Investigating conformation specific drug targets of human Thymidilate

synthase

Ukegbu B. IrohaDepartment of Pharmaceutical sciences, University of South Carolina, Columbia.

Mentor: Dr. Sondra Berger

December 10, 2004.

Introduction/Objective

• Cancer

• Thymidilate Synthase

• Background

• Aim of research

TS

N

NH

O

O

R

N

NH

O

O

R

CH3

dUMP dTMP

N

NN

N

NNHOH

O

NH2

O

H

OH

O

O

H

N

NN

N

NHNHOH

O

NH2

O

H

OH

O

O

H

mTHFDHF

Thymidylate Synthase (TS)Reaction Pathway

Role of Thymidylate Synthase

Thymidylate Synthase Structure

• Nucleophilic Cys is located in the active site cleft

Cys180°

Cys

Native Complexed

MethodConstructing Mutant TS proteins

PCRrxn

Dpn Idigest

TX61-

Growth Conditions+/- exogenous

Thymidine

Protein purificationFPLC

blue sepharosesepharose Q

catalyticactivity

LigandBindinganalysis

3-D structure determination

JM109

Creating V3A mutant

• dH2O -37.5l

• 10x Pfu Buffer -5.0l

• DtNps -1.0l

• Templates -1.0l

• Primer1(SB229) -2.5l

• Primer 2(SB230)-2.5l

• Pfu Turbo -0.5l

50l

Mutation primer sequence position

V3A SB229 5`-TTA TGC CTG CTG CTG GTT C-3`

SB230 5`-GAA CAA GCA GCA GGC ATA A-3`

DPNI digest

PCR

Transformation on [Tx61-] cells

Plasmid purification

• Grew 10ml overnight culture• Use Qiagen plasmid mini-prep purification kit

protocol

Western blot analysis of Active and inactive stabilized mutants of hts in TX61- cells.

• 10ml overnight culture were sonicated in Buffer A• Remove cellular debris by centrifugation• Use bradford assay to determine concentration of total

soluble protein• Load protein in well.

bradford assay

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2 4 6 8 10 12 14

concentration g/l

abso

rbsa

nce

59

5

Mutant protein SLB load

HtsTx61-V3A

232632

8 9 11

15 17 21

y = 0.0483x + 0.0007R2 = 0.9898

Future Direction

• Characterization of V3A

Activity assay

Ligand binding (DUMP ligands)

Reactivity of Catalytic cystine• Structural Determination

Crystallization of protein.

Acknowledgement

• Dr. Sondra Berger• Saphronia Johnson• Brittnaei Bell• Dr. Omar Bagasra• Twaina Harris• Claflin University• University of South Carolina, Columbia• National Cancer Institute