Acknowledgement

I would like to extend my sincere thanks to Dr. J. A. R. P. SARMA, Sr. Vice

President, GVK Biosciences pvt. Ltd., for giving me a chance to carry out my project
work at BioCampus.
My Sincere thanks to Dr. K. V. Radha Kishan, Associate Vice President,
Informatics Division, GVK Bio, Hyderabad for permitting me to carry out this work
and providing the necessary facilities in the GVK BioCampus.
My deepest sense of guidance and respect to Mr. Sudheer Kumar Reddy,
Manager, BioCampus for his excellent guidance and his innovative thoughts and
motivation throughout the period of this course.
It gives me immense pleasure to thank Dr. Rambabu Gundla, Miss Deepthi
Samala, Mr. Rohit Kotla for their timely help without which this project would be
incomplete. I would like to express my heart full thanks to all BioCampus staff
members of GVK Biosciences for their timely help and support.
I take this opportunity to express my warmest thanks to my beloved parents
and all my friends, who had been a source of inspiration to me at all steps in this
study. Last but not least, I owe my thanks to almighty god, who made all the things
possible.

DECLARATION

I, declare that the project work entitled Structure And

Analogue Based Studies On
Inhibitors is

an

Human Dihydrofolate Reductase

authenticated

BioCampus under the guidance of

fulfillment

of

the

work

carried

out

by

me at

MS. Deepthi Samala for the

professional Certificate

course titled

Protein

Modeling and Rational Drug designing , during September 2010 January 2011 in GVKBiosciences Pvt. Ltd Hyderabad.

DATE:
PLACE: Hyderabad

A. GOWTHAMI

DECLARATION

I, declare that the project work entitled Structure And

Analogue Based Studies On
Inhibitors is

an

Human Dihydrofolate Reductase

authenticated

BioCampus under the guidance of

fulfillment

of

the

work

carried

out

by

me at

MS. Deepthi Samala for the

professional Certificate

course titled

Protein

Modeling and Rational Drug designing , during September 2010 January 2011 in GVKBiosciences Pvt. Ltd Hyderabad.

DATE:
PLACE: Hyderabad

MAMILLA SUJATHA

DECLARATION
I, declare that the project work entitled Structure And
Analogue Based Studies On
Inhibitors is

an

Human Dihydrofolate Reductase

authenticated

BioCampus under the guidance of

fulfillment

of

the

work

carried

out

by

me at

MS. Deepthi Samala for the

professional Certificate

course titled

Protein

Modeling and Rational Drug designing , during September 2010 January 2011 in GVKBiosciences Pvt. Ltd Hyderabad.

ABSTRACT
DHFR is an enzyme that in humans is encoded by the DHFR gene. The enzyme
reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor,
which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon
transfer chemistry. DHFR plays a central role in the synthesis of nucleic acid
precursors, and it has been shown that mutant cells that completely lack DHFR
require glycine, a purine, and thymidine to grow.
DHFR can be target in the treatment of cancer. DHFR is responsible for the levels of
tetrahydrofolate in a cell, and the inhibition of DHFR can limit the growth and
proliferation of cells that are characteristic of cancer. Methotrexate, a competitive
inhibitor of DHFR, is one such anticancer drug that inhibits DHFR. Other drugs
include trimethoprim and pyrimethamine. These 3 are widely used as antitumor and
antimicrobial agents. Folic acid is necessary for growth, and the pathway of the
metabolism of folic acid is a target in developing treatments for cancer. DHFR is one
such target. A regimen of fluorouracil, doxorubicin, and methotrexate was shown to
prolong survival in patients with advanced gastric cancer. Further studies into
inhibitors of DHFR can lead to more ways to treat cancer. Insilico studies of
Dihydrofolate reductase inhibitors were found to be highly promising in further
improvement and development of new lead compounds.
The active site residues of hDHFR include Val 115,Glu 30, Ile 7 and the binding site
residues include aminoacid residues such as Ala 9, Val 8, Pro 61, phe 31, phe 34. In
Structure based studies, the newly designed ligand using LUDI showed an interaction
with Val 115. Using Cdocker protocol denovo ligand was docked into the active site
of DHFR. The denovo ligand showed an interaction with the active site residue Val
115 with a Cdocker energy of 11.2922 obtained by pose number 1. The crystal ligand
LIH showed interactions withVal 115, Glu 30, Ile 7.The highly active molecule 31
(IC50= 0.4nM) in Ligand fit, dock score of highly active molecule 31 (IC50= 0.4nM)
is 58.929. In case of Libdock, the dock score of highly active molecule 31 (IC50 =
0.4nM) is 135.224.

In Analogue based Drug Design (ABDD), pharmacophore studies on hDHFR

inhibitors were carried out using HipHop and Hypogen. In HipHop the first 6 highly
active compounds were taken to generate common feature hypothesis. Out of 10
catalyst features the pharmacophore model showed five features namely Hydrogen
bond acceptor lipid (H), Hydrophobic (Z), Hydrophobic aliphatic (Y), Hydrophobic
aromatic (X) and Positive ionizable (W).
Hypogen training set was prepared using 18 compounds .The best hypothesis
generated by Hypogen showed an Root Mean Square (RMS) value of 1.63497,
relative error of 80.0593, weight of 1.12499, configuration of 10.363 with a
correlation value 0.9177. Point plot of Log active Vs Log Estimate values of the test
set compounds resulted in an R2 value of 0.627.

CONTENTS
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHOD
3. STRUCTURE BASED DRUG DESIGNING

Ludi
Ligand fit
C Docker
Structure Based pharmacophore

ANALOGUE BASED DRUG DESIGNING

Pharmacophore modeling
1. Hip-Hop
2. Hypogen
4. RESULTS

Ludi
Ligand fit
C-Docker
Lib dock
Structure based pharmacophore
Hip-Hop
Hypogen
5. CONCLUSION
6. REFERENCES

List of Tables
S.No

Title

Page No
46

Cdocker energy of all poses generated for highest active

Molecule 30
Ligand fit score of all the poses generated for highest
Active molecule 31
Libdock scores of all the poses generated for highest active
Molecule 31
Hip Hop training set

Pharmacophore feature definitions

54

Rank file of the generated Pharmacophore model

54

Training set molecules taken for Hypogen

56

Input features considered for Hypogen

57

Best pharmacophore statistics

57

10

Ligand Pharmacophore mapped compounds showing

Log active and Log Estimate values

60

1
2
3

49
52
53

S.No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

List of Figures
Title
Potential areas for In Silico intervention in Drug discovery
process
Traditional Drug Discovery
Two strategies of Drug design
Role of computer aided Drug design
Crystal structure of Human Dihydrofolate reductase
Obtained from PDB
Mechanism of reduction of Dihydrofolate to
Tetrahydrofolate
Discovery Studio Client 2.5
Target Protein hDHFR with LIH crystal ligand
Inhibitor
Chemical structure of LIH crystal ligand
Ligand showing interactions with binding site residues
Chemical structure of 64 bioactive molecules
Docking work flow
Hypogen process flow
LUDI interaction map showing HB donar (blue), HB
acceptor (red), and hydrophobic atoms
Fragment placed on interaction map
Linker S50 placed on the fragment
Denovo ligand obtained by joining the fragment with
Linker using LUDI
Closeup view of LUDI molecule showing
Interaction with Val 115

Page No
1
3
4
5
7
9
10
13
13
14
15
33
40
42
43
43
44
45

Full view of LUDI molecule showing interaction with

Val 115
Grid showing the active site of the protein
Close up view of highly active molecule 31 showing
Interaction with Val 115 in Ligand fit
Full view of highly active molecule 31 showing
Interaction with Val 115 in Ligand fit
Libdock active protein grid

45

Closeup view of highly active molecule 31 showing

Interaction with Glu 30 in Libdock
Full view of highly active molecule 31 showing

51

47
48
48
50

51

26
27
28
29
30
31
32

Interaction with Glu 30 in Libdock

Hotspots file of highly active molecule 31 showing
Polar atoms
Closeup view of highly active molecule 30 placed on the
Pharmacophore in hiphop
Full view of highly active molecule 30 placed on the
Pharmacophore in hiphop
Hypogen showing best pharmacophore model
Full view of highly active molecule placed on the
Pharmacophore in hypogen
Close up view of highly active molecule placed on the
Pharmacophore in hypogen
Hypogen test set validation graph showing an r2 value of
0.627

52
55
55
58
59
59
60

INTRODUCTION

OBJECTIVE OF
THE STUDY

MATERIALS AND
METHODS

RESULTS AND
DISCUSSIONS

CONCLUSION

REFERENCES