Identification of New Inhibitor against Mycobacterium tuberculosis using structure based Drug Designing and Docking Studies

 

Subramaniam Sivakumar^*, Sangeetha. D

Department of Biochemistry, Sri Sankara Arts and Science College (Autonomous), Enathur – 631 561.

 

ABSTRACT:

Bioinformatics is playing a vital role in all aspects of drug discovery, drug assessment and drug development. It is intensively utilized for the development new drugs against several dreadful diseases. Mycobacterium tuberculosis is the etiologic agent of tuberculosis in humans.  This disease affects 1.8 billion people per year which is equal to one-third of the entire population of world. To eradicate the tuberculosis the following steps are followed, target and drug for the Mycobacterium tuberculosis identified from drug bank, homology modelling prediction carried out for the prediction of tertiary structure of fatty acyl CoA synthetase by using CPH Model server, similar chemical compounds to drug were retrieved from PubChem database, docking of enzyme with ligands achieved, molecular property using molinspiration online tool was calculated and the best ligand was finally selected as 2-pyrazinoylguanidine.

 

 

INTRODUCTION:

Bioinformatics is an interdisciplinary area mainly involving computer science, mathematics, life sciences and physics. Bioinformatics is playing an increasingly important role in nearly all aspects of drug discovery, drug assessment, and drug development¹. Protein structures are three-dimensional data and the associated problems are secondary and tertiary structure prediction, analysis of protein structures for clues regarding function, and structural alignment.  This field is intensively utilized for the development new drugs against several dreadful diseases.

 

Mycobacterium tuberculosis and Tuberculosis:

Mycobacterium tuberculosis is the etiologic agent of tuberculosis in humans. Humans are the only reservoir for the bacterium. Primarily a pathogen of the mammalian lungs. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test and chest radiographs.  Tuberculosis is the leading cause of death from a bacterial infectious disease in the world. Tuberculosis is caused by bacteria that spread through microscopic droplets released into the air from person to person. It can happen when contaminated patients coughs, speaks, sneezes, spits, laughs or sings. This disease affects 1.8 billion people per year which is equal to one-third of the entire population of world.  Another reason tuberculosis remains a major killer is due to the increase in the drug-resistant strains of the bacterium. Since the first antibiotics were used to fight tuberculosis more than 60 years ago, some tuberculosis germs have developed the ability to survive against them, and gets passed that ability on to their descendants. When an antibiotic fails to kill all of the bacteria it targets, drug-resistant strains of tuberculosis emerge. The surviving bacteria become resistant to that particular drug and frequently other antibiotics as well.

 

The major reasons behind the emergence of drug-resistant strains of the pathogen worldwide is the latent infection with dormant Mycobacterium tuberculosis. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. Acyl-CoA synthetase resembles that of eukaryotic fatty acid transport proteins and is able to stimulate fatty acid uptake. FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids².  The antibiotics include isoniazid, rifampin, pyrazinamide, and ethambutol are most commonly used. If a particular type of tuberculosis infection is resistant to regular antibiotic treatment then it is known as multidrug resistant tuberculosis or MDRTB, and to treat such condition a combination of different medications must be taken for 18 to 24 months. Thus, Fatty acyl-CoA synthetase used as target enzyme in the present study.  To find more efficient ligand for Mycobacterium tuberculosis, to eradicate the tuberculosis the following steps are followed, target and drug for the Mycobacterium tuberculosis identified from drug bank, homology modelling prediction carried out for the prediction of tertiary structure by using CPH Model server, similar chemical compounds to drug were retrieved from PubChem database, docking of enzyme with ligands achieved, calculation of molecular property using molinspiration online tool and the best ligand was finally selected.

 

Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA), whose target of action has never been identified as fatty acyl CoA synthetase. Further characterization of fatty acid synthase type I (FASI) as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis^3,4.  Drug resistance was developed against anti-M.tuberculosis treatment with isoniazid (INH),  rifampicin (RFP),  ethambutol (EB), and pyrazinamide (PZA) combination⁵. Pyrazinamide (PZA) drug have low plasma levels after oral administration, due to their low water solubility, poor permeability and ability to be rapidly metabolized by the liver and at high concentrations. Furthermore, they have short half-life only 1-4 hours, indicating a short residence in the plasma and the need for multiple high doses, which can result in neurotoxicity and hepatotoxicity⁶.  Thus, the present study targeted for the alternative drug to pyrazinamide.

 

METHODOLOGY:

Target and drug retrieved from Drugbank:

Fatty acyl CoA synthetase is a target of Mycobacterium tuberculosis and its drug is pyrazinamide were identified from Drugbank⁷.

 

Retrieval of Protein sequence of fatty acyl CoA synthetase:

Protein sequence of fatty acyl CoA synthetase was retrieved from Genpept database.

 

Structure prediction:

The prediction of three-dimensional structure of fatty acyl CoA synthetase using homology CPH Model⁸ server.

 

Similar chemical compounds retrieval:

Similar chemical compounds to drug were retrieved from PubChem database.

 

Docking of enzyme fatty acyl CoA synthetase with ligands:

The drug and similar structures were docked with fatty acyl CoA synthetase using PatchDock⁹ server. It gives the docking scores of the enzyme and ligands.

 

Calculation of molecular property using molinspiration:

Calculation of molecular property of the drug and ligand using molinspiration^{10, 11} online tool.

 

Selection of best ligands:

Best ligand was selected on the basis of docking score and Lipinski’s rule of five¹².

 

RESULTS AND DISCUSSION:

The target fatty acyl CoA synthetase of Mycobacterium tuberculosis and its drug is pyrazinamide were identified from Drugbank. Protein sequence for Fatty acyl CoA synthetase was retrieved from genpept database with Id 3R44_A.  The tertiary structure for fatty acyl CoA synthetase was predicted using CPH Model which was shown in figure-1.  The tertiary structures for four chemical constituents namely 2-pyrazinoylguanidine, 5,6-Dimethyl pyrazine-2-carboxamide, 3-tritiopyrazine-2-carboxamide, and 4-methylpyraine-4-ium carboxamide and drug Pyrazinamide, were retrieved from Pubchem database.

 

Figure-1: Tertiary structure of fatty acyl CoA synthetase

 

Figure -2: Docked structures of chemical 2-pyrazinoylguanidine

 

Table-1: Docked score of phytochemicals from the plant Plectanthus amboinicus

Sl. No.	Pubchem CID	Phytochemicals	PatchDock scores
01	1046	Pyrazinamide	2522
02	124704	2-pyrazinoyl guanidine	2918
03	12571040	5,6-Dimethyl pyrazine-2-carboxamide	2858
04	91256690	3-tritiopyrazine-2-carboxamide	2430
05	171647	4-methylpyrain-4-ium carboxamide	2752

 

Table - 2: Molinspiration results of drug and chemical compound.

Sl. No.	Drug name	Log P value	TPSA	N atoms	Molecular Weight	Hydrogen bond acceptor	Hydrogen bond donor	No. of violation of Lipinski’s rule of five
01	Pyrazinamide	-0.71	68.88	9	123.11	4	2	0
02	2-pyrazinoyl guanidine	-1.39	107.27	12	165.16	6	4	0

 

Docking was carried out between fatty acyl CoA synthetase and four chemical compounds and pyrazinamide using PatchDock server.  The docking scores represented in table-1. From the docking scores of four chemical compounds, one chemical compounds namely 2-pyrazinoyl guanidine compound selected with highest score 2918 compared with drug docking score of 2522.  The docked images of the selected chemical 2-pyrazinoyl guanidine depicted in figure-2. Molinspiration¹⁰ online tool was utilized to predict molecular properties of 2-pyrazinoyl guanidine compound and pyrazinamide drug.  From the molinspiration results, it was concluded that 2-pyrazinoyl guanidine is the best inhibitor of fatty acyl CoA synthetase because none of the violation regarding Lipinski’s rule of five¹² rules exhibited by it. 

 

Pyrazinamide is a drug which is used to treat tuberculosis in six months. Its target of action is fatty acyl coA synthetase enzyme. The drug pyrazinamide binds with fatty acyl coA synthetase and kill the bacterium. The side effects and treatment longevity stimulates present study to identify more effective drug against tuberculsosis. Based on the PatchDock results, high scoring chemical compound 2-Pyrazinoylguanidine was selected and subjected to molinspiration prediction to calculate the molecular properties of the chemicals compound.  The molinspiration result showed that selected ligand was best one with zero number of violations to the Lipinsk’s Rule of Five.  So, it was selected as best lead against Mycobacterium tuberculosis. Since, 2-pyrazinoyl guanidine identified only through in-silico study, these findings should be confirmed with wetlab studies before ascertaining the effectiveness of chemical compound as drug.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1      Wishart DS. Bioinformatics in drug development and assessment. Drug Metabol Rev. 2005; 37: 279–310. doi: 10.1081/DMR-55225.

2.     Daniel J, Sirakova T, Kolattukudy P. An Acyl-CoA Synthetase in Mycobacterium tuberculosis involved in Triacylglycerol Accumulation during Dormancy. PLoS ONE. 2014; 9(12): e114877. https://doi.org/10.1371/journal.pone.0114877.

3.     Zimhony O, Cox JS, Welch JT, Vilchèze C and Jacobs WR Jr. Pyrazinamide inhibits the eukaryotic-like fatty acyl coA synthetase of Mycobacterium tuberculosis. Nat med. 2000; 6(9): 1043-1047 PMID:10973326.

4.     Zimhony O1, Vilcheze C, Arai M, Welch JT, Jacobs WR Jr. Pyrazinoic acid and its n-propyl ester inhibit fatty acyl coA synthase in replicating tubercle bacilli. Antimicrob Agents Chemother. 2007; 51(2): 752-754. PMID:17101678.

5.     Seital I, Watanabe H, Uruma T, Chikasawa Y, Kikuchi R, Itoh M, Aoshiba K, Nakamura H, Oishi T. Successful desensitization therapy involving fluoroquinolone for the treatment of a solitary tubercloma. Mol Clin Oncol. 2016; 5(1): 117-120. PMID:27330780.

6.     Da silva PB, de freitas ES, Bernegossi J, Goncalez ML, Sato MR, Pavan FR, Chorilli M. Nanotechnology - Based drug delivary systems for tretment of tuberculosis. J Biomed Nanotechnol. 2016; 12(2): 241-260. PMID : 273O5759.

7.     Law V, Knox C, Djoumbou Y, Jewison T, Guo AC, Liu Y, Maciejewski A, Arndt D, Wilson M, Neveu V, Tang A, Gabriel G, Ly C, Adamjee S, Dame ZT, Han B, Zhou Y, Wishart DS. DrugBank 4.0: shedding new light on drug metabolism. Nucleic Acids Res. 2014; 42(1): D1091-1097.

8.     Nielsen M, Lundegaard C, Lund O, and Petersen TN. CPHmodels-3.0 - Remote homology modeling using structure guided sequence profiles. Nucleic Acids Research. 2010; 38:  doi:10.1093/nar/gkq535.

9.     Schneidman-Duhovny D, Inbar Y, Nussinov R, Wolfson HJ. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucl. Acids. Res. 2005; 33: W363-367.

10.   Ertl P, Rohde B, Selzer P. Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J. Med. Chem. 2000; 43: 3714-3717.

11.   Suresh MX. Computer aided drug design strategies for the development of novel beta lactam analogs targeting penicillin binding proteins. Trendz in Information Sciences & Computing (TISC). 2010; 88-93.

12.   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. Delivery Rev. 1997; 23: 4-25.

 

 

 

 

 

Received on 14.08.2017          Modified on 29.08.2017

Accepted on 14.09.2017       ©A&V Publications All right reserved