Molecular docking based approach for the design of Novel Flavone Analogues as inhibitor of Beta-Hydroxyacyl-ACP Dehydratase HadAB complex

 

 

Lalit Kumar¹, Ruchi Verma²*

¹Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University,

Madhav Nagar – 576 104, Manipal, Udupi, Karnataka, India.

²Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal University, Madhav Nagar – 576 104, Manipal, Udupi, Karnataka, India.

 

ABSTRACT:

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis and millions of people are suffering with this disease. Drug resistance has further worsened the situation by decreasing the potency of the drug regimen. Overcoming the resistance problem has been a challenging task for the researchers. Currently the drug regimen followed for tuberculosis contains first line and second line drugs but resistance towards some of these drugs is reducing their efficacy towards Mycobacterium. As per the reported literature flavones have been found to be active against Mycobacterium tuberculosis. In the present research work we have tried to design few flavones analogues using molecular modelling software. The 3-dimensional structure of 4RLT was retrieved from the Protein Data Bank, prepared and docked with designed flavones using Schrodinger software. The protein contained bound flavone and molecules were docked at that particular site only to compare the interaction between ligand and residue. About fifteen flavone molecules were docked against beta-hydroxyacyl-ACP dehydratase HadAB complex. Flavone m and flavone k showed the best binding affinity with good docking score. These molecules can be considered a good candidate for further structural modification, synthesis and evaluation.

 

 

 

INTRODUCTION:

Tuberculosis (TB) is one of the most infectious disease alongside with HIV affecting a wide range of population. As per World Health Organization report 2015 around 9.6 million people were affected by tuberculosis in 2014 and among them 12 percent were HIV positive¹. Though the mortality rate due to tuberculosis has fallen since 1990 but still there is a long way to go to eradicate this disease completely. In the past 15 years there has been a tremendous progress related to research for the tuberculosis management in the area of diagnostic tools, novel molecules in the drug discovery pipeline as well as the newer vaccines which are under various phases of the clinical trials².

 

The novel molecules in drug discovery pipeline targets various enzymes and proteins to inhibit mycobacterium tuberculosis and inhibition of cell wall synthesis of the organism is one of the fascinating target³. The cell wall of mycobacteria is very unique due to its lipid content. Fatty acid synthases I and Fatty acid synthases II promotes the formation of long chain fatty acid. FAS-II is not present in humans and hence is very attractive specific target for novel anti-tubercular drug discovery⁴.

Various enzymes play a major role in the elongation of fatty acid in the organism: the β-ketoacyl-ACP synthetase (KasA and KasB, Rv2245 and Rv2246, respectively. Enzyme Classification No.: 2.3.1.41), the β-ketoacyl-ACP reductase (MabA, Rv1483. EC: 1.1.1.100) and β-hydroxyacyl-ACP dehydratase (HadAB and HadBC Rv0635-Rv0636 and Rv0636-Rv0637. EC Number is not registered) is among one of them. The (3R)-hydroxyacyl-ACP dehydratase HadAB, consist of two subunits HadA (Rv0635) and HadB (Rv0636) and catalyzes the third step in fatty acid chain elongation cycle by dehydrating β-hydroxyacyl-ACP to trans-2-enoyl-ACP. HadAB elongates meromycolic chains, while HadBC, elongate chains to full-size molecules during the late elongation cycles. Some types of flavonoids and an anti-TB prodrug Thiacetazone (TAC) is found to inhibit the activity of HadB (Rv0636) hence HadAB complex is a potential drug target^5,6,7.

Therefore we have designed few flavonoids which could prove to be a potent drug molecule for antitubercular drug discovery.

 

MATERIAL AND METHODS:

Molecular docking studies depicts the strength of the interactions and gives a picture of best orientation of ligand which would form a complex with protein with minimum energy⁸. Schrodinger small molecule design suite with Maestro 10.4 interface was used for all the molecular docking simulations. The structure were drawn using 2D sketch and converted to 3D.

 

Fig. 1: Structure of  3,7,3׳,4׳-Tetrahydroxy flavone.

 

Fig.2. Structure of novel flavone analogues.

 

Ligand Preparation:

This convert, correct the structures, produce the structures variants, delete the structures which are not required, and optimize the structures. It performs the following function⁹. 

 

1      Add the hydrogen atoms.

2      According to the property it choose the molecules.

3      Delete the molecules which are unwanted like water, small ions.

4      Neutralizes the groups which contain charge and generates ionized and tautomer form of the structure.

5      Generates the stereoisomers.

6      Generation ring conformations with lower energy.

7      Geometry optimization.

 

Thus ligands were prepared:

The prepared ligands were aligned using Flexible ligand alignment option. PDB CODE: 4RLT Crystal Structure of (3R)-hydroxyacyl-ACP dehydratase HadAB hetero-dimer from Mycobacterium tuberculosis complexed with Fisetin was obtained from protein data bank  with the resolution of 2.05 angstroms.

 

Protein preparation:

The downloaded protein was processed using protein preparation wizard tool involving deletion of B chain. The water molecules that were in coordination with A chain were deleted. Metal binding states were generated and finally protein structure was optimized and minimized. Finally the prepared protein was ready for docking¹⁰.

 

Receptor Grid generation:

This step is done to locate the most appropriate interactions between ligand and a receptor molecule. The receptor grid was generated from the Receptor Grid Generation panel.

 

Molecular Docking:

Docking of the ligands was done at the active site to study the affinity of molecule to the protein. The extra precision docking tool was used for better accuracy. Various conformers are generated by the glide which are subjected to various filters like docking score, docking pose, docking energy, hydrogen bonding, etc. These determine the ligand and receptor interaction. The docking results were analyzed based on the Glide g score, Docking score and XP G Score¹¹.

 

RESULTS AND DISCUSSION:

Molecular modelling studies:

The Glide XP docking results suggests that the analogue flavone 1k showed a docking score of -8.584 and analogue 1m showed a docking score of -8.148 when compared to co-crystallized ligand 3,7,3׳,4׳-Tetrahydroxy flavone (-10.540).  Remaining analogues showed docking score -4.888 to -8.000. The docking score may be accredited to the hydrogen bonding interactions with Asn 125, Gln 86 at the active site and π-π stacking interaction with Tyr 65.

 

Table 1. Docking scores of standard 3,7,3׳,4׳-Tetrahydroxyflavone and analogues

Title	Docking Score	Glide G Score	XP G Score
3,7,3׳,4׳-Tetrahydroxy flavone	-10.540	-10.540	-10.540
1a.	-4.535	-6.916	-6.916
1b.	-6.595	-6.595	-6.595
1c.	-7.524	-7.524	-7.524
1d.	-5.432	-5.495	-5.495
1e.	-7.591	-7.591	-7.591
1f.	-7.006	-7.006	-7.006
1g.	-4.888	-4.888	-4.888
1h.	-6.079	-6.079	-6.079
1i.	-7.006	-7.006	-7.006
1j.	-6.495	-6.495	-6.495
1k.	-8.584	-8.584	-8.584
1l.	-7.916	-7.916	-7.916
1m.	-8.148	-8.148	-8.148
1n.	-5.646	-6.222	-6.222
1o.	-7.698	-7.698	-7.698
1p.	-7.589	-7.589	-7.589

 

Fig.3. 2D interaction diagram of flavone 1a and flavone 1c.

 

Flavone 1a and 1c showed molecular interaction with Asn 125 and Gln 86 and π-π stacking interaction with Tyr 65. Hydrogen bonding interaction was found with keto group of chromen ring with Asn 125 and oxygen of chromene ring with Gln 86 residue whereas π-π stacking interaction of substituted phenyl was found with Tyr 65 in both the molecules.

 

Fig.4. 2D interaction diagram of flavone 1d and 1k.

 

Flavone 1d and 1k showed molecular interaction with Asn 125. Hydrogen bonding interaction was found with nitrogen of pyridine ring with Asn 125 in molecule 1d and in molecule 1k keto group of chromene ring showed hydrogen bond interaction with Asn 125. There was no π-π stacking interaction with Tyr 65 neither these molecules showed any interaction with Gln 86.

 

Fig.5. 2D interaction diagram of flavone 1e and flavone 1n.

 

Flavone 1e and 1n showed molecular interaction with Asn 125 and Gln 86 and π-π stacking interaction with Tyr 65. Hydrogen bonding interaction was found with keto group of chromene ring with Asn 125 and oxygen of chromene ring with Gln 86 residue whereas π-π stacking interaction of substituted phenyl was found with Tyr 65 in both the molecules.

 

Fig.6. 2D interaction diagram of flavone 1g.

 

Flavone 1g showed hydrogen bonding interaction with Gln 86 only with oxygen of chromene ring.

 

Fig.7. 2D interaction diagram of flavone 1h and 1j.

 

Flavone 1h and 1j showed molecular interaction with Gln 86 and π-π stacking interaction with Tyr 65. In molecule 1h hydrogen bonding interaction was found with oxygen of methoxy group with Gln 86 and in 1j molecule hydrogen bonding interaction was found between oxygen of methoxy group and oxygen of phenoxy group whereas π-π stacking interaction of benzene ring of benzopyran was found with Tyr 65 in both the molecules.

 

Fig.8. 2D interaction diagram of flavone 1l.

 

Flavone 1l showed molecular interaction with Asn 125 and Gln 86. Hydrogen bonding interaction of molecule 1l could be seen with one of the nitrogen of triazole ring with Asn 125 residue and another of oxygen with Gln 86 residue.

 

Fig.9: 2D interaction diagram of flavone 1m and 1p.

 

Flavone 1m and 1p showed molecular interaction with Asn 125 and π-π stacking interaction with Tyr 65. Hydrogen bonding interaction was found with keto group of chromene ring with Asn 125 residue whereas π-π stacking interaction of substituted phenyl was found with Tyr 65 in both the molecules. No interaction was found with Gln 86 residue with both the molecules.

 

Fig.10. 2D interaction diagram of flavone 1o.

 

Flavone 1o showed π-π stacking interaction of substituted phenyl with Tyr 65.

 

The 2D interaction diagrams represent that there is no specific group responsible for the interaction as it could be seen that in most of the flavones hydrogen bond interaction with Asn 125 residue is found with keto of benzopyran ring in case 1m, 1p, 1e, 1n, 1a, 1c and 1k whereas in flavone 1l and 1d hydrogen bond interaction with Asn 125 residue is with substituents at the side chain. Based on these interaction and docking score it could be said that keto of benzopyran ring is very important for interaction with Asn 125 in most of the cases. Similarly most of the molecules showed hydrogen bonding interaction of oxygen of benzopyran ring with Gln 86 residue. π-π stacking interaction of Tyr 65 was found mostly with substituted phenyl ring at 2^nd position of chromene ring.

 

Fig. 11: Protein 4RLT Crystal Structure of (3R)-hydroxyacyl-ACP dehydratase HadAB hetero-dimer from Mycobacterium tuberculosis complexed with Fisetin.

 

Fig 12: Flavone 1m hydrogen bonding interactions with Asn 125 and π-π stacking interaction with Tyr 65.

 

This figure shows hydrogen bonding interaction of keto group of chromene ring with Asn 125 residue and π-π stacking interaction of substituted phenyl with Tyr 65. The dock score of this molecule was found to be -8.148.

 

Fig 13: Flavone 1k hydrogen bonding interactions with Asn 125.

This figure shows hydrogen bonding interaction of keto group of chromene ring with Asn 125 residue. The dock score of this molecule was found to be -8.52. Docking study of most active flavone 1m and 1k showed that it penetrated and positioned at the same binding site of the receptor as of 3,7,3׳,4׳-Tetrahydroxyflavone.

 

Fig. 14: Flavone 1m position in the binding pocket of beta-hydroxyacyl-ACP dehydratase HadAB complex.

 

Following figure shows that molecule 1m could very well accommodate and got accommodated at the same position in the cavity created in the protein residue where the initial ligand was placed.

 

Fig. 15: Flavone 1k position in the binding pocket of beta-hydroxyacyl-ACP dehydratase HadAB complex.

 

Similarly molecule 1k could very well accommodate and got placed at the same position in the cavity created in the protein residue where the initial ligand was placed.

 

Fig. 16. Superposition of the active molecules flavone 1m and 1k at the binding site.

 

The most active molecules flavone 1m and 1k superpose each other, binding with the same protein residues and occupy the same position where the standard flavone occupied the position.

 

CONCLUSIONS:

Two analogues showed good dock scores but was not comparable to 3,7,3׳,4׳-Tetrahydroxyflavone. Further our focus will be to further modify the structure by incorporating polar groups, synthesize the active analogues and validate the simulation studies by carrying out in-vitro and in-vivo studies. The study will help in identifying antitubercular agents with novel mechanism of action active at new target. Our focus would be to prepare the molecules which would avoid the activation by the enzyme and thus could reduce the resistance problem.

 

ACKNOWLEDGEMENT:

The authors would like to acknowledge the software facility provided by Manipal College of Pharmaceutical Sciences and DBT-BioCARE funding agency to execute this research work.

 

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Accepted on 20.07.2017           © RJPT All right reserved