INTRODUCTION:

Mycobacterium tuberculosis is the single most deadly human pathogen and is responsible for nearly three million deaths every year¹. Recent elucidation of the mode of action of frontline antimycobacterial drug, suggests that NAD metabolism is extremely critical for this micro-organism². M. Tuberculosis depends solely on the de novo pathway to meet its NAD demand. Quinolinic acid phosphoribosyltransferase (QAPRTase) [PDB code: 1QPQ], a key enzyme in the de novo biosynthesis of NAD, provides an attractive target for designing novel antitubercular agents^3,4.

The substituted Pyridine based molecules are found to be biological active⁵ against mycobacterium tuberculosis, whereas their molecular level interactions are not studied with Quinolinic acid Phosphoribosyltransferase (1QPQ). The knowledge of exact interactions between ligand and enzyme helps in enhancing the biological activities by designing the new and potent molecules.

Using computer simulation techniques, it is now possible to study the interaction of the ligand and enzyme for elucidating the binding energy. This process provides in-depth understanding about the inhibition strength of various ligands. This is generally achieved by designing series of derivatives of lead compound by varying the functional groups at various locations in the lead compound⁵.

Now days, various computational tools like autodock, amber, CHARMM etc are used to study the inhibition activities. These tools use ab initio or semi-empirical techniques such as QM/MM. They help in understanding three major types of interactions; viz electrostatic interaction, hydrophobic interaction and steric interactions^6-11. The combination of these interaction helps in understanding the strength of binding ligand with enzyme¹². The computational tools provide the value of ΔG Kcal/mole as a binding energy between ligand and enzyme. The value of ΔG represents the inhibition strength of ligand with enzyme^{6, 12}.

The class of Mycobacterium such as M. tuberculosis are characterized by lipid biosynthesis. They are synthesizes a large number of lipids with unknown function. The sequencing of the M. tuberculosis genome has also revealed that a large number of individual enzymes are dedicated to lipid metabolism. These bacteria are also posses at least two distinct sets of machinery for fatty acid biosynthesis, FAS-I and FAS-II¹³.

The fatty acids synthesized by FAS-I are then converted into the mycolic acids by FAS-II. This mycolic acid comprises half the mass of the mycobacterial envelope. Mycolic acids are supposed to form well-ordered monolayer in the envelope to form an efficient permeability barrier against hydrophilic molecules. This well ordered monolayer barrier is likely to account for the high resistance of mycobacterium to toxic substances¹³. Hence, FAS-I can be considered as a rational target for development of potential inhibitor. Therefore the small molecules which selectively inhibit mycobacterial FAS-I mechanism can be safe and effective potential inhibitors.

In FAS I pathway, M. tuberculosis depends exclusively on the de novo pathway to meet its NAD demand. Quinolinic acid phosphoribosyltransferase (QAPRTase) is a key enzyme in the de novo biosynthesis of NAD. The enzyme quinolinic acid phosphoribosyltransferase (QAPRTase) provides the only route for Quinolic Acid metabolism and is also an essential step in de novo NAD biosynthesis.

Hence the inhibition of the QAPRTase enzyme can stop the FAS I pathway as it will make it deficient of NAD. Therefore the Quinolinic acid phosphoribosyltransferase (QAPRTase) enzyme provides an attractive target for designing novel potential inhibitor for tuberculosis¹³.

The details of 2-d and 3-d designed molecules are listed in table1.

Table: 1. Substituted Pyridine.

Mol. No.	R₁	R₂	R₃	R₄
1	-CH₃	-CONH₂	-OH	-CH₃
2	-CH₃	-CONH₂	-OH	-OCH₃
3	-CH₃	-CONH₂	-OCH₃	-CH₃
4	-CH₃	-CONH₂	-CH₃	-NH₂
5	-NH₂	-CONH₂	-OH	-CH₃
6	-NH₂	-CONH₂	-OC₆H₅	-CH₃
7	-NH₂	-CONH₂	-OH	-C₆H₅
8	-NH₂	-CONH₂	-OH	-C₂H₅
9	-NH₂	-CONH₂	-OH	-Cl
10	-NH₂	-CONH₂	-OH	-F
11	-NH₂	-CONH₂	-OC₆H₄OH	-CH₃
12	-CH₃	-CONH₂	-OC₆H₄OH	-OH
13	-NH₂	-CONH₂	-OH	-OH
14	-NH₂	-CONH₂	-Cl	-OH
15	-NH₂	-CH₃	-Cl	-OH
16	-NH₂	-CH₃	-CN	-OH

Table: 2. MM2 Parameters for Pyridine based molecules

Mol no.	Stretch	Bend	Stretch-Bend	Torsion	Non VDW	1,4 VDW	Dipole-Dipole	TOTAL
1	0.3481	2.1954	0.0739	1.7406	-2.5134	0.4014	-3.8069	-1.5610
2	0.5420	3.2194	0.0818	0.5515	-2.8907	2.3132	-2.5628	1.2545
3	0.5041	2.6332	0.0875	5.6653	-3.1272	4.3307	-3.7021	6.3914
4	0.3267	1.8974	0.1052	1.8379	-2.1152	0.8182	-3.3257	-0.4555
5	0.2837	2.5178	0.0223	2.7857	-2.1412	-0.7690	-4.3522	-1.6492
6	0.6212	3.5042	-.0053	-2.6144	-3.3207	5.6821	-3.9567	-0.0897
7	0.8684	4.7607	0.0672	-2.7877	-0.2206	2.7114	-3.9893	1.4100
8	0.3843	3.0626	0.0614	2.6454	-2.1694	0.0919	-4.3429	-0.2667
9	0.2888	2.5898	0.0046	4.1632	-1.8781	-1.3127	-2.5433	1.3123
10	0.2640	2.5700	0.0024	4.6562	-1.7003	-1.6419	-2.1389	2.0115
11	0.6203	3.9177	-0.9261	-2.5516	-3.8843	4.4649	-3.9898	-1.4488
12	0.5252	3.9735	-0.0788	-2.6343	-7.8211	2.7986	-3.0251	-6.2620
13	0.2570	2.9699	-0.0077	2.6793	-5.1336	-2.3888	-3.5857	-5.2097
14	0.3012	2.3976	0.0257	4.1190	-2.2927	-0.7823	-1.4477	2.3208
15	0.2794	1.8961	-0.0042	-1.0789	-1.3756	2.8363	1.3145	3.8675
16	0.3454	2.0591	0.0513	-1.8996	-1.2771	4.0221	1.5688	4.8700

The aim of the present study is to understand the inhibition of QAPRTase enzyme with the derivatives of substituted Pyridine. For the same computational tools were used to design the series of substituted Pyridine molecules. These molecules were docked with the QAPRTase enzyme for in-silico studies.

EXPERIMENTAL:

Quinolinic acid Phosphoribosyltransferase (QAPRTase) having PDB code 1QPQ was selected as the target enzyme. Its 3D electronic structure having natural inhibitor was procured from protein repository databank. The position of natural inhibitor was selected as the centre of active site and it was removed before docking the ligand.

The series of compounds (also called as small molecules), which are the derivatives of substituted Pyridine, were designed using computer based design tools ChemOffice¹¹ and their 3D geometries were finalized by minimizing the total energy content using molecular mechanics techniques. While finalizing the geometry of small molecules, global minima were achieved and confirmed.

Pyridine which is selected as lead compound provides three substitution sites viz. R1, R2, R3 & R4 as shown in figure 1.

Fig 1 : General Formula for Substituted pyridine.

This compound provides total 4 substitution sites from R₁to R₄. The various pharmacophore used to substitutes these places were:

-H, -F, -CI, -Br, -OCH₃, -OH, -NH₂, -CH₂CH₃, -C₃H₇, -OCH₂CH₃, -NH₂, -CONH₂, -COCH₃, -C₆H₄CI, -C₆H₄(OH), -C₆H₅, -C₆H₄OC₂H₅, -OC₆H₅, -OC₆H₄OH, -CH₃, -C₂H₅.

The list of designed small molecules along with substituted pharmacophores is listed in table1. The table 1 shows only those molecules, which were successfully achieved global minima. The database of these molecules prepared and stored as an electronic library for docking process.

To ascertain the validity of the 3D design, various thermodynamic properties was calculated in-Silico using ChemDraw.

These molecules then subjected to docking studies with the binding site of QAPRTase enzymes using computer based tools Argus and Autodock.

RESULT:

The selected mode of docking was performed using genetical algorithm, which provides the most intelligent docking positions. The process gives the binding energy as the major of strength of interactions between small molecules and enzyme. Table 3 shows the results of binding energy of various docked molecules, the number and distance of hydrogen bonding involved in the complex formation between enzyme and small molecules.

Table: 3. The selected Pyridine based molecules with their docked values, H-bond information with Quinolinic acid Phosphoribosyltransferase.

Mol. No.	Molecular formula	Binding Energy in Kcal/mol.	Total Hydrogen Bonding	Amino Acids involved in Hydrogen bonding	Hydrogen bonding distance in A⁰.
1	C₈H₁₀N₂O₂	-6.92445	3	161 HIS, 62 ARG, 139 ARG	2.794158, 2.976157, 2.778591
2	C₈H₁₀N₂O₃	-7.4355	5	172 LYS, 605 ARG, 139 ARG, 162 ARG	2.884960, 2.721376, 2.919134, 2.999905, 2.664219
3	C₉H₁₂N₂O₂	-6.86433	2	139 ARG, 161 HIS	2.300237, 2.999767
4	C₈H₁₁N₃O	-6.46138	4	605 ARG, 139 ARG	2.220678, 2.999821, 2.790309, 2.939806
5	C₇H₉N₃O₂	-10.6487	3	139 ARG, 172 LYS, 248 SER	2.942337, 2.996365, 2.568471
6	C₁₃H₁₃N₃O₂	-7.74635	4	140 LYS, 605 ARG, 248 SER, 172 LYS	2.791829, 2.999489, 2.612801, 2.334158
7	C₁₂H₁₁N₃O₂	-7.22304	2	161 HIS, 139 ARG	2,798845, 2.994684
8	C₈H₁₁N₃O₂	-9.96639	3	161 HIS, 605 ARG, 139 ARG	2.999995, 2.999995, 900007
9	C₆H₆ClN₃O₂	-6.90993	2	172 LYS, 605 ARG	2.887324, 2.889778
10	C₆H₆FN₃O₂	-6.41817	3	139 ARG, 248 SER, 140 LYS	2.888346, 2.724458, 2.989948
11	C₁₃H₁₃N₃O₃	-8.4519	5	161 HIS, 248 SER, 140 LYS, 605 ARG, 172 LYS,	2.573241, 2.659292, 2.951146, 2.999308, 2.385103
12	C₁₂H₁₁N₃O₄	-7.77176	4	139 ARG, 605 ARG, 161 HIS, 173 ASP	2.718750, 2.924414, 2.998402, 2.237033
13	C₆H₅N₂O₃	-7.81639	6	139 ARG, 162 ARG, 161 HIS, 172 LYS	2.687629, 2.786095, 2.866649, 2.999347, 2.472646, 2.915051
14	C₆H₆ClN₃O₂	-6.90517	3	139 ARG, 162 ARG, 248 SER	2.918445, 2.839581, 2.834943
15	C₆H₇ClN₂O	-6.68548	3	137 ASP, 162 ARG, 161 HIS	2.898909, 2.839581, 2.125209
16	C₇H₈ClNO	-6.92726	2	162 ARG, 161 HIS	2.989823, 2.887392

Fig. 2: Substituted Pyridine based molecule no. 5 docked with protein Quinolinic acid Phosphoribosyltransferase.

Fig. 3: Substituted Pyridine based molecule no. 8 docked with protein Quinolinic acid Phosphoribosyltransferase.

Fig. 4: Substituted Pyridine based molecule no.11 docked with protein Quinolinic acid Phosphoribosyltransferase.

From the series of small molecules docked, the docking results of best three molecules are shown in Wireframe modes in Figure 2, 3, 4.

The figure 2 shows that the ligand is in the centre of the binding pocket oriented in 3 dimensions as well as it also shows the better fitting between the molecule number 5 and the selected protein Quinolinic acid Phosphoribosyltransferase.

The figure 3 shows that the ligand is in centre of the binding pocket oriented in 3 D orientation as well as better fitting between the molecule number 8 and the selected protein Quinolinic acid Phosphoribosyltransferase.

The figure 4 visualizes the 3D interaction of the molecule 11 with the binding site of protein. It also shows the better fitting between the molecule number 11 and the selected protein Quinolinic acid Phosphoribosyltransferase.

DISCUSSION:

In case of substituted Pyridine with Quinolinic acid Phosphoribosyltransferase interaction, the hydrophobic nature of the nucleus of Pyridine makes it sterically acceptable and reduces void space. Further the presence of highly electron rich and activating groups at R1 and R3, and hydrophobic group at R4 increases binding capabilities of the molecule. As well as the –CONH2 group particularly at R2, also increases the binding capabilities of molecule.

Further it is found that these small molecules have negative ∆G values, which indicates the stabilities of complexes formed between the small molecule and protein. In these cases a very less amount of void space i.e. about 28% was present indicting positive interactions. A few interactions which show highest values for docking are discussed in Table 4, 5 and 6.

The figure 5 shows that in case of Pyridine based molecule number 5, the-O*H group at R3_,-CO* NH₂group at R2and the >N*- atom of the ring forms the hydrogen bonds with the amino acids 139 ARG, 172 LYS, 248 SER of the binding site of the enzyme Quinolinic acid Phosphoribosyltransferase.

The figure 6 shows that in case of Pyridine based molecule number 8, the-O*H group at R3_,-CO* NH₂group at R2and the >N*- atom of the ring forms the hydrogen bonds with the amino acids 161 HIS, 605 ARG, 139 ARG of the binding site of the enzyme Quinolinic acid Phosphoribosyltransferase.

Fig.5: Graphical representation of Pyridine based molecule no. 5 interactions with amino acids 139 ARG, 172 LYS, 248 SER of enzyme Quinolinic acid Phosphoribosyltransferase.

Fig. 6: Graphical representation of Pyridine based molecule no. 8 interactions with amino acids 161 HIS, 605 ARG, 139 ARG of enzyme Quinolinic acid Phosphoribosyltransferase.

Table 4: Pyridine based molecule no. 5 interactions with amino acids 139 ARG, 172 LYS, 248 SER of enzyme Quinolinic acid Phosphoribosyltransferase.

Sr. no.	Molecule no.	Amino acid type	Hydrogen bond length(A0)	Group interactions of the molecules	Group Interactions of the Protein
5	C₇H₉N₃O₂	139 ARG	2.999790	-O*H	>N*H
		172 LYS	2.275942	>N*- [Ter. N of ring]	-NH₂
		248 SER	2.762835	-CO*NH₂	-O*H

* Represent the atom involved in bonding.

Table 5: Pyridine based molecule no. 8 interactions with amino acids 161 HIS, 605 ARG, 139 ARG of enzyme Quinolinic acid Phosphoribosyltransferase.

Sr. no.	Molecule no.	Amino acid type	Hydrogen bond length(A0)	Group interactions of the molecules	Group Interactions of the Protein
8	C₈H₁₁N₃O₂	161 HIS	2.942337	-O*H	>N*H
		605 ARG	2.996365	-CO*NH₂	-N*H₂
		139 ARG	2.568471	>N*- [Ter. N of ring]	-N*H₂

* Represent the atom involved in bonding.

Fig. 7 : Graphical representation of Pyridine based molecule no. 11 interactions with amino acids 161 HIS, 248 SER, 140 LYS, 605 ARG, 172 LYS of enzyme Quinolinic acid Phosphoribosyltransferase.

Table 6: Pyridine based molecule no. 11 interactions with amino acids 161 HIS, 248 SER, 140 LYS, 605 ARG, 172 LYS of enzyme Quinolinic acid Phosphoribosyltransferase.

Sr. no.	Molecule no.	Amino acid type	Hydrogen bond length(A0)	Group interactions of the molecules	Group Interactions of the Protein
11	C₁₃H₁₃N₃O₃	161 HIS	2.573241	-OC₆H₄O*H	>N*H
		248 SER	2.659292	- O*C₆H₄OH	-O*H
		140 LYS	2.951146	-CO*NH₂	-N*H₂
		605 ARG	2.999308	-CO*NH₂	-N*H₂
		172 LYS	2.385103	> N* - [Ter. N of ring]	-N*H₂

* Represent the atom involved in bonding.

The figure 7 shows that in case of Pyridine based molecule number 11, the - O*C₆H₄OH group at R3_,-CO* NH₂group at R2and the >N*- atom of the ring forms the hydrogen bonds with the amino acids 161 HIS, 248 SER, 140 LYS, 605 ARG, 172 LYS of the binding site of the enzyme Quinolinic acid Phosphoribosyltransferase.

CONCLUSION:

The comprehensive study of docking interaction and binding energies of the enzyme 1QPQ unveils that the newly designed compounds were in good conformity with the concept of in-silico drug design. These molecules gave favourable binding interactions and docking energy values which are superior as compared to the reference inhibitor.

In case of substituted Pyridines, different substitutions were made at R1, R2, R3 and R4 position. When the electrons reach groups such as –NH2 are substituted at R1 and then it increases the electron density on nucleus of molecule which in turn increases the electrostatic interaction of the molecule with protein binding site. Similarly, the substitution of highly activating group such as –OH or –NH2 at R2 and electron withdrawing groups such as -OC6H5 or -OC6H4OH at R3 increases the steric acceptance of molecule as well as the presence of hydrophobic group such as –CH3 or –C2H5 which is responsible for hydrophobic interactions with the binding site.

Further it is found that these small molecules have negative ∆G values, which indicates the stability of complexes formed between the small molecule and protein. Due to these favourable interactions, a small amount of void space was present indicting acceptance of these small molecules by the binding pocket of protein.

The substituted Pyridine based molecules shows the hydrogen bond interaction with N atom and O atom(>NH, -NH2, -OH, =N-) of 139 ARG, 605 ARG, 161 HIS, 140 LYS, 172 LYS, 248 SER, amino acids of binding site of Quinolinic acid Phosphoribosyltransferase protein which plays an important role in de novo pathway of NAD metabolism of mycobacterium. Hence, these facts confirmed that these molecules inhibit the Quinolinic acid Phosphoribosyltransferase protein.

Thus this study reports that that all these Pyridine based molecules, especially molecule No. 5, 8 and 11 can inhibitthe enzyme Quinolinic acid phosphoribosyltransferase (QAPRTase), a key enzyme in the de novo biosynthesis of NAD,provides an attractive target for designing novel antituberculosis drugs.

Hence, these Pyridine molecules can be potent inhibitors against Mycobacterium Tuberculosis.

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Received on 16.08.2012 Modified on 05.09.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1159-1165