Evaluation of Drug Candidature of some Benzimidazole Derivatives as Biotin Carboxylase Inhibitors: Molecular docking and Insilico studies

K. Hemalatha, K. Girija

Department of Pharmaceutical Chemistry, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, (A Govt. of Puducherry Institution), Indira Nagar, Gorimedu, Puducherry-06

*Corresponding Author E-mail: hemalathampharm@gmail.com

ABSTRACT:

A series of mannich bases of 1,2-disubstituted benzimidazole derivatives were designed and optimized with Auto Dock 4.2 to investigate the interaction between the target compounds and the amino acid residues of Biotin Carboxylase. Molecular descriptor properties were predicted by Molinspiration software. The free energies of binding and inhibition constants (Ki) of the docked ligands were calculated by the Lamarckian Genetic Algorithm (LGA).Among all the designed compounds, the compound 4e and 6d showed more binding energy-8.85 and -8.56 kcal/mol respectively when compared with the binding energy of the standard drug Mebendazole (-7.24 kcal/mol). Whereas the remaining compounds showed the binding energy in the range between -3.09 to -6.61 kcal/mol. These values suggested that the designed benzimidazole derivatives 4e and 6d are an excellent inhibitor of Biotin Carboxylase.

KEYWORDS: Benzimidazole, Mannich Base, Auto dock, Biotin carboxylase, Molinspiration.

INTRODUCTION:

Antibiotic resistance continues to be a major health problem in last few decades, which underlines the development of new novel antimicrobial agents¹. In the genomic revolution thousands of targets were identified against bacterial pathogens². Inhibition of fatty acid biosynthesis could be one of the best ways to control the microbial agents. Fatty acid biosynthesis is a multi-component system comprising of biotin carboxyl carrier protein (BCCP or AccB). Biotin carboxylase (AccC) is an excellent target for anti-bacterial agents, which involves in first step of fatty acid biosynthesis^3,4. The catalytic reaction of ACCase is mainly divided into two half reactions (Fig. 1).

Benzimidazole⁵ nucleus is a constituent of many bioactive heterocyclic compounds that are of wide interest because of their diverse biological and clinical applications like antimicrobial⁶ antisecretory⁷, anticancer⁸, antiHIV⁹, antihypertensive¹⁰, antitumor¹¹, anthelmintic¹², antidiabetic¹³, antioxidant¹⁴, antifungal¹⁵, analgesic¹⁶ and anti inflammatory¹⁷ and anti protozoal¹⁸ activities. A good number of them have been also marked as drugs, albendazole (antihelmintic), carbenadazim (fungicide), emedastine (antihistamine), omeprazole (proton pump inhibitor), Droperidol and pimozide (Psychopharmacological agent), etc. In view of pharmacological significance of benzimidazole derivatives, the present study involves the molecular docking study was done for some 1, 2-disubstiuted benzimidazole derivatives against Biotin Carboxylase. This was followed by molecular descriptor properties and Drug likeness analysis.

Fig. 1: Pictorial representation of Biotin Carboxylase pathway

MATERIALS AND METHODS:

The study comprised of fifteen compounds belonging to benzimidazole (Fig.2) along with one standard drug Mebendazole. The selected compounds have different substituents as shown in Table 1. Molinspiration were used to calculate log P, Polar surface area, Molecular weight, number of atoms, number of rotatable bonds, volume and number of violations to Lipinski’s rule of Five. Automated Molecular Docking was performed using the Autodock 4.2 version.

Fig. 2: Structure of 1,2 – disubstituted bezimidazole

Drug Likeness Score of Designed Molecules:

Druglikeness may be defined as a complex balance of various molecular properties and structure features which determine whether particular molecule is similar to the known drugs. These properties, mainly hydrophobicity, electronic distribution, hydrogen bonding characteristics, molecule size and flexibility and of course presence of various pharmacophoric features influence the behavior of molecule in a living organism, including bioavailability, transport properties, affinity to proteins, reactivity, toxicity, metabolic stability and many others.

Table 1: Various Substituent’s of the Designed ligands

Sl. No.	Compound Code	R	R^1	R^2
1	4a	-CH(OH)-CH3	-C6H5	-C6H5
2	4b	-C6H4OH	-C6H5	-C6H5
3	4c	-C6H5	-C6H5	-C6H5
4	4d	-C6H4NH2	-C6H5	-C6H5
5	4e	-C6H4CH2NH2	-C6H5	-C6H5
6	4f	-CH(OH)-CH3	-CH3	-CH3
7	4g	-(C=O) CH3	-C6H5	-C6H5
8	4h	-(C=O) CH3	-CH3	-CH3
9	6a	-CH(OH)-CH=CH- C6H4(2-OH)	-CH3	-CH3
10	6b	-CH(OH)-CH=CH- C6H4(4-Cl)	-CH3	-CH3
11	6c	-CH(OH)-CH=CH- C6H4(4-NO2)	-CH3	-CH3
12	6d	-CH(OH)-CH=CH- C6H4-N(CH3)2	-CH3	-CH3
13	6e	-CH(OH)-CH=CH- C6H4(2-OCH3)	-CH3	-CH3
14	6f	-CH(OH)-CH=CH- C6H3(2-OH, 4- OCH3)	-CH3	-CH3
15	6g	-C6H5	-CH3	-CH3

The Molinspiration virtual screening is fast (100,000 molecules may be screened in about 30minutes) and therefore allows processing of very large molecular libraries. Validation tests performed on various target classes (including kinase inhibitors, various GPCR targets, different enzymes etc.,) show 10 to 20-fold increases in hit rate in comparison with standard /random selection of molecules for screening. The score allows efficient separation of active and inactive molecules.

Table 2: Lipinski rule of five properties of synthesized compounds:

Sl. No.	Compound code	Log P	TPSA	Molecular Weight (in gms)	Hydrogen bond acceptor	Hydrogen bond donors	No. of rotatable bonds
1	4a	4.578	41.292	343.43	4	1	5
2	4b	6.757	41.292	391.474	4	1	5
3	4c	7.024	21.064	375.475	3	0	5
4	4d	6.1	47.087	390.49	4	2	5
5	4e	4.198	47.087	328.419	4	2	5
6	4f	1.18	41.29	219.29	4	1	3
7	4g	4.91	38.13	341.41	4	0	5
8	4h	1.52	38.13	217.27	4	0	3
9	6a	3.25	58.36	321.38	5	1	5
10	6b	4.15	38.13	339.83	4	0	5
11	6c	3.43	83.96	350.38	7	0	6
12	6d	3.57	41.37	348.45	5	0	6
13	6e	3.53	47.37	335.41	5	0	6
14	6f	2.83	67.60	351.41	6	1	6
15	6g	3.49	38.13	305.38	4	0	5
16	Mebendazole	2.89	84.09	295.30	6	2	4

_{Table 3: Drug likeness score of the synthesized derivatives:}

Sl. No	Compound code	GPCR ligand	Ion channel modulator	Kinase inhibitor	Nuclear receptor ligand	Protease inhibitor	Enzyme Inhibitor
1	4a	0.04	-0.17	-0.08	-0.21	-0.34	-0.01
2	4b	0.07	-0.01	0.15	-0.03	-0.18	0.08
3	4c	0.07	0.02	0.16	-0.09	-0.18	0.06
4	4d	0.11	0.07	0.23	-0.13	-0.10	0.14
5	4e	0.06	0.07	0.05	-0.44	-0.09	0.01
6	4f	-0.46	-0.48	-0.61	-1.05	-1.16	-0.26
7	4g	-0.11	-0.11	-0.07	-0.24	-0.18	0.01
8	4h	-0.34	-0.39	-0.59	-1.09	-0.89	-0.22
9	6a	-0.01	-0.26	-0.16	-0.34	-0.32	0.02
10	6b	-0.01	-0.24	-0.20	-0.45	-0.36	-0.04
11	6c	-0.18	-0.30	-0.30	-0.49	-0.44	-0.13
12	6d	-0.04	-0.28	-0.14	-0.38	-0.33	-0.04
13	6e	-0.06	-0.32	-0.22	-0.41	-0.37	-0.05
14	6f	-0.03	-0.27	-0.16	-0.36	-0.37	0.00
15	6g	-0.02	-0.26	-0.18	-0.44	-0.35	0.00
16	Mebendazole	0.20	0.18	0.51	-0.15	0.02	0.18

Molecular Docking Study:

Protein Preparation:

The three dimensional structure of protein, Biotin Carboxylase (Fig 3) were retrieved from the RCSB Protein data bank (PDB ID: 3JZI). All the water molecules and ligands were removed from the PDB file prior to docking. The receptor molecule was prepared by adding missing hydrogen and side chain atoms, using the graphic user interface of Autodock Tools 4.2 (ADT). The active site were calculated using Pdbsum and the active site of the protein Biotin Carboxylase was found to be Lys 159 (A), Gly 165 (A), Glu201(A), Lys 202(A),Leu 204 (A)

Ligand Preparation:

ChemSketch, the chemically intelligent drawing interface freeware (http://www.acdlabs.com/download) was used to draw the structures of Benzimidazole derivatives, followed by generation of 3Dstructure in PDB format using Marvin sketch.

Automated docking was used to locate the appropriate binding orientations and conformations of various inhibitors into the 3JZI binding pocket. To perform the task, the powerful genetic algorithm method implemented in the program Auto Dock 4.2 was employed. Grid maps were generated by Auto Grid program. Each grid was centered at the crystal structure of the corresponding 3JZI. Lamarckian Genetic Algorithm was employed as the docking algorithm. The grid dimensions were 60Å X 60Å X 60Å with points separated by 0.375Å. For all ligands, random starting positions, random orientations and torsions were used. During docking, grid parameters were specified for x, y and z axes as 40, 40 and 40 respectively. The Docking parameters, Number of Genetic Algorithm (GA) runs: 10, Population size: 150, Maximum number of evaluation: 2,500,000, Maximum number of generation:27,000 were used for this study. The structure with the lowest binding free energy and the most cluster members was chosen for the optimum docking conformation.

Fig. 3: Crystal structure of Biotin carboxylase (AccC), (PDB ID: 3JZI)

Fig. 4: Docking of Mebendazole in the active site of Biotin Carboxylase viewed through Auto Dock software

Table4: Interactions of the synthesized compounds with amino acids at the active site of the protein (Biotin Carboxylase)

Sl. No.	Derivatives	No. of Hydrogen bonds formed	Aminoacid involved in hydrogen bond interactions	Distance between Donor and Acceptor (Å)	Amino acid involved In Vanzder waals interactions
1	4a	1	Lys 202 (O)	2.828	His236, Gln233, His 438, Ile 437, Tyr 203
2	4b	0	……..	……..	Asp 419, Arg 423, Ile 422, Asn 423, Lys 443
3	4c	0	……..	……..	Arg 423, Ile 422, Asn 423, Lys 443
4	4d	0	……..	……..	Asp 419, Arg 423, Ile 422, Asn 423, Lys 443
5	4e	2	Asn 281 (N) Tyr 285 (O)	6.694 2.776	His 438, Lys 202, Ile 422, Asn 423, Ile 437, Tyr 203, Glu 201
6	4f	0	……..	……..	Ile 157, Met 169, His 216, Val171, Tyr203, Ile437, His438, Lys 202
7	4g	0	……..	……..	Tyr 203, Ile 437, His 438, Lys202, Ile422, Asn 423
8	4h	0	……..	……..	Lys 202, Asn 423, Ile 422, His 438, Lys 443
9	6a	0	……..	……..	His 236, Gln 233, His 438, Ile 437, Tyr 203, Glu 201
10	6b	0	……..	……..	His 216, Val 171, Tyr 203, Ile 437, His 438, Lys 202
11	6c	0	……..	……..	Asp 419, Arg 423, Ile 422, Asn 423, Lys 443
12	6d	1	Glu 428 (O)	2.806	Lys 443, Asp 427, Asn429, Tyr 439, Lys 442
13	6e	1	Tyr 203 (O)	2.755	Arg 235, His 226, Gln 233, Glu 441, His 438, Ile 437, Tyr 203
14	6f	1	Glu 441 (O)	2.901	His 236, Gln 233, His 438, Ile 437, Tyr 203
15	6g	0	……..	……..	Ile 157, Met 169, His 216, Val 171, Tyr 203, Ile 437, His 438, Lys 202, Glu 201
16	Mebendazole	1	Gly 165 (N)	2.563	Met 169, Tyr 203, Leu 204, Leu 278, Ile 287, Gly288, Tyr 199, Lys 159, Lys 116, Ala 160, Gly 164,

Table 5: Binding Energy and Inhibitory constant of Ligand-Biotin Carboxylase Interaction

Sl. No	Compound code	Binding energy (kcal/mol)	Inhibitory constant (μM)	Vdw. Desolvation Energy	Intermolecular Energies
1	4a	-7.36	6.62	-6	-5.99
2	4b	-5.97	42.27	-6.46	-6.86
3	4c	-6.54	15.99	-7.19	-7.44
4	4d	-6.61	14.18	-7.1	-7.51
5	4e	-8.85	5.2	-5.2	-5.2
6	4f	-3.09	5.46	-4.06	-3.98
7	4g	-3.74	1.82	-4.06	-4.63
8	4h	-4.38	20.52	-5.01	-5.27
9	6a	-5.09	3.99	-8.09	-8.86
10	6b	-5.31	129	-6.45	-6.8
11	6c	-3.9	11.39	-4.48	-5.69
12	6d	-8.56	5.7	-5.4	-6.35
13	6e	-7.68	8.63	-6.15	-7.47
14	6f	-7.53	8.39	-7.19	-7.32
15	6g	-5.98	41.54	-7.24	-7.47
16	Mebendazole	-7.24	4.97	-7.81	-7.83

Fig. 5: Docking of Ligand 4a and 6d in the active site of Biotin Carboxylase viewed through Auto Dock software.

Fig. 6: Docking of Ligand 4e and 6e in the active site of Biotin Carboxylase viewed through Chimera software

RESULTS AND DISCUSSION:

Molecular descriptor properties:

The selected compounds used in this study were evaluated as potential biotin decarboxylase inhibitors. The oral bioavailability of the compounds were evaluated by determining the molecular weight, number of rotatable bonds (nrotb), number of hydrogen bonds (nON and nOHNH) and drug’s polar surface (TPSA). Since the individual molecular weights of all the compounds were less than 500, the number of rotatable bond were <10, the number of hydrogen bond donors and acceptors were <12, and TPSA values being <140, log P value were < 5, they qualified to be an ideal oral drug (Table 2).The designed compounds were act as a ligand for various receptors like G-Protein Coupled Receptor (GPCR), Ion Channel Modulator, Kinase receptor and neuron receptor. The results were within the limits (-3 to +3) (Table 3).

Docking results:

The docking results showed that all the fifteen compounds showed low binding energy and inhibition constant (Table 4 and 5). The minimum binding energy (maximum stability) was found in case of the compound 4e (-8.85 kcal/mol). The NH₂ in second position of compound 4e forms hydrogen bond with Asn 281 with a distance of 6.694 A° and Tyr 285 with a distance of 2.776A°. The amino acids His 438, Lys 202, Ile 422,Asn 423, Ile 437, Tyr 203, Glu 201 are found to be involved in making hydrophobic interactions with 4e. The ligand 6d (-8.56 kcal/mol) has also formed significant stable complex on docking. Similar to 4e, the nitrogen atom of 6d formed hydrogen bond with Glu 428 with a distance of 2.806A°. Compound 6e with score of-7.68 kcal/mol, 6f with score of -7.53 kcal/mol, 4a with score of-7.36 kcal/mol, each with formation of one hydrogen bond with the target protein. Whereas the remaining compounds showed the binding energy in the range between -3.09 to -6.61 kcal/mol.

CONCLUSION:

Molecular docking is routinely used for understanding drug-receptor interaction in modern drug design. The molecular docking study signified that the designed compounds can act as a inhibitor of Bacterial Biotin decarboxylase. This study has enabled to broaden the vision for the generation of more specific drugs for bacterial infections and may pave the way for the production and identification of more effective drugs. Further we planned to synthesize these benzimidazole derivatives and screen for their in-vitro anti-bacterial activity.

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Received on 05.03.2016 Accepted on 19.03.2016

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Asian J. Res. Pharm. Sci. 6(1): Jan.-Mar., 2016; Page 15-20

DOI: 10.5958/2231-5659.2016.00002.3