server predicts bioactivity score for the most important therapeutic targets such as GPCR receptors, kinase inhibitors, ion channel modulators, enzymes, and nuclear receptors ²⁰. SwissADME database (Swissadme.ch/ index. php) was used to compare physiochemical properties, ADME parameters and pharmacokinetic properties of hydroxychloroquine with certain anti-inflammatory drugs. Smiles (chemical structure) for each structure were downloaded from the Drug Bank database (Drugbank.ca/). Statistical analyses (mean and standard deviation) were performed using Minitab statistical software Version 16. Mann-Whitney test was used to comparison of median Docking values.

RESULTS AND DISCUSSION:

Identification of the COX2 Interaction Site: Each COX isoform is considered as heterodimer with a catalytic site and an allosteric site ²¹. Inhibitors may act at one or both sites, depending on the structure and concentration of the inhibitor ²². Whatever the site, binding requires that a small molecule must first enter through the binding domain to the four-helix membrane in an open area called the "lobby" ²³. As shown in Fig. 1a and 1b, Ibuprofen was first identified in the pocket of the COX2 enzyme binding site.

FIG. 1: COX2 POCKET (1A: BINDING IBUPROFEN, 1B FREE)

Interactions between COX2-Ibuprofen have been illustrated in Fig. 2a and 2b.

FIG. 2: A AND B: RECEIVER-LIGAND INTERACTIONS ON A 2D DIAGRAM

Coordinates of the Grid Box (X, Y, Z points, dimensions and spacing) used for the Ibuprofen-COX2 model (shown in Fig. 3) have been noted to help identify the Hydroxychloroquine-COX2 interaction. Prepared hydroxychloroquine must first enter through the open area called the “hall” Fig. 4.

Molecular Docking Simulations: Molecular docking was chosen as the first choice as a method of discrimination of the hydroxychloroquine-COX2 interaction due to the ability to simulate the binding of small compounds in the active site of enzymes, based on in-silico prediction of putative competitors ²⁴.

In our work, the active site was identified in the A chain of COX2 of ibuprofen bound COX2 structure. Our simulations involved only a single monomer, as some works ²⁵, or others that have focused on a smaller fragment of protein, as the membrane-binding domain ²⁶, or the membrane binding domain and helices comprising active site of cyclooxygenase ²⁷. In addition, previous studies have indicated that many NSAIDs (including IBP) bind tightly to a single monomer of  COX-2 dimer and allosterically inhibit the oxygenation of the substrate in the partner monomer ^{28, 29}.

Binding affinities of the different poses in the COX2 active site A validation docking of crystal structure of murine (mu) COX-2 in complex with Ibuprofen (IBP) ¹⁴. (PDB ID: 4PH9) (http: / /www.rcsb.org/pdb) was performed on Autodock vina after viewing on discovery studio and treatment of the ligand and receptor.   Results are shown in Table 1.

TABLE 1: RESULTS OF THE BEST MOLECULAR DOCKING POSES: IBUPROFEN-COX2

Complex formed by Ibuprofen (pose 1) -COX 2 of experimental model of Orlando and al has the lowest energy value and shows the best mooring score (Affinity: -7.3 (kcal/mol) with a root mean square deviation of (RMSD = 23.884). Docking results of all hydroxychloroquine poses with the target (COX 2) are shown in Table 2.

TABLE 1: DOCKING RESULTS OF ALL HYDROXYCHLOROQUINE POSES WITH THE TARGET (COX 2)

Mann-Whitney test Table 3 reveals that the median affinity of the Hydroxychloroquine/COX2 complex (-6,350 kcal/mol) is significantly higher than the median affinity of the complex of the model crystallized by Orlando et al., in 2015: Ibuprofen /COX2 (-6,250 kcal/mol), ( P-Value= 0.6712), IC (95%): (-0.200; 1.200).

Pose that has the lowest binding energy of the docking score will be considered the best pose and inhibits the target receptor because lower binding energy corresponds to higher binding affinity ³⁰. Comparison of the pose-complex scores revealed that the complex formed by Hydrochloroquine (pose1) -COX 2 has the lowest energy value and gives the best docking score (Affinity: -7.6 (kcal/mol) with the best root mean square deviation (RMSD = 2.542). Hydroxychloroquine -COX 2 complex can be considered to be stable with a higher binding affinity.

Based on these docking results, we can classify hydroxychloroquine as a good inhibitor of the COX2 enzyme. 3D representations of the best pose interactions between poses and receptor were visualized using Discovery studio software. The COX2-hydroxychloroquine poses formed are shown in Fig. 5.

TABLE 3: PARAMETER OF THE MANN-WHITNEY TEST

FIG. 5: COX2-POSES COMPLEXES FORMED (COX2 BOUND TO HYDROXYCHLOROQUINE)

Binding mode observed for the best pose with receptor pocket is shown in Fig. 6, 7.

Types of interactions, bonds, and amino acids involved in the Hydroxychloroquine-COX2 complex are shown in Table 4. Hydroxychloroquine binds to COX2 through two hydrogen bonds (in H-GLY136 and H-GLY45) and four hydrophobic bonds: Cl -CYS36, 0 -CYS36, 0 -CYS47, and 0 -PRO154) at different distances (2.68043) A, 2.46642 A, 4.47781 A, 5.06953 A, 4.66876 A and 4.23862 A, respectively). Glycine with a hydrogen (H) radical is devoid of any hydrophobic or hydrophilic character. GLY45 and GLY136 bind to hydroxychloroquine through hydrogen bonds, H-GLY136 and H-GLY45) . Cysteine ​​is hydrophobic, but its SH group binds to another cysteine ​​to produce a strong (covalent) bond, the disulfide bridge. CYS36 and CYS47 are involved in hydrophobic bonds with hydroxychloroquine. Interestingly, CYS36 is doubly committed, with oxygen and chlorine atoms of hydroxychloroquine. Although the physiological significance of chloride binding at this position is unknown, chloride ions have previously been used to identify the molecular oxygen binding site in various proteins, including dioxygenases ^{31, 32}. This raises the possibility that the chloride ion may be indicative of the position of molecular oxygen prior to incorporation into COX substrates. In addition, proline (PRO154), which is highly hydrophobic, binds like CYS36 and CYS47 to the oxygen atom of hydroxychloroquine. Arylcarboxylic acid inhibitors bind in one of two orientations of the COX active site ¹². Flurbiprofen binds canonically with its hydrogen paired carbo-xylate fraction to the Arg-120 and Tyr-355 residues constriction site ¹². In contrast, diclofenac binds in an inverted orientation in which its carboxylate is hydrogen-bonded to the side chains of Tyr-385 and Ser-530 ¹¹. Ibuprofen occupies an area of the enzyme between the opening of the substrate channel and the top of the active site. A total of thirteen contacts is made between the isobutyl group of ibuprofen and Trp-387, Met-522, Val-523, Gly-526, Ala-527, and Ser-530. In addition, the α-methyl group of ibuprofen makes contact with Val-349 and Leu-359 ¹⁴. Rofecoxib binds to the active site of cyclooxygenase in the same general confor-mation as observed for celecoxib ¹⁶. Rofecoxib makes a total of 42 contacts with residues lining the cyclooxygenase channel ³³.

TABLE 4: TYPES OF INTERACTIONS, BONDS AND AMINO ACIDS INVOLVED: HYDROXYCHLOROQUINE-COX2

Prediction of Bioactivity: The diversity of possible drug targets (each of which requires a different combination of corresponding molecular characteristics) is so enormous. To predict the bioactivity of hydroxychloroquine for different receptors, we used Molinspiration (https://www. molinspiration.com/). This server predicts the bioactivity score for the most important therapeutic targets such as GPCR receptors, kinase inhibitors, ion channel modulators, enzymes, and nuclear receptors ²⁰. The bioactivity of a compound was decided based on the bioactivity score. If the bioactivity score is> 0, it is an active compound while <-5.0 is an inactive compound, and the range between -5.0 and 0.0 corresponds to moderately active compounds ³⁴.

TABLE 5: PREDICTION OF THE BIOACTIVITY OF HYDROXYCHLOROQUINE AND OTHER NONSTEROIDAL ANTI-INFLAMMATORY DRUGS

Hydroxychloroquine was the only one to show high activity against more than one target receptor. (GPCR, kinase inhibitors, ion channel modulators, protease inhibitor, and enzyme inhibitor) in Table 5 (bioactivity greater than zero). In drug discovery, activity against multiple targets means that a molecule will have multiple side effects. Further proof of our docking results, hydroxychloroquine is a COX2 enzyme inhibitor (bioactivity as an enzyme inhibitor : 0.15), like other NSAIDs whose enzymatic inhibitory biactivity is between 0.12 and 0.25. Hydroxychloroquine is active on GPCR ligand receptors (bioactivity : 0.35). Impact of GPCR structures on drug discovery is very important. Structure-based drug design has been applied to an increasing number of GPCR targets over the last decade ³⁵. Hydroxychloroquine was also active on modulating ion channel receptors (bioactivity : 0.30). Ligand-dependent ion channels are also pharmacological targets for the develop-ment of new therapies ³⁶. For receptors of kinase inhibitors, the bioactivity of hydroxychloroquine is 0.44). Receptor tyrosine kinases are key regulators of cellular processes and their role in the pathophysiology of many diseases is well recognized ³⁷. The structure of hydroxychloro-quine, which differs from that of nonsteroidal anti-inflammatory drugs, tends to show activity against a wide variety of biological targets. On this fact, it is sometimes said that such a compound has a promiscuous behavior. Several drugs may be promiscuous, especially in the case of drug reuse ³⁸. which could explain the cardiovascular effects related to the repositioning of hydroxychloroquine in hypertensive patients during COVID 19 infection.

Pharmacokinetics of Hydroxychloroquine: The SwissADME database (Swissadme.ch/index.php) was used to compare physicochemical data and pharmacokinetic properties of hydroxychloroquine with certain anti-inflammatory drugs. The smiles (chemical structure) of each structure were down-loaded from the Drugbank database (Drugbank.ca/). First, we checked whether the molecules comply with the conditions of the following five Lipinski rules ³⁹): molecular weight: ≤ 500, number of hydrogen bond donors: ≤ 5, number of hydrogen bond acceptors: ≤ 10, lipophilicity (in LogP): ≤5. Lipinky's rule analysis with SwssADME in Table 6.

TABLE 6: LIPINKY'S RULE FOR HYDROXYCHLOROQUINE AND CERTAIN NONSTEROIDAL ANTI-INFLAMMATORY DRUGS

As much as other NSAIDs, hydroxychloroquine respects the lipinski rule Table 6. Ibuprofen, diclofenac, naproxen, piroxicam and cecoxib have the following molecular weights, respectively: 335.87, 206.28, 296.15, 230.26, 331.35, 381.37), g /mol, they all have a molecular weight ≤ 500 g/ mol. By comparing the lipophilicity (LogP) values ​​of our ligands, we observed that they all have values ​​below 5 and therefore can be easily absorbed. However, all ligands have a number of hydrogen bond donors: ≤ 5, a number of hydrogen bond acceptors: ≤ 10.

The fact that hydroxychloroquine is taken into account for the management of COVID-19 patients clearly highlights the need to better understand its pharmacokinetics. The bioavailability after oral administration of hydroxychloroquine can reach 80% with a plasma peak of approximately 2 to 4 hours. Its large volume of distribution (200 to 800 L / kg) explains its long half-life, which could range from 30 to 60 days ^{40, 41}. Hydroxychloroquine are metabolized via CYP-450 enzymes into other active compounds, which are responsible for extensive pharmacological actions and increased toxicity.

Hydroxychloroquine is mainly excreted via the kidneys (60%) as unchanged or metabolized, and the remainder (40%) is usually eliminated by the liver, feces and skin or stored in other lean body tissues ⁴².

CONCLUSION: In view of the cardiovascular effects reported in hypertensive patients, particular attention should be paid to repositioned hydroxy-chloroquine in the treatment of COVID 19. Our results reveal for the first time in atomic detail how hydrochloroquine, one of the most controversial drugs binds to a new molecular target (COX2) with a mooring core (Affinity: -7.6 (kcal/mol) and a root mean square deviation (RMSD = 2.542). Hydroxychloroquine has been shown to be promiscuous. Its structure, which differs from that of non-steroidal anti-inflammatory drugs, tends to show activity against a wide variety of biological targets. This discovery improves the understanding of its mechanism of action and the adverse effects attributed to it.

ACKNOWLEDGEMENT: We would like to thank the General Directorate of Scientific Research and Technological Development (GDSRTD) who made the necessary budget available to PhD students to carry out this research.

CONFLICTS OF INTEREST: No direct or indirect interest (of a financial or other nature) with a private, industrial or commercial organization relationship with the subject presented.

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    How to cite this article:

    Merad-Boudia HN, Dali-Sahi M, Guermouche B and Dennouni-Medjati N: In-silico study: COX2 (cyclo-oxygenase 2), an unknown target of hydroxychloroquine repositioned for the treatment of Covid 19 infection and cardiovascular effects. Int J Pharm Sci & Res 2021; 12(2): 767-75. doi: 10.13040/IJPSR.0975-8232.12(2).767-75.

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