Research Article

Virtual screening for inhibitors of shikimate kinase of Mycobacterium tuberculosis

Pramod Kumar Sahu, Mukesh Kumar Raval*

Department of Chemistry, Gangadhar Meher College, Sambalpur, Odisha, India

*For correspondence

Prof. Mukesh Kumar Raval,

Retired Professor of Chemistry, Gangadhar Meher College, Sambalpur, 768004 Odisha, India.

Email: mraval@yahoo.com

 

 

 

 

 

 

 

 

 

 

 

Received: 22 April 2016

Accepted: 09 May 2016

ABSTRACT

Objective: The objective of the present work is to select a target in an essential metabolic pathway of Mycobacterium tuberculosis and identify the target specific inhibitor in the small molecular database to improve treatment of multi-drug resistant tuberculosis (MDR_TB). In the present work shikimate kinase is selected as target to develop novel drug against Mycobacterium tuberculosis.

Methods: Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing new anti-tuberculosis drugs, because this pathway is essential to bacteria but absent in mammals. Ligands in ZINC database are screened for the active site binding of this enzyme using i-dock and Arguslab.

Results: Screening of ZINC database taking molecular mass in 300-500 range and other physico-chemical properties suitable for drug-like molecules, yields 12855 numbers of ligands. Docking of these molecules to the active site of SK gives a list of hits in the descending order of binding energies. Best 10 out of 1000 hits are considered to select leads. These best ten hits are analysed through Molsoft online tool for drug-likeness and ProTox tool for oral toxicity. The best two from the list of hits passing the drug-likeness and toxicity test are considered as the lead molecules for the development of anti-tubercular drug.

Conclusions: Structure based virtual screening and examination of drug-likeness, toxicity and other molecular properties, suggest two molecules as leads. They require further validation studies.

Keywords: Shikimate kinase, Tubercle bacilli, i-dock, Arguslab, Molsoft, ProTox

Introduction

Tuberculosis (TB) is a bacterial disease caused by Mycobacterium tuberculosis bacteria. It continues to be one of the major diseases to mankind. TB primarily affects the lungs and slowly it affects the central nervous system.1 Currently one third of the world population, about 9 billion people, is latently infected with TB bacteria.2 About 9.6 million new TB cases were found in 2014 and 1.5 million people died worldwide. India is ranked as 17th country in the world with about 2 million population affected with TB.3

The objectives for TB drug development are set to shorten the overall duration of medication and/or reduce the total number of doses, to improve treatment of multi-drug resistant TB (MDR_TB).4 The new drug must be orally bioavailable with long term use. It must be free from significant side effects. Therefore, target-based approach is adopted for novel drug discovery.

The selected target should be participating in vital aspects of bacterial growth, metabolism, viability, survival and persistence in vivo. Recent development in mycobacterial molecular genetics tools (transposon mutagenesis, signature-tag mutagenesis and allelic exchange) will facilitate the identification and validation of new drug targets essential for tubercular bacilli not only in vitro but also for its survival and persistence in vivo.5,6

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing anti-tuberculosis drugs, because the pathway is essential to microorganisms but it is absent in mammals.7,8 In the present work SK is selected as target to develop novel drug against Mycobacterium tuberculosis.9

Materials and Methods

Structures of target protein- SK

Shikimate Pathway is a seven steps process and SK act in the fifth step of the process, which catalyse shikimate to shikimate -3-phosphate by AroK gene.10-12

The three dimensional structure of SK (PDB ID 3BAF) of Mycobacterium tuberculosis is obtained from RCSB Protein data bank (http://www.rcsb.org/pdb).13-17 The single chain protein contain 165 amino acid residues, bind with cofactor ANP (phosphoaminophosphonic acid-adenylate ester), substrate SKM ((3R,4S,5R)-3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid) and 136 water molecules.18 The water molecules are removed for the docking. SKM is the active site cavity of 3BAF as shown in Figure 1.

Figure 1: The 3D structure of protein (pdb id 3baf) with the substrate shikimic acid and cofactor ANP rendered as stick.

Database of compounds

ZINC, a free database (http://zinc.docking.org) consisting of over 2.7 million commercially-available compounds for virtual screening in 3D format, is used for docking process.19

Docking and identifying hit compounds

The compounds from ZINC database are subjected to a docking tool i-dock (http://istar.cse.cuhk.edu.hk/idock/) a structure-based virtual screening powered by fast and flexible ligand docking. The filter is set with Molecular weight (300-500 g/mol), Partition coefficient xlogP (1-3), Rotatable bonds (4-6), Hydrogen bond donors (2, 4), Hydrogen bond acceptors (4-6), Net charge(0), Apolar desolvation (0-10 kcal/mol), Polar desolvation (-40-0 kcal/mol), Polar surface area PSA (Å2): (60-80). The filter yields 12855 numbers of compounds, which are docked to the target for binding score. The screening is set to give 1000 hits.20

Identification of lead molecules

The hit molecules are screened for their drug-likeness by Molsoft and toxicity by ProTox.

Drug-likeness score is computed from the molecular properties, i.e. molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors, polar surface area, MolLogP, MolLogS, and number of stereo centers by Molsoft through online server (http://www.molsoft.com/products.html).

ProTox is an online web server for prediction of oral toxicities of small molecules in rodent.21 The median lethal dose (LD50) and toxicity class is calculated based on their similarities to toxic fragments of compounds. The toxic fragments are generated by ROTBOND and RECAP methods.22 Based on severity the compounds are classified into six different classes. Higher the class lower is the toxicity. Class 6 is the non toxic group.

Results and Discussion

The shikimate binding site contains the amino acids ASP34, PHE57, ARG58, GLY79, GLY80, GLY81, LEU119, ARG117 and ARG136. The ligand molecules from ZINC database are docked in the active binding site. The top 10 scoring ligands and the substrate of Shikimate (shikimic acid) again docked with Arguslab. The scoring result with the IUPAC name and ZINC id of ligands are shown in Table 1.

Table 1: Docking score of 10 top scoring ligands and shikimic acid.

Ligands

ZINC ID

IUPAC Name

i-dock score (kcal/ mol)

Argus Score (kcal/ mol)

Ligand 1

39353420

(4R)-2-oxo-N-[2-[(2S)-2-(3-thienyl)-3,5-dihydro-2H-1,4-benzoxazepin-4-yl]ethyl]-3,4-dihydro-1H-quino

-9.667

-10.74

Ligand 2

34797195

5-[2-(2,4-difluorophenyl)acetyl]-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1,4,6,7-tetrahydropyrazolo[4,3-c]pyridine-3-carboxamide

-9.667

-11.52

Ligand 3

45977224

1-(2,3-dihydro-1H-inden-5-yl)-5-oxo-N-[1-(2-oxo-3,4-dihydro-1H-quinolin-6-yl)ethyl]pyrrolidine-3-carboxamide

-9.666

-9.57

Ligand 4

71963683

N-[[4-(3-oxopiperazine-1-carbonyl)phenyl]methyl]-4,5-dihydrobenzo[g][1]benzothiole-2-carboxamide

-9.650

-9.83

Ligand 5

20720307

(3aS,4S,9bR)-4-(1H-indol-3-yl)-N-[[(2S)-tetrahydrofuran-2-yl]methyl]-3a,4,5,9b-tetrahydro-3H-cyclope

-9.623

-11.21

Ligand 6

20720315

(3aS,4S,9bS)-4-(1H-indol-3-yl)-N-[[(2R)-tetrahydrofuran-2-yl]methyl]-3a,4,5,9b-tetrahydro-3H-cyclope

-9.558

-11.84

Ligand 7

20453061

(4R)-2-oxo-N-[2-[(2R)-2-(3-thienyl)-3,5-dihydro-2H-1,4-benzoxazepin-4-yl]ethyl]-3,4-dihydro-1H-quino

-9.552

-11.64

Ligand 8

20720311

(3aS,4S,9bR)-4-(1H-indol-3-yl)-N-[[(2R)-tetrahydrofuran-2-yl]methyl]-3a,4,5,9b-tetrahydro-3H-cyclope

-9.538

-12.26

Ligand 9

72356318

N-[1-[3-[4-[[(3S)-2-oxo-3-piperidyl]methyl]phenyl]benzoyl]-4-piperidyl]acetamide

-9.518

-12.73

Ligand 10

44036990

N-[4-[2-(4-hydroxypiperidin-1-yl)-2-oxoethyl]phenyl]-9H-xanthene-9-carboxamide

-9.511

-6.26

Shikimic Acid

(3R,4S,5R)-3,4,5-trihydroxycyclohexene-1-carboxylic acid

.......

-7.74

The lower binding energy indicates the capacity of the compound to inhibit the enzyme at lower concentration. Since the Argus energy of ligand 10 is more than the natural substrate, so the ligand 1 to ligand 9 are identified as lead molecules. These ligands are assessed with molsoft online tool for their drug likeness score and along with logP, number of hydrogen bond acceptor, hydrogen bond donor and polar surface area with their range value. Out of the above ten ligands the molsoft drug likeness score of seven are good, called as lead and are listed in (Table 2).

Prediction of toxicity of lead compounds by ProTox

The lead molecules are again analysed through ProTox online tool for prediction of toxicity class and median oral lethal doses i.e. LD50 in mg per kg body weight in rodents. Out of seven only two score good as listed (Table 3).

Docking start with the protein chains will be rendered as lines, the ligands will be rendered as sticks, the ions and waters will be rendered as dots and the protein surface will be constructed asynchronously and can be seen by i-view.23 The I-dock protein-ligand interaction are shown in (Figure 2).

The binding site of shikimate kinase is hydrophilic as all the drug molecules which score best are relatively water soluble and form hydrogen bond. The interaction hydrogen bonds and hydrophobic bonds in protein and docked ligands are observed. The structure of ligands, in which number of Oxygen, Nitrogen and/or Fluorine atoms are numbered as shown in (Figure 3) to show the interaction of hydrogen bond and hydrophobic bonds.

Table 2: Prediction of molsoft drug-likeness score of lead molecules.

Compound

Mole. Wt. (g/mol)

Drug likeness score

Log p (-4.0 to 5.6)

HBA (≤10)

HBD (≤5)

PSA (0-150 Å2 )

Ligand 1

447.56

1.53

2.66

5

2

61.04

Ligand 2

450.489

1.94

3.71

3

2

65.31

Ligand 3

417.509

0.69

2.57

3

2

65.67

Ligand 4

445.544

0.96

3.61

4

2

66.92

Ligand 7

447.56

1.53

2.66

5

2

61.04

Ligand 9

433.552

1.15

3.61

3

2

65.73

Ligand 10

442.515

0.63

4.71

4

2

61.99

Table 3: Prediction of Toxicity class and median lethal dose LD50 of lead compounds by ProTox online tool.

Ligands

Toxicity Class(1-6)

Predicted LD50 mg/kg

Toxycity Target

Average Pharmacophore Fit

Average Similarity known Ligand

Ligand 2

5

4000

No tox related fragments was found

Ligand 9

4

2000

No tox related fragments was found

Figure 2: i-view of protein with ligand 2 (A) and with ligand 9 (B) docked using i-dock.

Figure 3: The structure of ligand 2 (A) and ligand 9 (B) in which number of Oxygen, Nitrogen and/or Fluorine atoms are numbered.

Figure 4: i-view of protein with ligand 2 (A) and with ligand 9 (B) showing interaction of hydrogen bond.

Interaction in i-dock

Interaction of protein with ligand 2, it has 4 hydrogen bonds LYS15:HZ1 - O2, 3.3Å, 142°, ARG117:HE - O2, 3.2Å, 163°, ARG117:2HH2 - O2, 3.2Å, 161° and ARG117:HN - O1, 3.1Å, 120° and 1 hydrophobic contacts LEU119:CD2 - C7, 3.4Å.

Interaction of protein with ligand 9, it has 4 hydrogen bonds ARG58:1HH1 - O3, 2.6Å, 140°, ARG58:1HH2 - O3, 2.1Å, 162°, GLY80:O - H31, 3.5Å, 164°, ARG117:2HH2 - O1, 2.7Å, 127° and 1 hydrophobic contacts ILE45:CG2 - C15, 3.5Å

The hydrogen bond interactions of ligand 2 and ligand 9 with amino acid groups of binding site are shown in (Figure 4).

Interaction in Arguslab

Interaction of protein with ligand 2, it has 6 hydrogen bonds, Gly 81 N-O1, 2.06 Å, LEU 119 N-O1, 3.81 Å, ARG136 NH2-O1, 2.06 Å, GLY 80 O-O2, 2.91 Å, GLY 81 N-N3, 2.44 Å, ASP34 OD2 –F2, 3.61 Å, ARG117 NH2-O2, 2.31 Å.

Interaction of protein with ligand 9, it has 6 hydrogen bonds, Gly 80 O-O1, 3.5Å, GLY81 N-O1, 3.66Å, ARG136 NH2-O1, 2.48Å, ARG136 NE-O1, 2.92Å, LUE119 N-O2, 2.65Å, ASP34 OD2-O3, 3.71Å, ARG117 O-O2, 2.31 Å.

Top 10 scoring ligands out of 1000 hits are docked to the target active site by Arguslab. It has been observed from the Arguslab docking score that nine of them (from ligand 1 to ligand 9) having free energy less than that of natural substrate shikimic acid. The lower binding energy indicates the capacity of compound to inhibit the enzyme at lower concentration. So these are considered for drug-likeness and toxicity test.

The molsoft result shows that all the ligands satisfies the Lipinsky's rule of five i.e. the molecular weight, the octane/water partition co-efficient logP, number of hydrogen bond acceptor, hydrogen bond donor and polar surface area are within the range as these are set in the docking process but out of ten ligands seven having drug-likeness score between 0.69 to 1.94 and other three are only 0.04. Hence these seven are consider as lead molecules.

All the ligands (1-10) belong to protox class 4 except ligand 2 which belongs to ProTox class 5. The LD50 values of all ligands are less than 2000 mg per kg except ligand 2 and ligand 9 which have LD50 2000 and 4000 mg per kg respectively. Hence these two are considered as leads. Further study on lead optimization and validation may lead to novel prodrugs for combating drug resistant TB.

Funding: No funding sources

Conflict of interest: None declared

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