MODULATION OF HUMAN PYRIDOXAL KINASE ACTIVITY BY DRUGS Samuel Aboagye Howard Hughes Medical Institute Scholar.

1. MODULATION OF HUMAN PYRIDOXAL KINASE ACTIVITY BY DRUGSSamuel AboagyeHoward Hughes Medical Institute Scholar.

2. Contents • Introduction • Expression and purification of human Pyridoxal Kinase (hPLK) • Effects of drugs on PL kinase activity

3. Vitamin B6 • Vitamin B6 is the most essential vitamin serving a vital role in the function of more than 100 enzymes, including: -Neurotransmitter metabolism – dopamine, serotonin, histamine, taurine, epinephrine -Red blood cell formation - synthesis of heme -Amino acid metabolism

4. Diseases associated with vitamin B6 deficiency • Neurological disorders • Anemia • Blood sugar levels • Cardiovascular disease • Immune disorders

5. Six forms of Vitamin B6

6. Salvage pathway of vitamin B6

7. Pyridoxal Kinase (PLK) PL Kinase catalyses the transfer of a phosphate group from ATP to PL, PN or PM to form PLP, PNP and PMP, respectively. Kinase PL + ATP PLP + ADP Cofactor for B6-Enzymes

8. Objectives • hPLK activity seems to be the key site for some disorders and side effects of many drugs. • - Protein production • - Kinetic studies • - X-ray crystallography

9. 3D Structure of hPLK

10. EXPRESSION AND PURIFICATION

11. PROTEIN EXPRESSION Preparation of Liquid broth (LB) medium - Bacto Tryptone; Yeast Extract; NaCl;K2HPO4 ; NaH2PO4 ;7L Deionized Water . Growing of the cells- The protein clone (E.coli PdxK gene) is added to the LB medium and incubated for a minimum of 24 hours. Grown cell culture incubated for at least an hour. Presence of grown cell cultures checked at absorbance of 1 or greater with a spectrophotometer at 600nm. IPTG added to cell culture and incubated for 5 hours - expressing the PL kinase protein. Cells broken using the cell breaker column to release protein, and the presence of protein checked by Gel-electrophoresis.

12. Expression of Protein After IPTG Before IPTG hPLK Band Corresponding to marker position at Band 6 ( Mol Wt. 34.)

13. hPLK Purification Procedures

14. First column purification • Sample loaded onto a 5x9cm TMAE column pre-equilibrated with 20mM KPi pH 7.2. Protein eluted after Abs of 278 is below 0.4 with the equilibration buffer and 40mM KPi 200mM Nacl pH 6.8. • Collected fractions checked at Abs of 278, followed by gel electrophoresis to determine the fraction containing the PL kinase. hPLK Band Corresponding to marker position at Band 6 ( Mol Wt. 34.)

15. Second Column Purification • Sample from 1st column loaded onto a phenyl sepharose column (2.5x5cm) that is pre-equilibrated with 20% ammonium sulfate in 20mM KPi pH 7.2 with 5mM Mercaptoethanol and 0.2mM EDTA. • Equilibration buffer used to wash away excess DNA until Abs 260nm is less than 0.1. • The enzyme eluted with the equilibration buffer and 20nM Na BES pH 7.5, 3% propylene glycol. hPLK Band Corresponding to marker position at Band 6 ( Mol Wt. 34.)

16. Purification columns

17. KINETIC STUDIES

18. How PL activity is measured • Spectrophotmetric Analysis Kinase • PL PLP MgATP Change in absorbance at 388 nm

19. Initial Activity of Protein

20. Activity after compound is added THEOPYLLINE.

21. Inhibition by Theophylline

22. Activity after compound is added CAFFIENE

23. Inhibition by Caffeine

24. X-RAY CRYSTALLOGRAPHY • Drugs that show inhibition such as theophylline are co-crystallized with PL Kinase to see where it binds. • I hope to work on this in the future.

25. CONCLUSION • These studies suggest that with theophylline, there is a significant drop in activity as compared to caffeine. • These finding suggests that certain drugs, such as theophylline has antagonistic effect on hPLK. • Other drugs with similar chemical structure as theophylline are known to cause neurological disorders. • We hypothesize that these drugs namely nicotine, theobromine and enprofylline may also inhibit PL Kinase activity.

26. References 1.Musayev et al,(1999) Crystallization and preliminary X-ray crystallographic analyses of pyridoxine 5’-phosphate oxidase complexed with flavin mononucleotide.J.Struct.Biol. 127,88-91. 2.Safo et al(2000) X-ray structure of Escherichia coli pyridoxine 5’-phosphate oxidase complexed with FMN at 1.8 A resolution. Structure 8, 751-762. 3.Safo et al(2001) X-ray structure of Escherichia coli pyridoxine 5’-phosphate oxidase complexed with Pyridoxal 5’-phosphate oxidase complexed with pyridoxal 5’-phosphate at 2.0 A resolution. J. Mol. Biol.310, 817-826 4.Di Salvo et al(2002) J.Mol.Bio. 315,385-397 5.Safo et al(2003) Structure and properties of Recombinant Human Pyridoxine 5’-phosphate Oxidase. Protein Sci.2003, In press. 6.Martino et al(2003) Structure and mechanism of Escherichia Coli pyridoxine 5’-phosphate Oxidase.Biochimica et Biopysica Acta Proteins& Proteomics, 1647, 76-82 7.Safo et al(2003) Role of Proline Residues in the Folding of Serine Hydroxymethyltransferase. J. Biol.Chem., In Press. 8.Cessac et al, Mechanisms of the Inhibition of Human Erythrocyte Pyridoxal Kinase by Drugs. Biochemical Pharmacology,Vol. 54 pp. 863-870,1997 9.The American Heritage Science Dictionary . 10.Linus Pauling Institute at Oregon State University. http://LPi.oregonstate.edu/infocenter/vitamins/vitaminB6/

27. ACKNOWLEDGEMENTS • Howard Hughes Medical Institute and organizers of the summer scholars program. Dr. Allison Johnson, Dr. Greg Buck • Dr. Martin Safo, Mentor, Assistant Professor Department of Medicinal Chemistry • Dr. Musayev Faik, Researcher, Institute of Structural Biology and Drug Discovery • Amit Gandhi , PHD candidate, Department of Medicinal Chemistry • Ashwani Goswami , PHD candidate, Department of Medicinal Chemistry.