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Enzyme Inhibitory and Molecular Docking Studies on Some Organic Molecules of Natural Occurrence.

Byline: Muhammad Athar Abbasi, Ghulam Hussain, Aziz-ur-Rehman, Durre Shahwar, Khalid Mohammed Khan, Ayesha Mohyuddin, Muhammad Ashraf, Jameel Rahman, Muhammad Arif Lodhi and Farman Ali Khan

Abstract: In the present study, in vitro enzyme inhibitory studies on cinchonidine (1), cinchonine (2), quinine (3), noscapine (narcotine, 4) and santonine (5) were carried out. The various enzymes included in the study were lipoxygenase, xanthine oxidase, acetyl cholinesterase, butyryl cholinesterase and protease. The results revealed that 2, 3, and 4 were moderate active against lipoxygenase and xanthine oxidase enzymes. The molecule 3 possessed weak activity against butyryl cholinesterase enzyme while remaining molecules were inactive against this enzyme. However, all these compounds were inactive against acetyl cholinestrase and protease enzymes. The synthesized compounds were computationally docked into the active site of lipoxygenase enzyme. The compounds 3 and 4 showed decent interactions, hence strengthening the observed results.

Keywords: Cinchonidine, cinchonine, quinine, noscapine, lipoxygenase, xanthine oxidase.


Lipoxygenase enzymes can be found in a wide variety of plant and animal tissues. In lipoxygenase type-1 (LOX), the iron is present in the divalent state. It is oxidized to the catalytically active Fe3+ by the reaction product 15-hydroperoxy-eicosatetraenoic acid (15-HPETE) and leukotrienes from arachidonic acid as a substrate. Its also oxidized by 13-hydroperoxy-octadecadienoic acid (13-HPODE) from linoleic acid as a substrate [1, 2]. Leukotrienes are important biologically active mediators in a variety of inflammatory events. It has been found that these LOX products play a key role in variety of disorders such as bronchial asthma, inflammation [3, 4].

Xanthine oxidase is considered to be an important biological source of superoxide radicals. These and other reactive oxygen species (ROS) participate to the oxidative stress on the organism. These are also involved in large number of pathological processes like inflammation, atherosclerosis, cancer, aging etc [5].

Acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE) comprise a family of enzymes which include serine hydrolases. The different specificities for substrates and inhibitors for these enzymes are due to the differences in amino acid residues of the active sites of AChE and BChE. The enzyme system is responsible for the termination of acetylcholine at cholinergic synapses. These are key components of cholinergic brain synapses and neuromuscular junctions. The major function of AChE and BChE is to catalyze the hydrolysis of the neurotransmitter acetylcholine and termination of the nerve impulse in cholinergic synapses [6]. In the Alzheimer's disease patients, a reduction in the Acetylcholine (ACh), a neurotransmitter, appear to be acute element in the development of dementia, hence Alzheimer's disease and other type of dementia could be administered by the use of agents that reinforce the level of acetylcholine.

The inhibition of AChE play a key role not only stimulating cholinergic transmission in the brain, but also decreasing the collection of amyloid b peptide (AB) and the formation of the neurotoxic fibrils in Alzheimer's disease [7]. The search for new cholinesterase inhibitors is considered an important and ongoing strategy to introduce new drug candidates for the treatment of Alzheimer's disease and other related diseases [8].

Serine protease inhibitor plays a key role in the natural defense system of plants against insect predation by restricting insect proteinases. Trypsin is a serine protease which has recently attracted much more importance and plays a role in the devastation of fibrous proteins [9]. Acute activity of trypsin causes cancer, hepatitis, muscular dysentery and arthritis. Nature has bestowed some of drugs for various illnesses. A number of metabolites obtained from natural sources, has moderate to efficient trypsin inhibitory activity [10]. Here, we report the screening of 1-5 against lipoxygenase, xanthine oxidase, acetyl cholinesterase, butyrl cholinesterase and protease enzymes to explore their therapeutic potentials. Additionally, a computational approach was adopted to find out the binding modes of these synthesized inhibitors against LOX enzymes. Molecular Operating Environment (MOE) was used for docking studies.

The results revealed that the designed inhibitors have decent affinity with the binding cavity of target enzymes [11].


Sample Materials

The compounds 1-5 are naturally occurring molecules in various plant sources but in the present studies these were purchased directly from Sigma-aldrich/merck Company and their structures were confirmed by comparison of their spectral data with reported data for 1 [12], 2, 3 [13], 4 [14] and 5 [15].

Lipoxygenase Assay

Lipoxygenase activity was assayed according to the method [16] with minor modifications. 200 u L lipoxygenase assay mixture containing 150 u L sodium phosphate buffer (100 mM, pH 8.0), 10 u L test compound and 15 u L purified lipoxygenase enzyme was prepared. The contents were mixed, pre-read at 234 nm and pre-incubated for 10 minutes at 25 C. The reaction was initiated by addition of 25 u L substrate solution. The change in absorbance noticed after 6 min at 234 nm was used as index for the inhibition. All reactions were performed in triplicates. The positive and negative controls were incorporated in the assay. Baicalein (0.5 mM well-1) was used as a positive control. The percentage inhibition (%) was calculated as,


where Control =Total enzyme activity without inhibitor Test = Activity in the presence of test compound

IC50 values (concentration at which there is 50% enzyme inhibition) of compounds was calculated using EZ-Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA). IC50 values were determined by serial dilution of the compounds from 0.5 mM to 0.25, 0.125, 0.0625, 0.03125, 0.015625 mM and from the graph. Values are mean of 3 independent experiments.

Xanthine Oxidase Assay

The XO activities with xanthine as the substrate were measured spectrophotometrically with the following modifications. The xanthine solution (0.15mM) was prepared by initially dissolving xanthine (Sigma) in a minimal volume of NaOH, and adjusting pH to 7.5. The XO solution was pre pared by diluting XO from cow's milk (Sigma) to a final concentration of 0.2 U:ml in cold 50 mM potassium phosphate buffer (pH 7.5). The assay mixture consisted of 0.250 ml plant extract solution (0.4 mg:ml 50 mM potassium phosphate buffer, pH 7.5), 0.385 ml 50 mM potassium phosphate buffer (pH 7.5) and 0.330 ml xanthine solution, giving a final concentration of 100 mg plant extract per ml assay mixture. The reaction was initiated by adding 0.035 ml XO solution, and the change in absorbance recorded at 295 nm for 3 min at room temperature.

Allopurinol (Sigma) was used as a standard inhibitor at a final concentration of 100 mg:ml in the assay mixture. Xanthine oxidase activity was expressed as percent inhibition of xanthine oxidase, calculated as (1 - B/A) x100, where A is the change in absorbance of the assay without the plant extract (Dabs. with enzyme_Dabs. without enzyme), and B is the change in absorbance of the assay with the plant extract (Dabs. with enzyme_Dabs.without enzyme) [17].

Cholinesterase Assays

The AChE and BChE inhibition study were done according to the method [18] with slight modifications. 100 u L reaction mixture contained 60 u L Na2HPO4 buffer having conc. of 50 mM with pH 7.7 was prepared. Test compound of volume ten u L and conc. of 0.5 mM well-1 was poured, accompanied by the accession of ten u L enzyme of conc. 0.005 unit well-1. All contents were immixed and pre-read at a wavelength of 405 nm. After that pre-incubation of the contents for 10 min at 37 C was performed and the initiation of the reaction was done through 10 u L of conc. 0.5 mM well-1 substrate i.e acetylthiocholine iodide (for AChE) or butyrylthiocholine chloride (for BChE). Then the ten u L of DTNB with conc. 0.5 mM well-1 were added. After incubation of 15 min at 37 C, absorbance at 405 nm was measured by 96-well plate reader Synergy HT, Biotek, USA. All the observations were carried out in triplicate with their respective controls.

Eserine of conc. 0.5 mM well-1 was applied as a positive control. The results were calculated as per formula mentioned for the lipoxygenase assay.

Protease Inhibition Assay

The protease inhibitory potential of isolated and its synthesized derivatives were evaluated using the colorimetric method with some modification. Tris buffer (100 mM) of pH 7.5 (1.0 mL),trypsin (0.3 mL), and the tested compound (0.1 mL) were incubated at room temperature for 10 min. BApNA (50 L) was added to the reaction and the absorbance read at 410 nm after an incubation period of 30 min at 37 C. Phenylmethylsulfonylfluoride (PMSF) was used as standard inhibitor. The % inhibition was calculated by using the following formula:

q %= A-B/A x 100

where A is the absorbance of blank and B is the absorbance of the tested compound.Protease [10].

Molecular Docking Study

Protein Preparation

The protein molecules included in our study, a-glucosidase and BChE were retrieved from Protein Data Bank. Water molecules were removed and the 3D protonation of the protein molecule was performed using MOE applications. The energy of the protein molecules were minimized via energy minimization algorithm of MOE tool. The following parameters were used for energy minimization; gradient: 0.05, Force Field: MMFF94X+Solvation, Chiral Constraint: Current Geometry. Energy minimization was terminated when the root mean square gradient falls below the 0.05. The minimized structure was used as the template for Docking.

Molecular Docking

The binding mode of the ligands into the binding pocket of protein molecule was predicted by MOE-Dock implemented in MOE. After the completion of docking, the best poses for Hydrogen Bonding/p-p interactions were analyzed by using MOE applications [11]. All the compounds were docked into the active site of lipoxygenase enzyme. The interaction analysis revealed that 3 and 4 have shown acceptable binding modes.

Results and Discussion

Cinchonidine (1), cinchonine (2), quinine (3), and noscapine (narcotine, 4) are the alkaloids which are used for the treatment of biological diseases like malaria, analgesic pain, anti-inflammatory, cancer, and stroke treatment [5]. The santonin (5) is a sesqui-terpene molecule (Fig. 1). The IC50 values of all these compounds have been illustrated in Table-1 against a series of enzymes (Table-1).

Enzyme Inhibition Activity

To find effective inhibitors of enzymes from natural sources, we tested 1-5 against aforementioned enzymes. Against lipoxygenase 2, 3 and 4 possessed moderate inhibitory potential with IC50 values 99.120.03, 189.120.11, 177.130.13 u moles/L relative to baicalein, a reference standard having IC50 value of 22.41.3 u moles/L (Table 1). The compounds 1 and 5 displayed very weak activities against this enzyme perhaps owing to changed stereochemistry of hydroxyl group in 1 as compared to 2 and a unique skeleton in 5. A moderate inhibitory potential was also ascribed by molecules 2-5 against xanthine oxidase with more than 50% inhibition except 1 which showed very less activity (Table 1). The altered stereochemistry of hydroxyl group in 1 as compared to 2 might be attributed here again for the loss of activity against this enzyme.

When the inhibitory potential of 1-5 against acetyl cholinesterase and protease were determined, it was revealed that none of these compounds has activity against these enzymes (Table 1). Quinine (3) was the only alkaloid which possessed weak inhibitory potential against butyryl cholinesterase enzyme with IC50 value of 61.25 0.01 u moles/L relative to Eserine, the reference standard having IC50 value of 0.850.001 u moles/L.

Molecular Docking Analysis

In case of compound 3, a total of 3 interactions were observed. The active site residues His518 and Gln716 interacts with the hydroxyl group of vinylquinuclidin methanol moiety whereas, His13 interacts with methoxyquinoline moiety respectively (Fig. 2). The analysis of the interaction of compound 4 showed that two of the active side residues interact with the docked molecule. His13 interacts with methoxy group of dimethoxybenzene moiety. The active site residue His518 interacts with double bonded oxygen of furan moiety as shown in Fig. 3.

Table-1: The enzyme inhibition studies on compounds 1-5.

###LOX###LOX###XO###XO###AChE###AChE###BChE###BChE###Protease Protease


###(%) at###(IC50)###(%)at###(IC50) (%) at###(IC50)###(%) at###(IC50)###(%) at###(IC50)


###0.5mM###umoles/L###0.5mM###umoles/L 0.5mM###umoles/L###0.5mM###umoles/L###0.5mM###umoles/L


###2###94.270.53###99.120.03 71.90.01###950.03###15.68###Nil###-0.37###Nil###39###Nil

###3###80.850.28###189.120.11 91.190.05 1700.11###25.49###Nil###86.291.53 61.250.01###32###Nil

###4###91.360.31###177.130.13 85.470.05###850.51###-1.02###Nil###20.580.25###Nil###0.34###Nil

###5###17.45###Nil###90.890.01 1800.51###5.83###Nil###1.34###Nil###24###Nil

Control###Baicalein###22.41.3 Allopurinol 6.6 0.13 Eserine 0.040.001 Eserine###0.850.001 PMSF###0.110.02


It was concluded from the present investigation that among the studied molecules, the compounds 2-4 are overall moderate inhibitors of lipoxygenase whereas, maximum inhibitory potential was shown by 2 with IC50 values 99.120.03 it is might be due to the presence of hydroxyl group. Minimum inhibitory potential was shown by 3 with IC50 values 189.120.11 u moles/L which might be due to the presence of hydroxyl and methoxy group relative to baicalein, a reference standard having IC50 value of 22.41.3 u moles/L. The compounds 2-5 are overall moderate inhibitors of xanthine oxidase whereas, maximum inhibitory potential was shown by 4 with IC50 values 850.051 it is might be due to the presence of three olkoxy, three ether and one ketonic group.

Minimum inhibitory potential was shown by 5 with IC50 values 1800.51 u moles/L which might be due to the presence of two ketonic and one ether group relative to allopurinol, a reference standard having IC50 value of 6.6.13 u moles/L. Lipoxygenase and xanthine oxidase enzymes can find their utility in a large number of pathological processes like inflammation, atherosclerosis, cancer and aging etc. These molecules can further be evaluated for in vivo studies by the pharmaceutical industries in drug discovery program.


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Author:Abbasi, Muhammad Athar; Hussain, Ghulam; Rehman, Aziz-ur-; Shahwar, Durre; Khan, Khalid Mohammed; Mo
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Feb 29, 2016
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