INTRODUCTION:

Alzheimer’s disease is a uniquely human disease whose single most important risk factor is age. Non-human models that mimic this disease with fidelity have not been discovered despite the old age that many animals achieve in captivity [Richard P.H et al., 1999]. Even though much progress has been made, a genetically engineered animal model that has most features of AD has been difficult to create [Brossi A et al., 1986]. The age risk factor is present in both sporadic (non-genetic) and genetic forms of the disorder. Increased amyloid peptide oligomer deposition is the ultimate manifestation of genetic forms of AD and is an important early event in the pathogenesis of AD [Richard P.H et al., 1999]. However, even though gene defects that increase amyloid production can accelerate the disease process in familial AD and disorders such as Down’s syndrome[Tarek M et al., 2010].

Detectable AD pathology does not develop until the early adult years (typically over age 30) and clinical symptoms do not appear until years to decades later despite the presence of the abnormal genes and their products from birth [Tarek M et al.,2010].The exact molecular basis of AD is complex but the evidence for possible mechanism of neurodegeneration is available. The human brain is the remarkable organ with complex, chemical and electrical process occurs[Pia, M.V et al., 2008,]. The various process like speaking, moving, seeing, remembering, feeling, emotions and taking decision were executed by different parts of the brain. In healthy brain, numerous number of cells called neurons communicate with one another.

The messages from each neurons travel along the axons of one end to the other and of the neuron as electric charges. Neurotransmitters are the chemical messengers released in respond to the electric charges, they move across the microscope gapes or synapses between neurons. They find receptors on dendrites of the post synaptic neuron (next neuron) and bind to it. Communications within the brain are performed by these cellular circuits. For the proper functioning of the brain, a healthy neurotransmission is necessary. In AD, memory and thinking skills are destroyed on overtime. The scientific research revealed some other brain changes that takes place in the brain, showing abnormal structures of biological hallmarks called beta amyloid plaques and tangles [Richard P.H et al., 1999] .In the forebrain (nucleus basalis of Meynert) the subcortical cholinergic neurons degeneration provide cholinergic innervations to the whole cerebral cortex and atrophy are anatomical basis of the cholinergic deficit [Maja et al., 2010]. The selective deficiency of acetylcholine as well as the observation that central cholinergic antagonist such as atropine can induce a confusion state that bears some resemblance to the dementia of AD, has given rise to the “cholinergic hypothesis”, which proposes that a deficiency of acetylcholine is critical in the genesis of the symptoms of AD[Todd, S.N et al., 2006].AChE contains 20 Å deep and narrow gorge, in which five regions that are involved in the substrate, the irreversible and reversible inhibitor binding (human and electric eel AChE numbering), can be distinguished:

Catalytic triad residues: Ser 203, His 447, and Glu 334, at the bottom of the gorge, which directly participate in catalytic cycle, by charge relay mechanism, as in other serine esterases[Pia, M.V et al., 2008]. Oxyanion hole represents the arrangement of hydrogen bond donors which stabilize the transient tetrahedral enzyme - substrate complex by accommodation of negatively charged carbonyl oxygen.This region inside of active center is formed by peptide –NH– groups of amino acid residues viz., Gly 121, Gly 122, and Ala 204 [Pia, M.V et al., 2008]. The ‘anionic site’ (AS), where Trp 86 is situated. This residue is conserved in all cholinesterases and it is involved in orientation and stabilization of trimethylammonium group of ACh, by forming cation–p interactions[Pia, M.V et al., 2008]. Acyl pocket comprises two phenylalanine residues in positions 295 and 297, which interacts with the substrate acyl group. They form the clamps around acetyl methyl group of acetylcholine, and decrease its degrees of freedom [Pia, M.V et al., 2008]. Peripheral anionic site (PAS) comprises residues which are located on the rim of the active site gorge, Tyr 72, Tyr 124, Trp 86, and Asp 74. Possible binding sites for the reversible inhibitors comprise anionic site and PAS[Pia, M.V et al., 2008].The so-called dimeric (dual) inhibitors bind to both sites [24].AChE inhibition represented the pure palliative treatment for AD. However in the past decade, another, non-catalytic roles of the enzyme have been established. AChE has a key role in the acceleration of Ab peptide deposition and promoting the formation of Ab-plaques. AChE inhibitors, which bind to PAS, could inhibit such processes. Butyrylcholinesterase (BChE), also rises as a pharmacological target for AD therapy. It was found that BChE is capable to compensate AchE catalytic functions in synaptic cleft and that its activity significantly increases, for 30–60%, during time course in AD. Among the four FDA approved AChE inhibitors, only donepzil binds simultaneously to active and peripheral anionic sites. FDA approved tacrine, rivastigmine, donepzil have been used for the management of Alzheimer’s disease[Richard P.H et al., 1999].Drug designing through modern scientific tools and high throughput screening lead to the discovery of many compounds. Chemically diverse compounds like terpenoids, flavonoids can be structurally optimized and employed for docking studies on Acetylcholinesterase using AutoDock 4.2.AutoDock 4.2 utilize Monte Carlo simulated annealing and Lamarckian genetic algorithm (LGA) to create a set of possible conformations [Garrett, M.M et al 2010]. LGA is used as a global optimizer and energy minimization as a local search method. For the evaluation of possible orientations, AMBER force field model in conjunction with free energy scoring function is used. Coordinate files preparation, atomic affinities (AutoGrid) calculation. Semi empirical free energy force field is used to evaluate conformations during docking. The Ligand and protein stay in an unbound conformation. Then binding is evaluated in two steps by force field. Force field evaluates intra-molecular energetics during the translation from their unbounded states to the conformation of both ligand and protein into the form of bound state. [Hay, D.V., et al 2010]

MATERIALS AND METHOD:

Softwares required

Python 2.7 - language downloaded from www.python.com,cygwin- a data storage c:\program downloaded from www.cygwin.com,MGL (Molecular Grap hics Laboratory) tools–AutoDock4.2 downloaded from www.scripps.edu, Discovery studio visualizer 2.5.5–downloaded from www.accelerys.com, chemsketch downloaded from www.acdlabs.com.Python 2.5 downloaded simultaneously during cygwin download, On-line smiles translatory notation using cactus.nci.nih.gov/translate

Chemicals required

Acetylcholine esterase (AchE) type IV-S from electric eel– 500 units, 5,5′-dithiobis[2] nitrobenzoicacid] DTNB), Acetyl thiocholine (AchI), Tacrine, Tris [hydroxyl methyl]methane(Tris buffer), Linalool, Geraniol, kaemp- ferol, ferulic acid, Limonene were purchased from Sigma Aldrich Pvt ltd, USA., and Quercetin, Curcumin, Catechin are purchased from hi-media labs, Mumbai. Ambrein purchased from M.M Chemicals and Perfumery Pvt Ltd Delhi. Luteolin purchased from S.G.Chemicals, Pondicherry.

METHODS:

1. In silico docking study on AchE using Auto dock 4.2 (www.scripps.edu, 2008)

2. In vitro acetyl choline esterase inhibition by terpenoids, flavonoids were evaluated by Ellman method (Ellman et al.,1961)

1. In Silico Docking Study on AChE Using Auto Dock 4.2:

The enzyme downloaded from the RCSB Protein Data Bank (PDB) and the protein should be refined in use for docking process.

Table 1: Insilico docking studies on AChE by flavonoids and terpenoids using AutoDock 4.2

LIGAND	DOCKED ENERGY(lowest binding energy)	*INHIBITION CONSTANT nM/ µM**	INTER MOLECULAR ENERGY	FINAL TOTAL INTERNAL ENERGY	TORSIONAL FREE ENERGY	UNBOUND SYSTEM ENERGY
TACRINE	-7.12	6.01	-7.42	+0.06	+0.30	+0.06
AMBREIN	-9.54	102.4*	-10.73	-0.50	+1.19	-0.50
GERANIOL	-5.22	148.3	-6.71	-0.23	+1.49	-0.23
LIMONENE	-5.46	99.63	-6.35	-0.47	+0.89	-0.47
LINALOOL	-5.16	164.6	-6.65	-0.22	+1.49	-0.22
FERULIC ACID	-5.17	163.3	6.66	-0.32	+1.49	-0.32
QUERCETIN	-8.34	764.30*	-10.13	-1.51	+1.79	-1.51
CURCUMIN	-8.21	957.9*	-11.19	-1.27	+2.98	-1.27
MYRICETIN	-8.28	845.3*	-10.37	-2.11	+2.09	-2.11
KAEMPFEROL	-7.16	5.64	-8.65	-0.09	+1.49	-0.09
LUTEOLIN	-8.56	529.9	-10.05	-1.22	+1.49	-1.22

The ligand is drawned using chem sketch software.Using Online smile translator, SMILES format will be converted into PDB,MOL, SDF and smile text file format. The selected ligand molecule of canonical smile formats was converted to pdb format. The protein and ligand files which are prepared by the above said procedures are taken for docking.

Docking With Auto Dock 4.2: Docking calculation in auto dock was performed using the refined protein and the desired ligand in .pdb format. The enzyme molecule is saved as AchE.pdb. Toggle the “Auto Dock” TOOLS, the ligand is from the file via input option. The torsions were analysed and finally the ligand is saved in the cygwin location as ligand.pdbqt. Then Auto Grid Calculation has to be performed and save it as “AchE.gpf” file. Then docking calculation is performed which involves search parameters, genetic algorithm, docking parameters and saving it as ligand.gpf. Programming for the docking simulation is done through cygwin interface by using commands. Once docking has completed,toggle the Auto Dock icon and see for the 10 different conformations. The docking performed here is of rigid docking which involves the confirmations of ligand only.

2. In-vitro Acetylcholinesterase inhibition by Terpenoids, Flavonoids.

Evaluation of Enzyme Inhibition: The assay is based on measurement of the change in absorbance at 405 nm. The method is described in detail by Ellman. The assay uses the thiol ester Acetylthiocholine instead of the oxy ester acetylcholine. AChE hydrolyses the Acetylthiocholine to produce Thiocholine and acetate. The Thiocholine in turn reduces the Dithiobis-Nitrobenzoic Acid liberating nitro benzoate, which absorbs at 405 nm (Ellman, 1961).

a) Measurement of AChE Activity: Mix 375 μl of Buffer, 100ul of Substrate, and 500 μl of DTNB in a ratio Of 150:2:5 to give a final concentration of Acetylthiocholine of 15 mM in a 2ml cuvette. Transfer 25 μl of the enzyme to the previously prepared solution and mix well, incubate it for 15 min.

b) Measurement of enzyme inhibition: Follow the 1st step as in measurement of enzyme activity. Then add respective quantities of test solutions to the test tube containing 25 μl of the enzyme and transfer these contents to the previously prepared substrate solution and mix well, incubate it for 15 min. The experiment is performed in triplicates for each of the concentration of test solutions in addition to the control absorbance. [Note: The negative controls are needed because thiol esters are unstable compared to oxy esters and there will be some spontaneous hydrolysis of the substrates.]

3. Data analysis

a)Measurement of Enzyme activity (Araujo, A.A et al., 2006)

Enzyme activity = % of the velocity compared to that of the assay using buffer instead of inhibitor

b)Measurement of enzyme inhibition

% inhibition = difference between Control absorbance and Sample absorbance to the control absorbance multiplied with 100 gives the percentage inhibition.

RESULTS and DISCUSSION:

Based upon the lowest binding energy and inhibition constants of terpenoids, and flavonoids obtained through docking studies are examined for their in vitro acetylcholine esterase inhibitory activity using tacrine as standard.Acetylcholine esterase (AchE) from electric eel for in silico docking studies possess 543amino acid.By using AutoDock, Tacrine (used as standard) was found to bind with Gly 120, Ser 125, Tyr 124, Tyr 337, Asp74, Asn 87 and Trp 86. Tyr 337 is found to be at the choline binding site, the amino acids Tyr 124, and Asp 74 is found to be at the peripheral anionic site.

Table 2: In vitro AChE inhibitory activity of flavonoids and terpenoids using Ellman method

S.No	LIGAND		IC50 µg/ml
1	Terpenoids	Ambrein	91± 0.12
2		Geraniol	200±0.21
3		Limonene	195±0.43
4		Linalool	200±0.24
5		Ferulic acid	185±0.56
6	Flavonoids	Quercetin	184±0.38
7		Curcumin	197±0.11
8		Myricetin	190±0.41
9		Kaempferol	205±0.08
10		Luteolin	135±0.18

Values are ± SEM (n=3),*P <0.05 when compared with standard

Note : Tacrine (STD) IC₅₀ = 92 µg/ml

These are the possible binding sites for the reversible inhibitors comprising anionic site and PAS. The lowest binding energy of Tacrine with rigid AchE with its possible conformation was found to be -7.16 kcal/mole possessing estimated inhibition constant of 5.65μM.

Tricyclic phenothiazine and acridine analogues are effective inhibitors and appear to associate with the choline subsite, located within the active center of the cholinesterase(Richard et al.,1999).Tacrine (1,2, 3, 4 - tetrahydroacridine) showed strong interactions with the choline binding site of AchE of Torpedo californica, however it does not interact with the peripheral anionic site. There is a pi-stacking interaction between the quinoline ring of the tautomer of Tacrine and the indolic ring of Trp 84. The nitrogen atom of the quinoline ring of this tautomer has hydrogen bond with the carbonyl group of the main chain of His 440, of the catalytic triad, and the quinoline ring is parallel and has contacts with the phenyl group of Phe 330^.(Richard et al., 1999)

Flavonoids like Quercetin, Luteolin, kaempferol, Myricetin, Catechin, Acacatechin, Curcumin, and Flavone were used for the investigation. The overall binding sites for flavonoids was found to be Gln 71, Tyr 72, Asn 87, Trp 86, Pro 88, Val 73, Asp 74, Ser 125, Gly 120, Tyr 124,Gly 121,Glu 202, Ser 203, Trp 286, Ile 294, Arg 296, Phe 295, Phe 297, Tyr 341, Tyr 337 , Phe 338, His 447, Gly 448, Ile 451. Among these Trp 86, Asp 74, Gly 120, Gly 121, Ser 125, Ser 203, Phe 295, Phe 297, Tyr 337,Phe 338, His 447, Gly 448 are the important amino acids with which flavonoids has high affinity of interaction and make the positioning of the atoms of the ligand inside the active site gorge providing lowest free energy of binding. The conformational changes shows differing DOF and hence the inhibition constant, Ki.

The main phenylchromen part of the quercetin showed several hydrogen bonds with the important amino acid residuesof anionic subsite (AS) of AChE, as O3 and O5 showed two hydrogen bonds of the OH atom of Tyr133; and O4 showed hydrogen bond with the O atom of Trp86. The entire quercetin showed hydrophobic interactions with Ser125, His447, and Glu202, which are some of the prominent residues of AS of AChE. The O atoms of the phenyl side chain of the same compound also showed hydrogen bonding to the important residues of peripheral anionic subsite (PAS) of AChE, as O6 atom of the quercetin showed hydrogen bonds with O atoms of the Tyr72 as well as Gln71, respectively; and O7 with the N atom of Asp74Analysis of the docking results reveal the overlapping binding energy for the kaempferol, flavone (-7.16 to -7.78 kcal/mole) as that of the value of Tacrine.

The estimated inhibition constant, Ki kaempferol lies in the range of 5.64 μM (of lowest binding energy, -7.16 kcal/mol) to 10.01μM. Whereas Myricetin, Quercetin, Luteolin, Curcumin showed lowest binding energy (-7.15 to -8.60kcal/ mole) than that of tacrine.

From Table2, it is evident to show that ,in-vitro evaluation of docked compounds shows supporting results. Among them Luteolin, Quercetin, Myricetin, Curcumin, Kaempferol possess IC50 value of 135μg /ml ,184 μg /ml, 190 μg /ml 197μg /ml, 205μg /ml, respectively. Luteolin was found to be most active against AchE and it shows 82.4%. Literature review shows screening on chemically diverse flavonoids derivatives for the AChE inhibitory effects at a concentration of 1mg/ml (Yang et al., 2006) and amongst them, quercetin was found to be the most active against AChE having 76.2% inhibition. Luteolin shows 79.7% of inhibition against AchE.

Terpenoids like Ambrein, geraniol, linalool, limonene, Euphol, Herolic acid, Lanosterol, ferulic acid were used for the investigation of AchE inhibition. The overall binding sites of terpenoids was found to be Trp 86, Asn 87, Asp 74, Pro 88, Tyr119,Trp 117, Gly 120, Gly 121, Tyr 124, Ala 127, Ser 125, Leu 130, Tyr 133, Glu 202, Ser 203, Ile 294, Phe 295, Phe 297, Phe 338,Tyr 337,Tyr 341, Gly 448, His 447,Ile 451.Among these Trp 86, Asp 74, Gly 120, Gly 121, Ser 125, Ser 203, Phe295, Phe 297, Tyr 337,Phe 338, His 447, Gly 448 are the important amino acids with which terpenoids has high affinity of interaction and make the positioning of the atoms of the ligand inside the active site gorge providing lowest free energy of binding. The conformational changes shows differing degree of freedom and hence the inhibition constant, Ki. The binding energy depends upon the hydrogen bond formed between the isoprene chain and the amino acid of the enzyme. If the length of the isoprene units increased there is an increase in binding sites. The structural rigidity makes increase in the number of binding sites. For eg: Ambrein, Ferulic acid, possess steroidal nucleus and hence the binding sites.

Analysis of the docking results reveals that the lowest binding energy of the terpenoids lies in the range of -9.55 to -5.91 kcal/mol. When compared with that of the tacrine, they possess better inhibition constant. Ambrein showed -9.55 kcal/mol as its lowest binding energy with its estimated inhibition constant of 100.3nM. This inhibitory constant in nano quantity shows its potency towards the AchE. With its diverse chemical structure the triterpenoid class of Ambrein has its inherent property of binding with the amino acids at the active site gorge of AchE.

The triterpenoid class of ferulic acid, showed its inhibition constant in the micro molar (163.3μM) with –5.17 kcal/mol where lanosterol being the triterpenoid shows the binding energy of -5.50 with estimated inhibition constant as 93.2mM.

The inhibitory constant overlaps in the range of milli molar (mM) to micro molar (μM) quantity. The main nucleus of the ferulic acid and lanosterol being the steroidal structure, where the proximal portion of the ferulic acid and lanosterol possess triterpenoidal chain structure which actively participate in the binding. The mono terpenoids class comprising Limonene, Linalool, Geraniol, with its simplest structure showed its lowest binding energy in the range of -5.91 to -5.05 kcal/mol with its inhibition constant ranges from 99.63 to 356.15μM.These mono terpenoidal structure has less number of binding sites, when compared to that of the triterpenoids. From Table2, it is evident to show that ,in-vitro evaluation of docked terpenoidal compounds showed supporting results. Among them Ambrein, geraniol, ferulic acid, linalool, limonene possess IC50 of 91μg/ml ,200μg/ml ,185μg/ml ,200μg/ml,195 μg/ml respectively. Ambrein was found to be most active against AchE and it shows 98.91%inhibition. Whereas ferulic acid showed 32.56% of inhibition with increase in concentration of ferulic acid from 25- 400mg/ml .The inhibitory activity of different concentrations of ferulic acid extracted from Impatiens bicolor, the inhibition of AChE was increased rapidly from 12.38 - 42.86% with the increase in the concentration of ferulic acid from 50 - 200 μg/ml(Shahawaret al.,2010)

CONCLUSION:

It is concluded that acetylcholine esterase (AChE) docking studies using flavonoids and terpenoids possessing lowest binding energy with its estimated inhibition constant (Ki) suggest that it is active against AchE and the evaluation of those docked compounds by in vitro study shows flavonoids and terpenoids has its inhibitory activity towards acetylcholinesterase. Further structural optimization studies and in vivo studies will pave for the discovery of new drugs for the treatment of Alzheimer’s disease.

REFERENCES:

1. Araujo AA, Ferreira C, Proenca M, Serralheiro L. The in vitro screening for acetyl cholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. Journal of ethno pharmacology, 108; 2006: 31–37.

2. Aatu K, Janne O. Protein docking-Basics of molecular docking and drug designing. 1st edition, pg no: 32-45, Elsevier publications,New York, 2002.

3. Brossi A, Schonenberger B, Clark OE, Ray R. Inhibition of acetylcholinesterase from electric eel by (-) and (+)-physostigmine and related compounds. Federation of european biochemical societies, 201(2); 1986:190-192.

4. Brigitte, K., Maurice, G., Christian, H., Ruedi, A., Jui, Y.C. Sequence determination of a peptide fragment from electric eel acetylcholinesterase, involved in the binding of quaternary ammonium . Federation of european biochemical societies , 202(1);1986:92-96.

5. Carlos, S., Vanessa, L.C., Ivone, C., Carlton, A.T. Molecular modelling, docking and ADMET studies applied to the design of a novel hybrid for treatment of alzheimer’s disease. Journal of molecular graphics and modelling,25; 2006: 169–175.

6. Daniel, K., Heather, G., Reetesh, K.P., Natilie, A.H., Israel, S., Joel, L.S., Palmer, T., Judith, G.V. Inactivation Studies of Acetylcholinesterase with phenylmethylsulfonyl Fluoride. Journal of molecular pharmacology, 57; 2000:1243–1248.

7. Girisha., Narendra, J., Sriramamurthy., Manish, M., Sadashiva, C., Rangappa. Active site directed docking studies: Synthesis and pharmacological evaluation of cis-2, 6-dimethyl piperidine sulphonamides as inhibitors of acetylcholinesterase. European journal of medicinal chemistry, 44; 2009: 4057–4062.

8. Garrett, M.M., David, S., Goodsell, Michael, E.P., William, L.L., Ruth, H., Stefano. F., William, E.H., Scott, H., Rik, B., Arthur, J.O. User Guide Autodock version 4.2 automated docking of flexible ligands to flexible receptors. [internet] 2010 [cited 2010 July 15].Available from: http://autodock.scripps.edu/faqs-help/tutorial

9. Hay, D.V., Israel, S., Michal, H., Terrone, L.R., Joel, L.S. Acetyl cholinesterase: From 3D structure to function; Chemico-biological interactions 2010; 187: 10–22.

10. Hamid, N., Morteza, H., Maryam, S., Mohammad, A., Vahid, S., Massoud A., Alireza, F. Design, synthesis and anticholinesterase activity of a novel series of 1-benzyl-4-((6-alkoxy-3-oxobenzofuran-2(3H)-ylidene) methyl) pyridinium derivatives. Journal of bioorganic and medicinal chemistry, 18; 2010: 6360–6366.

11. Helen, M.D., Laurent, P.R. The mechanism for the inhibition of acetyl cholineesterases by irinotecan. Journal of molecular pharmacology,56;1999:1346–1353.

12. Iqbal, C., Asaad, K., Zaheer, H., Nabeel, G., Fareeda, F., Atta-ur-Rahman., Gilani, A.H. Cholinesterase inhibitory and spasmolytic potential of steroidal alkaloids. Journal of steroid biochemistry and molecular biology, 92; 2004: 477–484.

13. Iqbal, C., Asaad, K., Kamran, M.A., Shehnaz, P., Rahman, A. Structural basis of acetylcholinesterase inhibition by triterpenoidal alkaloids. Biochemical and biophysical research communications,331; 2005:1528–1532.

14. Juan, F., Reciomaxi, M., Totrovruben, A. Screened charge electrostatic model in protein-protein docking Simulations. Pacific symposium on bio computing,07; 2002: 552-565.

15. Ken, S.L., Roger, A.A., Cameron, R.S., David, A.B., Soroushian., Brett, E., Rachael, S., Katherine, A.K., Kensaku, N. Dialkyl phenyl phosphates as novel selective inhibitors of butyrylcholinesterase. Biochemical and biophysical research communications, 355; 2007: 371–378.

16. Ling, H., Zonghua, L., Feng, H., Jing, L., Xingshu, L. Synthesis and biological evaluation of a new series of berberine derivatives as dual inhibitors of acetylcholinesterase and butyrylcholinesterase. Journal of bioorganic and medicinal chemistry,18; 2010: 1244-1251.

17. Li, P., Jia, HT., Jin, Q.H., Shi, LH., Lian, Q.G., Zhi, S.H. Design, synthesis and evaluation of isatin digotone deri vatives as acetylcholinesterase and butyrylcholinesterase inhibitors. Journal of bioorganic and medicinal chemistry, 18; 2008: 3790–3793.

18. Lilu, G., Alirica, I.S., Michael, R.B., John, M.G., Charles, M.T. Inhibition of acetylcholine esterase by chromophore-linked fluorophosphonates. Journal of bioorganic and medicinal chemistry, 20;2010:1194–1197.

19. Miguel, L.T., George, N.P., Lydia, E.K. Molecular docking: A problem with thousands of degrees of freedom. Portugese ministry of science, 12; 1999: 25-30.

20. Maja, V.T., Ivan, O.J., Ljuba, M.M., Branko, J.D. 4-Aryl-4-oxo-N-phenyl-2-aminyl butyramides as acetyl-and butyrylcholinesterase inhibitors. Preparation, anti choline esterase activity, docking study, and 3D structure–activity relationship based on molecular interaction fields. Bioorganic and medicinal chemistry, 18; 2010:1181–1193.

21. Marshall, L.Y., Quinn. In Vitro Acetylcholinesterase inhibition by type A botulinum toxin. Journal of bacteriology, 94(4); 1967:812-814

22. Pia, M.V., Adyary, F., Paivi, O., Shikhar, G., Pia, B., Anna, G., Gopi, M. Inhibition of acetylcholinesterase by coumarins: The case of coumarin 106. Journal of pharmacological research; 58; 2008:215–221.

23. Palmer, T. Anticholinesteraseagents .GoodmanandGilman's-ThePharmacological basis of therapeutics, 11th Ed, 125-129.McGraw-Hill medical publishing division, New York, 2006.

24. Recanatini., Cavalli, A., Hansch, C. A comparative QSAR analysis of acetylcholinesterase inhibitors currently studied for the treatment of Alzheimer’s disease. Journal of chemico-biological interactions, 105; 1997: 199-228.

25. Richard PH, Lin W, Craig AZ, Amy NE. Synthesis of Dihydro xanthone Derivatives and Evaluation of their Inhibitory activity against Acetylcholinesterase: Unique structural analogs of Tacrine based on the BCD-ring of Arisugacin. Bioorganic and Medicinal Chemistry, 9; 1999:973-978.

26. Robert, B.M., Robert, C.K. A Rapid spectrophotometric Method for the determination of Esterase Activity. Journal of biochemical and biophysical methods, 3; 1980: 345—354.

27. Sonkusare., Kaul, C.L., Ramarao, P. Dementia of Alzheimer’s disease and other neurodegenerative disorders - memantine, a new hope. Pharmacological Research,51; 2005: 1–17.

28. Sadhna T, Juergen P. Biochemical profiling in silico—predicting substrate specificities of large enzyme families. Journal of Biotechnology, 124; 2006:108–116.

29. Srinivas RA., Priyadarshini P, Praveen K, Pradeep H, Narahari S. Virtual Screening in drug discovery – A computational perspective current protein and peptide science. Current science,8; 2007:329-351.

30. Shahawar D, Shafiq UR, Muhammad AR. Acetyl cholinesterase inhibition potential and antioxidant activities of ferulic acid isolated from Impatiens bicolor Linn. Journal of medicinal plants research, 4(3); 2010: 260-266.

31. Sussman JL, Hay D, Israel S, Michal H, Terrone LR. Acetylcholinesterase: From 3D structure to function. Chemico-Biological Interactions 187; 2010: 10–22

32. Todd, S.N., Todd, O.Y. A Framework for describing topological frustration in models of protein folding. Journal of molecular biology, 362; 2006: 605–621.

33. Tarek M, Praveen PN. Design, synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors. Bioorganic and Medicinal Chemistry, 20; 2010: 3606–3609.

34. Tuba T, Kucukkilinc, Inci O. Inhibition of electric eel acetylcholinesterase by triarylmethane dyes. Chemico-biological interactions, 175; 2008: 309–311.

35. William GB, Charlotte MD. A Micro assay system for measuring esterase activity and protein concentration in small samples and in high-pressure liquid chromatography eluate fractions. Journal of analytical biochemistry, 131; 1983: 499-503.

36. Werner JG, David DA, Paul RL. 3-D-QSAR and docking studies on the neuronal choline transporter. Journal of bioorganic and medicinal chemistry, 20; 2010: 4870–4877.

37. Young SJ, Kamil M, Marketa K, Vlasta Z, Ondrej H, Martina H, Miroslav P, Vlastimil D, Florian N, Martin D, Kamil K. Preparation and in vitro screening of symmetrical bis-pyridinium cholinesterase inhibitors bearing different connecting linkage—initial study for myasthenia gravis implications. Journal of bioorganic and medicinal chemistry, 20; 2010: 1763–1766.

38. Yadin, D., Max, H., Israel, S. Molecular structures of acetylcholinesterase from electric organ tissue of the electric eel. Proc. Nat. Acad. Sci, 70(9); 1973: 2473-2476.

39. Zaheer UH, Waqasuddin K, Saima K, Farzana LA. In silico modeling of the specific inhibitory potential of thiophene-2,3-dihydro-1,5- benzothiazepine against BChE in the formation of β-amyloid plaques associated with Alzheimer's disease. Theoretical biology and molecular modelling; 7(22); 2010: 245-265.

Received on 18.07.2013 Modified on 01.08.2013

Asian J. Research Chem. 6(11): November 2013; Page 1011-1017