Details

In Silico Drug Design of Biofilm Inhibitors of Staphylococcus epidermidis

by Al-mulla, Aymen Faraoun, MS

Abstract (Summary)
In Silico drug design represents a new approach for drug discovery and industry. Structure based computer aided drug design (CADD) was used in this study to find an antibiofilm agents to suppress Staphylococcus epidermidis biofilm production which is considered the main virulence factor of this bacterium. The sarA protein was chosen as the target for this process as it stimulates icaADBC operon which is responsible for biofilm production. The first step was constructing a 3D structure of the protein which was obtained using the RaptorX homology modelling. Pharmacophore generation was performed using the Hip Hop generator from Discovery Studio package. One hundred seventy seven molecules were chosen by ligand based virtual screening using ZincPharmer. Thirty seven molecules were found suitable as having negative binding free energies with sarA protein in EADock engine from the Swissdock Website. Seven of them were tested in lab and four of which gave antibiofilm activity (acetaminophen, acetylsalicylic acid, ibuprofen and acetic acid).
Full Text Links

Main Document: View

10-page Sections: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Next >

Bibliographical Information:

Advisor:Prof. Dr. Zahra Al-khafaji

School:Jami'at Baghdad

School Location:Iraq

Source Type:Master's Thesis

Keywords:drug design, bioinformatics, antibiofilm, S. epidermidis, Homology Modelling, Pharmacophore.

ISBN:

Date of Publication:10/12/2013

Document Text (Pages 1-10)

Republic of Iraq
Ministry of Higher Education
and Scientific Research
University of Baghdad
Genetic Engineering and
Biotechnology Institute

In Silico Drug Design of Biofilm
Inhibitors of Staphylococcus epidermidis

A thesis
Submitted to the council of Genetic Engineering and
Biotechnology Institute for Postgraduate Studies
University of Baghdad
In Partial fulfillment of the Requirements for the Degree
of Master in Genetic Engineering and
Biotechnology

By

Aymen Faraoun Ahmed Al-Mulla

B.Sc. in Pharmacy / College of Pharmacy / University of
Baghdad
(2000)

Supervised by

Prof. Dr. Zahra M. Al-Khafaji

2013 A.D. 1434 A.H


Page 2

Page 3

Dedication

To her who had given me dreams to look
forward.


Page 4

ACKNOWLEDGMENT
First and foremost, thanks to the beneficent and merciful God who gives me
everything I have. This research project would not have been possible without
the support of many people.

I am deeply grateful to my supervisor Prof. Dr. Zahra Al-Khafaji for her
patience, support, encouragement, supervision, kind advice and interesting
follow up which gave me confidence and made the completion of this work
possible. I benefited from her advice and valuable discussion, particularly so
when exploring new ideas during this research project.

Special thanks also to all those who I learned from in Genetic Engineering and
Biotechnology Institute specially Prof. Dr. Esam Al-Jumaily, Dr. Maarib
Nazeeh, Mr. Kais Kasim and Mr. Hashim Mohammed.

I would also like to convey thanks to the Ministry and Faculty for providing
the financial means and laboratory facilities.

Deepest gratitude are also to: Dr. Abbas Mahmood from Central Public Health
Laboratory, Dr. Khalida Kareem from College of Medicine/ Baghdad
University, Dr. Khalid Al-Sahr from Agriculture College/ Tikrit University, Dr.
Diaa Jabbar from College of Pharmacy/ Baghdad University, Mr. Hasan Abo Al-
Maaly from College of Pharmacy/ Karbalaa University, Mr. Natheer Basheer
from Physics Department/ Science College/ Baghdad University, Miss Entissar
Faraoun from Biology Department/ Science College/ Baghdad University, Miss
Khairia Jaabir from Ibn Al-Baladi Hospital, without whose knowledge and
assistance this study would not have been successful.

Not forgetting my best friends Osama Mohammed and Ali Akram thanks for
their encouragement, assistance and love.

I wish to express my love and gratitude to my beloved family specially my
father, mother, my wife and my sister for their understanding & endless love,
through the duration of my study.


Page 5

Summary

In Silico drug design represents a new approach for drug discovery and
industry. Structure based computer aided drug design (CADD) was used in
this study to find an antibiofilm agent to suppress Staphylococcus
epidermidis biofilm production which is considered the main virulence
factor of this bacterium. The sarA protein was chosen as the target for this
process as it stimulates icaADBC operon which is responsible for biofilm
production. Ninty six isolates were taken from urine, ear and wound swaps
of Iraqi patients for bacterial identification from 18/10/2012 to 28/2/2013
intervals. Eighty seven of them were diagnosed as staphylococci by Gram
stain, seventy five percent (75 %) of which were diagnosed as
S.epidermidis by biochemical tests. Nine isolates were diagnosed as
biofilm producers according to the tissue culture plate method and the tube
method.
An in Silico drug design was made using a structure based drug design.
The first step was constructing a 3D structure of the protein which was
obtained using the RaptorX homology modelling. Pharmacophore
generation was performed using the Hip Hop generator from Discovery
Studio package. One hundred seventy seven molecules were chosen by
ligand based virtual screening using ZincPharmer.
Thirty seven molecules were found suitable as having negative binding
free energies with sarA protein in EADock engine from the Swissdock
Website. Seven molecules were chosen to determine their practical
antibiofilm activity by using tissue culture plates. Four of them gave
antibiofilm activity, as follows:
1. Acetaminophen inhibited biofilm production 100% at 11 mg/ml
concentration.
2. Acetylsalicylic Acid inhibition was 100% at 1.6 mg/ml concentration.


Page 6

3. Ibuprofen inhibited 35% of biofilm production at 0.6 mg/ml
concentration.
4. Acetic Acid inhibition was 25% at 1mg/ml concentration.
All the above concentrations were found to affect biofilm production
without significant change in bacterial growth.
It was concluded that a drug design strategy using Ligand Based Virtual
Screening had a success score of about 60%.
Acetaminophen, Acetylsalicylic Acid, Ibuprofen and Acetic Acid can be
used as antibiofilm molecules and Non-Steroidal Anti-Inflammatory Drugs
family can be a useful library for antibiofilm future investigations.
It was concluded also that the sarA protein can be used as a good target for
biofilm suppression.


Page 7

List of contents

Title

Summary

Page
No.
I

List of contents III

List of Figures VIII

List of Tables XII

List of Abbreviations XIII

Definitions of Key Terms XVI

Chapter One: Introduction 1

Chapter Two: Literature Review

2.1 Drug Discovery and Design 3

2.1.1 Drug Discovery 3

2.1.2 Drug Design 6

2.1.3 In Silico Drug Design 7


Page 8

2.1.4 Strategies of In Silico Design 9

2.2 Bacterial Studies 24

2.2.1 Staphylococcus epidermidis 24

2.2.2 Clinical importance of Staphylococcus epidermidis 25

2.2.3 Staphylococcus epidermidis virulence factors 26

2.2.4 Biofilms 27

2.2.5 Gene Regulation of Biofilm 30

2.2.6 Mechanism of Biofilm Formation 33

Chapter Three: Materials and Methods

3.1 Materials 35

3.1.1 Preparation of Media 37

3.1.1.I Media used for isolation and identification of bacteria 37

3.1.1.II Media used for detection of biofilm production 38

3.1.1.III Reagents and Solutions 38


Page 9

3.2 Methods 38

3.2.1 Bacteriological study 38

3.2.1.I Isolation of bacteria 39

3.2.1.II Identification of bacteria 39

3.2.1.III Preservation of the bacteria 40

3.2.1.IV Biofilm detection methods 41

3.2.1.V Bacterial growth curve estimation 42

3.2.1.VI Measurement of antibiofilm activity 43

3.2.2 Computer aided drug design programs and databases 43

3.3 Statistics 44

Chapter Four: Results and Discussion

4.1 Drug Design 45

4.1.1 Target Preparation 45

4.1.1.I Target Determination 45


Page 10

4.1.1.II Homology Modelling 51

4.1.2 Ligands Search 54

4.1.3 Docking 56

4.1.4 Pharmacophore Virtual Screening 58

4.2 Identification of Bacterial Isolates 76

4.3 Biofilm Producer S. epidermidis 77

4.4 Bacterial Growth Curve 82

4.5 Antibiofilm Activity Estimation 91

4.5.1 Molecules Results 92

Conclusions and Recommendations

5.1 Conclusions 110

5.2 Recommendations 111

References 112

© 2009 OpenThesis.org. All Rights Reserved.