INTRODUCTION:^[1]

Alzheimer's disease (AD)¹is a brain disease that slowly destroys memory and thinking skills and, eventually, the ability to carry out the simplest tasks. Memory problems are one of the first signs of Alzheimer's. It is the most common cause of dementia among older people. Dementia is generally defined as the ‘loss of intellectual abilities (medically called cognitive function) like loss of thinking, remembering, and reasoning skills that interferes with a person's daily life and activities.

Alzheimer's disease (AD)² is neurodegenerative diseases of the central nervous system associated with progressive memory loss resulting in dementia.

Two pathological characteristics are observed in AD patients at autopsy: extracellular plaques and intracellular tangles in the hippocampus, cerebral cortex, and other areas of the brain essential for cognitive function.³

Plaques are formed mostly from the deposition of amyloidal β (Aβ), a peptide derived from amyloidal precursor protein (APP). Filamentous tangles are formed from paired helical filaments composed of neuron filament and hyperphosphorylated tau protein, a microtubule-associated protein⁴. To date, genetic studies have revealed four genes that may be linked to autosomal dominant or familial early-onset AD. All mutations associated with APP and PS proteins can lead to an increase in the production of Aβ peptides, specifically the more amyloido genic form, Aβ₄₂. In addition to genetic influences on amyloidal plaque and intracellular tangle formation, environmental factors (e.g., cytokines, neurotoxins, etc.) may also play important roles in the development and progression of AD.

The molecular mechanisms and hypotheses of Alzheimer's disease (AD) can be incredibly complex. Rapid progress in deciphering the biological mechanism of Alzheimer's disease (AD) has arisen from the application of molecularand cell biology to this complex disorder of the limbic and associationcortices. All four genes definitively linkedto inherited forms of the disease to date have been shown to increasethe production and/or deposition of amyloidal -protein in the brain.

Tacrine⁵ is the first drug to be approved for the treatment of mild to moderate Alzheimer's disease. It is thought to increase concentrations of acetylcholine, one of the key neurotransmitters in Alzheimer's disease. As the first drug treatment for Alzheimer's disease, tacrine represents an advance; the pros and cons of prescribing it should be discussed fully with the patient and family.

Targets of tacrin therapy are Histamine N methyl transferase and Acetyl cholinesterases. Histamine N methyl transferase is 9-amino-1, 2, 3, 4-tetrahydroacridine can inhibit histamine-N-methyltransferase. Histamine-synthesizing neurons in the brain may play an important role in cognition, and a histaminergic deficit has been found in Alzheimer's disease (AD). The AD medication tacrine was inhibiting some forms of histamine-N-methyltransferase (HNMT).

Cerebral concentrations of most neurotransmitters and neuron peptides decrease with progression of Alzheimer's disease, particularly acetylcholine⁶, the major neurotransmitter for memory. The theoretical rationale for tacrine is that by blocking acetyl cholinesterase, the enzyme that degrades acetylcholine, there will be more neurotransmitter available.

AD is characterized by deficits in memory and cognition that are associated with significant losses of presynaptic cholinergic function in the brain. Donepezil⁷ enhances cholinergic transmission by reducing the enzymatic degradation of acetylcholine.

In the present study, we report few analogues of tacrine and donipezil that show higher affinity and better interaction with the Histamine N methyl transferase and Acetyl cholinesterase protein. These results have been confirmed by the fitness scores obtained from GOLD docking software.

MATERIALS AND METHODS:

Selection of target protein:

Histamine-N-methyltransferase protein and Acetyl cholinesterase protein was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/). Protein reports were obtained from RCSB Protein Data Bank (http://www.rcsb.org/pdb/). Active site identification is primarily done by using online tools like RCSB Ligand Explorer and PDBsum.

Selection of Lead moiety and designing of ligands:

A lead compound in drug discovery is a chemical compound that has pharmacological or biological activity and whose chemical structure is used as a starting point for modifications in order to improve potency, selectivity, pharmacokinetic properties.

Medications called cholinesterase inhibitors are prescribed for mild to moderate Alzheimer’s disease. These drugs may help delay or prevent symptoms from becoming worse for a limited time and may help control some behavioural symptoms. Tacrine is the first drug to be approved for the treatment of mild to moderate Alzheimer's disease⁷. It is thought to increase concentrations of acetylcholine, one of the key neurotransmitters in Alzheimer's disease. Donepezil enhances cholinergic transmission by reducing the enzymatic degradation of acetylcholine. So tacrine and donepezil is taken as lead moities.

Tacrine lead moiety

Donepezil lead moiety

Table: 1: newly designed tacrine and donepezil analogs:

S.N	R
T₁	-NH₂
T₂	-CH₃
T₃	-CH₂CH₃
T₄	-0H
T₅	-Cl
T₆	-H
D₁	-CH₃
D₂	-NH₂
D₃	-CH₂CH₃
D₄	-0H
D₅	-Cl
D₆	-H

Note:-

T_{1 – 6}----- Analogs of Tacrine

D_1-6----- Analogs of Donepezil.

Design of Ligands:

12 ligands were designed from the Lead compounds by modifying the non pharmacophoric parts like R, All the ligands were designed by using Chemsketch⁸. These ligands were designed according to the SAR properties of N-methyl D-aspartate (NMDA) antagonist and cholinesterase inhibitors respectively.

Major considerations during designing of ligands were given to increase the hydrophobic character of the molecule. Thus modifications of Rin all ligands increase the hydrophobicity by the presence of alkyl groups and hydroxylic, amine substitutions were made for some ligands to increases its Hydrophilic nature. Modifications were given in the Table: 1

Energy minimization of protein and ligands:

Energy minimization of protein and ligand is performed by using Hyperchem 8.0.

Proteins - The proteins Histamine-N-methyltransferase protein and Acetyl cholinesterase protein downloaded from PDB was prepared for docking by deleting all hetero atoms, ligands and water molecules and optimized by minimization of energy by using Hyperchem 8.0. Parameters were maintained as RMS Gradient-0.01 k.Cal/Mol, CHARMm- 27 force field with Polak-Ribiere (conjugate gradient) minimizer, in In Vaccoconditions⁹.

Ligands - Ligands designed by using Chemsketch were optimized by using Hyperchem 8.0 and parameters were maintained as RMS Gradient 0.01 k.Cal/Mol, OPLS force field, in vacco media and Polak-Ribiereminimizer. Later, all the optimized ligands were saved in .MOL format.

Docking:

All the 6 ligands of tacrine,were docked with Histamine-N-methyltransferase protein and 6 ligands of donepezil were docked with Acetyl cholinesterase protein in the GOLD version 3.01. GOLD (Genetic Optimization for Ligand Docking) uses a genetic algorithm to explore wide range of ligand conformational flexibility and rotational flexibility of selected receptor hydrogen¹⁰. The docking orientations were ranked based on a molecular mechanics–like scoring function known as Gold score. Gln-143 is set as active site residue for Histamine-N-methyltransferase protein and Tyr-146 is set as active site residue for Acetyl cholinesterase protein. In the aspect of producing binding energy estimates, the Gold score function appears to be more reliable than the Chemscore function. The compound binding site was defined within a 10 Å radius around the binding sites of protein. Standard default parameter settings were used to evaluate the protein-ligand interactions^{11, 12}.

RESULTS AND DISCUSSIONS:

Docking studies:

Protein - ligand interactions are studied by using GOLD 3.01 docking software. The Gold score function is a molecular mechanics-like function with four terms:

GOLD Fitness = Shb_ext + 1.375(Svdw_ext )+Shb_int + S(vdw_int)

Where, Shb_ext is the protein–ligand hydrogen-bond score and Svdw_ext is the protein-ligand van der Waals score. Shb_int is the contribution to the Fitness due to intramolecular hydrogen bonds in the ligand; this term is switched off in all calculations presented in this work: Svdw_int is the contribution due to intramolecular strain in the ligand. The analogue which is having the highest fitness score is having the highest binding affinity¹³.

Docking results are tabulated in the Table: 2. The analogue which is having the highest fitness score is having the highestbinding affinity.

From the table, ligand T₃ is having the best Gold Fitness score of 53.67 and ligand D₃ is having the best Gold Fitness score of 69.11. 9of 12 ligands are having better Gold Fitness score. Modifications to these ligands may result in better ligands. Ligands like T₆, T₂ and D₆ are having the least Dock scores.

Table: 2: GOLD Docking: fitness scores of tacrine and donepezil analogs with Histamine-N-methyltransferase protein and Acetyl cholinesterase protein respectively.

Ligands	R	fitness	s(hb_ext)	s(vdw_ext)	s(hb_int)	s(vdw_int)
T₃	-CH₂CH₃	53.67	3.94	37.62	0.00	-1.99
T₅	-Cl	48.61	0.00	35.35	0.00	0.00
T₄	-0H	47.95	0.40	34.91	0.00	-0.46
T₁	-NH₂	47.22	5.27	31.55	0.00	-1.44
T₆	-H	46.57	0.00	33.87	0.00	0.00
T₂	-CH₃	45.87	0.00	33.07	0.00	0.40
D₃	-CH₂CH₃	69.11	0.00	54.53	0.00	-5.86
D₂	-NH₂	65.47	4.63	48.90	0.00	-6.41
D₅	-Cl	61.11	5.55	46.26	0.00	-8.05
D₁	-CH₃	60.68	0.28	52.43	0.00	-11.69
D₄	-0H	52.62	4.10	47.12	0.00	-6.27
D₆	-H	49.54	0.01	50.72	0.00	-20.21

CONCLUSION:

Present study was conducted to design and identify the potent N-methyl D-aspartate (NMDA) antagonist and cholinesterase inhibitors for the treatment of Alzheimer’s disease using InSilico tools and techniques. The interactions between Histamine-N-methyltransferase protein and Acetyl cholinesterase protein with the respective ligands were studied by using GOLD 3.01 docking protocol. Based on Gold Fitness score docking results were analyzed.

The overall review of results concludes that ligand T₃ is having the best Gold Fitness score of 53.67 and ligand D₃ is having the best Gold Fitness score of 69.11. 9of 12 ligands are having better Gold Fitness score. Modifications to these ligands may result in better ligands. Ligands like T₆, T₂ and D₆ are having the least Dock scores. Further development and synthesis of these ligands may lead to be as better drugs for blocking Histamine-N-methyltransferase and Acetyl cholinesterase proteins in treatment of Alzheimer’s disease.

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Received on 08.11.2012 Modified on 17.11.2012

Research J. Pharm. and Tech. 6(1): Jan. 2013; Page 86-89