Molecular Modelling Techniques For Investigating Enzyme Interactions Biology Essay

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Computer Aided Drug Design-Investigating the interactions between an enzyme and a set of potential inhibitors using molecular modelling techniques


The work in this thesis mainly concerns about the computational molecular modelling techniques that are employed in the study of protein-substrate interactions. In this thesis the interaction of acetylcholinesterase with potential set of inhibitors were studied using the software SCIGRESS. Characterization of such interactions leads to the thorough understanding of the mechanism of action of the drug molecules, functions of the protein, and therapeutic effect of drugs. These approaches carried out influenced new ideas and reliable working hypotheses for molecular interactions in complexes of biological relevance. The application of these techniques is shown in the study of interaction of acetylcholinesterase with set of inhibitors.


1.1 Molecular modelling

Molecular modelling is a term used to describe the use of computers to construct and develop molecules and to perform a variety of calculations to predict their chemical characteristics and behaviour. Molecular modelling represents molecular structures numerically and simulating their behaviour with the equations of quantum and classical physics and it is one of the fastest growing fields in science. The modelling techniques are widespread in their use and have become more and more important in widely varying fields of chemistry, ranging from small organic molecules to proteins, polymers, inorganic solids, and liquids. Molecular modelling techniques has its impact on building, visualizing, and comparing molecules to performing complicated and time demanding calculations on rather big molecular systems. These tools are being successfully used, in conjunction with traditional research techniques, to examine the structural properties of existing compounds, develop and quantify a hypothesis which relates these properties to observed activity and utilize these "rules" to predict properties and activities for new chemical entities. The development of molecular modelling programs and their application in pharmaceutical research has been formalized as a field of study known as computer assisted drug design (CADD) or computer assisted molecular design (CAMD). As with all models, however, the intuition and training of the chemist is necessary to interpret the results appropriately. Comparison to experimental data, where available, is important to guide both laboratory and computational work.

Cholinergic synaptic mechanism

The neurotransmitter Acetylcholine is found throughout the body, in which it regulates various vital functions. In the CNS, cholinergic neurotransmission is involved in a number of processes including memory and learning, cognitive functions, arousal, and motor control. ACh exerts its physiological effects via signalling through two distinct receptors namely muscarnic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Once the ACh is released onto the synaptic cleft, acetylcholinesterase converts it into choline, which subsequently is taken up into the presynaptic terminal.

Biological Data of Acetylcholinesterase

Acetylcholinesterase (AChE, E.C is an enzyme associated with the cholinergic signal system, which performs the role of removing acetylcholine from the receptors. In vertebrates, two types of cholinesterases can be distinguished on the basis of their substrate and inhibitor sensitivity. The AChE belongs to the "α/β hydrolase fold protein" superfamily comprising of serine hydrolases such as cholinesterases, carboxylesterases, and lipases.

AChE, which is present in the central and peripheral nervous system and in skeletal muscles, plays a key role in terminating neurotransmission at cholinergic synapses by the hydrolysis of acetylcholine to choline and acetic acid. AChE may also participate in the development, differentiation, and pathogenic process such as Alzheimer's disease (AD) and involve in the deterioration of the cholinergic innervations in the cortex region of the brain leading to AD. Thus the introduction of AChE inhibitors came into effect for the symptomatic treatment of AD.

Alzheimer's disease

Clinical diagnosis of AD is based on the progressive impairment of memory, learning ability and other cognitive dysfunctions due to neurodegenerative disorder. It also decreases the ability of performing the basic daily activities. Other arrays of the disease are apathy, verbal and physical agitation, anxiety, depression, delusions and hallucinations. Aging is often regarded as the main factor in memory impairment and decline in other mental functions. Memory loss and other neuropsychological symptoms that include impairment of judgment, learning, abstract thinking, language, which are descriptive of AD, may be attributed to normal aging [1]. The fact that AD increases with advancing the age, nowadays represents a major public health problem and it is probably becoming the most important pathology of the 21st century in the developed countries [2].

Molecular level of AChE

At the molecular level AChE is a 537 amino acids long protein composed of a 12-stranded mixed β-sheet surrounded by 14 α-helices. The hydrolysis of ACh in AChE takes place at the bottom of a long and narrow gorge lined with numerous aromatic amino acid residues that penetrate half into the enzyme. The active site is located ~20 Šfrom the surface of the enzyme and is composed of two subsites. The catalytic triad located at the base of the narrow gorge comprises of three components namely His440, Glu327, and Ser200.

The peripheral anionic site was formed by the residues Tyr70 and Trp279. Furthermore, two sets of residues (270-278 and 251-266 in TcAChE) contribute to the peripheral anionic subsite, which are located near the rim of the gorge. Hence, ligand association with the peripheral site may prevent access of substrate to the gorge by physical hindrance to restrict entry to the gorge by an allosteric mechanism, in which the active center conformation is altered [11] [12]. Recently, evidence was presented that AChE accelerates assembly of amyloid-β-peptides into the amyloid fibrils with involvement of PAS [13].

Computational Molecular modeling

Molecular modeling has become a valuable and essential tool to medicinal chemists in the drug design process. Molecular modeling describes collection of techniques used for the generation, manipulation or representation of three-dimensional structures of molecules and associated physico-chemical properties. It involves a range of computerized techniques based on theoretical chemistry methods and experimental data to predict molecular and biological properties. Depending on the context and the rigor, the subject is often referred to as 'molecular graphics', 'molecular visualizations', 'computational chemistry', or 'computational quantum chemistry'.

Cholinesterase inhibitors

The physiological significance of AChE activity is reflected by the observation that it is targeted by number of natural and synthetic toxins. Based on the activity, the Acetyl cholinesterase inhibitors (AChEIs) are divided into two main classes: (1) irreversible organophosphorus inhibitors and (2) reversible inhibitors.

Reversible AChEIs

Reversible AChEIs binds to the binding site of the enzyme and inhibit of the activity of substrate. Aminoacridines (tacrine), N-benzylpiperidines (donepezil), edrophonium and alkaloids (galanthamine) are the well known examples of reversible AChEIs.


Tacrine hydrochloride (Cognex®) is a reversible cholinesterase inhibitor, known chemically as 1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate. Tacrine hydrochloride is commonly referred to in the clinical and pharmacological literature as THA. It has an empirical formula of C13H14N2•HCl•H2O and a molecular weight of 252.74. Tacrine blocks the sodium and potassium channels and has a direct effect on muscarnic receptors.


Galantamine (also called galanthamine) is an alkaloid containing tertiary amine isolated from several plants, including daffodil bulbs, but is now synthesized. Galantamine is a specific, competitive, and reversible inhibitor of AChE. It is also an allosteric modulator at nicotinic cholinergic receptor sites thereby potentiating and increasing the release of ACh neurotransmission. The IUPAC name is (4aS,6R,8aS)-4a,5,9,10,11,12-Hexahydro-3-methoxy-11-methyl- 6H-benzofuro[3a,3,2-ef] [2]benzazepin-6-ol.








Edrophonium is a rapid, short-acting reversible acetylcholinesterase inhibitor. It acts by inhibiting the action of acetylcholinesterase at sites of cholinergic transmission. IUPAC name of edrophonium is ethyl-(3-hydroxyphenyl)-dimethylazanium and its molecular formula is C10H16NO.


Rivastigmine is a reversible acetylcholinesterase inhibitor that exerts its cholinergic effect by increasing the function of cholinergic receptors. Rivastigmine increases the concentration of acetylcholine through reversible inhibition of its hydrolysis by cholinesterase. The IUPAC name of rivastigmine is (S)-N-Ethyl-N-methyl-3-[1-(dimethyl amino) ethyl] phenyl carbamate.


Huperzine A is a plant alkaloid derived from the Chinese club moss plant, Huperzia serrata. By reducing the activity of acetylcholinesterase, huperzine A reduces the breakdown of acetylcholine. Hence huperzine is a reversible acetylcholinesterase inhibitor. The IUPAC name of huperzine is (1R,9S,13E)- 1-Amino- 13-ethylidene- 11-methyl- 6 azatricyclo [] trideca- 2(7),3,10- trien- 5-one.

Irreversible organophosphate inhibitors

Molecular docking

The structure of receptor-ligand complexes is studied using the knowledge of molecular docking. The receptor usually be a protein and the ligand (inhibitors) is either a small molecule or another protein. Docking can be defined by placing the ligand in the most suitable conformation to interact with the protein.

Hence the structure of the intermolecular complex which is formed by two or more molecules to suggest binding modes of protein inhibitors can be predicted. The algorithms used in docking generate the possible structure conformations and thereby a mean score is calculated to identify the structure.