Comparative molecular docking studies of antimicrobial agents against native and mutant catalase peroxidase in Mycobacterium tuberculosis
Abstract M.Tuberculosis katG gene is responsible for producing catalase and peroxidase like enzymes. The antimicrobial agent interacts with heme pocket of enzymes. Point mutation on one point of the important residue was induced and the results of native and mutant were compared based on docking studies. The mutation of G279D present in the wild type of M.Tuberculosis katG peroxidase protein. Some generic antibiotics were used in this study as microbial resistant drugs which were firstly screened on the basis of Lipinski rule of five and then screened based upon ADMET and mutagenicity properties and the compounds nearby fulfilling all the criteria’s were selected for docking studies. Based upon best binding energy and non covalent interactions with different amino acids of both native protein (6.6kcal/mol) and mutant protein (-7.1 kcal/mol ) Trimethoprim drug was identified as best potent lead for inhibition and on the basis of this comparative study it was observed that mutant protein having more binding affinity towards this drug. And through this study while designing a new compound it would be a great significance for M.tuberculosis enzyme inhibition and the effect of mutation on all inclusive mechanism of protein binding. Keywords: katG, Peroxidase, Catalase, Lipinski rule of five, ADMET, Mutagenicity, Docking.
Discussion
Materials
Results
Protein Preparation
The following ten compounds were screened using Lipinski rule of Five as shown in table 1.
The Native protein M.tuberculosis katG (PDB ID:1SJ2) was retrieved from RCSB PDB. Since mutant structure(G279D) was not present in the database. So it was modelled using Pymol software by taking native structure as template. For further studies both proteins were prepared by removing all non standard residues, water molecules and then using DockPrep option alongwith polar hydrogen, Gastegier charges were also added to both proteins using Chimera software.
Sno.
Compounds name
PUBCHE M ID
HBD
HBA
LogP
Molecular weight
Lipinski rule of five (Y/N)
1
Amoxicillin
33613
4
6
0.02
365.41
Y
2
Doxycycline
54671203
6
9
-0.5
444.44
N
3
Cephalexin
27447
3
5
0.44
347.40
Y
4
Ciprofloxacin
2764
2
5
1.58
331.35
Y
All the ten compounds(Amoxicillin,Doxycycline,Cephalexin, Ciprofloxacin,Clindamycin,Metrnidazole,Clavulanic Acid,Levofloxacin, Sulfamethoxazole,Trimethoprim)were retrieved through PUBCHEM and using ADMET SAR 2.0 these ligands were screened on the basis of Lipinski rule of Five and different ADME properties. They were also screened on the basis of non toxicity and non mutagenicity.
5
Clindamycin
446598
4
7
0.39
424.99
Y
6
Metrnidazole
4173
1
5
0.09
171.16
Y
7
Clavulanic acid
5280980
2
4
-1.1
199.16
Y
8
Sulfamethoxa zole
5329
2
5
1.37
253.28
Y
9
Trimethoprim
5578
2
7
1.26
290.32
Y
Active site prediction
10
Levofloxacin
149096
1
6
1.54
361.37
Y
Ligand Preparation
For docking studies active pockets of proteins were predicted using ACTIVE SITE PREDICTION tool of SCFBIO software.
Docking Studies The screened compounds were docked to both proteins using AutoDock Vina software present in Chimera tool. The best docked structures were evaluated on the basis of binding energy (in kcal/mol). Further 2D interactions were also evaluated using Biovia Discovery Studio tool.
Methodology
Introduction
Further they were screened on the ADMET and non mutagenicity properties as shown in table 2. Compound name
Acute oral toxicity
Ames mutagenesis
HIA(+ )
BBB(+)
Caco2( -)
Trimethoprim
IV
no
Sulfamethazole
III
no
Clavulanic acid
IV
no
Levofloxacin
III
no
Metronidazole
III
yes
Clindamycin
III
no
Cephalaxin
IV
no
Amoxicillin
IV
no
Ciprofloxacin
III
yes
+ (0.98) + (0.98) + (0.76) + (0.99) + (0.98) + (0.92) (0.51) (0.69) + (0.98)
+ (0.99) + (0.98) + (0.935) + (0.96) + (0.99) (0.93) (0.93) (0.99) (0.33)
+ (0.93) (0.79) (0.80) + (0.93) + (0.51) (0.93) (0.88) (0.92) (0.94)
Mycobacterium tuberculosis is a causative agent for tuberculosis. katG genes is one of the set of genes found to be responsible for producing catalase peroxidase. A multifunctional heme enzyme Mycobacterium tuberculosis catalase peroxidase belongs to class 1 Peroxidases[2],[3]. Due to its catalase in nature which converts hydrogen peroxide to oxygen and water. And also the presence of peroxidase it oxidizes heme iron .
This mutation is also found to provide resistance to heme binding residues. Antimicrobial drugs such as antibiotics plays an important role in providing resistance against different diseases. They are unique class of compounds which helps to alter the metabolism of different microbes. In addition, they exhibits great oral bioavailability and thus provides an ease for parenteral use [11].
So, referring to literature survey G279D mutant was generated and targeted along with native catalase peroxidase protein to ten different generic antibiotics using different insilico tools for identifying a potent drug for inhibition of this enzyme and also the effect of this point mutation on overall binding process of the system.
+ + + + +
Binding Scores (kcal/mol)
Interaction with amino acids
Trimethoprim
-6.6
Hydrogen bond: GLN 190,ASN 615. Vander waal: GLN 50, THR 618,ASP 194,GLY 124,GLY 123, SER 486. PI-cation: LYS 488
Clavulanic acid
-6.2
Protein (1SJ2) and mutant protein modelling & preparation
Different generic antibiotics as ligands
Table 4: Docking and interactions results of mutant protein. Compound name
Binding Scores (kcal/mol)
Interaction with amino acids
Trimethoprim
-7.0
LYS 46,GLU 703, ARG 42,GLN 36,GLU 195,GLY 32. Vander waal :PRO 193, ASN 44, LEU 43, LEU 45, GLY 33,34,GLN 36. Pi cation: LYS 46
Clavulanic acid
-6.4
Hydrogen bond: ASN 701, SER 700, LEU 43, PRO 288.
Screening of all the compounds and ADMET
Figure 2: Methodology followed for the present study.
Our findings provided structural perception into great significance in designing a potent antibiotic drug for inhibition and effect of mutation on overall binding process of the system which can further may provide help during analysis through in vitro and in vivo process.
References • • • • • • • • • •
Aishwarya Singh, Aditi Singh, Sonam Grover, Bharati Pandey, Anchala Kumari, Abhinav Grover , Wild-type catalase peroxidase vs G279D mutant type: Molecular basis of Isoniazid drug resistance in Mycobacterium tuberculosis.Gene(2017), doi:10.1016/j.gene.2017.10.047. Johnsson, K. and Schultz, P.G., 1994. Mechanistic studies of the oxidation of isoniazid by the catalase peroxidase from Mycobacterium tuberculosis. Journal of the American Chemical Society 116, 7425-7426. Lee, B. and Richards, F.M., 1971. The interpretation of protein structures: estimation of static accessibility. Journal of molecular biology 55, 379-IN4. Lei, B., Wei, C.-J. and Tu, S.-C., 2000. Action mechanism of antitubercular isoniazid Activation by Mycobacterium tuberculosis KatG, isolation, and characterization of InhA inhibitor. Journal of Biological Chemistry 275, 2520-2526. Marri, P.R., Bannantine, J.P. and Golding, G.B., 2006. Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer. FEMS microbiology reviews 30, 906-925. Morlock, G.P., Metchock, B., Sikes, D., Crawford, J.T. and Cooksey, R.C., 2003. ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrobial agents and chemotherapy 47, 3799-3805. UCSF Chimera Pettersen EF, Goddard TD et al.2004. Pymol molecular graphics system Schrodinger,LLC.Delano,WL.2002. Biovia discovery studio Dassault systemes. Fasnacht M. 2016. Scfbio The Supercomputing Facility for Bioinformatics & Computational Biology,IIT Delhi .(2002).
Vander waal : ARG 653, ARG 42, GLU 607, GLY 297, SER 702.
Figure3:Docking results of both proteins.
Docking and binding energy calculation
Conclusion
Hydrogen bond: LEU 45,PRO 193,LYS 46,ARG 42 ASN 35. Vander waal: GLN 36, GLU 195,192,703.
So, taking into an account for present study different generic antibiotics were used for docking process in both native and mutant proteins. In this study using ligand docking mechanism and ADMET like properties for screening of compounds different bioinformatics tools were applied for finding a drug candidate based on its binding energy. The more negative the score is the best docked molecule.
+
(0.92) (0.88) ((0.92) (0.92) (0.90) (0.92) (0.93) (0.92) (0.92)
Compound name
During phagocytosis the presence of these both enzymes (catalase and peroxidase) provides protection to Mycobacterium species against oxygen and hydrogen peroxidase which are being used by macrophages[4],[5],[6].
It has being reported that mutation at codon 279 leading substitution of Glycine a neutral amino acid by Aspartic acid an acidic amino acid (G279D) which may lead to resistance of certain drugs like INH [10].
+
Cyp2d6 Inhibition(-)
Now, the screened compounds (Trimethoprim and Clavulanic Acid) were docked against both native and mutant proteins and were evaluated on basis of binding scores .The more negative the score is the best docked structure as shown in table 3 and 4 which was further evaluated by 2D different non covalent and pi-pi interactions with different amino acids of both proteins and a comparative study was performed. Table 3: Docking and interactions results of native protein.
Mycobacterium as one of the known species for being imposing a huge threat on the health of human beings causing different diseases like leprosy and tuberculosis [1].
Also during host defense NADPH oxidase generates peroxides which are being catabolized by it. During absence of this external influence katG gene are of no use and are totally expendable [7],[8],[9].
Human oral availabilit y(+) +
Lipinski rule of five which states that (Molecular weight <=500Da, Log P <=5, Hydrogen bond donor<=5,Hydrogen bond acceptor <=10) were evaluated and the compound was removed for violating the rule. Further other compounds were screened against ADMET properties (Absorption, Distribution, Metabolism ,Excretion and Toxicity) in which compounds which showed possible probabilities as mentioned along with non toxicity and non mutagenicity were selected for further studies. ADMET properties includes Human Intestinal Absorption (HIA) (>0.9),BBB (Blood Brain Barrier)permeability(>0.9),CaCO2 permeability were in range 0.5-0.98), Human oral availability were positive CYP2D6 (0.70.99) may lead to higher excretion of drugs. These compounds were also further screened on basis of acute oral toxicity (type 1V category) and non mutagenesis. The screened compounds then were docked against native and mutant proteins in provided active sites(x=42.096,y=28.765,z=24.774) and it was found that Trimethoprim with best docked in both native and mutant proteins with binding scores -6.6kcal/mol and 7.0kcal/mol respectively along with their interactions studies Hydrogen bond: GLN 190,ASN 615. Vander waal: GLN 50, THR 618,ASP 194,GLY 124,GLY 123, SER 486.PI-cation: LYS 488 of native protein and LYS 46,GLU 703, ARG 42,GLN 36,GLU 195,GLY 32. Vander waal :PRO 193, ASN 44, LEU 43, LEU 45, GLY 33,34,GLN 36. Pi cation: LYS 46 of mutant protein which reveals the compound strong binding nature with both the proteins. Thus it proved to be as potent drug against M.tuberculosis katG protein. Also , the mutant protein showed best docking score with Trimethoprim in comparison with native protein.
Figure4:2D interactions results of both proteins.
Abbreviations M.tuberculosis: Mycobacterium tuberculosis. BBB: Blood Brain Barrier PDB : Protein Data Bank
Conflict Of Interest There is no conflict of interest .