Potential of plant alkaloids as dengue NS3 protease inhibitors: Molecular docking and simulation approach
Abstract
Dengue infection has become a worldwide health problem and infection rate is increasing each year. Alkaloids are important phytochemicals of medicinal plant and can be used as vaccine candidates for viruses. Therefore, present study was designed to find potential alkaloids inhibitors against the Dengue virus NS2B/NS3 protease which can inhibit the viral replication inside the host cell. Through molecular docking it was investigated that most of the alkaloids bound deeply in the binding pocket of Dengue virus NS2B/NS3 protease and had potential interactions with catalytic triad. Five alkaloids (6'-desmethylthalifaboramin; 3,5-dihydroxythalifaboramine; Betanin; Reserpic acid and Tubulosine) successfully blocked the catalytic triad of NS2B/NS3 protease and these alkaloids can serve as a potential drug candidate to stop viral replication. It can be concluded from this study that these alkaloids could serve as important inhibitors to inhibit the replication of DENV and need further in vitro investigations to confirm their efficacy and drug ability.
Introduction
According to recent studies, it has been found that NS3 has a serine protease domain at the N terminal region and its activity depends on its interaction with cofactor (NS2B). These two forms a complex called NS2B-NS3pro complex. This complex is very important because it has ability to cleave viral proteins. Any disruption in functional activities of this region results into the inhibition of viral replication. Hence, to screen and evaluate effects of different drug candidates, NS2B-NS3 complex is considered an important target (Rothan et al., 2012). Currently, there is no vaccine and effective drug available for the treatment of DENV infection (Idrees and Ashfaq, 2012).
Recent computational techniques have opened new doors to drug development studies. Prediction of predominant binding mode of a ligand with a protein of known three-dimensional structure (Molecular docking) is considered as important technique in drug designing and screening of novel antiviral compounds against challenging diseases (Lengauer and Rarey, 1996). Therefore, this study has been designed to screen 1300 alkaloids of more than 80 antiviral medicinal plants against Dengue virus NS2B, NS3 protease using in silico techniques. The main theme of this study was to target the hydrophobic pockets of Dengue virus NS2B, NS3 Protease to screen novel alkaloids that could help in inhibition of the DENV infection. The result of this study will offer useful information about drug development and would help in computer aided screening of the drugs against DENV infection.
Materials and Methods
In this study alkaloids have been docked against Dengue virus NS2B/NS3 protease. Docking was carried out using the Molecular Operating Environment (MOE) software package (ul qamar et al., 2014).
Refinement of receptor protein
Three-dimensional (3D) structure of the Dengue virus NS2B NS3 protease was retrieved from the Protein Data Bank (PDB) using PDB ID:2FOM and was optimized by removing water molecules, 3D protonaion and Energy minimization using Molecular Operating Environment (MOE, 2012). Moreover, energy minimization was done using parameters, Force Field: MMFF94X + Solvation, gradient: 0.05, and Chiral Constraint: Current Geometry. This minimized structure was used as receptor for docking studies.
Refinement of receptor protein
Three-dimensional (3D) structure of the Dengue virus NS2B/NS3 protease was retrieved from the Protein Data Bank (PDB) using PDB ID:2FOM and was optimized by removing water molecules, 3D protonaion and Energy minimization using Molecular Operating Environment (MOE, 2012). Moreover, energy minimization was done using parameters, Force Field: MMFF94X + Solvation, gradient: 0.05, and Chiral Constraint: Current Geometry. This minimized structure was used as receptor for docking studies.
Molecular docking
The docking algorithm of the Molecular Operating Environment software was used to dock ligand database with catalytic triad (His 51, Asp 75, Ser 135) of Dengue virus NS2 NS3 protease. The parameters were set; Rescoring function: London dG, placement: triangle matcher, Retain: 10, Refinement: Force field, and Rescoring 2: London dG. Docking program of MOE provides correct conformation of the ligand so as to obtain minimum energy structure. After docking, top and best conformation for alkaloids was selected on the basis of S score to further study the hydrogen bonding pi-pi interactions.
Drug scan
Drug scan of final selected alkaloids was performed by using the ligand properties checking tool of MOE to make sure that the compounds possess appropriate molecular properties to be a drug candidate.
Results
The 3D-structure of DENV NS2,NS3 protease was retrieved from PDB. The PDB ID of 3D-structure was 2FOM, which had resolution of 1.50 Angstrom. All alkaloids were docked with the catalytic triad of Dengue virus NS2B,NS3 Protease.
Molecular Operating Environment software provided ten conformations for each alkaloid. All these conformations were sorted according to S score. Top six conformations for each alkaloid with minimum S score were selected for further analysis. 6'-Desmethylthalifa-boramin was ranked at top conformation followed by 3-hydroxy-6'-desmethyl-9-0-methylthalifaboramine, 3,5-dihydroxythalifaboramine, betanin, reserpic acid and tubulosine respectively. Plant names from which alkaloids were derived, S score, RMSD value and detail about interacting residues shown in (Table I). Chemical structures of selected alkaloids have shown (Figure 1).
Table I: Plant names from which alkaloids were derived, S score, RMSD value and detail about interacting residues is shown
| Plant name | Alkaloids | S Score | RMSD Value | Interacting Residues | Close Contact Residues |
|---|---|---|---|---|---|
| Thalictrum faberi | 6'-desmethyl-thalifaboramin | -12.24 | 2.21 | His51 | Asp75, Ser135, Leu128, Pro132, Gly135, Trp50, Arg54 |
| Thalictrum faberi | 3-hydroxy-6'-desmethyl-9-0-methylthali-faboramine | -11.96 | 2.09 | Asn152, Lys73 | Asp75, Asp129, His51, Thr120, Leu128, Gly153, Ser131 |
| Thalictrum faberi | 3,5-dihydroxythali-faboramine | -11.66 | 1.53 | His51, Asn152 | Asp75, Ser134, Arg54, Gly153, Pro132, Leu128, Val154, Ser135 |
| Hylocereus polyrhizus, Amaranthus powellii, Boerhavia erecta | Betanin | -11.45 | 3.00 | His51, Pro132, Gly151, Tyr150, Phe130, | Asp75, Leu128, Ser135, Trp50, Val72, Gly153 |
| Rauwolfia vomitoria | Reserpic acid | -11.15 | 2.31 | His51 | Asp75, Ser135, Gly135, Pro132, Trp50, Val72, Phe130 |
| Pogonopus speciosus, Alangium lamarckii | Tubulosine | -10.64 | 2.12 | Ser135 | Tyr161, Asp75, Gly135, His51, Leu128, Pro132, Tyr150 |
Figure 1: Chemical structures of selected alkaloids
Along with minimum S score, 6'-desmethylthalifaboramin also had potential interactions with His-51 and strong hydrophobic contact with Asp-75 and Ser-135 of catalytic triad and thus, it can be concluded that this alkaloid could use as potential drug against Dengue virus NS2B,NS3 Protease. All other alkaloids (3-hydro-xy-6'-desmethyl-9-0-methylthalifaboramine; 3,5-dihydroxythalifaboramine; betanin; reserpic acid; tubulosine) also have potential interaction and significant hydrophobic contact with active residues of catalytic triad. Interacting residues of the DENV NS2B,NS3 Protease are shown in (Table I). Interactions between Dengue virus NS2B,NS3 Protease catalytic triad and alkaloids are shown in (Figure 2). Binding mode of ligands with receptor is shown in (Figure 3).
Figure 2: Binding interactions of alkaloids with active residues of dengue virus NS2B/NS3 protease
Drug scan
Final selected alkaloids were further analyzed to check Lipinski's Rule of Five using the ligand properties checking tool of MOE which assessed the molecular properties and practicability of these compounds (Lipinski et al., 1997). The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism and excretion. These compounds were examined for their drug-suitableness and the results are shown in (Table II). Our results showed that all the alkaloids compounds used in this study fulfill the criteria of being drug candidates except 3-hydroxy-6'-desmethyl-9-0-methylthalifaboramine.
Table II: Molecular properties of flavonoids assessed through Ligand properties checking tool of MOE
| Alkaloids | Molecular weight | Log P | TPSA | Hydrogen bond donor | Hydrogen bond acceptor | Lipinski's rule of five |
|---|---|---|---|---|---|---|
| 6'-desmethylthalifaboramin | 640.5 | 3.8 | 95.5 | 4 | 6 | Suitable |
| 3-hydroxy-6'-desmethyl-9-0-methylthalifaboramine | 1035.3 | -3.7 | 342.4 | 13 | 21 | Not-suitable |
| 3,5-dihydroxythalifaboramine | 686.8 | 3.5 | 124.9 | 5 | 8 | Suitable |
| Betanin | 550.5 | -3.7 | 249.4 | 8 | 13 | Suitable |
| Reserpic acid | 401.5 | 0.9 | 96.2 | 4 | 5 | Suitable |
| Tubulosine | 477.6 | 2.9 | 75.5 | 4 | 3 | Suitable |
Figure 3: Docked alkaloids complexes with the binding pocket of dengue virus NS2B/NS3 protease
Discussion
Dengue is an appalling disease and requires urgent attention to develop new inhibitory compounds that could work against it. Genome of dengue encodes a single polyprotein which is cleaved into 10 viral proteins (Idrees et al., 2013). The cleavage of polyprotein precursor requires signal peptidase and NS3 serine protease which requires a cofactor named NS2B (Murthy et al., 1999). Dengue virus has four serotypes (Khan et al., 2008) but any inhibitor against the binding pocket of NS2,NS3 protease could work against all the serotypes (Li et al., 2005). Like other flaviviruses Dengue virus NS3 protease has been declared as significant drug target. Catalytic triad is important in viral replication therefore, any disruption in it may block the replication of virus (Van Hell et al., 2009.
In recent research, computational techniques have enabled researchers to estimate the binding affinity of different molecules before their synthesis and evaluation in lab. Molecular docking used to find out the binding orientation of the small molecules against their targets. Thus, molecular docking is considered as important technique in drug designing and screening of novel compounds against this dreadful and challenging diseases (Lengauer and Rarey, 1996). The current study focused on the docking of the plants phytochemicals against NS2B-NS3 protease.
We examined the potential of 1300 alkaloids against dengue virus NS2B-NS3 protease. Alkaloids were downloaded from different databases. In this study, 1300 alkaloids were docked with the catalytic triad of Dengue virus NS2,NS3 protease to find their affinity as inhibitors. Only top conformations after docking were selected on the basis of minimum S score. Our results showed potential and significant interactions of alkaloids with the active site residues of catalytic triad. Our results also showed that the final selected alkaloids fulfill the criteria of being drug candidates.
Through our study it was found that five alkaloids (6'-desmethylthalifaboramin; 3,5-dihydroxythalifaboramin; betanin; reserpic acid and tubulosine) have potential interaction and significant hydrophobic contact with active residues of catalytic triad thus, it can be concluded that these alkaloid could use as potential drug against dengue virus NS2B,NS3 protease. Further study needs to be conducted on the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) of alkaloids proposed as a drug.
This study has discovered potential binding of alkaloids from medicinal plants Thalictrum faberi, Hylocereus polyrhizus, Amaranthus powellii, Boerhavia erecta, Rauwolfia vomitoria, Pogonopus speciosus, Alangium lamarckii with active residues of NS2,NS3 protease.
Acknowledgment
The authors would like to acknowledge the Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, 38000, Pakistan.
References
Ashfaq UA, Mumtaz A, ul Qamar MT, Fatima T. MAPS Database: Medicinal plant activities, phytochemical and structural database. Bioinformation 2013; 9: 993-95.
Calixto JB. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res. 2000; 33: 179-89.
Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990; 44: 649-88.
Das S, Pingle MR, Munoz-Jordan J, Rundell MS, Rondini S, Granger K, Chang GJJ, Kelly E, Spier EG, Larone D, Spitzer E, Barany F, Golightly LM. Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay. J Clin Microbiol. 2008; 46: 3276-84.
Hakim ST, Tayyab SMH, Nadeem SG. An experience with dengue in Pakistan: An expanding problem. Ibnosina J Med BS. 2011; 3: 3-8.
Idrees S, Ashfaq UA, Khaliq S. RNAi: Antiviral therapy against dengue virus. Asian Pac J Trop Biomed. 2013; 3: 232-36.
Idrees S, Ashfaq UA. A brief review on dengue molecular virology, diagnosis, treatment aAnd prevalence in Pakistan. Genet Vaccines Ther. 2012; 10: 6.
Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG. ZINC: A free tool to discover chemistry for biology. J Chem Inf Model. 2005; 45: 177-82.
Jassim SA, Naji MA. Novel antiviral agents: A medicinal plant perspective. J Appl Microbiol. 2003; 95: 412-27.
Kubmarawa D, Khan ME, Punah AM, Hassan. Phytochemical screening and antibacterial activity of extracts from Parkia clappertoniana keay against human pathogenic bacteria. J Med Plants Res. 2008; 2: 352-55.
Khan AM, Miotto O, Nascimento EJ, Srinivasan KN, Heiny AT, Zhang GL, Marques AT, Tan TW, Brusic V, Salmon J, August JT. Conservation and variability of dengue virus proteins: Implications for vaccine design. PLoS Negl Trop Dis. 2008; 2: 272.
Lengauer T, Rarey M. Computational methods for biomolecular docking. Curr Opin Struct Biol. 1996; 6: 402-06.
Li J, Lim SP, Beer D, Patel V, Wen D, Tumanut C, Tully DC, Williams JA, Jiricek J, Priestle JP, Harris JL, Vasudevan SG. Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries. J Biol Chem. 2005; 280: 28766-74.
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 1997; 46: 3-26.
MOE: Molecular Operating Environment (MOE). Chemical Computing Group Inc, 1010 Sherbooke St West, Suite #910, Montreal, QC, Canada, H3A 2R7 2012.
Murthy HM, Clum S, Padmanabhan R. Dengue virus NS3 serine protease: Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. J Biol Chem. 1999; 274: 5573-80.
Rothan HA, Han HC, Ramasamy TS, Othman S, Rahman NA, Yusof R. Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1. BMC Infect Dis. 2012; 12: 314.
Thomas SJ, Strickman D, Vaughn DW. Dengue epidemiology: Virus epidemiology, ecology, and emergence. Adv Virus Res. 2003; 61: 235-89.
ul Qamar MT, Mumtaz A, Ashfaq UA, Azhar S, Fatima T, Hassan M, Hussain SS, Akram W, Idrees S. Computer aided screening of phytochemicals from Garcinia against the Dengue NS2B,NS3 Protease. Bioinformation 2014; 10: 115-18.
van Hell AJ, Crommelin DJ, Hennink WE, Mastrobattista E. Stabilization of peptide vesicles by introducing interpeptide disulfide bonds. Pharm Res. 2009; 26: 2186-93.
Watson AA, Fleet GW, Asano N, Molyneux RJ, Nash RJ. Polyhydroxylated alkaloids à natural occurrence and therapeutic applications. Phytochemistry 2001; 56: 265-95.
Weaver SC, Vasilakis N. Molecular evolution of dengue viruses: Contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease. Infect Genet Evol. 2009; 9: 523-40.
