BIOINFORMATICS

MOLECULAR MODELLING BY
VIRTUAL SCREENING METHOD
 Presentation Topics
 Bio Informatics Over view
 Drug Design based on Bioinformatics
 Virtual Screening method
 Asthma- Case Study
 Tools Used In Case Study
 Methodology
 Results
 Conclusion
 Bioinformatics- Overview
 Over the past few decades, major advances in the
field of molecular biology, coupled with advances
in genomic technologies, have led to an explosive
growth in the biological information generated by
the scientific community. This deluge of genomic
information has, in turn, led to an absolute
requirement for computerized databases to store,
organize, and index the data and for specialized
tools to view and analyze the data.
 BioInformatics-Definition
 An interdisciplinary area at the intersection of
biological, computer, and information sciences
necessary to manage, process, and understand
large amounts of data, for instance from the
sequencing of the human genome, or from large
databases containing information about plants and
animals for use in discovering and developing new
drugs. It is the science of informatics as applied to
biological research.
 Major Research Areas

 1.Sequence analysis
 2.Computational evolutionary biology
 3. Measuring biodiversity
 4.Gene expression analysis
 5. Regulation analysis
 6. Protein expression analysis
 7. Analysis of mutations in cancer
 8.Oligonucleotide microarrays,
 9. Structure prediction
 10. Modelling biological systems
 11. High-throughput image analysis
 Drug Design based on
Bioinformatics
 The processes of designing a new drug using bioinformatics tools have
open a new area of research. However, computational techniques assist
one in searching drug target and in designing drug in silico, but it takes
long time and money. In order to design a new drug one need to follow
the following path.
 Identify Target Disease
 Study Interesting Compounds
 Detect the Molecular Bases for Disease
 Rational drug design techniques
 Refinement of compounds
 Quantitative Structure Activity Relationships (QSAR):
 Solubility of Molecule
 Drug Testing
 Virtual Screening Method
 Screening of molecules from different data bases

 The data bases screened include LIGAND INFO which further constitute 7
database subsets & the molecules which are almost identical to original
ligands were selected &visualized in MERCURY (CCDC).

 These ligands are saved as PDB format &are used for docking in ARGUS
LAB with the active site determinants.

 The docked molecules which are having minimum of four inter molecular
bonds between the ligand & protein are viewed in PYMOL.These docked
molecules are considered as HIT molecules.

 Unknown ligand information ie., chemical name, molecular

weight,chemical structure, smiles formula is visulised in MARVIN VIEW
and ACCELERYS DS Visualiser
 Asthma (Case Study)
 Asthma affects more than 5% of the population of the
US, including children. It is a chronic inflammatory
disorder of the airways characterized by coughing,
shortness of breath, and chest tightness. A variety of
"triggers" may initiate or worsen an asthma attack,
including viral respiratory infections, exercise, and
exposure to irritants such as tobacco smoke. The
physiological symptoms of asthma are a narrowing of
the airways caused by edema (fluid in the intracellular
tissue space) and the influx of inflammatory cells into
the walls of the airways
 Types of Asthma
 1.Asthma related to underlying cause or disease process
 Allergic asthma
 Non-allergic asthma
 Occupational asthma
 Exercise-induced asthma
 Cough-variant asthma
 Medication-induced asthma

 2.Asthma classifications by severity

 Classification of asthma by severity is based on frequency and severity
of asthma symptoms, along with peak flow readings

 Mild Intermittent
 Mild Persistent
 Moderate Persistent.
 Severe Persistent

 3.Asthma types by degree of control

 Controlled
 Partly controlled
 Uncontrolled
 Symptoms
 Dyspnea, wheezing, coughing, a tightness and
itching of the chest or an inability for physical
exertion.

CAUSES:

 1.Environmental
 2.Genetic
 3.Gene-environment Interactions
 before asthma after asthma

Inflamed airways and bronchoconstriction in asthma.

Airways narrowed as a result of the inflammatory
response cause wheezing.
 Prevention
 Prevention medications such as an inhaled corticosteroid, which helps to
suppress inflammation and reduces the swelling of the lining of the airways, in
anyone who has frequent (greater than twice a week) need of relievers or who has
severe symptoms.

Treatment

 Medical: Bronchodilators are recommended for short-term relief in all patients.

 For those with mild persistent disease (more than two attacks a week), low-dose
inhaled glucocorticoids or alternatively, an oral leukotriene modifier, a mast-cell
stabilizer, or theophylline may be administered.
 For those who suffer daily attacks, a higher dose of glucocorticoid in conjunction
with a long-acting inhaled β-2 agonist may be prescribed; alternatively, a
leukotriene modifier or theophylline may substitute for the β-2 agonist. In severe
asthmatics, oral glucocorticoids may be added to these treatments during severe
attacks.

 Pharmaceutical: A nebulizer which provides a larger, continuous dose can also be

used.
 Short-acting, selective beta2-adrenoceptor agonists, such as salbutamol (albuterol
USAN), levalbuterol, terbutaline and bitolterol.

 Long-acting β2-agonists:Long-acting bronchodilators have much longer side

chains resulting in a 12-hour effect, and are used to give a smoothed symptomatic
relief
 HUMAN BETA-II TRYPTASE
(Enzyme involved in Asthma):
 Tryptase is a serine protease found almost exclusively in mast cells. It
has trypsin-like specificity, favoring cleavage of substrates with an
arginine (or lysine) at the P1 position, and has optimal catalytic activity
at neutral pH.

 Human beta tryptase hydrolyzes a number of peptide substrates using a

typical serine protease mechanism. It cleaves the peptide bonds
following Arg and Lys. The catalytic residues are Ser195, His57 and
Asp102. These are found at the junction between two beta barrels that
are perpendicular to each other. The His and Asp are on the loop of one
barrel and the Ser is on a loop of the other barrel. These three amino
acids make up the catalytic triad.

 In animal models tryptase provokes broncho-constriction and induces a

cellular inflammatory infiltrate characteristic of human asthma. Screening
of in-house inhibitors of factor Xa (a closely related serine protease)
identified β-amidoester benzamidines as potent inhibitors of recombinant
human βII tryptase.
Structure of Tryptase
 Tools Used In Case Study(Asthma)
 NCBI (National Center for Biotechnology Information)
 PDB (Protein Data Bank)
 Drug Bank
 Expasy Tools (Expert Protein Analysis System)–Proteomic Study
 Primary Structure Analysis: Prot Param
 Secondary Structure Analysis: HNN Predict,NNpredict,Sopma
 Tertiary Structure Analysis: 3D PSSM,HHpredict

VISUALISATION TOOLS

 RASMOL
 Swiss PDB Viewer
 Pymol
 Small Molecule Databases (LIGAND Info:)
 Mercury
 Marvin View

 Docking Tools: Argus lab

 Methodology
 Disease & Protein Structure:
 Asthma disease is selected as case study & the information is retrieved
from NCBI-GENES&DISEASES.
 The crystal structure of HUMAN BETA 2 TRYPTASE in complex with 4-(3-
Amino Methyl Phenyl)-Piperidine-1-yl-(5-Phenethyl- pyridine-3-yl)-
Methanone is the target protein for the disease.
 It was solved by X-ray Diffraction method& was retrieved from Protein
Data Bank (PDB)(id:2BM2)
 Ligand information ie.,chemical structure, molecular weight, smiles
formula,logP value, biological function in complex with protein etc., is
collected from DRUG BANK.
 Catalytic activity of the protein & its molecular activity in complex with
ligand is done from Literature.
 Proteomic study was performed using EXPASY Proteomics server.
ie.,analysed the primary, secondary & tertiary structures of the protein
throughPROT PARAM, HNN PRED, SOPMA, 3DPSSM, HHPRED
respectively.
 Structure of the protein is visualized using RASMOL.
 The receptor & ligand were saved separately using Swiss PDB Viewer.By
using Swiss PDB Viewer the residues within 4.5 vicinity of the ligand was
defined the active site of the target molecule.
 Virtual Screening Method
 Screening of database which contain biologically active molecules was
done to find lead molecules.

 The data bases screened include LIGAND INFO which further constitute 7
database subsets & the molecules which are almost identical to original
ligands were selected &visualized in MERCURY (CCDC).

 These ligands are saved as PDB format &are used for docking in ARGUS
LAB with the active site determinants of Tryptase.

 The docked molecules which are having minimum of four inter molecular
bonds between the ligand & protein are viewed in PYMOL.These docked
molecules are considered as HIT molecules.

 Unknown ligand information ie., chemical name, molecular

weight,chemical structure, smiles formula is visulised in MARVIN VIEW
and ACCELERYS DS Visualiser

 Based on this 6 HIT molecules are selected & are recommended for
clinical trials. If promising they can be released as Anti Asthmatic drugs.
 Results
Molecule name Chemical formula Molecular Log p
weight(g/mol)
Lavendustin AOr C21H19NO6 381.38 3.605
RG 14355

8 Bromo CAMP C10H10BrN5O6P 407.9

-0.5
(2(formyl-hydroxy- 169.07
amino)-ethyl)- C3H8N05P -2.6
phosphonic acid
1-O-[P- C12H15NO8 C12H15NO8 -0.5
NITROPHENYL]-
BETA-D-
GALACTOPYRANO
SE
PENTAETHYLENE C10H22O6 -2.6
238.28
GLYCOL
5-[4-(1-Carboxymethyl- C20H20N2O9S 464.45 1.8
2-Oxo-
Propylcarbamoyl)-
Benzylsulfamoyl]-2-
Hydroxy-Benzoic Acid
 Biological Activities of Hit Molecules
 1.LAVENDUSTIN A :Potent, cell-permeable inhibitor of epidermal growth
factor receptor (EGFR) tyrosine kinase (IC50 = 11 nM). Inhibits p60c-src
with an IC50 of 500 nM and is selective over PKA, PKC and PI 3-kinase
(IC50 > 100 μM
 2. 8 Bromo CAMP,Sodium salt : Cell-permeable CAMP analog; activator
of protein kinase A.
 3. (2(formyl-hydroxy-amino)-ethyl)-phosphonic acid :catalytic activity,
triose-phosphate isomerase activity, isomerase activity.
 4. 1-O-[P-NITROPHENYL]-BETA-D-GALACTOPYRANOSE : Catalytic
function, Protein binding
 5. PENTAETHYLENE GLYCOL:catalytic activity,hydrolase
activitypeptidase activityendopeptidase activity,serine-type
endopeptidase activit subtilase activity
 6. 5-[4-(1-Carboxymethyl-2-Oxo-Propylcarbamoyl)-Benzylsulfamoyl]-2-
Hydroxy-Benzoic Acid:catalytic activity
endopeptidase activitycysteine-type endopeptidase activity
Human Beta 2 Tryptase
 [4-(3-AMINOMETHYL-PHENYL)-
PIPERIDIN-1-YL]-(5-PHENETHYL-
PYRIDIN-3-YL)-METHANONE
LavendustinA
8 Bromo CAMP
Penta Ethylene Glycol
1-O-[P-NITROPHENYL]-BETA-D-
GALACTOPYRANOSE
(2(formyl-hydroxy-amino)-ethyl)-
phosphonic acid
 5-[4-(1-Carboxymethyl-2-Oxo-
Propylcarbamoyl)-Benzylsulfamoyl]-2-
Hydroxy-Benzoic Acid
 Conclusion
 Asthma as a result of (or worsened by) workplace exposures is
the world's most commonly reported occupational respiratory
disease. Still most cases of occupational asthma are not reported
or are not recognized as such.
 American Thoracic Society (2004) suggest that 15%–23% of new-
onset asthma cases in adults are work related.
• Tryptases have been implicated as mediators in the pathogenesis
of asthma and other allergic and inflammatory disorders.
• Tryptase inhibitors have the function of catalytic activity, Endo
peptidase activity where they preferentially cleave the molecule
near Arg & Lys residues.
 The study has found out six inhibitor molecules which docked
well into the active site of Human Beta 2 Tryptase (target
molecule) for the study.
 These molecules can be considered as the interesting molecules
which need to be further tested in the lab.