Final Project

ABSTRACT
Alzheimer¶s disease is a neurodegenerative disorder. The most common from of AD attacks 65 and above aged people. It is a condition where there is a loss of memory and an absolute change in personality. AD just persists for a long time about 8-10 years and the eventuality is critical illness leading to death in India, a growing number of people are affected by AD nowadays. There is enzyme called beta secretase, which plays a role in the formation of plaque. It is a key enzyme in the AD pathway, which is a potential target for drug therapy. Beta secretase is an enzyme that cleaves amyloid precursor protein to give C99 fragment, which on cleavage by another enzyme gamma secretase becomes insoluble, thus forming plaques. This enzyme can be said to be a predisposing factor for the formation plaques. Since it has no known biological function inhibition of this enzyme would actually help to treat or prevent AD. There is no cure for Alzheimer¶s at present, although cholinesterase inhibitors have shown promise for delaying or preventing the symptoms of mild to moderate forms of the disease, experts say. The newly isolated compound, nostocarboline, was shown to be a potent inhibitor of cholinesterase a brain chemical thought to be important for memory and thinking whose breakdown has been associated with the disease¶s progression. The natural compound¶s potency is comparable to galanthamine, a cholinesterase inhibitor already approved for the treatment of Alzheimer¶s, the researchers says. A compound isolated from a cyanobacterium, a type of blue-green algae known as Nostoc, shows promise of becoming a natural drug candidate for fighting Alzheimer¶s and other neurodegenerative disease. This compound has so far no known side effects and if approved as a drug, would be of low costs and thus efficacy of nostocarboline as an inhibitor can be checked. Thus, if the docking results are good, nostocarboline might just be the answer for AD.

INTRODUCTION
Dementia is a brain disorder that seriously affects a person¶s ability to carry out daily activities. The most common form of dementia among older people is Alzheimer¶s disease (AD), which initially involves the parts of the brain that control thought, memory, and language. Although scientists are learning more every day, right now they still do not know what causes AD, and there is no cure.

Scientists think that as many as 4.5 million Americans suffer from AD. The disease usually begins after age 60, and risk goes up with age. While younger people also may get AD, it is much less common. About 5 percent of men and women ages 65 to 74 have AD, and nearly half of those age 85 and older may have the disease. It is important to note, however, that AD is not a normal part of aging. AD is named after Dr. Alois Alzheimer, a German doctor. In 1906, Dr. Alzheimer noticed changes in the brain tissue of a woman who had died of an unusual mental illness. He found abnormal clumps (now called amyloid plaques) and tangled bundles of fibers (now called neurofibrillary tangles). Today, these plaques and tangles in the brain are considered signs of AD.

Scientists also have found other brain changes in people with AD. Nerve cells die in areas of the brain that are vital to memory and other mental abilities, and connections between nerve cells are disrupted. There also are lower levels of some of the chemicals in the brain that carry messages back and forth between nerve cells. AD may impair thinking and memory by disrupting these messages.

Country
US India Japan Brazil United Kingdom Canada Pakistan Australia Western Europe World

Number of Affected by Demntia/AD
4.5 million 3.5 million 1.7 million 1.5 million 750,000 354,000 157,000 100,000 6±7 million

People

Alzheimer¶s disease (AD) is a silent killer of brain and lives of world¶s elderly people. It is the fourth leading cause of death among the older adults in the developed world. Named after Alois Alzheimer,( Maelicke, A. et al. (2000). the German physician who identified it in 1907, it remains elusive as to its cause and is resistive to treatment. It starts as a robber of memory and slowly erodes the intellectual and functional abilities leaving the patients bed-ridden and ultimately leads to death, mostly by pneumonia (infection of the lungs). It enormously affects the patients as well as the caregivers considering the long period of suffering (8-20years). Over the last few decades AD has dramatically changed from an obscure disorder to a major public health problem affecting millions of people worldwide. Besides the emotional and social issues at stake the economic costs of AD to society is massive. This

disease is a major contributor to increasing health care, bankrupting families and draining. Age is a major risk factor: the longer one lives, the greater the possibility of getting AD (about 50 percent of Americans over 85 have AD). AD seldom occurs before the middle age in a clinically obvious form, and then the likelihood doubles exponentially every five years. Given the fact that the elderly are the fastest growing group in the world population AD is termed as the epidemics of the elderly. In Asia, China's elderly population over 60 is projected to increase from 130 million in 2000 to 370 million in 2050, from 11% to 26% of the total population, with an annual .In addition to being the world's second most populous country, India has one of the largest populations of older adults. India¶s 60-plus population (around 80 million) is increasing by 3 % annually and is likely to double over the next 25 years. Besides, a large section of the older population in India is illiterate and lives in rural areas which lack infrastructure. Due to lack of awareness of AD most patients/family members tend to ignore the symptoms of the disease as normal part of aging process. Clinical help is sought only after a drastic deterioration of patient¶s health. There are various researches being carried around the world to study this disease. One of the studies carried out by a group of scientists (okello et al, 2004) in university of Newcastle have found out that the extracts of camellia sine a very good treatment for inhibiting beta secretase which is a key enzyme playing a role in the pathogenesis of the disease (kowalska, 2004). In addition to (Gademann, 2005) others involved in this study include Friedrich Jüttner and Paul Becher of the University of Zurich and Julian Be chat, currently with the University de Luanne in Switzerland. There were other researches also conducted in vitro to show that cyanobacturiem are gi/180484/gb(cholinesterase) inhibitors (jeon SY, 2003 and Levites, 2003).

LITERATURE REVIEW
AD is named after Dr. Alois Alzheimer¶s, a German doctor. In 1906, Dr. Alzheimer noticed changes in the brain tissue of a woman who had died of an unusual mental illness. He found abnormal clumps (now called neuritic plaques) and tangled bundles of

fibers (now called neurofibrillary tangles). Today, these plaques and tangles in the brain are considered signs of AD. (Kostrzewa RM et al, 2003) Scientists also have found other brain changes in people with AD. Nerve cells die in areas of the brain that are vital to memory and other mental abilities, and connections between nerve cells are disrupted. There also are lower levels of some of the chemicals in the brain that carry messages back and forth between nerve cells. AD may impair thinking and memory by disrupting these messages in the brain.

RISK FACTORS
Age: The risk of developing Alzheimer¶s increase with age. One out of every 10 persons 65 year and older is victim of Alzheimer¶s disease, although early-onset victims may be in there 40s and 50s. Approximately Americans between the ages of 75 and 84,and almost half of those 85 years and older suffer from Alzheimer¶s disease. Genetics: early- onset Alzheimer¶s has been clearly shown to be genetic in origin. While a mutation on chromosome 19 has been linked with late onset Alzheimer¶s (Sabbagh, M. et al. (2002). not everyone with the mutation develops the disease. The relationship between genetics and late-onset Alzheimer¶s is not fully known. Other: some studies have implicated prior traumatic head injury, lower education level, and female gender as possible risk factors. However, at the present time, no definite causes have been identified. Scientists still need to learn a lot more about what cause might play in the development of this disease. AD. In addition to genetics, they are studying education, diet, environment, and infections to learn what role they

SYMPTOMS
Common early symptoms of Alzheimer¶s are: Confusion  Disturbances in short-term memory  Problems with attention and spatial orientation  Personality changes  Languages difficulties  Unexplained mood swings

It is important to understand that Alzheimer¶s disease does not affect every patient in the same way.

CURRENT THERAPIES AVAILABLE
No treatment can stop AD. However, for some people in the early and middle stages of the disease, the drugs tacrine (Cognex), donepezil (Aricept), rivastigmine (Exelon), or galantamine (Razadyne, previously known as Reminyl) may help prevent some symptoms from becoming worse for a limited time. Another drug, memantine (Namenda), has been approved to treat moderate to severe AD, although it also is limited in its effects. These drugs are cholinesterase inhibitors that slow the breakdown of acetylcholine in the brain. Acetylcholine is a chemical in the brain that carries message between nerve cells. These drugs may help some people with memory and thinking. Other kinds of medicines are used to help control behavioral symptoms of AD such as sleepleness, agitation, wandering, anxiety, and depression. Treating these symptoms often makes persons with AD more comfortable and makes their care easier for caregivers. Drugs used included antidepressants, antipsychotics, and anxiolytics. No drug treatment include assuring that the person with AD has a healthy diet, exercise, social activities, social activities, regular medical care, and written directions, can be helpful for people in the earlier stages of AD. Caregivers can learn behavioral management methods to help cope with problem behaviors.

ALTERNATIVE THERAPIES
Several herbal remedies and other dietary supplements are promoted as effective treatments for Alzheimer¶s disease and related disorders. Claims about the safety and effectiveness of these products, however, are based largely on testimonials, tradition, and a rather small body of scientific research. Law for the marketing of dietary supplements does not require the rigorous scientific research required by the U.S. Food and Drug Administration for the approval of a prescription drug.
y Effectiveness and safety are unknown. The maker of a dietary supplement is not

required to provide the U.S. Food and Drug Administration (FDA) with the evidence on which it bases its claims for safety and effectiveness.

y Purity is unknown. The FDA has no authority over supplement production. It is a

manufacturer¶s responsibility to develop and enforce its own guidelines for ensuring that its products are safe and contain the ingredients listed on the label in the specified amounts.
y Bad reactions are not routinely monitored. Manufacturers are not required to

report to the FDA any problems that consumers experience after taking their products. The agency does provide voluntary reporting channels for manufacturers, health care professionals, and consumers, and will issue warnings about products when there is cause for concern. Coenzyme Q10, or ubiquinone, is an antioxidant that occurs naturally in the body and is needed for normal cell reactions to occur. This compound has not been studied for its effectiveness in treating Alzheimer¶s disease. Ginkgo biloba is a plant extract containing several compounds that may have positive effects on cells within the brain and the body. Ginkgo biloba is thought to have both antioxidant and anti-inflammatory properties, to protect cell membranes, and to regulate neurotransmitter function. Ginkgo has been used for centuries in traditional Chinese medicine and currently is being used in Europe to alleviate cognitive symptoms associated with a number of neurologic factors. Dementia is a brain disorder that seriously affects a person¶s ability to carry out daily activities. The most common form of dementia among older people is Alzheimer¶s disease (AD), which initially involves the parts of the brain that control thought, memory, and language. Although scientists are learning more every day, right now they still do not know what causes AD, and there is no cure.

Scientists think that as many as 4.5 million Americans suffer from AD. The disease usually begins after age 60, and risk goes up with age. While younger people also may get AD, it is much less common. About 5 percent of men and women ages 65 to 74 have AD, and nearly half of those age 85 and older may have the disease. It is important to note, however, that AD is not a normal part of aging.

AD is named after Dr. Alois Alzheimer, a German doctor. In 1906, Dr. Alzheimer noticed changes in the brain tissue of a woman who had died of an unusual mental illness. He found abnormal clumps (now called amyloid plaques) and tangled bundles of fibers (now called neurofibrillary tangles). Today, these plaques and tangles in the brain are considered signs of AD. Scientists also have found other brain changes in people with AD. Nerve cells die in areas of the brain that are vital to memory and other mental abilities, and connections between nerve cells are disrupted. There also are lower levels of some of the chemicals in the brain that carry messages back and forth between nerve cells. AD may impair thinking and memory by disrupting these messages.

Country US India Japan Brazil United Kingdom Canada Pakistan Australia Western Europe World

Number of Affected by Demntia/AD 4.5 million 3.5 million 1.7 million 1.5 million 750,000 354,000 157,000 100,000 6±7 million

People

Alzheimer¶s disease (AD) is a silent killer of brain and lives of world¶s elderly people. It is the fourth leading cause of death among the older adults in the developed world. Named after Alois Alzheimer, the German physician who identified it in 1907, it remains elusive as to its cause and is resistive to treatment. It starts as a robber of memory and slowly erodes the intellectual and functional abilities leaving the patients bed-ridden and ultimately leads to death, mostly by pneumonia (infection of the lungs). It enormously affects the patients as well as the caregivers considering the long period of suffering (820years). Over the last few decades AD has dramatically changed from an obscure disorder to a major public health problem affecting millions of people worldwide. ( Nashmi, R. et al. (2003). Besides the emotional and social issues at stake the economic costs of AD to society is massive. This disease is a major contributor to increasing health care, bankrupting families and draining. Age is a major risk factor: the longer one lives, the greater the possibility of getting AD (about 50 percent of Americans over 85 have AD). AD seldom occurs before the middle age in a clinically obvious form, and then the likelihood doubles exponentially every five years. Given the fact that the elderly are the fastest growing group in the world population AD is termed as the epidemics of the elderly.

In Asia, China's elderly population over 60 is projected to increase from 130 million in 2000 to 370 million in 2050, from 11% to 26% of the total population, with an annual .In addition to being the world's second most populous country, India has one of the largest populations of older adults. India¶s 60-plus population (around 80 million) is increasing by 3 % annually and is likely to double over the next 25 years. Besides, a large section of the older population in India is illiterate and lives in rural areas which lack infrastructure. Due to lack of awareness of AD most patients/family members tend to ignore the symptoms of the disease as normal part of aging process. Clinical help is sought only after a drastic deterioration of patient¶s health. There are various researches being carried around the world to study this disease. One of the studies carried out by a group of scientists (okello et al, 2004) in university of Newcastle have found out that the extracts of camellia sine sis (green tea) is a very good treatment for inhibiting beta secretase which is a key enzyme playing a role in the pathogenesis of the disease (kowalska, 2004). In addition to (Gademann, 2005) others involved in this study include Friedrich Jüttner and Paul Becher of the University of Zurich and Julian Be chat, currently with the University de Luanne in Switzerland. There were other researches also conducted in vitro to show that cyanobacturiem are gi/180484/gb (cholinesterase) inhibitors (jeon SY, 2003 and Levites, 2003). Huperzine A (pronounced HOOP-ur-zeen) is a moss extract that has been used in traditional Chinese medicine for centuries. Because it has properties similar to those of FDA-approved Alzheimer medications, it is promoted as a treatment for Alzheimer¶s disease. Phosphatidylserine (pronounced FOS-fuh-TIE-dil-sair-een) is a kind of lipid, or fat, that is the primary component of cell membranes of neurons. In Alzheimer¶s disease and similar disorders, neurons degenerate for reasons that are not yet understood. The strategy behind the possible treatment with phosphatidylserine is to shore up the cell membrane and possibly protect cells from degenerating.

³Coral´ calcium supplements have been heavily marketed as a cure for Alzheimer¶s disease, cancer, and other serious illnesses. Coral calcium is a form of calcium carbonate claimed to be derived from the shells of formerly living organisms that once made up coral reefs.

MOLECULAR MECHANISM
The molecular mechanisms and hypotheses of Alzheimer's disease (AD) can be incredibly complex. A top-down cartoon-like overview followed by increasing depth & detail may be the best way to gain understanding. I begin this approach in this introductory section. The key event leading to AD appears to be the formation of a peptide (protein) known as amyloid beta (beta amyloidal, Aß) which clusters into amyloid plaques (senile plaques) on the blood vessels and on the outside surface of neurons of the brain -- which ultimately leads to the killing of neurons. A first sketch of the amyloid cascade of events in AD would therefore be: Aß formation => amyloid plaques => neuron death => dementia The first fact to know in creating a second sketch is that the amyloid beta peptide is created by enzyme clipping of the normal neuron membrane protein known as Amyloid Precursor Protein (APP). APP is actually thought to be a natural neuroprotective agent induced by neuronal stress or injury, which reduces Ca2+ concentration and protects neurons from glutamate excitotoxicity [NEURON 10:243-254 (1993)]. Injections of a 17peptide subunit of APP have been shown to significantly reduce isocheim damage [EXPERIMENTAL NEUROLOGY 129:112-119 (1994)]. Enzymes can clip APP in ways that do not result in amyloid beta formation. Moreover, there are two forms of amyloid beta peptide, one of which has 40 amino acids and one of which has 42 amino acids. The 42 amino acid amyloid beta peptide (Aß42) is more hydrophobic & "sticky" (and hence aggregates more readily) than the 40 amino acid amyloid beta peptide (Aß40). Fibrils of Aß42 clump together to form amyloid plaques. Aß40 & Aß42 are formed intracellular, but exert damaging effects when transported

outside of cells. Insulin accelerates extra cellular transport, which may be the reason why type-2 diabetics have a greatly increased incidence of AD [THE JOURNAL OF NEUROSCIENCE 21(8): 2561-2570 (2001)].

Following amyloid plaque formation two processes play an important role in causing the death of neurons: (1) inflammation and (2) Neurofibrillary Tangles (NFTs). The two major types of brain cells that participate in the immune/inflammatory response are astrocytes and microglia. Astrocytes become more numerous in AD and these cells become activated to produce prostaglandin/arachidonic acid mediated inflammation. Activated microglial cells produce damaging free radicals. The activities of astrocytes & microglia lead to the death of neurons.

Neurons can be very large -- with axons & dendrites extending large distances. Substances for nutrient & cell-regulation are transported along microtubules within neurons. Tau is an important protein that maintains the structural integrity of microtubules. But in AD the tau proteins become hyper-phosphorylated and lose the capacity to bind to microtubules. Instead, the phosphorylated tau proteins bind to each other, tying themselves in "knots" (paired helical filaments -- two threads of tau wound around each other) known as NeuroFibrillary Tangles (NFTs). Neurons full of NFTs rather than functional microtubules soon die. With these facts in mind, the second sketch of the amyloid cascade would be: APP => Aß42 => fibrillar Aß =>amyloid plaques => inflammation/NFTs => neuron death Some researchers have found evidence that beta-amyloid fibrils form pores in neurons leading to calcium influx and the neuron death associated with AD [NATURE 418:291 (2002)] and [CHEMICAL & ENGINEERING NEWS 80(32): 31-34 (2002)]. This would appear to contradict the assertion that amyloid is insufficient for neuron death. The question of whether the central mechanism of AD neurodegeneration is beta-amyloid or NeuroFibrillary Tangles (NFTs) of tau-protein has been characterized as a "religious war" between the "tauists" and the "ßaptists". It is possible those amyloid plaques are an early event and that NFTs are a late event of an underlying process of AD that makes each event independent. But application of amyloid plaque to cultured neurons and injection of amyloid plaque into the brains of non-human primates both lead to NFTs. Amyloid beta may facilitate Ca2+ entry into neurons, causing calcium-activated kinases to excessively phosphor late tau protein leading to NFTs. Fibrillar Aß can induce MitogenActivated Protein Kinase (MAPK) leading to tau phosphorylation and hence to NFTs [JOURNAL OF NEUROCHEMISTRY 74:125-133 (2000)], although other kinases may be involved as well [JOURNAL OF MOLECULAR NEUROSCIENCE 19:249-250 (2002)]. (MAPK activity -- which is crucial to T-cell activation -- normally, declines with aging of the immune system, but MAPK pathways are aberrantly increased in AD.) Moreover, Aß inhibits ubiquitin-dependent degradation of protein and ubiquitin is concentrated in NFTs [THE JOURNAL OF BIOLOGICAL CHEMISTRY 270(34): 19702-19708 (1995)].

Amyloid beta is always a feature of AD, but NFTs are not. Amyloid plaque is insufficient to cause the cell death of AD tau has been shown to be essential for AD neurodegeneration [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA) 99(9): 6364-6369 (2002)]. In cases of AD where NFTs are absent, cell death is almost invariably due to the Lewy bodies of Parkinson¶s dementia. Lewy bodies are similar to NFTs, but are composed of ubiquitin & phosphorylated neurofilament rather than tau. Lewy bodies are typically found in the amygdala & limbic areas of the cerebral cortex (particularly the anterior cingulated cortex) in contrast to NFTs, which are concentrated in the entorhinal cortex & hippocampus (particularly the CA1 region). On autopsy AD patients have lost most of their CA1 neurons, whereas normal elderly don't show CA1 neuron loss on autopsy [EXPERIMENTAL GERONTOLOGY 38:95-99 (2003)]. Neuron death is due to NFTs or Lewy bodies -- the role of amyloid plaque is at most indirect. Amyloid plaques typically appear first in the association areas of the cerebral cortex, whereas NFTs typically begin in the entorhinal cortex. NFTs develop most frequently in large pyramidal neurons with long cortical-cortical connections (allowing for influence by amyloid plaques located some distance away). NFTs are associated with cells of origin of corticocortical projections whereas amyloid plaques are associated with the termination of corticocortical projections [EUROPEAN NEUROLOGY 37:71-81 (1997)]. The neurons being killed in the greatest numbers by NFTs are (1) the large cholinergic (acetylcholine-transmitting) neurons in the basal nucleus of Meynart (2) the large pyramidal neurons in the entorhinal cortex forwarding inputs from association cortices to the hippocampus via the perforant path and (3) output neurons in the CA1 region of the hippocampus. All three classes are output neurons [SCIENCE 278:412-419 (1997)]. The process of Aß42 formation from APP can now be described in a bit more detail. The enzymes that cleave APP are known as surceases. The two enzymes that initially compete to cleave APP are alpha-secretase and beta-secretase. If alpha-secretase cleaves APP there is no formation of Aß42. If APP is cleaved by beta-secretase it can then be further cleaved by gamma-secretase to form either a 40 amino acid amyloid peptide (Aß40) which is soluble & innocuous -- or a 42 amino acid peptide (Aß42) which clumps together

to form insoluble amyloid plaques. Alpha-secretase cleavage occurs at the cell surface, whereas beta-secretase acts at the endoplasmic reticulum. Gamma-secretase produces Aß42 if cleavage occurs in the endoplasmic reticulum and Aß40 if the cleavage is in the trans-Golgi network [NATURE MEDICINE 3(9): 1016-1020 (1997)].

III. GENETIC DETERMINANTS OF ALZHEIMER¶S DISEASE
Alzheimer's Disease (AD) can be divided into forms that run in families (genetically inherited) [known as Familial Alzheimer's Disease (FAD)] and forms showing no clear inheritance pattern [known as Sporadic Alzheimer's Disease (SAD)]. FAD accounts for only a small portion (less than 10%) of AD. All FAD is early-onset -- usually occurring between ages 30 to 60 -- whereas SAD typically occurs after age 65. SAD affects roughly 2% of those 65 years of age, with the incidence roughly doubling every 5 years up to age 90 at which the incidence is over 50%. AD is much more prevalent in women than in men for any given age group. All FADs can be cited as evidence of the amyloid cascade interpretation of AD .The gene that encodes tau-protein is located on chromosome 17 and is not associated with any FAD. In fact, at least half of FAD cases can be accounted for by the PS1 (Pre-Senilin 1) gene located on chromosome 14. PS1 is the predominant enzyme cleaving the gammasecretase site. PS1 resides within the endoplasmic reticulum/Golgi complex. Abnormal proteins from the PS1 and PS2 genes apparently influence gamma-secretase enzyme causing more Aß42 peptide formation. The mutation on chromosome 21 (the chromosome that is present in triplicate in Down's syndrome) is on the Amyloid Precursor Protein (APP) gene itself, resulting in abnormal APP protein that is preferentially cleaved by secretases to form more Aß42. (Down's syndrome` victims frequently develop AD if they reach age 40.) Genetic causes of Alzheimer¶s disease PROTEIN Presenillin-1 Presenillin-2 Amyloid precursor protein Apolipoprotein e CHROMOSOME 14 1 21 9 GENE S182 STM2 APP APOE

The mutations on chromosome 19 to the APOE gene are more complicated -- more accurately described as a "risk factor" for SAD than as an FAD. APOE occurs in three common forms (alleles): APOE2, APOE3 & APOE4 representing in Caucasians 8%, 78% & 14% of total APOE, respectively. Although only 14% of Caucasians have one APOE4 allele and 2% will have two APOE4 alleles, 40% of AD patients will have at least one APOE4 allele. (Africans have higher APOE4 and Orientals have lower APOE4 concentrations.) The mean age of onset of AD for Caucasians with no APOE4 allele is 84.3, is 75.5 for one allele and 68.8 for two alleles. Nonetheless, many AD patients will have no APOE4 allele and many Caucasians with the APOE4 allele never develop AD. Because the APOE4 allele is neither a necessary nor a sufficient condition for AD, but is significantly correlated with AD, the APOE4 allele is described as a "risk factor" for Caucasians rather than as a genetic determinant. By contrast, the APOE4 allele has not been shown to be a "risk factor" for African-Americans or Hispanics (who have high risk of AD regardless of APOE4 allele) [JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION 279(10): 751-755 (1998)].

MECHANAISM OF DEMENTIA IN ALZHEIMER DISEASE
D ementia is defined as "the progressive deterioration of intellectual and cognitive abilities associated with (organic) damage to the brain". The deficits seen in Alzheimer's disease patients are a consequence of the massive and widespread death or atrophy of nerve cells (neurons) of the brain, but particularly affecting the cerebral cortex and hippocampus. Since cognitive function is the result of communication between the cells within the cerebrum and other parts of the brain, the loss of these cells and their inter-connections leads to the clinical features seen in Alzheimer's disease. The neuritic plaques and neurofibrillary tangles (which are the postmortem neuropathologic hallmarks of Alzheimer's disease) represent the "tombstones" of the dead and dying neurons and their connecting axons and dendrites. Since each area of the brain performs a unique function, the death of the neurons in any particular area results in loss of whatever function or ability is sub-served by that area.

All central nervous system neurons normally are formed by about the twentieth week of fetal life (mid-second trimester) and no new neurons can be produced thereafter. The subsequent growth of the brain from late fetal life through adulthood is the result of increasing complexity of the connections between neurons. This is associated with increasing number and size of neuronal processes (axons and dendrites) and an increase in the amount of myelin surrounding axonal processes. Since neurons are not produced after that twentieth week of fetal life, redundancy has been built into the system. There is approximately a five-fold excess of neurons produced during development, with the extra cells serving as a "backup" throughout life. These extra cells are not ordinarily used but serve as "spares" when other neurons in an area are lost (an analogy is the spare tire carried in an automobile - the spare tire is not functional until one of the other tires is damaged). The five-fold excess of neurons means that about 80% of neurons must be lost in a particular area before symptoms appear (this is the so called "80% safety margin for nervous system function"). Thus, symptoms and signs of nervous system dysfunction (such as motor, sensory, or cognitive deficits) will not be apparent until there are fewer than 20% of the neurons remaining in a particular area of the brain. Studies that have carefully compared neuronal counts in young (less than age 20 years) and old (over age 60 years) individuals have shown that normal aging (the wear-and-tear of normal life) produces about a 30% loss of neurons in many areas of the brain; however, since at least 70% of nerve cells remain, there are no clinical deficits.

A disease process such as Alzheimer's disease causes accelerated death of neurons, but the affected individual will be a symptomatic until the number of remaining nerve cells is less than the 20% safety margin. Since neuronal loss continues after the initial onset of symptoms, an increasing number of neurological deficits become evident, until death ensues from inanition, which usually occurs within 5 to 8 years after the onset of clinical symptoms. The nerve cell loss in Alzheimer's disease is thought to occur over a period of at least 30 years before clinical symptoms become apparent. These assumptions are based upon findings from the study of Down's syndrome (trisomy of chromosome 21) patients; despite being mentally retarded, nearly all individuals with Down's syndrome acquire some cognitive and social skills (e.g., self-care, sheltered-workshop skills, etc.), however, those who live beyond age 40 years develop a progressive dementia in which they lose those skills, and at autopsy have Alzheimer's disease. Careful examination of the brains

of Down's syndrome patients have demonstrated neuritic plaques and neurofibrillary tangles even in infancy which indicates that the destructive process of Alzheimer's disease in these patients begins in early childhood, even though clinical symptoms do not appear until 30 years later. Accordingly, it is assumed that non-Down's syndrome individuals, who are just beginning to show clinical symptoms of Alzheimer's disease, really began experiencing neuronal destruction 30 or more years previously. This long prodrome before the onset of clinical symptoms explains in part the overall increasing prevalence of Alzheimer's disease. In simple terms, we are living longer than we were in the past! The 30+-year destructive process of Alzheimer's disease generally does not begin until the brain has completed its growth at about age 20 years. Thus, if the average life expectancy were under 50 years, as it was until almost 1920, most individuals would die before clinical symptoms set in. With the current average life expectancy at 75 years (72 years for men and 78 years for women), many more individuals are living longer and therefore developing clinical dementia from Alzheimer's disease

CHOLINESTERASES
Dale's momentous 1914 paper, in which he differentiated between the muscarine- and nicotine- like actions of choline esters on different tissues, proposed: "it seems not improbable that an esterase contributes to the removal of [acetylcholine] from the circulation". This hypothesis was based on observations of the inactivation of acetylcholine (ACh) injected into cats. However, it wasn't until 1926 that Loewi and Navratil, working on isolated frog's hearts, experimentally demonstrated its existence by inhibition with physostigmine (eserine), thus prolonging the effect of administered ACh. In 1932 Stedman et al. prepared a crude extract of an ACh-splitting enzyme from horse serum, which they called "choline- esterase". Cholinesterases from different species were found to differ in their substrate specificity and susceptibility to inhibitors. This provoked numerous schemes for naming the various cholinesterases. They are now considered to constitute a family of enzymes, which fall broadly into two types depending on their substrate preference (Silver, 1974). This

division is not absolute and holds true more in mammalian than non-mammalian species. Those enzymes, which preferentially hydrolyze acetyl esters such as ACh, are called acetyl cholinesterase (AChE) or acetylcholine acetylhydrolase (EC 3.1.1.7), and those, which prefer other types of esters such as butyrylcholine are termed butyrylcholinesterase (BChE) or acylcholine acylhydrolase (EC 3.1.1.8). BChE is also known as pseudocholinesterase, non- specific cholinesterase, or simply cholinesterase. This last term has led to confusion, and in this thesis the term cholinesterase will refer to all choline ester-hydrolysing enzymes, irrespective of their substrate specificity. The main function of AChE is the rapid hydrolysis of the neurotransmitter ACh at cholinergic synapses. The hydrolysis reaction proceeds by nucleophilic attack of the carbonyl carbon, acylating the enzyme and liberating choline. This is followed by a rapid hydrolysis of the acylated enzyme yielding acetic acid, and the restoration of the esteratic site (Wilson et al., 1950). The function of BChE remains a puzzle; it has no known specific natural substrate, although it is capable of hydrolysing ACh. It has been suggested that BChE acts as a scavenging enzyme in the detoxification of natural compounds (Massoulié et al., 1993). Certain human individuals have a mutant BChE that lacks the ability to hydrolyse succinyl choline. In rare individuals the complete BChE gene is missing. Neither of these cases results in any apparent physiological consequence. There is however an important clinical implication; zucchinis choline is commonly used during tracheal intubation in the administration of inhalation anaesthetics, and causes post operative apnoea in these people (McGuire et al., 1989).

Active site structure
The traditional view of the active site of AChE was considered to consist of two sub sites; a negatively charged or 'anionic' site, to which the positively charged quaternary nitrogen moiety binds, and an esteratic site containing the actual catalytic residues, probably both an electrophilic and a nucleophilic group (Nachmansohn and Wilson, 1951). A second 'anionic' site, which became known as the 'peripheral anionic' site, around 14Å from the active site, was proposed on the basis of binding of bis quaternary compounds (Bergman et al., 1950). O'Brien (1969) proposed his "heretical thought" that no true anionic site

existed. He argued that the carbon analogue of ACh, which lacks the charge of the quaternary nitrogen atom, and other uncharged molecules, are good substrates for the enzyme. He also noted that if columbic forces were of major importance in substrate binding, then the ammonium ion should be a very good inhibitor, whereas it is one of the poorest. The nucleophile was assumed to be a serine residue, with a histidine residue enhancing its nucleophilicity (Cunningham, 1957). As other enzyme mechanisms became understood, AChE was classified as a serine hydrolyses, and therefore assumed to contain a catalytic triad of Asp-His-Ser at the esteratic sub site. AChE from the electric ray Torpedo californica has most recently been the centre of attention due to its high concentration in the electroplax. According to the agreement adopted at the Oholo Conference in 1992, residues are numbered from the first residue of the mature protein. All references to residue number are for AChE from T. californica unless otherwise stated, when the number of the homologous residue of T. californica AChE is given in italics, in parentheses (Massoulié et al., 1992a). The position of the active site serine in T. californica AChE was established by irreversibly labeling it with [3H] isopropyl fluorophosphates followed by tryptic digestion and analysis of the tryptic peptides (MacPhee-Quigley et al., 1985; Schumacher et al., 1986), localizing it to Ser-200. Mutagenesis studies identified the catalytic histidine (Gibney et al., 1990) as His-440. The aspartic acid group of the catalytic triad was assumed to be Asp-326.

Crystal structure
A great leap forward in the understanding of the catalytic mechanism, and mode of action of inhibitors, came in 1991 with the determination of the three dimensional structure of dimeric T. californica AChE (Sussman et al.). This enzyme was crystallized in 1988 (Sussman et al.), and its structure was determined to a resolution of 2.8Å (Sussman et al., 1991), which has more recently been refined to 2.2Å (J.L. Sussman, personal communication). The structure determination uncovered a number of interesting findings. The catalytic triad seemed to contain a glutamate residue, rather than the usual aspartate. This was

confirmed by site- directed mutagenesis on the closely related T. marmorata AChE; the mutations Glu-327Gln and Glu- 327Asp led to inactive products (Duval et al., 1992). The relation of this triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. The only other proteins known with a similar catalytic triad are fungal lipases from Geotrichum candidum (Schrag et al., 1991) and Candida rugosa (Grochulski et al., 1993). Both these enzymes show a high overall similarity with the primary and tertiary structures of AChE. These proteins have recently been classified as members of a new protein fold; the alpha/beta hydrolase fold (Olli¶s et al., 1992). Alpha/beta Hydrolase fold proteins are considered to be related to the trypsin family of serine proteases, Subtilisin and papa in by convergent evolution. The active site was found to be located 20Å from the enzyme surface at the bottom of a narrow gorge, lined with 14 aromatic residues, which may be important in guiding the substrate to the active site (Ripoll et al., 1993). There was no discernible 'anionic' site, the quaternary nitrogen of choline binds chiefly through interactions with the pi electrons of the residue Trp-84 (Sussman et al., 1991). The structures of AChE with the bound inhibitors; decamethonium, tetrahydroaminoacridine (Tacrine) and edrophonium (Harel et al., 1993), and 1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51; J.L. Sussman, personal communication), have been determined. These show that ligand binding at the peripheral site also do so by interaction with pi electrons, in this instance with the residue Trp-279.

Substrate Inhibition.
The inhibition of AChE by excess substrate is one of the key features that distinguish it from BChE. BChE exhibits the converse substrate activation, and both phenomena are likely to be related to the binding of substrate, and to the catalytic mechanism of the enzymes. It is not known whether substrate inhibition has a biological role, or is simply a consequence of the structure and mechanism of AChE. Various explanations have been proposed to account for the different responses of AChE and BChE to excess substrate. Rosenberry (1975) suggested that the rate-limiting step in catalysis was formation of an induced fit complex, and substrate inhibition was brought about by interference with this step. It has also been proposed that substrate inhibition is

due to deacylation being retarded by binding of a second ACh molecule to the anionic sub site of the acyl enzyme (Krupka, 1963). It has been suggested that AChE is allosterically regulated by the binding of ACh to the peripheral site through conformational changes at the active centre (Changeux, 1966). During development, the electrophoretic mobility and sedimentation coefficient of rat brain G4a AChE remain constant, but its kinetic parameters including substrate inhibition change (Inestrosa and Ruiz, 1985). Amphiphilic AChE from mosquito showed no substrate inhibition when freshly extracted, however upon conversion to its nonAmphiphilic derivatives, with thiocyanate (a chaotropic anion), the substrate inhibition returned (Dary and Wedding, 1990). These results indicate that the environment may confer a slight conformational change in AChE, which results in substrate inhibition. The importance of the peripheral site in substrate inhibition has been supported by Radic et al. (1991), based on studies of competition of substrate with the peripheral site ligand propidium. However studies on chicken AChE, which lacks the Tyr-70 and Trp-279 residues of the T. californica peripheral site, reveal substrate inhibition characteristics similar to those of T. californica AChE (Eichler et al., 1994). The mutations carried out so far, which abolish substrate inhibition, can be divided into two groups; those at the active site, and those at the peripheral site. Mutations of recombinant human AChE (rhAChE) at the active site have concentrated on the glutamate residue next to the catalytic serine. Mutations Glu-202 (199) Asp, Glu-202 (199) Gln and Glu-202 (199) Ala all abolish substrate inhibition (Shaffer man et al., 1992), the same effect has also been demonstrated by mutation Glu- 199Gln in T. marmorata AChE (Gibney et al., 1990). In rhAChE, mutants of an aspartate at the gorge entrance Asp-74 (72) Asn, Asp-74 (72) Gly and Asp-74 (72) Lys all abolish substrate inhibition, possibly by inducing a conformational change. Mutation of a gorge entrance tryptophan residue Trp-286 (279) Ala reduces substrate inhibition, probably by a similar mechanism (Shaffer man et al., 1992). Mutations on mouse AChE have also been studied, Phe-297 (290) Ile not only eliminates substrate inhibition, but confers elements of substrate activation characteristic of BChE (Radic et al., 1993).

NOSTOCARBOLINE
In collaboration with Prof. Jüttner's group at Zurich University, we isolate novel natural products possessing interesting biological activity from cyanobacteria and elucidate their structure. These molecules than serve as leads for the development of more active, synthetic. Compounds.

We have now isolated the cyanobacterium (blue-green algae)

-carbolinium alkaloid nostocarboline from the
Nostoc

78-12A. The definitive proof of its structure

was delivered by its total synthesis. (Grassi, F. et al. (2003). Nostocarboline.also is a potent inhibitor of butyrylcholinesterase (13.2 M). This value is comparable to the inhibitory power of galanthamine, an approved drug for the treatement of Alzheimer¶s disease.
Moreover, we found that the related deschlorocompound or 2-

methylnorharmane is also a butyrylcholinesterease inhibitor (11.2

M). This compound

might be endogenous in humans as it was found in post mortem human brain. Interestingly, the biological role of this compound is unknown. Based on our data, we propose that methylnorharmane is an endogenous ligand to proteins relevant to neurological disorders. Nostocarboline thus serves as an important lead structure for the development of novel neurochemicals. Substances from nature have provided mankind with fibres, food, drugs and medicines. These natural products contain the evolutionary wisdom of ages as they have been evolved to serve a special task such as inhibiting certain enzymes. These unique functions can be exploited to understand biological processes or even to treat diseases (bioprospecting).

CHEMICAL IDENTIFICATION
Gladstone Study Links AD with Toxic Protein Fragments - New research from the Gladstone Institute of Neurological Disease details exactly how a mutant forms of the protein apolipoprotein E, also known as apoE, is a causative factor for AD. It pinpoints mitochondria, the organelles within cells designed to turn glucose into energy, as a key site that specific fragments of a particular form of apoE attack, leading to the neuronal death characteristic of AD. Several years ago, Yadong Huang and his scientific team found that apoE (a protein comprised of 299 amino acids whose apoE4 isoform has been known for the last decade to be the most significant genetic risk factor for AD) is subject to cleavage that results in fragments that are toxic to neurons.³This study shows which parts of apoE are toxic and gives hints as to the site of its action,´ says Huang. The research team investigated the cellular and molecular mechanisms of the neuro-toxicity caused by apoE4 fragments, performing a series of studies in cultured mouse neuronal cells.( Scott, L. and K. Goa (2000). The cells expressed apoE fragments of various lengths and with mutations designed to enable the investigators to determine precisely which portions of the fragment ± that is, which of apoE4¶s 299 amino acids ± are responsible for its detrimental effects. Research findings showed that fragments containing both the lipid- and receptor-binding regions, but lacking the C-terminal 27 amino acids (273-299), were found to be neurotoxic. The toxic fragments appear in the mitochondria, where they impaired membrane integrity and mitochondrial function. ³Blocking interaction of apoE4 fragments with the mitochondria is a potential new strategy for inhibiting the detrimental effects of apoE4 in AD and other neurological diseases,´ Huang explains. ³AD is a complex condition,´ says co-author (Robert W. Mahley, MD, PhD, president of the Gladstone Institutes and UCSF professor of pathology and medicine. ³Many factors seem to be involved, and all need to be explored to help us find a way to combat this terrible disease. We are very excited about these particular results because they point to a new and potentially valuable therapeutic strategy.´ PR 12/14/05 Proceedings of the National Academy of Science 12/20/05 vol. 102, no. 51 18694-18599 Free copy available.

Drugs
Doctors Study Cancer Drug For AD Patients - A prostate cancer treatment may help slow and even stop the progression of AD. Researchers said they developed the treatment after an AD patient who developed prostate cancer started using a medicine called LUPR, which is also known as Lupron. Earlier clinical trials using Lupron injections on AD patients showed the drug stabilized the disease. (Banerjee, C. et al. (2000). Now, researchers are looking into another delivery method, including a tiny tube loaded with the medicine that¶s inserted just under the skin in the abdomen. Researchers said that method would slowly release the medication over eight weeks. ³You get a better delivery of medicine over that eight weeks than giving it by injection. When you give it by injection, your blood level goes up and then it goes down and it¶s gone. When you give it this way, it¶s slowly released and it¶s more available to the body to work, so it works over a longer period of time,´ AD researcher Dr. Stephen Salloway said. This study continues to determine the effectiveness of the treatment. WBALChannel.com 12/27/05. Natural Compound from ³Pond Scum´ Shows Potential Activity Against AD - A compound isolated from a cyanobacterium, a type of blue-green algae known as Nostoc, shows promise of becoming a natural drug candidate for fighting AD and other neurodegenerative diseases, according to an in vitro study by researchers in Switzerland. It is believed to be the first time that a potent agent against AD has been isolated from cyanobacteria, commonly known as ³pond scum.´ Cyanobacteria and other marine natural products have been increasingly found to be a promising source of drug candidates for fighting a variety of human diseases, including cancer and bacterial infections, but their chemistry has been largely unexplored, experts say. Now, a common marine organism could lead to yet another potential health benefit, says study leader Karl Gademann, Ph.D., an organic chemist at the Swiss Federal Institute of Technology (ETH) in Zürich. Gademann¶s lab specializes in identifying, synthesizing and studying new bioactive compounds from natural sources. There is no cure for AD at present, although

cholinesterase inhibitors have shown promise for delaying or preventing the symptoms of mild to moderate forms of the disease, experts say. The newly isolated compound, nostocarboline, was shown to be a potent inhibitor of cholinesterase -- a brain chemical thought to be important for memory and thinking -- whose breakdown has been associated with the disease¶s progression. The natural compound¶s potency is comparable to galanthamine, a cholinesterase inhibitor already approved for the treatment of AD, the researchers say. As with any promising structure, it could be many years before the new compound is tested as a drug candidate in humans, the scientist¶s caution. Innovations Report 12/29/05 Journal of Natural Products 68(12) 1793-1795 (12/26/05)

Testing
Study Links Fatty Myelin Breakdown to AD - The breakdown of myelin, a sheet of fat that insulates nerves and helps speed messages through the brain, appears to be a key contributor to the onset of AD, a study published on 1/2/06 said. Researchers at UCLA, who reported their findings in the journal Archives of General Psychiatry, used magnetic resonance imaging to assess the breakdown of myelin in 104 healthy adults between the ages of 55 and 75. They found the severity and rate of myelin breakdown was correlated with the type of APOE, or apolipo-protein-E, gene a person had. Previous research has shown APOE status to be the second biggest risk factor for the disease after aging, with a version called APOE-4 putting persons at highest risk. The study found the breakdown of myelin, a natural part of the aging process, proceeded most rapidly for those with APOE4, less so for those with APOE-3 and most slowly for those with APOE-2. APOE-2 is thought to offer some protection from AD, (Guan, Z. et al. (2003). while APOE-3 is seen as neutral. ³These new findings offer, for the first time, compelling genetic evidence that myelin breakdown underlies both the advanced age and the principal genetic risks for AD,´ said Dr. George Bartzokis, professor of neurology at UCLA¶s David Geffen School of Medicine. Myelin has very high cholesterol content. As it builds up, cholesterol levels in the brain increase and eventually promote the production of a toxin that attacks the myelin. Bartzokis said in an interview the study opened up a potential avenue to preventive care since it showed you could track one of the underlying factors for the progressive brain disorder, which afflicts an estimated 4.5 million Americans. Combining

genetic testing with an MRI evaluation of myelin breakdown could help assess potential preventive treatments, he said. Reuters 1/2/06 Archives of General Psychiatry 2006; 63:63-72

Chemical and physical properties
Description: nostocarboline is major component of cyanobacterium of extract. In its pure form, it is an odorless green colour or colourless.crystal melting point: 218 c Solubility: sol in water (clear, colourless solution at 5mg/ml), acetone, ethanol, pyridine, and tetrahydrofuran.

Toxicity:
Pond scum is nontoxic both in acute dosage and high long-term dosage. It has no potential for causing mutations of both defects, and has no adverse effect on fertility, pregnancy or nursing.

Chemical and physical properties
Description: nostocarboline is major component of cyanobacterium of extract. In its pure form, it is an odorless green colour or colourless.crystal melting point: 218 c Solubility: sol in water (clear, colourless solution at 5mg/ml), acetone, ethanol, pyridine, and tetrahydrofuran.

Toxicity:
Pond scum is nontoxic both in acute dosage and high long-term dosage. It has no potential for causing mutations of both defects, and has no adverse effect on fertility, pregnancy or nursing.

MATERIALS AND METHOD
Docking is a small object in to the groove of anther big object. In Bioinformatics, the fitness of a small molecule (a probable drug candidate), in the active site (a binding pocket) of a target protein structure is termed as docking. Here, its also possible to predict whether the small molecule can bind, its also possible to predict how well it binds, in other terms, how much stable is the binding. After docking a small molecule with a large molecule, the resulting docked structure is called as a complex. A target molecule, a drug lead molecule and a computer program docking are pre-requisites for any rational drug-discovery project. Now, having got the target protein and the drug of interest, its time to check, if this drug would bind to the `target and bring out the necessary effect. This binding is convent ally tested in the natural chemistry laboratory, wetting the hands but doing the same thing in the computers, in a simulated environment, using specialized software, without wetting the hands is the docking.

TOOLS FOR DOCKING
Docking is a bit cumbersome or costly affair. Either you have to be highly skilled enough to use open source docking tools, to save money or you have to spare several lakes if you need comfortable docking. There are several methods in docking. Generally all of them involve GRID generation and scoring techniques.

APPLICATION OF DOCKING
Docking is used for structure-based drug discovery, where docking it with the known well computationally tests the binding efficiency of a particular drug studied target structure. Docking is used for virtual screening. Where, every molecule in a given chemical compound database, is virtually docked against a given target structure and a best

compound with low energy is discovered. Docking could also be used to study the protein-protein interactions. Here its not the actual docking of a small molecule with a large molecule, but its the study of interaction of one protein with another protein. Of course, this also applies the same docking principles and methods, but with minor variations.

HEX
Hex is an interactive molecular graphics program for calculating and displaying feasible docking modes of pairs of protein and DNA molecules. Hex can also calculate smallligand/protein docking (provided the ligand is rigid), and it can superpose pairs of molecules using only knowledge of their 3D shapes. ] The main thing, which distinguishes Hex from other macromolecular docking programs and molecular graphics packages, is its use of spherical polar Fourier correlations to accelerate the docking and superposition calculations. The graphical nature of Hex came about largely because I wanted to visualize the results of such docking calculations in a natural and seamless way, without having to export unmanageably many (and usually quite big) coordinate files to one of the many existing molecular graphics packages. For this reason, the graphical capabilities in Hex are relatively primitive compared to commercial packages, although these days one can do quite a lot with a few calls to OpenGL. Nonetheless, if your main interest is in modeling macromolecular docking, then please read on. Hex may have something new to offer. In Hex's docking calculations, each molecule is modeled using 3D parametric functions that are used to encode both surface shape and electrostatic charge and potential distributions. The parametric functions are based on expansions of real orthogonal spherical polar basis functions. Essentially, this allows each property to be represented by a vector of coefficients. Hex's surface shape representation uses a novel 3D surface skin model of protein topology, whereas the electrostatic model is derived from classical electrostatic theory. By writing an expression for the overlap of pairs of parametric functions, one can derive an expression for a docking score as a function of the six degrees of freedom in a rigid body-docking search. With suitable scaling factors, this

docking score can be interpreted as an interaction energy, which we seek to minimize. Due to the special orthogonal property of the basis functions, the correlation (or overlap as a function of translation/rotation operations) between a pair of 3D functions can be calculated using expressions, which involve only the original expansion coefficients. In many respects, this approach is similar to conventional fast Fourier transform (FFT) docking methods that use a Cartesian grid to perform the Fourier transforms. However, the FFT approach only accelerates a docking search in three (transnational) degrees of freedom whereas with a spherical polar approach, we can both translate (with some effort) and rotate (relatively easily) the coefficient vectors to generate and evaluate candidate-docking orientations in what is effectively a six dimensional Fourier correlation. In the spherical polar approach, it is natural to assign the six rigid body degrees of freedom as five Euler rotation angles and an intermolecular separation. Thus, in complete contrast to the FFT approach, the rotational part of a docking search is the "easy bit" and modeling translations becomes the "hard part". Fortunately, however, only a few translations (typically about 40 steps of 0.75 Angstroms) are required to complete a six dimensional docking search. A further advantage of the spherical polar approach is that it is easy to constrain the docking search to one or both binding sites, when this knowledge is available, simply by constraining one or two of the angular degrees of freedom. This can reduce docking times to a matter of minutes on modern workstations. So, depending on how well a particular FFT algorithm is implemented (and on who you believe!), I claim that Hex is somewhere between 10 and 100 times faster than conventional FFT docking algorithms. Closely related to the protein-docking problem is the molecular similarity problem - i.e. how to find the relative orientation of a pair of similar molecules such that some measure of the similarity (difference) between the molecules is maximized (minimized). Both problems involve translating and rotating one or both molecules into the desired orientation. However, to a first approximation, the similarity problem can be reduced to a three dimensional rotational search by initially placing both molecules in a common coordinate system.

In fact, much of the early development of Hex concentrated on displaying and superposing protein surface shapes using two-dimensional spherical harmonic expansions to represent surface shapes parametrically. This proved to be a fast and accurate way to superpose pairs of similar protein molecules but this type of 2D surface approach does not encode sufficient detail to give a viable docking algorithm. It was this observation that prompted the development of our 3D density model of molecular shape.

SOLID SURFACES
Solid molecular surfaces may be drawn using the Graphics ... Solid Surfaces control panel. These surfaces are calculated using a novel marching tetragons algorithm to contour a Gaussian density representation of the atoms of each molecule. The surface skins used in the docking correlations are calculated using this Gaussian density approach. The Colour Mode selector allows surfaces to be colored by atom colour (the default), electrostatic potential or charge density, or by using the "classic" blue-red colour scheme used in earlier versions of Hex. For electrostatic surfaces, a colour ramp is calculated which initially shows positive potentials/charges in blue, negative values in red, and intermediate values in white. The Surfaces ... Colour Ramp button may be used to activate a simple graphical controller for the colour ramp. The electrostatic potential and charge density displays are calculated from the in vacuo global charge density expansion using the current spherical polar docking expansion order, N. The colours in a charge density display may appear somewhat "washed-out" compared to (perhaps more familiar) potential displays. This is because the potential is calculated directly from the charge density using Poisson's equation, and the del-squared form of this equation strongly emphasises any local variation in the charge density. So if the potentials "look right" then the charge density is also correct. NB. Hex uses a relative permittivity value of 8, instead of the more usual value of around 80. So, in addition to Hex's in vacuo assumption, the numerical values for the potential are likely to differ from other software. Some molecules may have internal cavities, and these can produce one or more contour surfaces in additional to the primary external surface. Other molecular structures (e.g.

structures with many waters, or well-separated domains) may also give multiple surfaces when contoured. As the contouring algorithm implicitly produces positively oriented surfaces (outward normals, positive total volumes), it is convenient to assign each calculated surface to one of two possible classes: Primary (positive volumes, outward normals), or Secondary (negative volumes, inward normals). By default, only Primary surfaces are displayed, but this may be changed using the Draw Surface control. In addition to the default Gaussian surface, the Surface Type selector provides the option to draw contoured density functions of the Sigma and Tau surface functions as used by the docking algorithm. These two density functions allow the shape functions used in docking calculations to be visualized. Sigma is the external skin density function, typically calculated with a probe radius of 1.4Å, and Tau is the interior density function (effectively, the van der Waals volume). For relatively small molecules, and when using high expansion orders (N), the Tau density can be seen to give a remarkably good representation of the initial (atomic) Gaussian density. The order of the 3D expansion (default N=25) is taken from the Docking ... Final Search parameter in the Docking Control panel. Both the Sigma and Tau surfaces are contoured using a hard-wired density value of 0.25. However, reconstructing each 3D density from the shape expansion coefficient vectors is a relatively expensive calculation. This display mode is mainly intended to illustrate the internal representations used in docking. The Sigma/Tau Shift slider controls how the shape (and electrostatic) expansion coefficients are computationally translated using the T(R) translation matrices prior to reconstructing the surface from translated expansion coefficients. Thus it is possible to view graphically how the representation used in the docking correlation degrades with increasing distance from the origin. In this display mode, the receptor surface is translated in the negative z direction, and the ligand surface is translated in positive z for ease of viewing. You should see a blurring of those portions of each molecule furthest from the origin, although the shapes of the surface regions near the origin are very well preserved after translation. NB. In Hex's docking correlations, all computational translations are applied entirely to the receptor. Be aware that drawing surfaces can use a lot of memory. Drawing the surface of a large molecule using a fine (0.25-0.5 Ångstrom) grid can use hundreds of megabytes of

memory. Attempting to draw a complex surface on a machine with insufficient memory could cause the machine to hang.

SPHERICAL HARMONIC SURFACES
The Harmonic Surface control panel controls the way in which spherical harmonic molecular surfaces are calculated and displayed. Spherical harmonic molecular surfaces are generated from the dot surfaces calculated by Hex's internal dot surface algorithm, according to the parameters set in the Harmonic Surface control panel. These parameters are distinct from similar parameters in the Dot Surfaces control panel, which generates dot surfaces purely for visualization (see section 3.6 Dot Surfaces). When Hex calculates a spherical harmonic molecular surface, it first finds the shape of an angular envelope, which just enclosed all dots of the dot surface. This envelope is made up of a list of approximately evenly spaced angular sample points (derived from the vertices of a tessellated icosahedrons), along with a radial distance from the origin to the surface at the corresponding angular sample. Note that the surface envelope does not properly handle multi-valued, or re-entrant, surfaces. That is, if a ray from the origin cuts the molecular surface more than once, only the most extreme cut point is recorded for the given angular sample. Nonetheless, this type of global envelope often gives a remarkably good low-resolution representation of most protein surfaces.

DOT SURFACES
These days, dot surfaces are somewhat of a historical remnant in Hex. They used to be used as an approximate but fast way to calculate the molecular surface, the solventaccessible surface and the van der Waals surface for docking. However, docking and superposition calculations now use a much more accurate method based on contouring Gaussian density functions to calculate these two surfaces. Nonetheless, its still sometimes useful to be able to draw dot surfaces. If nothing else, dot surfaces give a fast way to verify that Hex is recognising all the atoms in a PDB file because sometimesunrecognised atoms may not be drawn in the other drawing modes, whereas the dot

surface calculation always uses all atoms, regardless of their type. Spherical harmonic surface envelopes are also calculated from the sampled dot surface. The calculation and display of dot surfaces is controlled by settings in the Dot Surface control panel. The short-cut Dots button on the right-hand border of the main window provides a fast way to toggle the display of dot surfaces. By default, Hex displays a molecular surface calculated using a 1.4 Ångstrom radius probe sphere. Dot surfaces are calculated by covering the surface of each atom with a set of sample points generated from an icosahedral tessellation of the sphere. The default dot density is 162 dots/atom, but this may be changed using the Dot Density selector. Any dots, which are occluded by neighboring atoms, are immediately discarded. If the Probe Radius is zero, then this gives the van der Waals surface. Otherwise, the program calculates the molecular surface and solvent-accessible surface by rolling a probe sphere over the surface (non-occluded) atoms of the molecule. When Hex calculates a molecular surface, non-occluded atom dots are classified as one of convex-accessible, toroidal-reentrant or singular-reentrant. These classifications correspond to the type of contact the dot should make with a probe sphere being rolled over the molecule. The convex-accessible dots are those, which may be touched by the probe when the probe sphere is not simultaneously in contact with any other atom. All

ANIMATION
The Animation Control panel allows you to run a "movie" of either the results of a docking calculation, or a sequence of models from an NMR structure, for example, and it controls how the scene "spins" if you perform a mouse drag-release action. After a docking run, pressing Start in the Graphics. Animation control panel will cause Hex to draw each docking solution in turn. The Frame Rate slider controls the rate at which orientations are drawn. Similarly, if you have loaded a PDB file that contains several NMR model structures, setting the Movie Type to Receptor Models or Ligand Models, as appropriate, and pressing Start will show a movie of the sequence of models. The orientations, or frames, of both types of movie may be shown just once, or cycled forever. The movie can be stopped at the current frame by pressing the Stop button. The Frame Rate slider controls the speed of the movie.

The maximum frame rate achievable will depend on the speed of the CPU and on whether your machine has hardware-accelerated graphics. The Frame Rate thus defines a requested rate. Actual performance will vary. But please note, Hex never hogs the CPU in a tight loop on input events, even during an animation, as do many other programs (which shall remain nameless). Thus it should still be possible to perform other activities while a movie is running without things becoming sluggish. You can spin the scene by dragging with Button 1 or Button 2 to start a rotation and by releasing the button while still moving the mouse to initiate a continuous rotation (spinning). When spinning, the rotatation angle is incremented by the current value in the Spin Angle slider, and new rotational increments are drawn at the current Frame Rate. If desired, the mouse button action that initiates spinning can be enabled/disabled with the Enable Spinning toggle. Spinning is enabled by default, and it is possible to spin a movie.

DOCKING MOLECULES
In order to run a docking calculation in Hex, you will need to load a receptor and a ligand PDB structure using the File pull-down menu. If you want to test the docking algorithm by docking two separately determined sub-units of a complex for which the crystal structure is also available, you can also load the complex structure which will be used as a reference orientation to evaluate the accuracy of the docking prediction. Generally, you will have to remove water molecules and any other hetero molecules prior to docking. You can do this globally using the Hetero Control menu panel. If more detailed control is required, you will probably have to edit each PDB file manually. It may also be necessary to remove other chains in the PDB file or to shorten a chain to the domain of interest in docking. For example, when docking an antigen to an antibody it is usually advisable to delete all but the Fv fragment of the antibody structure (although the program has been used to dock a protein G molecule to a complete Fab fragment). Having edited your PDB files, you should have a receptor and a ligand file which contain only the receptor and ligand molecules, respectively, and (optionally) a complex file, which contains both molecules in the docked orientation. When using a complex structure, you should ensure that the chain names are consistent with those of the receptor and ligand because Hex uses the chain labels to identify and hence superpose

corresponding pairs of alpha-carbon atoms from each chain in order to calculate RMS deviations between the docked position of the ligand and its position in the known complex.

CLUSTERING DOCKING RESULTS

As Hex uses essentially a brute-force search approach to the docking problem, it is advisable to over-sample the search space rather than to risk missing a good solution by under-sampling the space. However, this can cause multiple similar but incorrect orientations (false-positives) to push good solutions down the list. By default, Hex uses a simple clustering algorithm to group spatially similar docking orientations. Each docking solution is first ordered by energy, and the lowest energy solution is made the seed orientation for the first cluster. The list is then searched for other similar orientations whose main-chain alpha-Carbon RMS deviation is within a given threshold (default 2Å RMS) of the seed orientation, and these orientations are then assigned to the first cluster. The process is then repeated starting from the next lowest unassigned orientation, until all solutions have been assigned to a cluster.

CONCLUSION.
The inhibition of cholinesterase by nostocarboline was determined by Insilco method. The carboline showed a high affinity towards the binding site of beta secretase. The internal energy was found to be minimum, thus indicating that a good docking had taken place. The enzyme beta secretase is a quaternary protein with four chains interlinked and all the chains have a similar amino acid sequence. Based on the active site pattern nostocarboline was docked to the receptor enzyme. Alzheimer¶s disease is a neurodegenerative disorder with a high fatality rate. Usually aged members of the society suffer with Alzheimer¶s disease. It is so prevalent that a cure or prevention for this disorder is a need of the hour. So on this basis, this project was done. Based on literature review the ligand [nostocarboline] was chosen and docked to the active site of cholinesterase [which is involved in the pathogenesis of Alzheimer¶s disease]. The future scope of this study is that more in vivo and in vitro studies can be performed to determine id nostocarboline is really effective in the prevention or cure of Alzheimer¶s disease. Further drugs can be designed on the active components of nostocarboline.also docking results can be used as reference for other experiments. Thus, cure for Alzheimer¶s disease has a lot of scope in the future.

SUMMARY
Alzheimer¶s disease is disorder of the brain, which causes memory loss in aged people. It is due to genetic mutation as well as sporadic occurrences. y Since there are no existing drugs that can stop the manifestation of Alzheimer¶s disease, much research is required in this field. y There is an enzyme named cholinesterase, which plays a very important role in

y

the formation, or plaques, which are the predisposing factors to Alzheimer¶s disease. y On sequence analysis of cholinesterase it was found that it belongs to the peptidase AI family with a asp domain and a eukaryotic aspartyl protease active site. y On studying the secondary structure of the enzyme a maximum of random coils were predicted. y The region between amino acids 458 and 478 is a transmembranic region. Thus the protein is a transmembranic region. Thus the protein is Tran membrane region. y Based on literature study a cyanobacturiem isolated named nostocarboline was converted to a 3D structure and then docked with nostocarboline. y y The conformation with the lowest energy is the best conformation. The idea is that based on this study, further studies can be carried out to find an effective inhibitor for cholinesterase and thus prevent Alzheimer¶s disease.

REFERENCES
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3. 3Kausler, Donald H., and Barry C. Kausler. Alzheimer's disease. In there the graying of America: an encyclopedia of aging, health, mind, and behavior. Urbana, University of Illinois Press, c1996. p. 23-28. 4. Gifford, D. R. and Cummings, J. L., "Rating dementia screening test: methodological Standards to rate their performance, Neurology, 52, 224-227, 1999. 5. Relkin, N., "Screening and early diagnosis and dementia", The American Journal of Managed Care, 6(22) supplement: S1111-S1124, 2000. 6. Relkin, N., "Screening and early diagnosis and dementia", The American Journal of Managed Care, 6(22) supplement: S1111-S1124, 2000. 7. Banerjee, C. et al. (2000). Cellular Expression of alpha-7 Nicotinic Acetylcholine Receptor Protein in the Temporal Cortex in Alzheimer¶s and Parkinson¶s DiseaseA StereologicalApproach.NeurobiologyofDisease7,666-672. 8. Bourin, M. et al. (2000). Nicotinic receptors and Alzheimer's disease. Current Medical ResearchandOpinion19,169-177. 9. Doody, R. S. et al., "Donepezil Study Group. Open-Label, multicenter, phase 3 extensionStudy of the safety and efficacy of donepezil in patients with Alzheimer's disease", Archives of Neurology, 58, 427-433, 2001. 10. Auriacombe, S. et al., "Efficacy and safety of rivastigmine in patients with Alzheimer¶s Disease who failed to benefit from treatment with donepezil", Current Medical Research and Opinions, 18(3): 129-138, 2002. 11. Boustani, M., et al., "Screening for dementia in primary care: a summary of the evidence for The U.S. preventative services task force", Annals of Internal Medicine, 138(11), 927-937, 2003. 12. Trinh, N. et al., "Efficacy of cholinesterase inhibitors in the treatment of neuropsychiatricSymptoms and functional impairment in Alzheimer's disease: A meta-analysis", JAMA, 289(2): 210-216, 2003. 13. Shankle, W. R. et al., "Method to improve the detection of mild cognitive impairment´, Proceedings of National Academy of Science, 102 (13), 4919-4924, 2005.

14. Shankle, W. R. et al., "Method to improve the detection of mild cognitive impairment", Proceedings Of National Academy of Science, 102 (13), 4919-4924, 2005.Bjorklund M, Jakala P, Schmidt B, Riekkinen M, Koivisto E, Riekkinen P JarAn indirect cholinesterase inhibitor, metrifonate, increases neocortical EEG arousal in rats. Neuroreport. 1996 Apr 10; 7(5): 1097-101. 15. Bjorklund M, Jakala P, Schmidt B, Riekkinen M, Koivisto E, Riekkinen P Jr. An indirect cholinesterase inhibitor, metrifonate, increases neocortical EEG arousal in rats. Neuroreport. 1996 Apr 10; 7(5): 1097-101. 16. D Maciejewski, PD Crowe, EB De Souza, and DP Behan Regulation of corticotrophin releasing factor-binding protein expression in cultured rat astrocytes J Pharmacol Exp There 1996 278: 455-461. 17. AU Trendelenburg, N Limberger, and K Starke The presynaptic alpha-2 autoreceptorin pig brain cortex are alpha-2A J Pharmacol Exp There 1996 278: 462-467. 18. N Macrez-Lepretre, JL Morel, and J Mironneau Angiotensin II-mediated activation of L-type calcium channels involves phosphatidylinositol hydrolysisindependent activation of protein kinase C in Rat portal vein myocytes J Pharmacol Exp There 1996 278: 468-475. 19. LL Devaud, RH Purdy, DA Finn, and AL Morrow Sensitization of gamma-amino butyric acid A receptors to neuroactive steroids in rats during ethanol withdrawal J Pharmacol Exp There 1996 278: 510-517. 20. L Diao and TV Dunwiddie Interactions between ethanol, endogenous adenosine and adenosine uptake in hippocampal brain slice J Pharmacol Exp There 1996 278: 542-546. 21. Conroy, W. et al. (2003). Potentiation of alpha-7-Containing Nicotinic Acetylcholine Receptors by Select Albumins. Molecular Pharmacology 63, 419428. 22. Dineley, K. et al. (2002). Beta-Amyloid Peptide Activates alpha-7 Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes. The Journal of Biological Chemistry 277, 25056-25061.

23. Doggrell, S. and S. Evans (2003). Treatment of dementia with neurotransmission modulation. Expert Opinion on Investigational Drugs 12, 1633-1654. 24. Dougherty, J. et al. (2003). Beta-Amyloid Regulation of Presynaptic Nicotinic Receptors in Rat Hippocampus and Neocortex. The Journal of Neuroscience 23, 6740-6747. 25. Frier, D. and C. Herron (2003). Nicotine Enhances the Depressive Actions of ABeta1-40 on Long-Term Potentiation in the Rat Hippocampal CA1 Region In Vivo. Journal of Neurophysiology 89, 2917-2922. 26. Fu, W. and J. Jhamandas (2003). Beta-Amyloid Peptide Activates Non-alpha-7 Nicotinic Acetycholine Receptors in Rat Basal Forebrain Neurons. Journal of Neurophysiology 90, 3130-3136. 27. Giacobini, E .(2003). Cholinergic function and Alzheimer's disease. International Journal of Geriatric Psychiatry 18, S1-S5. Grassi, F. et al. (2003). Amyloid Beta1-42 peptide alters the gating of human and mouse alpha--bungarotoxin-sensitive nicotinic receptors. The Journal of Physiology 547, Disease. Drugs 147-157. 61, 41-52. 28. Grutzendler, J. and J. Morris (2001). Cholinesterase Inhibitors for Alzheimer's 29. Guan, Z. et al. (2000). Decreased Protein Levels of Nicotinic Receptor Subunits in the Hippocampus and Temporal Cortex of Patients with Alzheimer's Disease. Journal of Neurochemistry 74, 237-243. 30. Guan, Z. et al. (2003). Loss of Nicotinic Receptors Induced by Beta-Amyloid Peptides in PC12 Cells: Possible Mechanism Involving Lipid Peroxidation. Journal of Neuroscience Research 71, 397-406. 31. Kern, W. (2000). The brain alpha-7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer¶s disease: studies with DMXBA (GTS-21).. Behavioural Brain Research 113, 169-181. 32. Maelicke, A. et al. (2000). Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer¶s disease. Behavioural Brain Research 113, 199-206.

33. Nashmi, R. et al. (2003). Assembly of alpha-4Beta2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons. The Journal of Neuroscience 23, 11554-11567. 34. O'Neill, M. et al. (2002). The Role of Neuronal Nicotinic Acetylcholine Receptors in Acute and Chronic Neurodegeneration. Current Drug Targets. CNS and Neurological Disorders 1, 399-411. 35. Pettit, D. et al. (2001). Beta-Amyloid1-42 Peptide Directly Modulates Nicotinic Receptors in the Rat Hippocampal Slice. The Journal of Neuroscience 21(RC120)., 36. Sabbagh, M. et al. (2002). The nicotinic acetylcholine receptor, smoking, and Alzheimer'sdiease.JournalofAlzheimer¶sDisease4,317-325. 37. Scott, L. and K. Goa (2000). Galantamine: A Review of its Use in Alzheimers Disease. Drugs 60, 1095-1122. 38. Dougherty, J. et al. (2003). Beta-Amyloid Regulation of Presynaptic Nicotinic Receptors in Rat Hippocampus and Neocortex. The Journal of Neuroscience 23, 6740-6747. 39. Frier, D. and C. Herron (2003). Nicotine Enhances the Depressive Actions of ABeta1-40 on Long-Term Potentiation in the Rat Hippocampal CA1 Region In Vivo. Journal of Neurophysiology 89, 2917-2922. 40. Fu, W. and J. Jhamandas (2003). Beta-Amyloid Peptide Activates Non-alpha-7 Nicotinic Acetycholine Receptors in Rat Basal Forebrain Neurons. Journal of Neurophysiology 90, 3130-3136. 41. Giacobini, E .(2003). Cholinergic function and Alzheimer's disease. International Journal of Geriatric Psychiatry 18, S1-S5. 42. Grassi, F. et al. (2003). Amyloid Beta1-42 peptide alters the gating of human and mouse alpha--bungarotoxin-sensitive nicotinic receptors. The Journal of Physiology 547, Disease. Drugs 147-157. 61, 41-52. 43. Grutzendler, J. and J. Morris (2001). Cholinesterase Inhibitors for Alzheimer's

44. Guan, Z. et al. (2000). Decreased Protein Levels of Nicotinic Receptor Subunits in the Hippocampus and Temporal Cortex of Patients with Alzheimer's Disease. Journal of Neurochemistry 74, 237-243. 45. Guan, Z. et al. (2003). Loss of Nicotinic Receptors Induced by Beta-Amyloid Peptides in PC12 Cells: Possible Mechanism Involving Lipid Peroxidation. Journal of Neuroscience Research 71, 397-406. 46. Kern, W. (2000). The brain alpha-7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer¶s disease: studies with DMXBA (GTS-21).. Behavioural Brain Research 113, 169-181. 47. Kern, W. (2000). The brain alpha-7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer¶s disease: studies with DMXBA (GTS-21).. BehaviouralBrainResearch113,169-181. 48. Maelicke, A. et al. (2000). Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer¶s disease. Behavioural Brain Research 113, 199-206. 49. Nashmi, R. et al. (2003). Assembly of alpha-4Beta2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons. The Journal of Neuroscience 23, 11554-11567. 50. O'Neill, M. et al. (2002). The Role of Neuronal Nicotinic Acetylcholine Receptors in Acute and Chronic Neurodegeneration. Current Drug Targets. CNS and Neurological Disorders 1, 399-411. 51. Pettit, D. et al. (2001). Beta-Amyloid1-42 Peptide Directly Modulates Nicotinic Receptors in the Rat Hippocampal Slice. The Journal of Neuroscience 21(RC120)., 52. Sabbagh, M. et al. (2002). The nicotinic acetylcholine receptor, smoking, and Alzheimer's diease. Journal of Alzheimer¶s Disease 4, 317-325. 53. Scott, L. and K. Goa (2000). Galantamine: A Review of its Use in Alzheimers Disease. Drugs 60, 1095-1122.

54. Dougherty, J. et al. (2003). Beta-Amyloid Regulation of Presynaptic Nicotinic Receptors in Rat Hippocampus and Neocortex. The Journal of Neuroscience 23, 6740-6747. 55. Frier, D. and C. Herron (2003). Nicotine Enhances the Depressive Actions of ABeta1-40 on Long-Term Potentiation in the Rat Hippocampal CA1 Region In Vivo. Journal of Neurophysiology 89, 2917-2922. 56. Fu, W. and J. Jhamandas (2003). Beta-Amyloid Peptide Activates Non-alpha-7 Nicotinic Acetycholine Receptors in Rat Basal Forebrain Neurons. Journal of Neurophysiology Journal of 90, 3130-3136. Psychiatry 18, S1-S5. 57. Giacobini, E .(2003). Cholinergic function and Alzheimer's disease. International Geriatric 58. Grassi, F. et al. (2003). Amyloid Beta1-42 peptide alters the gating of human and mouse alpha--bungarotoxin-sensitive nicotinic receptors. The Journal of Physiology Disease. Drugs 547, 147-157. 61, 41-52. 59. Grutzendler, J. and J. Morris (2001). Cholinesterase Inhibitors for Alzheimer's 60. Guan, Z. et al. (2000). Decreased Protein Levels of Nicotinic Receptor Subunits in the Hippocampus and Temporal Cortex of Patients with Alzheimer's Disease. Journal of Neurochemistry 74, 237-243. 61. Guan, Z. et al. (2003). Loss of Nicotinic Receptors Induced by Beta-Amyloid Peptides in PC12 Cells: Possible Mechanism Involving Lipid Peroxidation. Journal of Neuroscience Research 71, 397-406. 62. Kern, W. (2000). The brain alpha-7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer¶s disease: studies with DMXBA (GTS-21).. Behavioural Brain Research 113, 169-181. 63. Maelicke, A. et al. (2000). Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer¶s disease. Behavioural Brain Research 113, 199-206. 64. Nashmi, R. et al. (2003). Assembly of alpha-4Beta2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal

Mammalian Cells and in Cultured Midbrain Neurons. The Journal of Neuroscience 23, 11554-11567. 65. O'Neill, M. et al. (2002). The Role of Neuronal Nicotinic Acetylcholine Receptors in Acute and Chronic Neurodegeneration. Current Drug Targets. CNS and Neurological Disorders 1, 399-411. 66. Maelicke, A. et al. (2000). Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer¶s disease. Behavioural Brain Research 113, 199-206. 67. Nashmi, R. et al. (2003). Assembly of alpha-4Beta2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons. The Journal of Neuroscience 23, 11554-11567. 68. O'Neill, M. et al. (2002). The Role of Neuronal Nicotinic Acetylcholine Receptors in Acute and Chronic Neurodegeneration. Current Drug Targets. CNS and Neurological Disorders 1, 399-411. 69. Pettit, D. et al. (2001). Beta-Amyloid1-42 Peptide Directly Modulates Nicotinic Receptors in the Rat Hippocampal Slice. The Journal of Neuroscience 21(RC120)., 1-5. 70. Sabbagh, M. et al. (2002). The nicotinic acetylcholine receptor, smoking, and Alzheimer's diease. Journal of Alzheimer¶s Disease 4, 317-325. 71. Wilkinson, D. et al., "The role of general practitioners in the diagnosis and treatment of Alzheimer¶s disease: a multinational survey", Journal of International Medical Research, 32(2), 149-159, 2004. 72. Winblad, B. and Jelic, V., "Long-term treatment of Alzheimer's disease: Efficacy and safety of Acetyl cholinesterase inhibitors", Alzheimer's disease and Associated Disorders, 18(supl.1): S2-S8, 2004. 73. Karl Gademann, Ph.D, Zürich Friedrich Jüttner and Paul Becher, of the University of Zürich and Julien Beuchat, Natural Compound From 'Pond Scum' Shows Potential Activity Against Alzheimer¶s Source: American chemical society, December 27,2005.

APPENDEX
Hex 4.5 starting at Mon Mar 06 16:23:01 2006 on host IBM12. Running HEX_STARTUP file: C:\Program Files\Hex 4.5\data\startup_v4.mac Disc Cache enabled. Using directory: C:\Program Files\Hex 4.5\cache Opened PDB file: C:\pro\protein.pdb, Code = 1XN3 PDB structure has crystal symmetry elements. PDB structure has biological symmetry elements. Loaded PDB file: C:\pro\protein.pdb, (2319 residues, 15700 atoms, 1 models) ASN:N Radius = 1.40, Charge = -0.52 ASN:CA Radius = 1.50, Charge = 0.22 ASN:C Radius = 1.40, Charge = 0.53 ASN:O Radius = 1.50, Charge = -0.50 ASN:CB Radius = 1.70, Charge = 0.00 ASN:CG Radius = 1.40, Charge = 0.68 ASN:OD1 Radius = 1.50, Charge = -0.47 ASN:ND2 Radius = 1.40, Charge = -0.87 ASN:OXT Radius = 1.50, Charge = -0.57 ASN:H Radius = 0.00, Charge = 0.25 ASN:1HD2 Radius = 0.00, Charge = 0.34 ASN:2HD2 Radius = 0.00, Charge = 0.34 GSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGV YVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWE GILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGS LWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRL PKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLR PVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAV SACHVHDEFRTAAVEGPFVTLDMEDCGYN >1XN3 B GSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGV YVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWE GILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGS LWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRL PKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLR PVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAV SACHVHDEFRTAAVEGPFVTLDMEDCGYN >1XN3 C GSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGV YVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWE GILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGS LWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRL PKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLR PVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAV SACHVHDEFRTAAVEGPFVTLDMEDCGYN >1XN3 D GSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGV YVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWE GILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGS LWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRL

PKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLR PVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAV SACHVHDEFRTAAVEGPFVTLDMEDCGYN >1XN3 I KTEEISEVNXVAEF Opened PDB file: C:\pro\ligand.pdb, Code = MOL2 Loaded PDB file: C:\pro\ligand.pdb, (1 residues, 17 atoms, 1 models) [255,36,63] = Firebrick1 Found 1023 MB main memory: setting N_MAX=32. Check threefold = 0 Docking search mode = 6D rotation + translation (optimal). Using intermolecular distance R12 = 81.20, rounded to 81.00 Setting distance range = 61.00-101.00, with steps of 1.00 Calculating surface skins: Grid = 0.60A Contouring surface for molecule 1XN3. Gaussian sampling over 12329 atoms done in 7.48 seconds. Vim: 50.00 MB. Contoured 637548 triangles (318754 vertices) in 2.75 seconds. Culled 241961 short edges in 6 cycles in 8.14 seconds. Surface traversal done in 0.33 seconds - Found 1 surface segments. Primary surface: Area = 49451.75, Volume = 312206.55. Culled 0 small segments in 0.36 seconds. Culling reduced surface complexity by 75 per cent (153612 triangles, 76785 vertices). Total contouring time: 11.58 seconds. Contouring surface for molecule MOL2. Gaussian sampling over 17 atoms done in 0.02 seconds. Contoured 4984 triangles (2494 vertices) in 0.00 seconds. Culled 1863 short edges in 5 cycles in 0.03 seconds. Surface traversal done in 0.00 seconds - Found 1 surface segments. Primary surface: Area = 395.92, Volume = 652.25. Culled 0 small segments in 0.00 seconds. Culling reduced surface complexity by 74 per cent (1258 triangles, 631 vertices). Total contouring time: 0.03 seconds. Calculating radial envelope for molecule 1XN3. Radial envelope done in 0.25 seconds. Sampling surface skins for molecule 1XN3. Generated 121407 exterior and 178625 interior skin grid cells. Exterior skin volume = 26223.91; interior skin volume = 38583.00. Volume sampling done in 0.73 seconds. Calculating radial envelope for molecule MOL2. Radial envelope done in 0.01 seconds. Sampling surface skins for molecule MOL2. Generated 2446 exterior and 375 interior skin grid cells. Exterior skin volume = 528.34; interior skin volume = 81.00. Volume sampling done in 0.02 seconds. Calculating skin coefficients to N = 25...

Integration applied to 301298 cells: 18.81 per cent of the total grid volume. Skin integration to N = 25 done in 26.23 seconds. Calculating surface envelopes to L = 12... Surface envelopes to L = 12 done in 0.03 seconds. Docking will output a maximum of 500 solutions per pair... Nr, Nl, Na, Nd = 642 642 64 41 Total no. Orientations to scan = 1081518336; working buffer size = 1000000. -----------------------------------------------------------------------------Docking 1 pair of starting orientations... Docking receptor: 1XN3 and ligand: MOL2... Receptor 1XN3: Tag = 0 Ligand MOL2: Tag = 0 Working buffer for 1000000 orientations: (27Mb) Initial rotational increments (N=16) Receptor: 642 (15Mb), Ligand: 642 (15Mb) Loading all coefficient vectors into memory... Coefficient rotations done in 1.52 seconds. Starting docking search with N=16, Nalpha=64/64. Estart = -0.00 KJ/mol (Escape=-0.00, Eforce=0.00) R = 61.00 R = 62.00 R = 63.00 R = 64.00 R = 65.00 R = 66.00 R = 67.00 R = 68.00 R = 69.00 R = 70.00 R = 71.00 R = 72.00 R = 73.00 R = 74.00 R = 75.00 R = 76.00 R = 77.00 R = 78.00 R = 79.00 R = 80.00 R = 81.00 R = 82.00 R = 83.00 R = 84.00 R = 85.00 R = 86.00

R = 87.00 R = 88.00 R = 89.00 R = 90.00 R = 91.00 R = 92.00 R = 93.00 R = 94.00 R = 95.00 R = 96.00 R = 97.00 R = 98.00 R = 99.00 R = 100.00 R = 101.00 Estart = -0.00 -> rank 5723497 First pass found 2337994/1081518336 within threshold but *not* including start guess. First pass done in 7 min, 48 sec: 2309165 orientations/sec. Starting guess not found in top 1000000 solutions. Energy range: Emin = -23.59, Emax = -2.22 Top 25000 orientations -> 87386 after distance sub-sampling. Working buffer for 87386 orientations: (3Mb) Surviving rotational steps (N=25) Receptor: 332 (28Mb), Ligand: 97 (9Mb) Loading all coefficient vectors into memory... Coefficient rotations done in 3.34 seconds. Starting docking search with N=25, Nalpha=128/128. Estart = 0.06 KJ/mol (Escape=0.06, Eforce=0.00) R = 60.00 R = 60.50 R = 61.00 R = 61.50 R = 62.00 R = 62.50 R = 63.00 R = 63.50 R = 64.00 R = 64.50 R = 65.00 Estart = 0.06 -> rank 160063 Main pass found 116311 minima within threshold but *not* including start guess. Main pass done in 0 min, 60 sec: 187740 orientations/sec. Starting orientation [alpha=0] (Energy=0.06) ranked 160063 in the search. Starting guess not found in top 87386 solutions. Energy range: Emin = -20.99, Emax = -3.47 Docking correlations for 1XN3:MOL2 done in 8 min, 55 sec.

Docking correlation summary by RMS deviation and steric clashes --------------------------------------------------------------Soln Etotal Eshape Eforce RMS Bumps ---- --------- --------- ------------------------ ----Docked structures 1XN3:MOL2 in a total of 8 min, 55 sec. -----------------------------------------------------------------------------Saving top 500 orientations. Docking done in a total of 9 min, 42 sec. Evaluated 1081518336 orientations at N=16 and 11185408 at N=25. Average: 1878811.72 orientations/sec.

-----------------------------------------------------------------------------Clustering found 180 clusters from 500 docking solutions in 0.03 seconds. ---- ---- ------- ------- ------- ------- ------- ------ --- ----Clst Soln Models Etotal Eshape Eforce Shape Clash Bmp RMS ---- ---- ------- ------- ------- ------- ------- ------ --- ----1 1 000:000 -21.0 -21.0 0.0 0.0 0.0 -1 -1.00 1 2 000:000 -20.8 -20.8 0.0 0.0 0.0 -1 -1.00 1 53 000:000 -17.9 -17.9 0.0 0.0 0.0 -1 -1.00 1 71 000:000 -17.5 -17.5 0.0 0.0 0.0 -1 -1.00 1 93 000:000 -17.2 -17.2 0.0 0.0 0.0 -1 -1.00 1 122 000:000 -16.9 -16.9 0.0 0.0 0.0 -1 -1.00 1 170 000:000 -16.4 -16.4 0.0 0.0 0.0 -1 -1.00 2 3 000:000 -19.9 -19.9 0.0 0.0 0.0 -1 -1.00 2 22 000:000 -18.8 -18.8 0.0 0.0 0.0 -1 -1.00 2 110 000:000 -17.1 -17.1 0.0 0.0 0.0 -1 -1.00 2 125 000:000 -16.9 -16.9 0.0 0.0 0.0 -1 -1.00 2 179 000:000 -16.3 -16.3 0.0 0.0 0.0 -1 -1.00 3 4 000:000 -19.9 -19.9 0.0 0.0 0.0 -1 -1.00 3 20 000:000 -18.9 -18.9 0.0 0.0 0.0 -1 -1.00 3 29 000:000 -18.4 -18.4 0.0 0.0 0.0 -1 -1.00 3 38 000:000 -18.2 -18.2 0.0 0.0 0.0 -1 -1.00 3 48 000:000 -18.0 -18.0 0.0 0.0 0.0 -1 -1.00 3 79 000:000 -17.4 -17.4 0.0 0.0 0.0 -1 -1.00 3 114 000:000 -17.0 -17.0 0.0 0.0 0.0 -1 -1.00 3 121 000:000 -16.9 -16.9 0.0 0.0 0.0 -1 -1.00 3 131 000:000 -16.8 -16.8 0.0 0.0 0.0 -1 -1.00 3 183 000:000 -16.3 -16.3 0.0 0.0 0.0 -1 -1.00 4 5 000:000 -19.7 -19.7 0.0 0.0 0.0 -1 -1.00 4 10 000:000 -19.5 -19.5 0.0 0.0 0.0 -1 -1.00 4 14 000:000 -19.0 -19.0 0.0 0.0 0.0 -1 -1.00 4 21 000:000 -18.9 -18.9 0.0 0.0 0.0 -1 -1.00 4 128 000:000 -16.8 -16.8 0.0 0.0 0.0 -1 -1.00 4 201 000:000 -16.2 -16.2 0.0 0.0 0.0 -1 -1.00 5 6 000:000 -19.5 -19.5 0.0 0.0 0.0 -1 -1.00 5 8 000:000 -19.5 -19.5 0.0 0.0 0.0 -1 -1.00 5 47 000:000 -18.0 -18.0 0.0 0.0 0.0 -1 -1.00

5 6 6 6 6 7 7 7 7 7 7 8 8 8 8 9 9 9 9 9 9 10 10 10 10 10 10 11 11 11 11 12 12 12 12 12 12 12 12 12 13 13 13 13 13 13 14 14 14 14 14 14 15 15 15 15 15

56 7 12 15 42 9 52 59 97 115 169 11 13 24 25 16 45 51 61 85 188 17 35 43 67 148 215 18 28 86 100 19 46 63 64 84 144 178 199 200 23 30 32 90 98 108 26 58 65 94 134 210 27 34 69 81 89

000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000 000:000

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29 29 29 29 30 30 30 30 30 31 31 31 31 32 32 32 32 33 33 33 34 34 34 34 34 34 34 35 35 35 36 36 36 36 37 37 37 37 38 38 39 39 39 40 40 40 40 40 41 41 41 42 42 43 43 43 43

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43 44 44 44 44 45 45 46 46 46 47 47 47 47 47 47 48 48 48 49 49 50 50 50 51 52 52 53 53 54 54 54 54 55 55 55 55 55 55 55 55 55 56 56 56 57 57 57 58 58 58 58 58 58 59 60

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243 000:000

60 60 60 61 61 62 62 62 62 63 63 64 64 65 65 66 66 67 67 67 68 68 69 69 70 70 70 70 71 71 72 72 72 72 73 74 75 75 75 76 77 78 78 78 78 78 78 78 79 79 79 79 79 80 80 81 81

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82 83 83 84 84 84 85 85 86 86 87 87 87 88 88 89 89 89 89 90 90 90 90 90 90 91 91 91 91 91 91 91 92 92 93 93 94 94 95 95 95 95 96 96 97 98 98 99 99 100 100 101 101 101 102 102 103

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103 104 104 104 104 104 105 106 106 107 108 109 109 109 110 111 111 111 111 112 112 113 114 114 115 115 116 116 116 117 117 118 118 119 119 120 120 121 121 121 122 122 123 124 125 126 126 127 128 129 129 129 130 131 131 132 133

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134 135 135 135 135 135 136 136 136 136 137 138 139 139 140 140 140 140 141 141 142 143 144 145 146 146 147 147 147 147 148 149 150 151 151 152 153 153 154 155 156 157 158 159 160 160 161 162 163 164 164 165 166 167 168 169 170

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43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

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100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156

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33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

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90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146

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WEBSITES: www.alzheimersabout.com www.alzheimers.org www.alzheimersdisease.com www.dementia.com www.agingsearch.com www.sciencedaily.com www.alzheimersjournal.com