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 Human Dihydrofolate Reductase

Inhibitors ” is an authenticated work carried out by me at

BioCampus under the guidance of MS. Deepthi Samala for the

fulfillment of 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 Human Dihydrofolate Reductase

Inhibitors ” is an authenticated work carried out by me at

BioCampus under the guidance of MS. Deepthi Samala for the

fulfillment of 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 Human Dihydrofolate Reductase

Inhibitors ” is an authenticated work carried out by me at

BioCampus under the guidance of MS. Deepthi Samala for the

fulfillment of 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

ABSTRACT1. INTRODUCTION2. MATERIALS AND METHOD3. 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

1 Cdocker energy of all poses generated for highest activeMolecule 30

46

2 Ligand fit score of all the poses generated for highestActive molecule 31

49

3 Libdock scores of all the poses generated for highest activeMolecule 31

52

4 Hip Hop training set 53

5 Pharmacophore feature definitions 54

6 Rank file of the generated Pharmacophore model 54

7 Training set molecules taken for Hypogen 56

8 Input features considered for Hypogen 57

9 Best pharmacophore statistics 57

10 Ligand Pharmacophore mapped compounds showingLog active and Log Estimate values

60

List of FiguresS.No Title Page No

1 Potential areas for In Silico intervention in Drug discoveryprocess

1

2 Traditional Drug Discovery 33 Two strategies of Drug design 44 Role of computer aided Drug design 55 Crystal structure of Human Dihydrofolate reductase

Obtained from PDB7

6 Mechanism of reduction of Dihydrofolate toTetrahydrofolate

9

7 Discovery Studio Client 2.5 108 Target Protein hDHFR with LIH crystal ligand

Inhibitor13

9 Chemical structure of LIH crystal ligand 1310 Ligand showing interactions with binding site residues 1411 Chemical structure of 64 bioactive molecules 1512 Docking work flow 3313 Hypogen process flow 4014 LUDI interaction map showing HB donar (blue), HB

acceptor (red), and hydrophobic atoms42

15 Fragment placed on interaction map 4316 Linker S50 placed on the fragment 4317 Denovo ligand obtained by joining the fragment with

Linker using LUDI44

18 Closeup view of LUDI molecule showingInteraction with Val 115

45

19 Full view of LUDI molecule showing interaction withVal 115

45

20 Grid showing the active site of the protein 4721 Close up view of highly active molecule 31 showing

Interaction with Val 115 in Ligand fit48

22 Full view of highly active molecule 31 showingInteraction with Val 115 in Ligand fit

48

23 Libdock active protein grid 50

24 Closeup view of highly active molecule 31 showingInteraction with Glu 30 in Libdock

51

25 Full view of highly active molecule 31 showingInteraction with Glu 30 in Libdock

51

26 Hotspots file of highly active molecule 31 showingPolar atoms

52

27 Closeup view of highly active molecule 30 placed on thePharmacophore in hiphop

55

28 Full view of highly active molecule 30 placed on thePharmacophore in hiphop

55

29 Hypogen showing best pharmacophore model 5830 Full view of highly active molecule placed on the

Pharmacophore in hypogen59

31 Close up view of highly active molecule placed on thePharmacophore in hypogen

59

32 Hypogen test set validation graph showing an r2 value of0.627

60

INTRODUCTION

OBJECTIVE OF THE STUDY

MATERIALS AND METHODS

RESULTS AND DISCUSSIONS

CONCLUSION

REFERENCES