INSILICO COMBINATORIAL ANALYSIS OF

LEAD COMPOUNDS AGAINST ACIDIC

MAMMALIAN CHITINASE

ACKNOWLDEGEMENT:

First of all I thank Almighty God, for all his blessings and I ever thankful to my celestial guide, for
their supportive guidance through out in my project.

I heartfully thank Miss Akilandeshwari and Mrs.Lesitha who helped me to carry out this project for
six months by providing all facilities in lab and also would like to add a few heart felt words for the
people who have always lent a never-ending hand to complete this project successfully.

I like to thank Mr.Magendaran manager (R &D) of Alchymas chemical industry for helping me design
drug molecules for this project.

I express my heart felt thanks to Mrs.D.Malathi, Director-Technical and Sai’s Biosciences Research
Institute Pvt.Ltd., for providing all facilities during the project work.

I take this opportunity to thank Mr.Jesu sudhan, Mrs.Navya, Mrs.Hema and Mr.Kishore, Faculty,
SAI’S BIOSCIENCES INSTITUTE PVT.LTD, CHENNAI. For their valuable guidance and all Staff
members of the Institution for their keen interest shown for the completion of the project.
I inhale my immense pleasure in expressing our gratitude to our parents, life long friends and relatives
for their economic, physical support and encouragement, without which I would have ever
accomplished the task.

ABSTRACT
ABSTRACT:

Myocardial infarction (MI) or acute myocardial infarction (AMI), commonly known as a

heart attack, is the interruption of blood supply to a part of the heart, causing heart cells to die. This
is due to blockage of a coronary artery following the rupture of a vulnerable atherosclerotic plaque,
an unstable collection of lipids and white blood cells. The resulting ischemia (restriction in blood
supply) and oxygen shortage, if left untreated for a sufficient period of time can cause damage or
death (infarction) of heart muscle tissue (myocardium).The protein responsible for the formation of
plaques and fatty deposites in the walls of the coronary artery is LOW DENSITY
LIPOPROTEIN[LDL].WARFARIN is the effective drug used for myocardial infarction. Aim of this
study is to predict the active binding sites of LDL by using the tools Q SITE FINDER and POCKET
FINDER,to retrieve the analogs of the drug warfarin by using ZINC database and to discover a
potential drug for myocardial infarction from the analogs which do not causes any toxicitic effects in
our body.
 INTRODUCTION

BIOINFORMATICS:

Bioinformatics is the combination of biology and information technology. The discipline

encompasses any computational tools and methods used to manage, analyze and manipulate large sets
of biological data. Essentially, bioinformatics has three components .The creation of databases
allowing the storage and management of large biological data sets.The development of algorithms and
statistics to determine relationships among members of large data sets. The use of these tools for the
analysis and interpretation of various types of biological data,Including DNA, RNA and protein
sequences, protein structures, gene expression profiles, and biochemical pathways.

The term bioinformatics first came into use in the 1990s and was originally synonymous with
the management and analysis of DNA, RNA and protein sequence data. Computational tools for
sequence analysis had been available since the 1960s, but this was a minority interest until advances
in sequencing technology led to a rapid expansion in the number of stored sequences in databases such
as GenBank.Now, the term has expanded to incorporate many other types of biological data, for
example protein structures, gene expression profiles and protein interactions. Each of these areas
requires its own set of databases, algorithms and statistical methods.Bioinformatics is largely, although
not exclusively, a computer-based discipline.
 Bioinformatics is the use of IT in biotechnology for the data storage, data warehousing and
analyzing the DNA sequences. In Bioinfomatics knowledge of many branches are required like
biology, mathematics, computer science, laws of physics & chemistry, and of course sound knowledge
of IT to analyze biotech data. Bioinformatics is not limited to the computing data, but in reality it can
be used to solve many biological problems and find out how living things works.

It is the comprehensive application of mathematics (e.g., probability and statistics), science (e.g.,
biochemistry), and a core set of problem-solving methods (e.g., computer algorithms) to the
understanding of living systems.

CHEMINFORMATICS:

Cheminformatics is a relatively new field of information technology that focuses on the

collection, storage, analysis, and manipulation of chemical data. The chemical data of interest typically
includes information on small molecule formulas, structures, properties, spectra, and activities
(biological or industrial). Cheminformatics originally emerged as a vehicle to help the drug discovery
and development process; however cheminformatics now plays an increasingly important role in many
areas of biology, chemistry, and biochemistry. The intent of this unit is to give readers some
introduction into the field of cheminformatics and to show how cheminformatics not only shares many
similarities with the field of bioinformatics, but that it can also enhance much of what is currently done
in bioinformatics.

MYOCARDIAL INFARCTION:

Acute myocardial infarction (MI) is defined as death or necrosis of myocardial cells. It is a diagnosis at
the end of the spectrum of myocardial ischemia or acute coronary syndromes. Myocardial infarction occurs
when myocardial ischemia exceeds a critical threshold and overwhelms myocardial cellular repair me designed
to maintain normal operating function and hemostasis. Ischemia at this critical threshold level for an extended
time period results in irreversible myocardial cell damage or death.
 CAUSES:
myocardial infarction is a gradual process by which plaques (collections) of cholesterol are deposited
in the walls of arteries. Cholesterol plaques cause hardening of the arterial walls and narrowing of the inner
channel (lumen) of the artery. Arteries that are narrowed by myocardial infarction cannot deliver enough blood to
maintain normal function of the parts of the body they supply. For example, myocardial infarction of the arteries in
the legs causes reduced blood flow to the legs. Reduced blood flow to the legs can lead to pain in the legs while
walking or exercising, leg ulcers, or a delay in the healing of wounds to the legs. myocardial infarction of the
arteries that furnish blood to the brain can lead to vascular dementia (mental deterioration due to gradual death of
brain tissue over many years) orstroke (sudden death of brain tissue).
In many people, myocardial infarction can remain silent (causing no symptoms or health problems) for
years or decades. Atherosclerosis can begin as early as the teenage years, but symptoms or health problems usually
do not arise until later in adulthood when the arterial narrowing becomes severe. Smoking cigarettes, high blood
pressure, elevated cholesterol, and diabetes mellitus can accelerate atherosclerosis and lead to the earlier onset of
symptoms and complications, particularly in those people who have a family history of early myocardial infarction.
Coronary (or coronary artery disease) refers to the myocardial infarction that causes hardening and
narrowing of the coronary arteries. Diseases caused by the reduced blood supply to the heart muscle from coronary
atherosclerosis are called coronary heart diseases (CHD). Coronary heart diseases include heart attacks, sudden
unexpected death, chest pain (angina), abnormal heart rhythms, and heart failure due to weakening of the heart
muscle.
Myocardial infarction and angina pectoris

Angina pectoris(also referred to as angina) is chest pain or pressure that occurs when the blood and
oxygen supply to the heart muscle cannot keep up with the needs of the muscle. When coronary arteries are
narrowed by more than 50 to 70 percent, the arteries may not be able to increase the supply of blood to the heart
muscle during exercise or other periods of high demand for oxygen. An insufficient supply of oxygen to the heart
muscle causes angina. Angina that occurs with exercise or exertion is called exertional angina. In some patients,
especially diabetics, the progressive decrease in blood flow to the heart may occur without any pain or with just
shortness of breath or unusually early fatigue.
Exertional angina usually feels like a pressure, heaviness, squeezing, or aching across the chest. This pain
may travel to the neck, jaw, arms, back, or even the teeth, and may be accompanied by shortness of breath, nausea,
or a cold sweat. Exertional angina typically lasts from one to 15 minutes and is relieved by rest or by
taking nitroglycerin by placing a tablet under the tongue. Both resting and nitroglycerin decrease the heart muscle's
demand for oxygen, thus relieving angina. Exertional angina may be the first warning sign of advanced coronary
artery disease. Chest pains that just last a few seconds rarely are due to coronary artery disease.
Angina also can occur at rest. Angina at rest more commonly indicates that a coronary artery has narrowed
to such a critical degree that the heart is not receiving enough oxygen even at rest. Angina at rest infrequently may
be due to spasm of a coronary artery (a condition called Prinzmetal's or variant angina). Unlike a heart attack, there
is no permanent muscle damage with either exertional or rest angina.
Myocardial infarction and heart attack

Occasionally the surface of a cholesterol plaque in a coronary artery may rupture, and a blood clot forms on
the surface of the plaque. The clot blocks the flow of blood through the artery and results in a heart attack (see
picture below). The cause of rupture that leads to the formation of a clot is largely unknown, but contributing
factors may include cigarette smoking or other nicotine exposure, elevated LDL cholesterol, elevated levels of
blood catecholamines (adrenaline), high blood pressure, and other mechanical and biochemical forces.

Unlike exertional or rest angina, heart muscle dies during a heart attack and loss of the muscle is
permanent, unless blood flow can be promptly restored, usually within one to six hours.
SYMPTOMS:
Although chest pain or pressure is the most common symptom of a heart attack, heart attack victims may
experience a variety of symptoms including:
 • Pain, fullness, and/or squeezing sensation of the chest
• Jaw pain, toothache, headache
• Shortness of breath
• Nausea, vomiting, and/or general epigastric (upper middle abdomen) discomfort
• Sweating
• Heartburn and/or indigestion
• Arm pain (more commonly the left arm, but may be either arm)
• Upper back pain
• General malaise (vague feeling of illness)

• No symptoms (Approximately one quarter of all heart attacks are silent, without chest pain or new
symptoms. Silent heart attacks are especially common among patients with diabetes mellitus.)

Even though the symptoms of a heart attack at times can be vague and mild, it is important to remember that
heart attacks producing no symptoms or only mild symptoms can be just as serious and life-threatening as heart
attacks that cause severe chest pain. Too often patients attribute heart attack symptoms to "indigestion," "fatigue,"
or "stress," and consequently delay seeking prompt medical attention. One cannot overemphasize the
importance of seeking prompt medical attention in the presence of symptoms that suggest a heart attack.
Early diagnosis and treatment saves lives, and delays in reaching medical assistance can be fatal. A delay in
treatment can lead to permanently reduced function of the heart due to more extensive damage to the heart muscle.
Death also may occur as a result of the sudden onset of arrhythmias such as ventricular fibrillation

DIAGNOSIS:
When there is severe chest pain, suspicion that a heart attack is occurring usually is high, and tests can be
performed quickly that will confirm the heart attack. A problem arises, however, when the symptoms of a heart
attack do not include chest pain. A heart attack may not be suspected, and the appropriate tests may not be
performed. Therefore, the initial step in diagnosing a heart attack is to be suspicious that one has occurred.
Electrocardiogram. An electrocardiogram(ECG) is a recording of the electrical activity of the heart.
Abnormalities in the electrical activity usually occur with heart attacks and can identify the areas of heart muscle
that are deprived of oxygen and/or areas of muscle that have died. In a patient with typical symptoms of heart
attack (such as crushing chest pain) and characteristic changes of heart attack on the ECG, a secure diagnosis of
heart attack can be made quickly in the emergency room and treatment can be started immediately. If a patient's
symptoms are vague or atypical and if there are pre-existing ECG abnormalities, for example, from old heart
attacks or abnormal electrical patterns that make interpretation of the ECG difficult, the diagnosis of a heart attack
may be less secure. In these patients, the diagnosis can be made only hours later through detection of elevated
cardiac enzymes in the blood.
Blood tests. Cardiac enzymes are proteins that are released into the blood by dying heart muscles. These
cardiac enzymes are creatine phosphokinase (CPK), special sub-fractions of CPK (specifically, the MB fraction of
CPK), and troponin, and their levels can be measured in blood. These cardiac enzymes typically are elevated in the
blood several hours after the onset of a heart attack. A series of blood tests for the enzymes performed over a 24-
hour period are useful not only in confirming the diagnosis of heart attack, but the changes in their levels over time
also correlates with the amount of heart muscle that has died.
The most important factor in diagnosing and treating a heart attack is prompt medical
attention. Rapid evaluation allows early treatment of potentially life-threatening abnormal rhythms such as
ventricular fibrillation and allows early reperfusion (return of blood flow to the heart muscle) by procedures that
unclog the blocked coronary arteries. The more rapidly blood flow is reestablished, the more heart muscle that is
saved.
Large and active medical centers often have a "chest pain unit" where patients suspected of having heart attacks are
rapidly evaluated. If a heart attack is diagnosed, prompt therapy is initiated. If the diagnosis of heart attack is
initially unclear, the patient is placed under continuous monitoring until the results of further testing are available

PREVENTION AND TREATMENT:

An array of drugs can be used to prevent and treat asthma. Most of the drugs used to prevent asthma are also
used to treat an asthma attack but in higher doses or in different forms. Some people need to use more than one
drug to prevent and treat their symptoms.

Therapy is based on two classes of drugs: anti-inflammatory drugs and bronchodilators. Anti-inflammatory
drugs suppress the inflammation that narrows the airways. Bronchodilators help to relax and widen (dilate) the
airways. Anti-inflammatory drugs include corticosteroids (which can be inhaled, taken by mouth, or given
intravenously), leukotriene modifiers, and mast cell stabilizers. Bronchodilators include beta-adrenergic agonists
(both those for quick relief of symptoms and those for long-term control), drugs with anticholinergic effects, and
methylxanthines.

THEOPHYLLINE USE IN ASTHMA:

 Therapeutic actions — Though traditionally classified as a bronchodilator, the ability of theophylline to
control chronic asthma appears disproportionately greater than is explainable by its modest degree of
bronchodilator activity alone.Theophylline has antiinflammatory, immunomodulatory, and bronchoprotective
effects that potentially contribute to its efficacy as a prophylactic anti-asthma drug .

Theophylline down-regulates inflammatory and immune cell function in vitro and in vivo in animals with
airway inflammation . In patients with allergic asthma, it attenuates the late phase increase in airway obstruction
and airway responsiveness to histamine, decreases allergen-induced migration of activated eosinophils into the
bronchial mucosa, and decreases the sputum eosinophil count . Moreover, withdrawal of theophylline from patients
with severe chronic asthma receiving high-doses of inhaled glucocorticoid therapy results in increased symptoms
of asthma accompanied by an increase in the number of activated cytotoxic T-lymphocytes in the bronchial
mucosa and an increase in helper T-lymphocytes in the airway epithelium . The reduction in nocturnal worsening
of lung function when theophylline is used is associated with both a decrease in the percentage of neutrophils and a
decrease in stimulated leukotriene B4 from macrophages in early morning bronchoalveolar lavage fluid . An in
depth review of in vitro and in vivo studies demonstrating the immunomodulatory, anti-inflammatory, and
glucocorticoid-sparing effects of theophylline has been published .

Although several molecular mechanisms have been proposed to explain the actions of theophylline, the non-
specific inhibition of phosphodiesterase that occurs at clinically relevant drug concentrations appears to be the
most important. Theophylline increases the intracellular concentration of cyclic nucleotides in airway smooth
muscle and inflammatory cells by inhibiting phosphodiesterase-mediated hydrolysis. However, inhibition of
specific isozymes may also be important; inhibition of phosphodiesterase type III and type IV relaxes smooth
muscles in pulmonary arteries and in airways , while antiinflammatory and/or immunomodulatory actions probably
result from inhibition of the type IV isozymes. This information has stimulated interest in investigating more
specific inhibitors of phosphodiesterase type IV for asthma therapy.

PENTOXIFYLLINE:
 Pentoxifylline is a tri-substituted methylxanthine derivative, the biologic activities of which are numerous.
This includes increasing red cell deformability, leukocyte chemotaxis, antithrombin and anti- plasmin activities,
and more importantly to the present context, its fibrinolytic activity. Pentoxifylline decreases red cell and platelet
aggregation, granulocyte adhesion, fibrinogen levels, and whole blood viscosity . Recent work has delineated
pentoxifylline's ability to decrease production of tumor necrosis factor alpha and reduce some of the systemic
toxicities mediated by interleukin-2 . These two cytokines are important mediators of the inflammatory response.

PHARMACOKINETICS AND DYNAMICS:

Pharmacokinetics, sometimes described as what the body does to a drug, refers to the movement of drug into,
through, and out of the body—the time course of its absorption, bioavailability, distribution, metabolism, and
excretion. Pharmacodynamics describes what drug does to the body.

ABSORPTION:

The drug compound not given directly into the bloodstream by intravenous administration, the compounds
need to be transported from the site of administration into the systemic circulation. A drug is only considered to be
absorbed once it has entered the blood capillaries. The transport of drugs across membranes involves one or more
of the following processes: 1) passive diffusion, 2) filtration, 3) bulk flow, 4) active transport, 5) facilitated
transport, 6) ion-pair transport, 7) endocytosis, and 8) exocytosis. Drug absorption also depends on a number of
physicochemical factors, the two most important of which are lipophilicity and solubility

A simple measure of absorption is the ratio of the concentration of a compound in blood following
intravenous and nonintravenous (e.g., oral) administration. This value (also called bioavailability) is usually
expressed as a percentage and is defined as the fraction of its oral dose that reaches the systemic circulation as
follows:
Where Doral is the distribution of a compound after oral administration and Di is its distribution after intravenous
administration

DISTRIBUTION:
Once in the bloodstream, the drug then distributes across the body. It is not possible to determine this
directly; there is a relationship between drug concentration in blood and its volume of distribution (Vd):

Where Dbody is the amount of drug in the body at any time and D blood is the corresponding amount of drug in blood
at the same time

METABOLISM:
The purpose of drug metabolism is to make drugs more water soluble so they can be more easily excreted
from the body. There are three general types of biotransformation reactions involved in the metabolism of drugs
and other foreign compounds (xenobiotics):

• Oxidation-reduction

• Hydrolysis

• Conjugation.

Most of these drug-metabolizing enzymes are found in the liver, but they are also found in other tissues,
particularly the lung. Oxidative metabolism by cytochrome p450 enzymes is the primary method of drug
metabolism

SEX RELATION WITH METABOLISM:

There are also profound sex-related differences in drug metabolism. Thus, sexual dimorphism in rats, and
possibly in other species, results from the differential expression of sex-dependent hepatic cytochrome P-450s.
Such sex-related differences in the levels of cytochrome P-450 expression can be expected to give rise to profound
differences in toxicological response because the susceptibility of a tissue to the toxic and/or carcinogenic effects
of drugs often is determined by the rate of metabolic inactivation and/or activation by cytochrome P-450. For this
reason, regulatory agencies require that equal numbers of males and females of each species be used in toxicity
studies of drugs

EXCRETION:
The kidney serves as the major organ responsible for the removal of most drugs, especially those that are water
soluble.Another key organ of excretion is the liver, which secretes about 1 liter of bile daily. Because the passage
of xenobiotics from the blood into liver is normally not restricted, they often reach the hepatic extracellular fluid
from the plasma. The passage of substances into the bile, however, is much more restrictive. Other routes of
elimination include the lungs, sweat, and saliva

PHARMACOPHORE:

A pharmacophore model, according to IUPAC definition, is "an ensemble of steric and electronic features
that is necessary to ensure the optimal intermolecular interactions with a specific biological target and to trigger (or
block) its biological response". In Discovery Studio, a pharmacophore is defined as the essential features or
chemical substructures and their corresponding 3D locations that are responsible for the similar biological
activities of a set of compounds. Typically, pharmacophore features include hydrophobic, aromatic, hydrogen bond
acceptor, hydrogen bond donor, and positive ionizable and negative ionizable.
A pharmacophore is the ensemble of steric and electronic features necessary to ensure the optimal
supramolecular interactions with a specific biological target structure, and to trigger (or to block) its biological
response. Typical pharmacophore features indicate regions in a molecule that are hydrophobic or aromatic, can
participate in hydrogen bonding, or are positively or negatively ionizable.
 The following picture shows a typical pharmacophore model with three types of chemical features and their
3D location constraints:

The pharmacophore model consists of one Hydrogen Bond Acceptor (HBA, in green), three Hydrophobic
centers (in light blue), and one Negative Ionizable center (in dark blue). Since a hydrogen bond is directional, each
HBA feature is specified with two points: one is the location of the associated heavy atom, and the other is the
projection point, which defines the direction for forming a hydrogen bond with a target. Each sphere defines the
location tolerance of a particular feature point.

MOLECULAR TARGET:

PROTEINS AS CELLULAR TARGETS OF MEDICAL DRUGS:

The cellular targets (or receptors) of many drugs used for medical treatment are proteins. By binding to the
receptor, drugs either enhance or inhibit its activity. Basically there are two major groups of receptor proteins:
proteins that "float" around in the cytoplasm of the cell, and proteins that are incorporated into the cell membrane.
In the latter case, a drug does not even need to enter the cell, it can bind simply to an extracellular binding site of
the protein and control intracellular reactions from the outside.

DRUG SPECIFICITY AND SIDE EFFECTS:

An important criterion to determine the medical value of a drug is specificity: the physiological effect of the
drug should be as clearly defined as possible. It has to specifically bind to the target protein in order to minimize
undesired side-effects. The idea that molecules can interact in a highly specific manner has a long history in
medical chemistry. A century ago, Fischer and Ehrlich already used a "lock-and-key" analogy. Undesired side-
effects, however, are not always an indication for insufficient specificity of drugs as these effects might also result
from a reaction of our body to the desired and therefore successful regulation of the malfunctioning biochemical
process.

RATIONAL DRUG DESIGN (RDD):

Rational drug design is a process used in the biopharmaceutical industry to discover and develop new drug
compounds .The input of biocomputing in drug discovery is two steps :

• Firstly the computer may help to optimize the pharmacological profile of existing drugs by guiding
the synthesis of new and "better" compounds.
• Secondly, as more and more structural information on possible protein targets and their biochemical
role in the cell becomes available, completely new therapeutic concepts can be developed.
• The computer helps in both steps: to find out about possible biological functions of a protein by
comparing its amino acid sequence to databases of proteins with known function, and to understand the
molecular workings of a given protein structure. Understanding the biological or biochemical
mechanism of a disease then often suggests the types of molecules needed for new drugs. RDD uses a
variety of computational methods to identify novel compounds, design compounds for selectivity,
efficacy and safety, and develop compounds into clinical trial candidates. These methods fall into
several natural categories – structure-based drug design, ligand-based drug design, de novo design and
homology modeling – depending on how much information is available about drug targets and
potential drug compounds. My project is entirely focused on the focus on structure-based drug design.

TECHNIQUES APPLIED:
 The aim of using the computer for drug design is to analyze the interactions between the drug and its receptor
site and to "design" molecules that give an optimal fit. The central assumption is that a good fit results from
structural and chemical complementarity to the target receptor. The techniques provided by computational methods
include computer graphics for visualization and the methodology of theoretical chemistry. By means of quantum
mechanics the structure of small molecules can be predicted to experimental accuracy. Statistical mechanics
permits molecular motion and solvent effects to be incorporated. Basically statistical mechanics is a three-
dimensional equivalent of describing the position of billiard balls using Newton's law of motion.

Structure-Based Drug Design (SBDD). Structure-based drug design is one of several methods in the rational
drug design toolbox. Drug targets are typically key molecules involved in a specific metabolic or cell signaling
pathway that is known, or believed, to be related to a particular disease state. Drug targets are most often proteins
and enzymes in these pathways. Drug compounds are designed to inhibit, restore or otherwise modify the structure
and behavior of disease-related proteins and enzymes. SBDD uses the known 3D geometrical shape or structure of
proteins to assist in the development of new drug compounds. The 3D structure of protein targets is most often
derived from x-ray crystallography or nuclear magnetic resonance (NMR) techniques. X-ray and NMR methods
can resolve the structure of proteins to a resolution of a few angstroms (about 500,000 times smaller than the
diameter of a human hair). At this level of resolution, researchers can precisely examine the interactions between
atoms in protein targets and atoms in potential drug compounds that bind to the proteins. This ability to work at
high resolution with both proteins and drug compounds makes SBDD one of the most powerful methods in drug
design.

DOCKING LIGANDS:

One of the key benefits of SBDD methods is the exceptional capability it provides for docking putative drug
compounds (ligands) in the active site of target proteins. Most proteins contain pockets, cavities, surface
depressions and other geometrical regions where small-molecule compounds can easily bind. With high-resolution
x-ray and NMR structures for proteins and ligands, one can show precisely how ligands orient themselves in
protein active sites.

LEAD OPTIMIZATION:

Once a number of lead compounds have been found, SBDD techniques are especially effective in refining
their 3D structures to improve binding to protein active sites, a process known as lead optimization.

In lead optimization one can systematically modify the structure of the lead compound, docking each
specific configuration of a drug compound in a protein’s active site, and then testing how well each configuration
binds to the site.

In a common lead optimization method known as bioisosteric replacement, specific functional groups in a
ligand are substituted for other groups to improve the binding characteristics of the ligand. With SBDD one can
examine the various bioisosteres and their docking configurations, choosing only those that bind well in the active
site.
 REVIEW

OF

LITERATURE
ANTIHYPERALGESIC EFFECT OF PENTOXIFYLLINE ON EXPERIMENTAL

INFLAMMATORY PAIN

Mariana L Vale,1 Verônica M Benevides,1 Daniela Sachs,2 Gerly A C Brito,1 Francisco A C daRocha,1
Stephen Poole,3 Sérgio H Ferreira,2 Fernando Q Cunha,2 and Ronaldo A Ribeiro1*11Departamento de Fisiologia e
Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Rua Cel, Nunes de Melo, 1127, CEP 60430-
270, Fortaleza, CE, Brazil22Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade
de São Paulo, São Paulo, SP, Brazil 33Endocrinology Section, National Institute for Biological Standards and Control
(NIBSC), London.

POTENTIAL ROLE OF CHITINASE 3-LIKE-1 IN INFLAMMATION-ASSOCIATED

CARCINOGENIC CHANGES OF EPITHELIAL CELLS

Katrin Eurich, Mayuko Segawa, Satoko Toei-zShimizu, and Emiko Mizoguchi Katrin Eurich Mayuko
Segawa, Satoko Toei-Shimizu, Emiko Mizoguchi, Gastrointestinal Unit, Department of Medicine, Massachusetts
General Hospital, Harvard Medical School, Boston, MA 02114, United StatesEmiko Mizoguchi, Center for the Study
of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114,
United States

Author contributions: Mizoguchi E designed the research; Eurich K, Segawa M, Toei-Shimizu S and
Mizoguchi E performed the research, analyzed and summarized the data; Eurich K and Mizoguchi E

ACIDIC MAMMALIAN CHITINASE IS SECRETED VIA ADAM17/EPIDERMAL GROWTH

FACTOR RECEPTOR-DEPENDENT PATHWAY AND STIMULATES CHEMOKINE
PRODUCTION BY PULMONARY EPITHELIAL CELLS

Dominik Hartl,1 Chuan Hua He,1 Barbara Koller, Carla A. Da Silva, Robert Homer, Chun G.
 SOFTWARES
AND
DATABASE

DRUG BANK:
The DrugBank database is a unique bioinformatics and cheminformatics resource that combines
detailed drug (i.e. chemical, pharmacological and pharmaceutical) data with comprehensive drug target
(i.e. sequence, structure, and pathway) information. Each DrugCard entry contains more than 100 data
fields with half of the information being devoted to drug/chemical data and the other half devoted to
drug target or protein data.
 .

ACCLERYS DISCOVERY STUDIO 2.5

 Acclerys is premier leading company providing the software’s for life sciences, it as unique
features when compared to other commercial software’s .the algorithm and protocols are designed in
such way with the help of wet lab results hence one can analyze the life science data effectively and it
is user friendly available in all platforms .my project is entirely done with the help of acclerys inherited
tools and protocols the home page of acclerys discovery studio 2.5 is shown below

PUBMED:

PubMed is a free search engine for accessing the MEDLINE database of citations, abstracts
and some full text articles on life sciences and biomedical topics. The United States National Library
of Medicine at the National Institutes of Health maintains PubMed as part of the Entrez information
retrieval system.

PDB:
 The PDB archive contains information about experimentally-determined structures of proteins,
nucleic acids, and complex assemblies.The RCSB PDB also provides a variety of tools and resources.
Users can perform simple and advanced searches based on annotations relating to sequence, structure
and function. These molecules are visualized, downloaded, and analyzed by users who range from
students to specialized scientists.
METHODOLOGY
METHODOLOGY

 Selection of disease related protein structure from PDB

 Using drug bank ,finding the target
 Prediction of pharmacokinetics properties
 Prediction of pharmacodynamaics properties
 Calculating general property

 Checking for Lipinski rule of 5

 Qualitative structure activity relationship

 Structure based drug designing

SELECTION OF DISEASE RELATED PROTEIN STRUCTURE FROM PDB:

The PDB archive contains information about experimentally-determined structures of proteins,
nucleic acids, and complex assemblies. These molecules are visualized, downloaded, and analyzed by
users who range from students to specialized scientists.
USING DRUG BANK ,FINDING THE TARGET:
The DrugBank database is a unique bioinformatics and cheminformatics resource that combines
detailed drug (i.e. chemical, pharmacological and pharmaceutical) data with comprehensive drug target
(i.e. sequence, structure, and pathway) information.
PREDICTION OF PHARMACOKINETICS PROPERTIES:
ADME is the one of the protocol which is available through acclerys for the pharmacokinetics
studies, which explains what drug does to the body? The parameters as Absorption, Distribution,
Metabolism, Excretion, is determined by protocol in two ways such as probability and levels. The
ligands/lead molecules which falls with in the limit of levels is taken in consideration for further
analysis since this is Insilico prediction .I dint filtered the lead compounds but the values are kept for
reference and can be cross verified through wet lab screening.
PREDICTION OF PHARMACODYNAMICS PROPERTIES:
TOPKAT is used to analyze what body does to the drug? It uses the animal models and sub
models to predict important parameters like mutagen, carcinogenicity, irritancy etc.., based on QSTR,
Bayesian methods and fingerprints of chemical molecule.
CALCULATING GENERAL PROPERTY:

It is generally used to determine the molecular properties of ligand /lead such 1D, 2D, 3D, 4D
descriptors which is used for QSAR studies knowing that IC50 value in wet lab screening.
CHECKING FOR LIPINSKI RULE OF 5:

The Lipinski rule of 5 is very important rule for drug designing it explains that lead molecule
should have certain properties to behave as a drug. This can be easily performed in prepare ligand
protocol available in acclerys.

QUALITATIVE STRUCTURE ACTIVITY RELATIONSHIP:

In the application, common feature pharmacophores are generated using the HipHop algorithm.
Hip Hop identifies configurations or three-dimensional spatial arrangements of chemical features that
are common to molecules in a training set. The configurations are identified by a pruned exhaustive
search, starting with small sets of features and extending them until no larger common configuration is
found. Training set members are evaluated on the basis of the types of chemical features they contain,
along with the ability to adopt a conformation that allows those features to be superimposed on a
particular configurations. One can define how many molecules must map completely or partially to the
pharmacophore. This option allows broader and more diverse pharmacophores to be generated. The
 resultant pharmacophores are ranked as they are built. The ranking is a measure of how well the
molecules map onto the proposed pharmacophores, as well as the rarity of the pharmacophore model

LIGANDS FROM DRUG BANK:

In this docking both receptor and ligand is known followed by performing the respective steps for

NAME FORMULA CANONICAL SMILE MOL WT

PENTOXIFYLLINE C13H18N4O3 CN1C=NC2=C1C(=O)N(CCCCC(C)=O)C(=O)N2C 278.30
ENPROFYLLINE C8H10N4O2 CCCN1C(=O)NC(=O)C2=C1N=CN2 194.19
DYPHYLLINE C10H14N4O4 CN1C(=O)N(C)C2=C(N(CC(O)CO)C=N2)C1=O 254.24
PENCICLOVIR C10H15N5O3 NC1=NC(=O)C2=C(N1)N(CCC(CO)CO)C=N2 253.25
CAFFEINE C8H10N4O2 CN1C=NC2=C1C(=O)N(C)C(=O)N2C 194.19
THEOBROMINE C7H8N4O2 CN1C=NC2=C1C(=O)NC(=O)N2C 180.16
AMINOPHYLLINE C16H24N10O4 NCCN.CN1C(=O)N(C)C2=C(NC=N2)C1=O.CN1C(=O 140.42
)N(C)C2=C(NC=N2)C1=O
ENTECAVIR C12H15N5O3 NC1=NC(=O)C2=C(N1)N(C=N2)C1CC(O)C(CO)C1=C 277.27
THEOPHYLLINE C7H8N4O2 CN1C(=O)N(C)C2=C(NC=N2)C1=O 180.16
FAMCICLOVIR C14H19N5O4 CC(=O)OCC(CCN1C=NC2=CN=C(N)N=C12)COC(C) 321.33
=O
RIBOFLAVIN C17H20N4O6 CC1=CC2=C(C=C1C)N(CC(O)C(O)C(O)CO)C1=NC( 376.36
=O)NC(=O)C1=N2
DIDANOSINE C10H12N4O3 OCC1CCC(O1)N1C=NC2=C1NC=NC2=O 236.22
PIMOZIDE C28H29F2N3O FC1=CC=C(C=C1)C(CCCN1CCC(CC1)N1C(=O)NC2= 461.54
CC=CC=C12)C1=CC=C(F)C=C1

LIGAND FIT ALGORITHM IS USED FOR DOCKING THE PROTEIN AND RECEPTOR
WITH LEAD /LIGAND MOLECULES.

Figure 1: Flowchart of the major steps followed by the LigandFit docking algorithm is shown below
 RES
ULTS
 PENTOXIFYLLINE:

ADME RESULT FOR PENTOXIFYLLINE:

SL.NO BBB ABSORPTION SOLUBILITY HEPATOTOXICITY CYP2D6 PPB

LEVEL LEVEL LEVEL LEVEL LEVEL

MOL 1 3 0 3 0 0 0

MOL 2 3 0 4 0 0 2

MOL 3 3 0 4 0 0 0

MOL 4 4 2 5 1 0 0

MOL 5 3 0 4 0 0 0

MOL 6 3 0 4 0 0 2

MOL 7 3 0 4 0 0 1

MOL 8 4 1 4 0 0 0

MOL 9 3 0 4 0 0 1

MOL 10 4 0 4 0 0 0

MOL 11 4 3 4 0 0 2

MOL 12 3 0 4 1 0 0

MOL 13 0 0 0 0 1 2
THE BELOW TABLES WITH LEVELS SHOWS THE SIGNIFICANCE OF LEAD
MOLECULE OF PHARMACO KINETICS:

BLOOD BRAIN BARRIER:

There are four prediction levels within the 95% and 99% confidence:

LEVEL DESCRIPTION
0 Very high penetrant
1 High
2 Medium
3 Low
4 Undefined

ABSORPTION LEVEL:

LEVEL DESCRIPTION
0 Good absorption
1 Moderate absorption
2 Low absorption
3 Very low absorption

SOLUBILITY LEVEL:

LEVEL DRUG –LIKENESS

0 Extremely low
1 No, very low, but possible
2 Yes, low
3 Yes, good
4 Yes, optimal
5 No, too soluble
6 Warning: molecules with one or more unknown AlogP98 types

HEPATOTOXICITY:
 LEVEL DESCRIPTION

0 Nontoxic

1 Toxic

CYP2D6:

LEVEL DESCRIPTION

0 Non-inhibitor

1 Inhibitor

PPB LEVEL:

LEVEL DESCRIPTION

0 Binding is < 90% (No markers flagged and AlogP98 < 4.0)

1 Binding is > 90% (flagged at 90% or AlogP98 > 4.0)

2 Binding is > 95% (flagged at 95% or AlogP98 > 5.0)

BBB = Blood Brain Barrier, PPB = Plasma Protein Binding

THE GRAPHICAL FORM OF ADMET PROPERTIES FOR PENTOXIFYLLINE:

PHARMACODYNAMICS STUDIED USING TOPKAT AND BELOW TABLE
EXPLAINS THE LEVEL :
 NTP CARCINOGENICITY FDA CARCINOGENICITY
MODEL FEMALE MALE FEMALE MALE FEMALE MALE FEMALE MALE
MOUSE MOUSE RAT RAT MOUSE MOUSE RAT RAT
Mol1 0.4 0.05 0.3 0.4 0.2 0.1 0.2 0.2
Mol 2 0.6 0.04 0.3 0.5 0.2 0.2 0.3 0.2
Mol 3 0.3 0.05 0.3 0.4 0.2 0.1 0.2 0.2
Mol 4 0.5 0.4 0.3 0.5 0.2 0.2 0.2 0.2
Mol 5 0.4 0.1 0.4 0.5 0.2 0.2 0.2 0.2
Mol 6 0.5 0.2 0.4 0.5 0.2 0.2 0.2 0.2
Mol 7 0.2 0.0002 0.3 0.4 0.2 0.1 0.2 0.2
Mol 8 0.5 0.3 0.3 0.5 0.2 0.2 0.2 0.2
Mol 9 0.3 0.006 0.3 0.4 0.2 0.2 0.2 0.2
Mol10 0.5 0.6 0.3 0.5 0.2 0.1 0.9 0.2
Mol 11 0.3 0.2 0.3 0.4 0.2 0.2 0.2 0.2
Mol 12 0.6 0.4 0.4 0.6 0.2 0.2 0.2 0.2
Mol 13 0.5 0.2 0.2 0.4 0.8 0.1 0.2 0.1

LIMIT VALUES FOR ABOVE TABLE

<=0.3 indicates a non-carcinogen

0.3 And 0.7 refers to the "indeterminate" zone

>=0.7 signifies a carcinogen

Model
DOSAGE EFFECT ON TISSUES
RAT MAXIMUM DAPHNIA INHALATION SKIN SKIN OCCULAR
LD50 TOLERATE MAGNA LC50 IRRITATION SENSITIZATION IRRITANCY
(GM/KG) D DOSAGE EC50 (G/M3/H)
(GM/KG) (UG/L)

LEAD

MOL1 2.73 5.06 5.94 4.18 0.99 0.64 0.99

MOL 2 3.49 3.49 4.91 77.56 0.96 0.66 0.99
MOL 3 1.88 0.94 614.7 13.49 0.97 0.73 0.99
MOL 4 2.73 82.71 51.89 1.94 0.97 0.79 0.99
MOL 5 2.49 4.68 42.09 10.60 0.99 0.78 0.99
MOL 6 4.67 3.15 7.37 9.81 0.97 0.76 0.99
MOL 7 0.68 0.15 125.99 0.97 0.97 0.66 0.99
MOL 8 1.67 6.58 76 1.45 0.97 0.79 0.99
MOL 9 1.63 1.71 38.53 16.63 0.97 0.68 0.99
MOL 10 1.36 10.28 4.34 1.71 0.99 0.70 0.96
MOL 11 5.06 2.23 1.10 7.58 0.93 0.65 0.99
MOL 12 3.31 33.48 217.2 6.51 0.97 0.76 0.99
MOL 13 0.58 1.32 0.58 31.59 0.88 0.85 0.99

LIPINSKI’s RULE OF 5:

SL.NO ALOGP HBA HBD MOL.WT

Mol1 0.507 4 0 278.3

Mol2 0.36 3 2 194.1

Mol3 -1.15 5 1 254.2

Mol4 -1.53 7 3 253.2

Mol5 -0.1 3 0 194.1

Mol6 -0.306 3 0 180.1

Mol7 -2.089 8 2 420.4

Mol8 -1.404 7 2 277.2

Mol9 -0.306 3 3 180.1

Mol10 -0.294 8 1 321.3

Mol11 -0.255 9 1 376.3

Mol12 -0.844 6 0 236.2

 Mol13 5.521 2 5 461.5

QUALITATIVE STRUCTURE ACTIVITY RELATIONSHIP:

It shows how the ligand binds with the target by acceptor and donor values

MOLECULE 2: ENPROFYLLINE

MOLECULE 6: THEOBROMINE
MOLECULE 9: THEOPHYLLINE
 DICUSSION

STRUCTURE BASED LIGAND AND PROTEIN DOCKING

TARGET =ACIDIC MAMMALIAN CHITINASE(PDB ID-3FXY)

The following table shows the docking of lead molecules against ACIDIC MAMMALIAN
CHITINASE:
3FXY AMINO ANIMO ATOMS LIGAND LENGTH LIG1 LIG2 -PLP1 -PLP2 JAIN -PMF POS DOCK ROT INT
ACID ACID IN OF NO SCORE BON ENERG
POSITIO AMINO BOND DS Y
N ACID
Mol 2 GLU 297 OE1 N7 3.034 3.06 4.15 35.55 35.15 0.35 92.75 1 32.86 1 -2.50
Mol6 GLU 297 OE1 N9 3.195 3.42 3.27 32.83 27.2 - 102.6 1 26.96 0 -0.97
0.69
Mol9 GLU 297 OE1 N9 3.169 3.44 3.38 32.43 25.15 - 98.37 1 27.03 0 -1.59
1.01

MOLECULE:2 MOLECULE:6 MOLECULE:9

From the above table ,the molecule 2,6,9 of pentoxifylline derivatives shows the docking value which
suits the target to be appropriate ligand
PHARMACOPHORE FEATURE:

SL.NO CONFIG FIT HBA HBD HYDROPHOBIC HYDROPHYLIC LENGTH LENGTH

VALUE
NO OF OF

HBA HBD

MOL2 1 3 0 1 1 0 0 3.015

MOL6 1 3.9 1 1 1 1 3.03 3.00

MOL9 1 3 3 0 0 0 3.01 0

The above value says how the interaction inbetween the ligand and target .These molecules has good
interaction with target .therefore from 13 moleules which is taken for the studies,only 3 molecule has the
interaction with target so it can be taken has effective feature to act as drug.

ADME RESULT FOR 3 LIGAND:

SL.NO BBB ABSORPTION SOLUBILITY HEPATOTOXICITY CYP2D6 PPB

LEVEL LEVEL LEVEL LEVEL LEVEL

MOL 2 3 0 4 0 0 2

MOL 6 3 0 4 0 0 2

MOL 9 3 0 4 0 0 1

THE LEVELS FOR ABOVE TABLE :

NAME DESCRIPTION
BBB Low (3)
ABSORPTION Good absorption (0)
SOLUBILITY Yes, optimal (4)
 HEPTATOTOXICITY Nontoxic (0)
CYP2D6 Non inhibitor (0)
PPB Binding is > 90% (flagged at 90% or AlogP98 > 4.0) (1)
Binding is > 95% (flagged at 95% or AlogP98 > 5.0) (2)

THE TOPKAT VALUES FOR 3 LIGAND:

NTP CARCINOGENICITY FDA CARCINOGENICITY

MODEL FEMALE MALE FEMALE MALE FEMALE MALE FEMALE MALE
MOUSE MOUSE RAT RAT MOUSE MOUSE RAT RAT
Mol 2 0.6 0.04 0.3 0.5 0.2 0.2 0.3 0.2
Mol 6 0.5 0.2 0.4 0.5 0.2 0.2 0.2 0.2
Mol 9 0.3 0.006 0.3 0.4 0.2 0.2 0.2 0.2

LIMIT VALUES FOR ABOVE TABLE

<=0.3 indicates a non-carcinogen

0.3 And 0.7 refers to the "indeterminate" zone

>=0.7 signifies a carcinogen

From the above values ,shows that these 3 molecule values come under the limits .Therefore these
molecule can be taken as best ligand for the target that can act as the drug for affected protein

CONCLUSION
 Asthma is a condition in which the airways narrow usually reversibly in response to certain
stimuli. Asthma affects more than 20 million people in the US, and it is becoming more common.
Although it is one of the most common chronic diseases of childhood, adults can also develop asthma,
even at an old age. The reason for the increase in asthma in children is not known. Increased use of
vaccines and antibiotics may have shifted the activity of a special subgroup of white blood cells (called
lymphocytes) in the body from fighting infection to releasing chemical substances that promote the
development of allergies.

Initially the 13lead molecules were screened for Pharmaco dynamics and kinetics properties, the
pharmaco kinetics limit values as stated in the respective tables of absorption, solubility, hepatoxicity,
blood brain barrier, and plasma binding protein values are obtained using suitable descriptors and
training set molecules was validated against several dataset. In order to ensure the leads to behave as
drugs the Lipinski’s rule of 5 is performed to screen the lead molecule. This analysis sounds good to
narrow down the work to few candidate drug molecules. Typically the molecules like 2,6,9 satisfies
insilico lead property; on the other hand the qualitative Pharmacophore is generated to perform ligand-
pharmacophore comparisons and fitting. Compared to various experimental techniques, including
combinatorial chemistry, the correct ranking of the results obtained seems to be the largest unsolved
problem of computer-aided design techniques. Experimental and theoretical approaches complement
each other, especially in the early stages of drug research, where mass screening and De novo design
independently provides new leads which can be optimized by computer-aided design, and can be
supplemented by the intuition and creativity of the human mind.

Thus by final analysis of lead molecule results that lead number 2,6,9 can serve as potential
drug candidate in future and can be serve as novel drug to treat the patients and also its proven that
these molecules can be synthesised in wet lab technique with maximum yield of lead molecules with
ACIDIC MAMMALIAN CHITINASE