Computational Method on Hydroxychloroquine and Azithromycin for SARS-CoV-2: Binding Affinity Studies

 

Yogesh Vaishnav¹*, Laxmi Banjare², Shekhar Verma³, Govind Sharma¹, Deepak Biswas¹,

Arpan Tripathi¹, Afzal B. Shaik⁴, Richie R. Bhandare^5,6, Arvinder Kaur⁷, Kavya Manjunath⁸

¹Faculty of Pharmaceutical Sciences, Shri Shankaracharya Technical Campus, Junwani, Bhilai, 490020.

²School of Pharmaceutical Sciences, Guru Ghasidas Central University, Bilaspur – 495009 (C.G.) India.

³University College of Pharmacy, Pt Deendayal Upadhyay Memorial Health Sciences and Ayush University, Raipur, Chhattisgarh 493661.

⁴St. Mary’s College of Pharmacy, St. Mary’s Group of Institutions Guntur, Affiliated to Jawaharlal Nehru Technological University Kakinada, Chebrolu, Guntur, Andhra Pradesh, 522212.

⁵Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences,

Ajman University, Ajman, United Arab Emirates.

⁶Centre of Medical and Bio-allied Health Sciences Research, Ajman Uniersity, Ajman, United Arab Emirates.

⁷Department of Pharmaceutics, KLE College of Pharmacy, Bengaluru, Constituent unit of KLE Academy of Higher Education and Research (Deemed to be University), Rajajinagar 560010 Karnataka, India.

⁸Department of Pharmacology, KLE College of Pharmacy, Bengaluru, Constituent unit of KLE Academy of Higher Education and Research (Deemed to be University), Rajajinagar 560010 Karnataka, India.

 

ABSTRACT:

World is facing a new pandemic called covid-19SARS-CoV-2) since a year ago. Unfortunately there is no treatment for Covid 19 nowadays as well as no potential therapies has been developed to overcome from coronavirus pandemic. Some potential drug molecules with combination have ability to respond for covid19 virus. From the research it was found that the reduction of viral load can be treated with hydroxychloroquine and azithromycin combination. We evaluate the mode of interactions of hydroxychloroquine and azithromycin with the dynamic site of SARS-CoV-2 coronavirus main protease. Molecular Structure-based computational approach viz. molecular docking simulations were performed to scale up their affinity and binding fitness of the docked complex of novel SARS-CoV-2 coronavirus protease and hydroxychloroquine and azithromycin. The natural inhibitor N3 of novel SARS-CoV-2 coronavirus protease were exhibited highest affinity in terms of MolDock score (-167.203Kcal/mol), and hydroxychloroquine was found with lowest target affinity (-55.917 Kcal/mol).The amino acid residue cysteine 145 and histidine 41 is bound covalently and formed hydrogen bond interaction with SARS-CoV-2 inhibitor known as inhibitor N3 as such, hydroxychloroquine and azithromycin also formed hydrogen bond interaction. The binding patterns of the inhibitor N3 of SARS-CoV-2 coronavirus main protease could be used as a guideline for medicinal chemist to explore their SARS-CoV-2 inhibitory potential.

 

 

1. INTRODUCTION:

Pandemic viruses hit the whole world at the beginning of twenty first century and were named as COVID-19. One of the major city of China, Wuhan has witnessed this emerging infection in late December 2019^1,2. Covid-19 was declared as Severe Acute Respiratory Syndrome Coronavirus 2 with abbreviation SARS-CoV-2 by International Virus Classification Commission (ICTV) on 11^th February 2020. As the infection of epidemic COVID 19 increased day by day all over the World, later on it has been declared as Pandemic on 12^th March 2020 by WHO³. Recent study on covid-19 reveals that the infection is more in geriatric persons and the fatality rate is around 23 percent⁴. World is facing a worst pandemic of the century and has affected more than 5 lakh active cases with 25 thousand death so far and more likely to increase in coming days. After Wuhan, France has reported more than thirty thousand active cases as well as two thousands of mortality till end of March 2020⁵. Respiratory problems associated with fever, cough, shortness of breath were observed in patients infected with coronavirus. Infected person in severe cases diagnosed with pneumonia, kidney failure, SARS and even death. The SARS-CoV-2 comes under enveloped virus having a positive RNA genome. Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) proteins are the four different proteins found in SARS-CoV-2. Coronaviruses are a persistent threat to health of the global community. SARS-CoV-2 arises from animals such as other viruses like SARS and MERS. The structure of coronavirus consists of extremely large genome which encodes numerous dozen viral proteins. Protein structures helps to develop novel approach as well as new drug for creating vaccines to fight against coronavirus. Coronaviruses contain a genome composed of a long RNA strand—one of the largest of all RNA viruses. The genome behaves as a messenger RNA when genome infects a cell and leads the production of two long polyproteins as machinery that the virus desires to replicate new viruses. The long polyproteins consist of transcription or replication complex which makes more RNA also helps to construct new virions and two proteases. The proteases plays a crucial roles in cutting the polyproteins into all of these functional pieces. In February 2020, Zihe Rao and Haitao Yang's research team at ShanghaiTech University determined the first high resolution crystal structure of COVID-19 coronavirus 3CL hydrolase (Mpro) with PDB code of 6LU7 (Figure 1)⁶. The structure consist of two identical molecules linked together to form two active sites. The protein structure reveals that folding of protein is analogous toward serine proteases like trypsin, but a cysteine amino acid and a close histidine perform the protein-cutting reaction and an additional domain stabilizes the dimer (Figure 2). This structure has a peptide-like inhibitor bound in the active site.

 

Researchers are actively using these structures to search for compounds that block the action of the proteases, for use as antiviral drugs. The diversity of coronaviruses poses a great challenge with this effort. In four distinct genera coronaviruses have been classified. The sequence and structural studies have shown that the proteases of these viruses can be very different, so drugs designed to fight one may not be effective against others. One possible way to address this challenge is to try to design a broad-spectrum inhibitor targeted against the progenitor bat coronavirus, which may then provide a head-start for discovering inhibitors against newly emerging viruses. Based on target selection, potential anticoronavirus therapies has been classified into two categories with respect to immune response of human beings and in targeting the corona virus itself. To control replication mechanism and infection of coronavirus, innate immune system plays a crucial role⁷. Different therapies were involved, action on genetic material of virus prevents the synthesis or production of viral RNA. Action on virus enzymes results in inhibition of virus replication process, also by the action on some structural proteins, inhibition of virus self-assembly process or blocking the binding of virus to human cell receptors. At present no standard drug, vaccine or even therapy has been approved for the treatment of novel corona virus⁸. Therefore there is an urgent need of vaccine, effective drug or effective therapies to fight against corona virus infection. The fastest or efficient way to develop new medicine is to follow the potential molecule which were effective against many virus infection. The benefit of following potential molecule is that there drug interaction, posology, side effects are well known and already established^{9, 10}. Research findings provide an insight that the analogues of chloroquine inhibits endosomes acidification and it also exhibits in vitro nonspecific antiviral activity at higher micro molar concentration against broad range of different viruses like dengue, HIV, Hepatitis C, influenza, EBOLA, SARS and MERS virus. Recent study showed the effectiveness of chloroquine against COVID 19^{11, 12}. Hydroxychloroquine has been reported as an effective drug against SARS-CoV¹³. Hydroxychloroquine is better than that of chloroquine in terms of clinical safety profile, long term use and higher daily dose. Drug –drug interaction is minimum in Hydroxychloroquine. A report has been published by researchers which showed that chloroquine and hydroxychloroquine inhibits Covid 19 virus in vitro in which hydroxychloroquine found to be the most potent than chloroquine^14,15.

 

Hydroxychloroquine in combination with azithromycin showed significant result against Zika, Ebola virus and also effective for severe respiratory tract infection (Figure 3)¹⁶. Some studies showed that combination of hydroxychloroquine and azithromycin significantly clear 100 percent viral consent in nasopharyngeal swabs in more than five patients at period of 5-6 days¹⁷. In present research work, authors have performed molecular docking to determine the binding fitness and nature of interactions between hydroxychloroquine and azithromycin with SARS-CoV-2 coronavirus main protease to achieve binding affinities and binding mode of interactions.

 

 

Figure 1 The crystal structure of SARS-CoV-2 main protease protein with inhibitor N3 (inhibitor in green colour, helices in red, strands in blue and loop in white).

 

 

Figure 2 The close key amino acid residue (cysteine 145 and histidine 41) in SARS-CoV-2 protease enzyme (natural inhibitors in green colour and close amino acid residue in ball and stick form clolour by amino acid type).

 

Figure 3 Chemical structure of Natural inhibitor main protease enzyme, hydroxychloroquine and azithromycin.

 

2. COMPUTATIONAL METHODOLOGY:

Chem Draw Ultra of version 8.0 was used to sketch the structure of hydroxychloroquine and azithromycin¹⁸. The two-dimensional structures were again subjected into Chem Three-Dimensional Ultra software of version 8.0 in order to minimize the energy and generate stable energy conformers. MM2 (Molecular Mechanics) and AM1 (Austin Model 1) were the two specific optimizers which comes under semi-empirical process calculated the minimization. All the operations were done in MOPAC. RMSD parameters plays a key role for minimization of energy and was set at value of 0.001 kcal/mol. Another important parameter for prediction is the interaction and binding affinity among hydroxychloroquine, azithromycin and protease of SARS-CoV-2 virus^19-22. The whole process was carried out by the aid of software named as MVD 2013 of version 6.0. A PDB (Protein Data Bank) id was required for further study. PDB provide crystal structure of SARS-CoV-2 main protease. In present study, 6LU7 was the PDB id for SARS-CoV-2 main protease which was retrieved from bank which consist data of proteins. All the three essential things, hydroxychloroquine, azithromycin and structure of main protease in the form of PDB id were subjected into MVD software workstation. In the beginning, the first step was removal of water molecules from protease enzyme structure. Structural errors were corrected then the targeted molecules and ligands were subjected for the establishment of molecule. To find the promising dynamic position in the target molecule, MVD software provide cavity prophecy tool. Following parameters were fixed to found dynamic position such as Maximum number of cavities were fixed to 5, resolution of grid at 0.80 angstrom with probe size at 1.2 angstrom and other parameters were fixed as per standard conditions. Other parameters like iterations fixed at 1500 and resolution of grid maintained at 0.3 angstrom with radius of binding at 15 angstrom^23-25. MolDock SE (Simplex Evolution) was fixed for docking algorithm. In this the value of MolDock Simplex Evolution, population size was in the range of 50 also RMSD was in the range of 1.00 angstrom for cluster poses. The same parameter of RMSD was fixed for ignore similar poses for numerous run. The value 10 was selected for number of independent runs and after each run, a final solution came in the form of pose. Negative value represents the best docking score while similar poses were considered as negligible. The clusters obtained in the docking study were categorized depending upon the conformation of lowest binding energy value. For the examination of docking result and alignment, the first minimum binding free energy pose was selected^26-27.

 

3. RESULT AND DISCUSSION:

SARS-CoV-2 structure was instituted with inhibitor known as inhibitor N3, covalently bound to residue cysteine 145 at the protease dynamic site and adjacent active site with residue histidine 41. To detect binding mode, binding affinity and binding interaction, structure guided molecular docking was successfully performed on energy minimized structures of hydroxychloroquine and azithromycin and natural inhibitors of SARS-CoV-2 within the active site of target protease enzyme. The cavity 1 was found with largest surface area and highest volume among all detected cavities (1-5) (Figure 4, Table 1).

 

After successful execution of molecular docking, the values or scores of parameters MolDock, Re-rank, H-bond and Steric scores were generated. The score of MolDockre presents capability of ligand to find appropriate spot inside the active site of target and it represents the affinity of ligands towards active site of target.

 

Figure (4) The major cavities (1- 5) within SARS-CoV-2 main protease protein with inhibitor N3 green in colour, Hydroxychloroquine in turquoise colour, azithromycin in yellow colour)

 

Table 1: Predicted binding cavities (1-5) in SARS-CoV-2 main protease complex along with their volume, surface area, and position

 

Table 2:Comparative docking scores results of inhibitor N3 with Hydroxychloroquine and Azithromycin

 

The value of Re-rank identifies most promising solution from docking algorithm and improve docking accuracy. The best docking pose was with highest MolDock score (-167.203) and highest re-rank score (-129.19) associated to inhibitor N3. Hydroxychloroquine has shown better MolDock score, Re-rank, H-Bond (-108.46, -82.7944, -7.29492Kcal/mol, respectively) compared to azithromycin (-55.9178, -29.4545, -5.13897Kcal/mol, respectively). The docking score of the hydroxychloroquine and azithromycin was compared with main protease protein with inhibitor N3 in terms of binding affinity was depicted in Table2.Moreover, the composite binding affinity and fitness of inhibitor N3 with target SARS-CoV-2 protease enzyme was better compared to hydroxychloroquine and azithromycin. Docking views of natural inhibitor within cavity 1 of target SARS-CoV-2 protease enzyme is depicted in (Figure 5) which showed, prominent H-bond interactions (Id. 1-4, (Figure 5A, Table 3)), and steric interactions (Id. 1-13 (Figure 5B, Table 3)). The Docking views of hydroxychloroquine within cavity 1 of target SARS-CoV-2 protease enzyme is depicted in (Figure 6) which showed, H-bond interactions (Id. 1-4, (Figure 6A, Table 4), and steric interactions (Id. 1-4 (Figure 6B, Table 4). Azithromycin displayed lesser extent presented in (Figure 7) the H-bonding (Id. 1-4, (Figure 7A, Table 5), as well as steric interactions (Id. 1-14, (Figure 7B, Table 5).

 

  

Figure (5) Docking view of natural inhibitor N3 (colour by CPK) with SARS-CoV-2 main protease enzyme (5A) H-bond interactions (yellow dotted bonds) (5B) Steric interactions (red dotted bonds).

 

Table 3: Properties of h-bond and steric interactions displayed by natural inhibitor N3 with SARS-CoV-2 main protease enzyme

 

 

Table 4: Properties of h-bond and steric interactions of hydroxychloroquine with SARS-CoV-2 main protease enzyme

 

Table 5: Properties of h-bond and steric interactions of azithromycin with SARS-CoV-2 main protease enzyme

 

 

Figure (6) Docking views of hydroxychloroquine within cavity 1 of target SARS-CoV-2 protease enzyme

(6A) H-bond interactions (6B) steric interactions

 

Figure (7) Docking view of azithromycin (colour by CPK) with SARS-CoV-2 main protease enzyme (7A) H-bond interactions (yellow dotted bonds) (7B) Steric interactions (red dotted bonds).

 

CONCLUSION:

The present research work reveals and support the work done previously that the combination of hydroxychloroquine and azithromycin produces synergistic effect, we have chosen these drugs in our study for molecular docking because these drugs shown prominent effect individually against some viruses as a result the combination of both the drugs in docking studies reveals that they were active against SARS-CoV-2 coronavirus but target of action was not known. So docking study suggested that hydroxychloroquine and azithromycin has good binding affinity to amino acid residueCys 145 and His 41 which is an important characteristic of the SARS-CoV-2 main protease enzyme and its natural inhibitor N3. Study also revealed that N3 inhibitor also interact with the same amino acid residue. We therefore suggests that the combination therapy with hydroxychloroquine and azithromycin would be of great help to overcome from this SARS-CoV-2 coronavirus. The overall research outputs were promising and will help to develop a new prototype against this pandemic causing virus.

 

CONFLICT OF INTEREST:

The authors verify that the content related to current research article has no conflict of interest.

 

ACKNOWLEDGEMENTS:

Authors would like to thank Shri Shankaracharya Technical Campus for providing necessary facilities. One of the author Laxmi Banjare would like to acknowledge UGC for providing her Senior Research Fellowship (SRF) under National Fellowship Scheme (NFSC). We are also thankful to Dr. Rene Thomsen for Molegro Virtual Docker (MVD) software.

 

REFERENCES:

1.     Lai CC, Shih TP, Ko WC, Tang HJ, Hsueh PR. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int J Antimicrob Agents. 2020 Feb 17: 4 doi.org/10.1016/j.ijantimicag.2020.105924

2.     Wang LS, Wang YR, Ye DW, Liu QQ. A review of the 2019 Novel Coronavirus (COVID-19) based on current evidence”. Int J Antimicrob Agents. doi.org/10.1016/j.ijantimicag.2020.105948

3.     WHO Director-General's opening remarks at the media briefing on COVID-19 - 11 March 2020. https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-atthe-media-briefing-on-covid-19-11-march-2020.

4.     Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese Center for Disease Control and Prevention. JAMA. 2020; 323(13):1239-1242. doi.org/10.1001/jama.2020.2648

5.     www.who.int/covid-19/information.

6.     Zhenming Jin, Xiaoyu Du, […]Haitao Yang Structure of Mpro from COVID-19 virus and discovery of its inhibitors, Nature. doi.org/10.1038/s41586-020-2223

7.     Omrani AS, Saad MM, Baig K, Bahloul A, Abdul-Matin M, Alaidaroos AY, et al. Ribavirin and interferonalfa-2a for severe Middle East respiratory syndrome coronavirus infection: a retrospective cohort study.Lancet Infect Dis 2014; 14: 1090-5. doi.org/10.1016/S1473-3099(14)70920-X

8.     Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet 2020; 395: 497-506.doi.org/10.1016/S0140-6736 (20)30183-5

9.     Colson P, Rolain JM, Raoult D. Chloroquine for the 2019 novel coronavirus SARS-CoV-2. Int J Antimicrob Agents. 2020 Feb 15:105923. doi.org/10.1016/j.ijantimicag.2020.105923.

10.  Colson P, Rolain JM, Lagier JC, Brouqui P, Raoult D. Chloroquine and hydroxychloroquine as available weapons to fight COVID-19. Int J Antimicrob Agents. doi.org/ 10.1016/j.ijantimicag.2020.105932

11.  Wang M, Cao R, Zhang L, et al. Remdesivir and chloroquineeffectivelyinhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res2020; 30:269–71.doi.org/10.1038/s41422-020-0282-0.

12.  Al-Bari AA. Targeting endosomal acidification by chloroquine analogs as apromising strategy for the treatment of emerging viral diseases. PharmacolResPerspect 2017; 5: doi.org/10.1002/prp2.293

13.  Biot C, Daher W, Chavain N, Fandeur T, Khalife J, Dive D, et al. Design and synthesisof hydroxyferroquine derivatives with antimalarial and antiviral activities. J Med Chem 2006; 49:2845-2849. doi.org/10.1021/jm0601856

14.  Marmor MF, Kellner U, Lai TY, Melles RB, Mieler WF; American Academy of Ophthalmology. Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision). Ophthalmology. 2016 Jun;123 (6):1386-94. Doi.org/10.1016/j.ophtha.2016.01.058.

15.  Yao X, Ye F, Zhang M, Cui C, Huang B, Niu P, et al. In Vitro Antiviral Activity andProjection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Clin Infect Dis. 2020 Mar 9. Doi.org/10.1093/cid/ciaa237

16.  Bacharier LB, Guilbert TW, Mauger DT, Boehmer S, Beigelman A, Fitzpatrick AM, et al. Early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: A randomized clinical trial. JAMA. 2015 Nov 17; 314(19):2034-2044. doi.org/10.1001/jama.2015.13896

17.  Gautret P, Lagier JC, Parola P, et al. Hydroxychloroquine and azithromycin as atreatment of COVID-19: results of an open-label non-randomised clinical trial. Int J Antimicrob Agents 2020. Doi.org/10.1016/j.ijantimicag.2020.105949

18.  Yogesh Vaishnav, Aloksingh Thakur, Chanchal Deep Kaur, Shekhar Verma, Achal Mishra, Sanmati Kumar Jain , Piyush Ghode. QSAR Analysis of some N, N-diphenyl urea derivatives as CCR5 Receptor Antagonist. Research J. Pharm. and Tech 2018; 11(9): 3802-3810.doi.org/10.5958/0974-360X.2018.00697.2

19.  Navjot Kaur, Monika, Kulwinder Singh. 3D-QSAR and Molecular Docking Studies of N-(2-Aminophenyl)-Benzamide Derivatives as Inhibitors of HDAC2. Research J. Pharm. and Tech. 7(7): July 2014 Page 760-770. Doi.org/10.5958/0974-360X

20.  E. Shanmugapriya, V. Ravichandiran, M. Vijey Aanandhi. Molecular docking studies on naturally occurring selected flavones against protease enzyme of Dengue virus. Research J. Pharm. and Tech. 2016; 9(7):929-932.doi.org/:10.5958/0974-360X.2016.00178.5

21.  Neenapriya Prasad Nair, Josna Joy, Silpa. S. Kumar, S.Sathianarayanan, Asha Ashokan Manakadan, Saranya T.S. In-silico Docking Studies of Coumarin Derivatives as Caspase 8 and PDE4 Antagonist. Research J. Pharm. and Tech 2016; 9(12):2199-2204. doi.org/10.5958/0974-360X.2016.00445.5

22.  Pavlo V. Zadorozhnii, Vadym V. Kiselev, Anastasia E. Titova, Aleksandr V. Kharchenko, Ihor O. Pokotylo, Oxana V. Okhtina. Molecular Docking Studies of N-5-Aryl-1, 3, 4-oxadiazolo-2, 2-dichloroacet-amidines as Inhibitors of Enoyl-ACP Reductase Mycobacterium tuberculosi. Research J. Pharm. and Tech. 2017; 10(4): 1091-1097.doi.org/10.5958/0974-360X.2017.00198.6

23.  Animisha Mokkapati, Radha Krishna Nagumantri, Chinna Babu Pydi, Ramakrishna Chintala, Satyanarayana Rentala. Docking Studies of Piperine-Vitamin a Conjugate to Study the Increase in Bioavailability of Vitamin A. Research J. Pharm. and Tech. 2017; 10(7): 2189-2193.doi.org/10.5958/0974-360X.2017.00386.9

24.  T. Arunkumar, Ann Feba Ebby, G. Narendrakumar. Docking studies of Benzisoxazole analogues in White Spot Syndrome Virus. Research J. Pharm. and Tech. 2017; 10(8): 2497-2500. Doi.org/10.5958/0974-360X.2017.00441.3

25.  Jeyabaskar Suganya, Viswanathan T, Mahendran Radha, Nishandhini Marimuthu. In silico Molecular Docking studies to investigate interactions of natural Camptothecin molecule with diabetic enzymes. Research J. Pharm. and Tech. 2017; 10(9): 2917-2922. Doi.org/10.5958/0974-360X.2017.00515.7

26.  H. S. Baul, M. Rajiniraja. Molecular Docking Studies of Selected Flavonoids on Inducible Nitric Oxide Synthase (INOS) in Parkinson’s Disease. Research J. Pharm. and Tech 2018; 11(8): 3685-3688. doi.org/10.5958/0974-360X.2018.00676.5

27.  Naresh Panigrahi, Swastika Ganguly, Jagadeesh Panda. Synthesis, Antimicrobial Evaluation and Molecular Docking Studies of Novel Oxazolidinone-Thiophene Chalcone Hybrid Derivatives. Research J. Pharm. and Tech 2018; 11(12): 5611-5622. doi.org/10.5958/0974-360X.2018.01019.3

 

 

 

Accepted on 24.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5467-5472.