In Silico Discovery of Novel Ligands for Anti-Tubercular Targets using Computer Aided Drug Design

 

Vaishnav Bhaskar¹, Krishnan Namboori², Dr Leena K Pappachen*¹

¹Department of Pharmaceutical Chemistry & Analysis, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi-682041, Kerala, India.

²Amrita Molecular Modelling and Synthesis (Ammas) Lab, Amrita Vishwa Vidyapeetham, Amritanagar Coimbatore-641112, Tamil Nadu, India.

 

ABSTRACT:

Tuberculosis still remain one of the most burdened infectious disease in the world especially with the emergence of drug resistance to the current drug regimen. It is the second most leading reason for infectious disease related death and with the emergence of MDR and XDR TB there is a consistent need to develop novel drugs with disparate modes of actions. Our objective is to design novel benzimidazole derivatives with anti-tubercular activity. A series 4-(1H-benzimidazole-2-ylmethyl) aniline derivatives were designed using ACD/Chem Sketch and their molecular properties and ADMET properties were designed using BIOVIA Discovery Studio. Target proteins were chosen in comparison to the standard drug isoniazid which are (Enoyl-{acyl-carrier-protein} reductase, Catalase-peroxidase, Dihydrofolate reductase) and a newly developed target protein MmpL3 were taken. CDOCKER energy and CDOCKER INTERACTION energy of both ligand and standard drug on all the target candidates were determined using BIOVIA Discovery Studio. The CDOCKER energy and CDOCKER INTERACTION energy of the newly developed ligand on the above mentioned targets were found to be higher in compared to standard drug, which signifies that the ligand molecules have higher specificity towards the target than the standard drug. Newly designed derivatives were found to have a better docking score towards the target protein enoyl-acyl carrier protein reductase and MmpL3 both of which aids to the development of mycobacterial cell wall synthesis therefore blocking both or either one of the protein will result in the depletion of mycolic acid concentration in mycobacterial cell wall resulting in loss of structural integrity of bacterial cell wall resulting in mycobacterial death.

 

 

1. INTRODUCTION:

Tuberculosis also known as TB is a multi-systemic infectious disease caused by exposure to a rod shaped bacterium called Mycobacterium tuberculosis. It remains one of today’s global health challenges, ranking as the second leading infectious cause of death of almost 3 million people each year and one of the most burden-infecting disease in the world¹. The leading reason for the widespread of the disease is primarily due to development of resistance to already existing drugs.

 

These drug resistant strains of TB becomes difficult to treat as they show resistance to two or more first line antibiotics like MDR (Multiple drug resistant) and XDR (Extensive drug resistant) TB. TB is the lead cause of death for HIV infected patients².

 

Benzimidazole and its derivatives are reported to be physiologically and pharmacologically active and find application in treatment of several diseases like eplilepsy, diabetes, anti-fertility³. It is an important heterocyclic pharmacophore and privileged structure in medicinal chemistry encompassing a diverse range of biological activity including antibacterial, anti-inflammatory, analgesic, antihistamine, anti ulcerative, antioxidant, anti-proliferative, anticancer etc.

 

 

Isoniazid (INH) is one of the first-line anti-TB drug and a chief component of global tuberculosis control programmes⁴. INH has been used as a prophylactic drug for individuals with latent Mycobacterium tuberculosis (MTB) infections to prevent the reactivation of disease. The very definition of MDR (multiple drug resistance) in tuberculosis is based on the resistance the TB bacterial strain show towards isoniazid and rifampicin⁵. The mechanism behind INH resistance involve multiple genes involved in multiple biosynthetic pathways and networks. Mutation in the KatG gene is the major cause for showing isoniazid resistance, followed by inhA, ahpC, KasA, ndh, iniABC and fadE. Association of efflux genes with isoniazid resistance has also gained attention.

 

In silico drug design and homology modelling talks about the interaction between ligands and the binding sites⁶. In past few years, computational modelling and design has become an exciting area of discipline in search for new therapeutically active molecules. Faster synthetic techniques and reduced failure rate makes computational drug designing more advantageous⁷.

 

Lipinski's rule of five also known as the pfizer's rule of five or simply rule of five (RO5) is a rule of thumb to evaluate drug likeness or determine if a chemical compound with certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism and exceretion (ADME)^8,9.

 

Enoyl-ACP reductase is a type II fatty acid synthase (FAS-II) system responsible for mycolic acid biosynthesis. Mycolic acid is a major component of mycobacterial cell wall. It is the primary target of first line antitubercular drug Isoniazid (INH) and second line drug Ethionamide. MmpL3 belongs to the Resistance, Nodulation and Division (RND) super family proteins responsible for translocation of complex (glyco) lipids and siderophores across the cell envelop of mycobacterium and functions in biogenesis of the outer membrane of the organism. They also play a role in transporting mycolic acid needed for bacterial cell wall formation⁹.

 

2 MATERIALS AND METHODS

2.1 Target Identification:

Standard drug isoniazid was selected as it is a widely used frontline anti-tubercular drug for treating tuberculosis. Its chemical and physical properties as well as its mechanism of action was determined using drug bank. From the data collected drug target is identified¹.

 

2.2 Protein Characterization:

The proteins were downloaded from Protein Data Bank. The protein PDB ID has been characterized based upon the sequence and sub-cellular location using PROTPARAM and SOPMA. This include theoretical PI, estimated half life, aliphatic index, instability index, gravy value, alpha-helix, beta-turn, random coil. The PDB ID that showed favourable results were selected for further studies¹.

 

Fig 1 Proteins selected for docking studies

2NV6 4Y0L:

 

2.3 Ligand Modelling:

Ligand molecules were generated using ACD/Chem Sketch v14.1.1. The molecules were geometrically optimized within the QM level till the PE values are converged.

 

BI-1

BI-2

BI-3

BI-4

BI-5

Fig 2 Structure of ligand molecules generated for docking.

 

2.4 Ligand characterization:

Molecular pharmacophoric properties of ligand molecules such as (alogp, molecular weight, HBD, HBA) were determined using 'Discovery studio' and compared with the standard drug isoniazid.

 

ADMET properties of the ligand molecules were determined using Discovery studio and compared with the standard drug¹.

 

2.5 Molecular Mechanism:

Molecular mechanisms of the standard drug molecule and the ligand molecules were determined using way2drug pass online. Effective Drug Response(EDR) of each mechanism were determined and compared¹.

 

2.6 Docking:

Ligand and drug molecule were docked with the selected target proteins and their CDOCKER Energy and Cdocker Interaction Energy were determined and compared with the results of the standard drug molecule. The default conditions have been set up for the docking and 10 poses of each ligands have been used for each docking^1,2.

 

3 RESULTS AND DISCUSSION:

3.1 Target Identification:

Out of seven targets identified, three mycobacterial targets were selected along with a newly developed target proteins MmpL3 in order to identify the interaction between the newly developed ligand molecules with these targets.

 

3.2 Protein Characterization:

All the selected PDB ID shows high estimated half life, low instability index and a negative gravy value witch suggest that the target protein are hydrophilic in nature. By analysing the above data we can conclude that the selected target proteins are favourable candidates for carrying out docking studies.

 

3.3 Ligand Characterization:

1) Drug likeness (LIPINSKI'S Rule of Five)

 

Table 1 Molecular properties of designed molecules

SI. No	Ligand	Alog P	Molecular weight	Hydrogen bond donor	Hydrogen bond acceptor
1.	BI-1	3.531	251.326	2	2
2.	BI-2	3.307	302.169	2	2
3.	BI-3	3.223	257.718	2	2
4.	BI-4	2.764	241.264	2	2
5.	BI-5	2.542	241.299	3	2
6.	Isoniazid	-0.543	137.13	1	3

 

All the developed ligand molecules satisfy lipinski's rule of five and hence we can conclude that the above designed molecules shows drug like properties.

 

2) ADMET Properties:

Table 2 ADMET properties of designed molecules and isoniazid

Ligand	ADMET_BBB	BBB level	Absorption level	Solubility	Hepatotoxicity	Hepatotoxicity probability	ADMET_AlogP98
BI-1	0.101	1	0	-4.576	1	0.827	3.531
BI-2	0.032	1	0	-4.536	1	0.986	3.307
BI-3	0.006	1	0	-4.445	1	0.986	3.223
BI-4	-0.136	2	0	-3.995	1	0.986	2.764
BI-5	-0.346	2	0	-3.794	1	0.94	2.542
Isoniazid	-1.479	3	0	-0.033	0	0.443	2.764

 

3.4 Molecular Mechanism:

Table 3 Molecular mechanism of the developed ligand

SI. No	Ligands	Pa	Pi	Activity	Pa-Pi (EDR)
1.	BI-1	0.403	0.03	Anti-tubercular	0.373
2.	BI-2	0.402	0.03	Anti-tubercular	0.372
3.	BI-3	0.388	0.035	Anti-tubercular	0.353
4.	BI-4	0.313	0.064	Anti-tubercular	0.249
5.	BI-5	0.365	0.043	Anti-tubercular	0.322
6.	isoniazid	0.813	0.003	Anti-tubercular	0.81

 

All the developed ligand molecules shows anti-tubercular mechanism. When compared to the standard drug isoniazid, the designed molecules show moderate effective drug response.

 

 

 

3.5 Docking:

 

Fig 3 Docking of ligands on the selected proteins

 

Table 4 Docking score of designed ligands on protein, enoyl-acyl carrier protein reductase

Protein	PDB	Ligand	-CDOCKER ENERGY (kcal/mol)	-CDOCKER INTERACTION ENERGY (kcal/mol)
Enoyl-{acyl-carrier-protein} reductase	2nv6	BI-1	28.516	31.398
		BI-2	26.742	30.294
		BI-3	24.807	28.487
		BI-4	24.352	28.007
		BI-5	25.317	31.481
		isoniazid	20.551	23.952
MmpL3	4yol	BI-1	30.822	35.324
		BI-2	29.812	34.358
		BI-3	29.699	34.626
		BI-4	28.232	32.376
		BI-5	28.587	34.984
		isoniazid	11.705	23.212

 

The Cdocker Energy and Cdocker Interaction Energy of the above ligands on all the target was found to be appreciably high compared to the standard and the difference between the Cdocker Energy and Cdocker Interaction Energy was found to be lower which renders the ligands good binding ability on the above mentioned targets.

 

4. CONCLUSION:

The above mentioned newly designed ligands were found to have higher -Cdocker energy and -Cdocker INTERACTION energy compared to the standard drug isoniazid which signifies that the ligand molecules have higher specificity towards the target than the standard drug isoniazid. The difference between -CdOCKER energy and -CDOCKER INTERACTION energy was found to be less for newly designed ligands, which shows that these compounds don't act as a pseudo drug. Since newly designed ligand shows affinity towards both enoyl-acyl carrier protein reductase and MmpL3 both of which contributes to the depletion of mycolic acid for mycobacterial cell wall formation, hence resistance develop for either one of the mechanism the ligands will still utilize the other mechanism resulting in depletion of mycolic acid in bacterial cell wall resulting in cell wall loosing its structural integrity and finally cell death.

 

5. REFERENCES:

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Received on 04.06.2019           Modified on 18.08.2019

Accepted on 05.09.2019         © RJPT All right reserved