In silico binding study of bioactive Hispolon and its Analogues to mycobacterial mtfabH

 

Muthukumaran P, Rajiniraja M*

School of Bio-Sciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India.

 

ABSTRACT:

The emergence of drug resistant mycobacterial strains has urged us to find potential targets against Tuberculosis. Mycobacterium tuberculosis H37Rv β- ketoacyl acyl carrier protein synthase III (mtfabH) is one of the most promising target against Tuberculosis. This research focuses on understanding the binding site of hispolon (a polyphenolic bioactive compound) and its analogues in to the target mtfabH. Docking study was performed to position the bioactive ligands into the mtfabH binding site using AutoDock 4.0. The docking result was shows that the Hispolon (Hc analogue) and dihydrohispolons (DHd and DHe analogues) were efficiently bound to mtfabH. The consistency of binding of these analogues with the same site was also observed using cluster analysis (RMS tolerance of 4Å). Hence these analogues may study further to serves as a new promising candidate to combat Mycobacterium tuberculosis resistant strains.

 

 

 

 

INTRODUCTION:

Tuberculosis is one of the deadliest communicable diseases caused by Mycobacterium tuberculosis (gram-positive bacteria) killing millions of people around the world. According to World Health Organization (WHO), over 4000 men, women, and children die each day due to lack of diagnosis, quality care or due to drug resistance¹. It is evident that bacteria evolve into different strains and thus making it resistible against many antibiotics. Even though there were developed drugs such as isoniazid, ethambutol, rifampicin, pyrazinamide, streptomycin, etc. against such strains, several demerits were found in concern with the period of effect and resistance towards the target². The current study focuses on the search for the potent drug molecule against the notorious target responsible for causing Tuberculosis. Mycolic acid plays a significant role in cell structural stability, cell wall fluidity and membrane permeability of Mycobacterium sp.².

 

The biosynthesis of mycolic acid is catalyzed by two fatty acid synthase (FAS-I and FAS-II) systems, considerably differing in eukaryotes which have only FAS-I. FAS-II system consists of dissociating enzyme components, which act on the substrate that binds to an acyl carrier protein (ACP)³. Several studies have been carried out on ligands based interactions against FAS-II enzymes. M. tuberculosis β-ketoacyl acyl carrier protein synthase III (mtfabH) is the initiator enzyme of FAS II system playing a regulatory role by catalyzing the condensation of acetyl malonyl-CoA and malonyl-ACP⁴. This enzyme has been found to be essential for organism survival, and performs a single function. This unique characteristic paved a way to develop novel drugs against Tuberculosis⁵.

 

Hispolon is a polyphenolic compound present as a yellow pigment in the fruit bodies of Phellinus linteus, an herbal mushroom which has been used to treat cancer in East Asia. Previous research showed its biological activity with curcumin and cinnamic acid which contains the same pharmacophore (α,β-unsaturated carbonyl/acid attached to an aromatic ring). The dihydrohispolon (DH) is a hispolon derivative which possesses a double bond at the C7-C8 position of its ethylene chain. Experimental evidence showed the antimycobacterial activity of hispolon and dihydrohispolon against M. tuberculosis H37Rv strain⁶. It is evident from the fact that the binding mode is based on unsaturated alkyl chain slender into the catalytic binding site leading the catalytic triad of amino acids Cys112, His244 and Asn274 in mtfabH⁷. Docking and pharmacophore studies have become a staple of drug discovery and are universally applied in various researches⁸. This research has been done to understand the interaction of hispolon and dihydrohispolon analogues at the active site of the target protein, to inhibit its function. Emphasis has been given to identify the conserved amino acids of the target that is responsible for ligand binding and enzyme activity^5,6.

 

In the present study, synthesized hispolon and dihydrohispolon analogs reported from the literature were used to predict their interaction against the mtfabH target.

 

MATERIALS AND METHODS:

Preparation of ligands:

A series of dihydrohispolon and hispolon analogues (labelled as DHa, DHb, DHc, DHd, DHe and Hc) having antimycobacterial activity were considered in this study⁹. The 2D structures of the docked ligands were built using ChemSketch. Ligands were prepared by addition of hydrogen atoms at standard geometry, optical isomers and 3D conformations were optimized using Argus Lab tool with UFF force field (Table 1).

 

Preparation of target:

The three dimensional (3D) structure of protein mtfabH was obtained from the protein data bank (PDB). X-ray crystallographic 3D structure of mtfabH with 2.1 Å resolution (PDB: 1HZP) was used for the current investigation.

 

 

Molecular Docking:

All molecular modelling calculations and docking studies were carried using AutoDock 4.0 following Lamarckian genetic algorithm (LGA)¹⁰. The active site definition was located using Autogrid, with size set to 60×60×60 points and a spacing was set to 0.375 Å centred on the binding pocket of the protein¹¹. Step sizes were set to 1 Å for translation and 50° for rotation. The maximum number of energy evaluations were set to 250,00,000. A total of one hundred independent runs were performed for each ligand. For each run, a maximum of 27000 genetic algorithm operations were generated on a single population of 150 individuals. The operator weights of crossover, mutation and elitism were set to 0.80, 0.02, and 1.00, respectively. The clusters of all docked conformations were generated based on the root mean square (RMS) tolerance (4Å) at each ligand conformations¹². Subsequently, the best conformation among the each cluster was selected and visualized using Pymol 3D viewer. The best scoring pose of the docked compounds was recognized and interactions of the complexes were examined in 2D and 3D styles.

 

RESULTS AND DISCUSSION:

The appropriate optimized ligands (hispolon analogues) were used for the docking study. Docking result shows that, the efficient binding site of hispolon analogues was identified to interact with the binding groove of mtfabH (Figure 1). The best conformation was selected based on the least binding energy and more number of conformations in the binding pocket. It was found that the amino acid residues Asn274 and His244 are consistently present in all clusters with the highest number of conformations 50, 48 and 58 of DHd, DHe and Hc respectively out of hundred runs. It was also observed that the ligands DHd, DHe and Hc efficiently bound (binding energy of -6.09, -5.66 and -6.91) to the target site of mtfabH (Table 1).

 

Figure 1. Structure of dihydrohispolon and hispolon analogues

Figure 2 show the modelled interaction of the hispolon and dihydrohispolon with mtfabH complex, the acetylene chain of hispolon extends to the small hydrophobic pocket directing to the catalytic triad Cys112, His244 and Asn274. The side chain of dihydrohispolon possesses conjugative double bonds to form hydrophobic interactions with the hydrophobic channel of mtfabH.

 

Table 1. Docking results of hispolon analogues (DHa, DHb, DHc, DHd, DHe and Hc) with mtfabH using AutoDock 4.0

	Hispolon analogues
	DHa	DHb	DHc	DHd	DHe	Hc
Lowest binding energy (kcal/mol)	-4.25	-5.33	-4.2	-6.09	-5.66	-6.91
Inhibitory constants (µM)	524.88	124.13	832.85	997.33	2.08	253.35
Mean binding energy(SD)	-3.4(0.61)	-4.09(0.37)	-3.58(0.41)	-4.76(0.76)	-3.78(1.17)	-4.43(0.33)
Number of conformations in cluster	30	10	10	50	48	58

 

It can be observed that the acetylene-based side chain of the compound Hc precisely fits onto the hydrophobic channel, with three hydrogen bonds formed between the methyl group and carbonyl group residues Asn274 and His244 located on the surface of the catalytic site (Figure 2).

 

Figure 2. Proposed interaction of ligands (a) DHd, (b) DHe, and (c) Hc at the active site of mtfabH  

 

The frequency of occurrence of the catalytic amino acids at the ligand binding site of mtfabH is given in Table 2. Evidently, the amino acids Cys112, His244 and Asn274 were found to be projected at the predicted binding site of mtfabH (Figure 2) [7]. The results also indicated that DHd and Hc interact with His244 and Asn274 more efficiently when compared with other analogues.

 

Table 2. Frequency of occurrence of Binding site residues at the binding site

Binding site residues	Ligand	ASN274	HIS244
Frequency of occurrence (%)	DHd	98	32
	DHe	95	31
	Hc	100	7

 

In the docking analysis it was found that, phenolic ring of the hispolon Hc showed strong hydrogen bond interactions with Asn274 and His244 (Figure 2). It was observed that two strong hydrogen bond interactions were observed between the enolic hydroxyl and keto group of the hispolon to the amino acids Asn274 and His244.

 

CONCLUSION:

In conclusion, a series of dihydrohispolon and hispolon analogues were studied for their interactions with the target mtfabH. Among the selected compounds, docking study with the enzyme mtfabH clearly showed that the ligands DHd, DHe, and Hc had better ligand-protein interactions to their observed bioactivity, indicating the potency of these analogues. Inhibitor combination studies of hispolon against the protein target involved in mycolic acid biosynthesis may evident to show these candidates may further screen to acts as a potent drug to combat mycobacterial resistant strains.

 

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Accepted on 07.06.2017          © RJPT All right reserved