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Investigating conformation specific drug targets of human Thymidilate synthase

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.

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Investigating conformation specific drug targets of human Thymidilate synthase

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  1. 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.

  2. Introduction/Objective • Cancer • Thymidilate Synthase • Background • Aim of research

  3. Thymidylate Synthase (TS)Reaction Pathway

  4. Role of Thymidylate Synthase

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

  6. Native Complexed

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

  8. Creating V3A mutant DPNI digest • 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 PCR 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`

  9. Transformation on [Tx61-] cells

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

  11. 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

  12. Future Direction • Characterization of V3A Activity assay Ligand binding (DUMP ligands) Reactivity of Catalytic cystine • Structural Determination Crystallization of protein.

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

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