Investigating conformation specific drug targets of human Thymidilate synthase Ukegbu B. Iroha Department of Pharmaceutical sciences, University of South Carolina, Columbia. Mentor: Dr. Sondra Berger December 10, 2004.

Introduction/Objective Cancer Thymidilate Synthase Background Aim of research

Thymidylate Synthase (TS) Reaction Pathway

Role of Thymidylate Synthase

Thymidylate Synthase Structure Cys 180° Cys Nucleophilic Cys is located in the active site cleft

Native Complexed

Constructing Mutant TS proteins Method Constructing Mutant TS proteins JM109 Dpn I digest PCR rxn TX61- catalytic activity Growth Conditions +/- exogenous Thymidine Protein purification FPLC blue sepharose sepharose Q 3-D structure determination Ligand Binding analysis

Creating V3A mutant dH2O -37.5ml 10x Pfu Buffer -5.0ml DtNps -1.0ml DPNI digest PCR dH2O -37.5ml 10x Pfu Buffer -5.0ml DtNps -1.0ml Templates -1.0ml Primer1(SB229) -2.5ml Primer 2(SB230)-2.5ml Pfu Turbo -0.5ml 50ml 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`

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. y = 0.0483x + 0.0007 R2 = 0.9898 Mutant protein SLB load 23 26 32 8 9 11 15 17 21 Hts Tx61- V3A

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