key: cord-0946825-uwr29i45
authors: Thirumalaisamy, R.; Aroulmoji, V.; Iqbal, Muhammad Nasir; Deepa, M.; Sivasankar, C.; Khan, Riaz; Selvankumar, T.
title: Molecular Insights of Hyaluronic Acid-Hydroxychloroquine Conjugate as a Promising Drug in Targeting SARS-CoV-2 Viral Proteins
date: 2021-04-13
journal: J Mol Struct
DOI: 10.1016/j.molstruc.2021.130457
sha: 57d0146b4e82cb95634de9ca30d62bc36e9e0d04
doc_id: 946825
cord_uid: uwr29i45

In-silico anti-viral activity of Hydroxychloroquine (HCQ) and its Hyaluronic Acid-derivative (HA-HCQ) towards different SARS-CoV-2 protein molecular targets were studied. Four different SARS-CoV-2 proteins molecular target i.e., three different main proteases and one helicase were chosen for In-silico anti-viral analysis. The HA-HCQ conjugates exhibited superior binding affinity and interactions with all the screened SAR-CoV-2 molecular target proteins with the exception of a few targets. The study also revealed that the HA-HCQ conjugate has multiple advantages of efficient drug delivery to its CD44 variant isoform receptors of the lower respiratory tract, highest interactive binding affinity with SARS-CoV-2 protein target. Moreover, the HA-HCQ drug conjugate possesses added advantages of good biodegradability, biocompatibility, non-toxicity and non-immunogenicity. The prominent binding ability of HA-HCQ conjugate towards Mpro (PDB ID 5R82) and Helicase (PDB ID 6ZSL) target protein as compared with HCQ alone was proven through MD simulation analysis. In conclusion, our study suggested that further in-vitro and in-vivo examination of HA-HCQ drug conjugate will be useful to establish a promising early stage antiviral drug for the novel treatment of COVID-19.

The treatment of COVID-19 viral disease, remains challenging; it is a global issue, which requires both the national and the global approaches. To save lives an efficient and safe prescription drug for the disease is urgently needed. Several antiviral drugs have been considered for the treatment of the disease such as Hydroxychloroquine (HCQ), Lopinavir, Ritonavir, Favipiravi, and Remdesivir; the later originally used for the Ebola virus disease, MERS and SARS viruses have been recommended for COVID-19. Although the above-mentioned drugs show positive activity towards the disease, almost all of these drugs are associated with few disadvantages such as insolubility, some toxicity, instability, and kidney clearance [1, 2] .

Based on our research work on pharmacologically important drugs such as camptothecin, methotrexate, methylprednisolone, propofol to improve their efficacy and targeting delivery for the treatment of diseases such as cancer, arthritis, osteoporosis and anaesthetics respectively; we have developed a unique technology, according to which, the drugs were specifically and covalently linked to the hyaluronic acid (HA) molecule, a natural biopolymer, biocompatible, ubiquitously present in the human and animal body, to afford new drugs, new chemical entities, possessing the unique biological functionalities and properties of both the components synergistically; the evidence is available that the conjugation of drugs with macromolecules enhances the pharmacokinetic profiles of the drugs themselves [3] [4] [5] .

Hyaluronic acid (HA) generally referred to as Hyaluronan is an anionic, non-sulfated mucopolysaccharide spreading widely throughout the connective and epithelial tissues of animals. HA is one of the main components of the extracellular matrix (ECM) and contributes significantly to the activation of signalling pathways that regulate cell proliferation, differentiation, adhesion, and migration [6] [7] [8] [9] . It mediates its biological functions through various protein receptors present on different cell surfaces including CD44 [10] , HARE [11] , RHAMM [12] , and LYVE [13] .

HA is suitable for various chemical modifications easily in the position of hydroxyl, carboxyl, and N-acetyl functional groups; for example, HA-drug conjugates are used as drug carriers to sustained release, transdermal absorption, and to improve drug targeting [14] . In order to exploite its unique biological properties we have developed a technology, according to which, the drugs were specifically and covalently linked to the hyaluronic acid (HA) molecule of a specific molecular weight and at a specific position to afford a new drug contributing synergistically the pharmacological properties of both the components. We have employed this principle to produce a number of pharmacologically important drugs such as HA-Camptothecin, HA-Methotrexate, HA-Methylprednisolone, and HA-Propofol with improved efficacy and targeted delivery for the treatment of diseases such as cancer, arthritis, osteoporosis and anaesthetics respectively [14, 15] .

Hydroxychloroquine (HCQ) is a conventional anti-malarial drug with efficiency in several clinical disease conditions like Q fever, rheumatoid arthritis, lupus, sarcoidosis and so on. The mechanism of action by hydroxychloroquine is that of increasing the lysosomal pH of antigenpresenting cells thereby inhibiting the autophagy process. The SARS CoV-2 anti-viral effects of HCQ have also been linked to a mechanism involving interference of SARS-CoV-2 cellular receptor ACE-2 (angiotensin-converting enzyme-2) glycosylation [16] . The observed major sideeffects are diarrhoea, headache and muscle fatigue. The HCQ produces supplemented early virological immune response against hepatitis C and also reduces HIV-1 cell count [17, 18] .

Importantly, it has immune-suppressive properties which may be helpful to reduce the risk associated with severe COVID cytokine storm [19] .

Our objective is to propose a novel and efficient drug for COVID-19 disease, using an old and well-known antiviral drug, Hydroxychloroquine (HCQ), covalently linking it at a specific position with the Hyaluronic acid (HA) to afford HA-HCQ conjugate to increase its bioavailability, safe, improve its localization, controlled release in the body, enhance its overall efficiency, and eliminate or reduce HCQ's systemic toxicity [14, 20, 21] . The conjugation of HCQ to hyaluronic acid can be accomplished through the HCQ hydroxyl group to the hyaluronic acid C5'carboxylic group of the glucuronic moiety via an ester linkage. In this scenario, the proposed HA-HCQ conjugate and HCQ are tested separately through molecular docking studies for against four different SARS-CoV-2 target proteins, an efficient drug target for the treatment of COVID-19.

The 2D structure of Hydroxychloroquine (HCQ) and Hyaluronic Acid-Hydroxychloroquine Conjugate (HA-HCQ) are drawn in ACD-Chemsketch and saved as mol format file ( Fig. 1 a & b) [22] . The retrieved 2D SDF file format of HCQ and HA-HCQ conjugate structure was submitted to "Online SMILES convertor and Structure file generator", and converted into 3D PDB format [23] .

PreADMET online server was used to calculate bioavailability and the metabolic score of drug HCQ and its conjugate form (HA-HCQ) to proven "Hyaluronan Drug Delivery (HDD)" technology. Since hyaluronic acid is one of the main components of extracellular matrix (ECM) serving as the ligand of receptor glycoprotein CD44 (cluster of differentiation 44) and RHAMM (receptor for hyaluronan mediated motility), we used hyaluronic acid as a targeting drug carrier for new drug development, HCQ and its conjugate form (HA-HCQ) to show that the drug-carrier conjugate form (HA-HCQ) is less harmful, it is more stable and bio-available as it can be tested for pharmacokinetic properties. It is important to mention here that the drug and its conjugate form, having many differences in molecular weight and its complex structure, produce somewhat different results on computational drug profile results and therefore this study is designed to screen these two parameters and omitting other drug profiles.

Two different types (Mpro and Helicase) of molecular target proteins from novel coronavirus (SARS COV-2 or nCoV-2019) are the key viral molecules involved in the attachment, replication and reproduction of viral particles in the human host cells. These protein molecules served as targets to inhibit the viral lifecycle in human host cells. Four three dimensional crystal structures of SARS-CoV-2, of which three molecules of main protease complex with different inhibitors (5R80, 5R82, 6Y84) and one Helicase protein (6ZSL) were retrieved from the RCSB PDB database and used for the docking studies (https://www.rcsb.org/) [24] . To determine the binding affinities between the ligand and COVID-19 receptors, the amino acids with the binding pockets were predicted at the Q-site finder server [25] .

The generated HCQ and HA-HCQ conjugate SDF structures were docked with the predicted binding site of all selected protein target binding site by using FlexX docking software with the 

The interactions of HCQ and HA-HCQ with sixteen SARS COV-2 protein targets in the docked complexes were analyzed by the pose-view of LeadIT [25] . The 2D and 3D pose views of SARS COV-2 target protein-ligand complexes were generated and analyzed using LeadIT.

The Molecular Dynamic (MD) Simulations method was employed to predict the ligand binding status of HCQ and HA-HCQ in ligand-protein complexes of HCQ-Mpro (PDB ID 5R82), HAHCQ-Mpro (PDB ID 5R82), HCQ-Helicase (PDB ID 6ZSL) and HAHCQ-Helicase (PDB ID 6ZSL). The MD simulations were run using the Desmond Simulation Package of Schrodinger Suite [26] . The best ligand conformers obtained from molecular docking studies were subjected to molecular dynamics simulations. Protein-ligand complexes were prepared for simulations by using the default parameters of Schrodinger Maestro through the Protein Preparation Wizard tool [27] . Transferable Intermolecular Interaction Potential 3 Points was selected as the solvent model with an orthorhombic box of 10x10x10 Å. The system was made electrically neutral by adding appropriate counter ions. OPLS_2005 force field parameters [28] were utilized for the simulations study. The model systems were relaxed before the simulations.

At 300 K temperature and 1 atm pressure with NPT ensemble was applied for all MD simulations. Also, salt at the concentration of 0.15M was added to mimic physiological conditions. The complexes were subjected to molecular dynamics simulation for 1-50 ns. The Cut-off radius for the Coulombic interactions was set at 9 Å. Nos_e-Hoover chain thermostat (with a relaxation time of 1 ps) used for controlling the temperature and the Martyna-Tobias-Klein chain barostat (with a relaxation time of 2 ps) were used for pressure. The RESPA integrator utilized for calculating non-bonded forces. The trajectories of MD simulation saved at every 50 ps intervals and the simulations stabilities were evaluated using RMSD of the proteins and ligands over time. The MD simulations were repeated thrice for each complex using the same parameters. All MD simulations studies were carried out using Dell Precision T5810 Workstation with an Nvidia GeForce GTX 1070 8GB graphics processing unit (GPU).

The prediction of the drug and its conjugate form metabolism and bioavailability profile were screened through preADMET web server and the results are presented in Table 1 . It is quite interesting that the bioavailability parameters such as buffer and pure water solubility of HA-HCQ conjugate are found to be 22.04 mg/l and 261.32 mg/l respectively, which are more than its HCQ drug bioavailability scores of 12.91 mg/l and 224.78 mg/l respectively. It is also interesting to mention here that the metabolic profile of HA-HCQ shows no inhibition against all screened cytochrome drug-metabolizing xenobiotic enzymes such as CYP_1A2, CYP_2C19, CYP_2C9, CYP_2D6, CYP_3A4 whereas HCQ shows its inhibitory action against three out of five screened mitochondrial enzymes such as CYP_1A2, CYP_2D6, CYP_3A4. The above results revealed that HA-HCQ conjugate shown more bioavailability, less toxicity and ease of clearance from the body when compared to its free form of drug HCQ. It is worth mentioning here that conjugation of drug with hyaluronic acid enhances the pharmacokinetic profiles of the drugs themselves.

Four different COVID-19 target proteins including three main proteases, one helicase, their docking score and 3D crystal structure are reported in Table 2 , and their detailed molecular interaction between the viral target protein and its ligands (HCQ and HA-HCQ conjugate) are tabulated and presented in Table 3 & Fig. 2-5 .

The results of flexible docking study using FlexX software between COVID-19 viral targets and HA-HCQ showed the binding affinity and docking score ranging from -13.2046 KJ/mol to -23.1778 KJ/mol whereas HCQ against COVID-19 viral targets shown binding affinity and docking score ranging from -12.2217 KJ/mol to -13.6327 KJ/mol. It should be noted that HA-HCQ conjugate shows the highest binding affinity and docking score with SARS-CoV-2 viral target protein over HCQ (Table 3) . The HA is predominately present throughout the body including lung tissue and shields the respiratory organ scleroprotein from inflammation [36, 37] . The earlier scientific evidence confirms that HA is utilized in the distribution of drugs to the respiratory organ and also its sustained release of the drug makes biocompatibility, half-life extension, solubility enhancement, the possibility of high drug loading, less toxicity and increased drug retention time. Moreover, it is proposed that HA-HCQ drug conjugate enters into the lower respiratory tract cell through CD44 receptor, then under the influence of hydrolytic enzymes released from the lysosome, splits the HA-HCQ ester covalent bond and releases the HCQ drug into the target cell to interact with the SARS-COV-2 viral protein targets [37] [38] [39] .

The degree of HCQ substitution on hyaluronic acid impact the uptake of HA-HCQ conjugates by CD44 variant isoforms of the lower respiratory tract. Another important way to increase the uptake of HA-HCQ conjugates by CD44 is through lipopolysaccharide (LPS) treatment. The interactions between hyaluronic acid and CD44 receptor is strictly regulated to maintain a quiescent state revealing little appreciable binding between them [40] . During inflammation, the pro-inflammatory cytokine such as TNF alpha induces sulfation and subsequent conformational changes in the CD44 receptor, which transforms it into a greater HA affinity [41] . Kamat et al [42] reported the synthesis of iron oxide-based magnetic nanoparticles bearing hyaluronic acid on the surface to target activated macrophages. The low-molecularweight of HA is a key player in macrophage activation, which could activate an innate immune response via Toll-Like Receptor TLR2 and TLR4 [43] [44] [45] .

Moreover, earlier studies have proven that the delivery of drug rifampicin through a low degree of substitution of hyaluronic acid-Tocopherol succinate to alveolar macrophage cells.

Further, these RIF-HA-TS conjugate shows enhanced interaction with CD44 receptor that leads to the entry of drug-conjugate complex inside the MH-S murine alveolar macrophage cells [46] . Hydroxychloroquine (HCQ) is an anti-malarial drug that shows its efficiency by saving millions of lives during the COVID-19 outbreak. Chloroquine is a versatile bioactive agent that possess anti-viral action against numerous RNA viruses such as HIV [47] [48] [49] , Hepatitis A virus [50, 51] , Hepatitis C virus [52] , Poliovirus [53] , Rabies virus [54] , Influenza A & B virus [55] [56] [57] [58] , SARS-CoV-1 [59] . It also inhibits in vitro replication of HCoV-229E [60] and also found to inhibit MERS-CoV [61] in in vitro condition. The HCQ is a fairly safe drug and offer potentially useful treatment options for COVID-19 disease of the respiratory tract. Hydroxychloroquine has been known to possess in vitro anti-SARS-CoV effect. The prolonged usage of the clinical safety profile of Hydroxychloroquine is greater than that of Chloroquine for prolonged usage of drugs and makes a higher daily dose and has fewer questions regarding drug-drug interactions [62] .

The HCQ anti-viral mechanism such as inhibition of viral attachment and entry into the host cell, inhibition of N-glycosylation of cell surface viral receptor ACE2 [63, 64] , inhibition of Nglycosylation of viral spike protein [65] , endosomal alkalization [66] , inhibition of phospholipase A2 and membranous structure are essential for replication and translation of viral particles [67] . The HCQ also possess immunosuppressive properties that may help to reduce the cytokine storm in severe COVID-19 [68, 69] . This plot shows that by the end of the simulation, the ligand structures were found to be stable at 2.2 Å after 45 ns but proteins were undergoing continuous conformational changes and they might need longer simulation to be stable because of their large structure (Fig. 6b) whereas HCQ-Mpro (PDB ID 5R82) complex shown stable at 1.8 Å from 1-10ns and 45-50ns (Fig. 6a) .

The changes in the order 1-3 Å is perfectly acceptable for small and globular shape proteins.

The HAHCQ-Helicase (PDB ID 6ZSL) complex (Fig. 6d) shows stability after 45ns at 3.0 Å. A slight deviation from 1.5 Å to 2.0 Å happened up to 30 ns whereas HCQ-Helicase (PDB ID 6ZSL) complex has shown stable at 3.5 Å from 1-50ns (Fig. 6c) 

The molecular docking study was used to evaluate the safety of drug delivery, targeting receptors, toxicity, solubility and efficacy of Hyaluronic acid-Hydroxychloroquine (HA-HCQ) conjugate over free Hydroxychloroquine (HCQ) drug. Four SARS-CoV-2 viral molecular protein targets have been analyzed with HA-HCQ and the results obtained reveal better interactions with viral protein targets than the free HCQ drug. The HA-HCQ drug conjugate showed maximal drug delivery to lower respiratory tract through CD44 receptors with increased drug clearance and less toxicity to host cells. The results of MD simulation trajectories study reveal that HA-HCQ-SARS-CoV-2 protein complexes found superior complex stability over HCQ-SARS-CoV-2 protein complexes. Further studies are in progress to the synthesis of HA-HCQ conjugates with different degrees of substitutions of drugs to the polymer and to evaluate the antiviral activity both in in-vitro and in-vivo towards Mpro and Helicase SAR-CoV-2 viral protein targets. This will leads to the establishment of a promising and novel treatment option for COVID-19 and other related diseases through its versatile drug conjugation technology.

Please indicate the specific contributions made by each author (list the authors' initials followed by their surnames, e.g., V. Aroulmoji). The name of each author must appear at least once in each of the three categories below.

Conception and design of study: Riaz Khan, V. Aroulmoji, C. Sivasankar Acquisition of data: R. Thirumalaisamy, Muhammad Nasir Iqbal , T.Selvankumar, Analysis and/or interpretation of data: T.Selvankumar, R. Thirumalaisamy, Riaz Khan 

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