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New pterin chemistry for the development of small molecule therapeutics

New pterin chemistry for the development of small molecule therapeutics. Jeffrey Pruet, Ph.D Franklin & Marshall Research Seminar. Overview. Pteridine heterocycles Background Significance Pterins targeting Ricin Toxin A Pterins targeting Methionine Synthase

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New pterin chemistry for the development of small molecule therapeutics

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  1. New pterin chemistry for the development of small molecule therapeutics Jeffrey Pruet, Ph.D Franklin & Marshall Research Seminar

  2. Overview • Pteridine heterocycles • Background • Significance • Pterins targeting Ricin Toxin A • Pterins targeting Methionine Synthase • Cobalamin dependant vs independent • Candida albicans • Developing new pterin chemistry • Inhibitor design

  3. Pteridines • Structure • 2-amino-4(3H)-pteridinone • Biologically relevant pterins • Folates • Biopterin

  4. Pterin Synthesis • Gabriel/Colman (Isay condensation) • Boon synthesis • Taylor method Brown, D. Pteridines; Wiley: New York, 1988

  5. Insolubility Ciuchi, F; Nicola, G; Franz, H; Mariani, P Bossi, M; Spada, G. JACS1994, 116, 7064-7071

  6. Ricin • Ricin is a type 2 ribosome inactivating protein (RIP) • A chain inactivates Ribosome (RTA) • B chain assists in uptake into cell • Extracted from castor oil byproducts • Toxic at <1 µg/Kg • Currently no antidote • Potential weapon • Georgi Markov • Tainted letters sent to Washington Yan, X.; Hollis, T.; Monzingo, F.; Robertus, J. J Mol Biol1997, 266, 1043-1049

  7. Proposed method of action

  8. Carboxy pterins No Inhibition IC50= 230µM

  9. Direct synthesis of new 7-substituted pterins via acyl-radical insertion • Reactions over in minutes • Products often precipitate out • Products unattainable via Friedel-Crafts acylation • Regiospecific Pruet, J. M.; Robertus, J. D.; Anslyn, E. V. Tet Lett.2010, 51,2539-2540

  10. Too insoluble to be synthetically useful Pruet, J; Jasheway, K; Manzano, L; Bai, Y; Anslyn, E; Robertus, J. Eur J Med Chem2011, 46, 3608-3615

  11. IC50= 700 µM No inhibition No inhibition IC50= 1.6 mM 500 µM (35%) IC50= 210 µM No inhibition IC50= 200 µM IC50= 570 µM IC50= 500 µM IC50= 380 µM Pruet, J; Jasheway, K; Manzano, L; Bai, Y; Anslyn, E; Robertus, J. Eur J Med Chem2011, 46, 3608-3615

  12. Two Routes for Optimizations • Furans • Ethylene diamine/glycine linkers

  13. Soluble organic salt Pruet, J; Saito, R; Manzano, L; Jasheway, K; Wiget, P; Kamat, I; Anslyn, E; Robertus, J. J. ACS Med Chem Lett2012, 3, 588-591

  14. DBU expands the scope of pterin chemistry IC50= 30 µM Pruet, J; Saito, R; Manzano, L; Jasheway, K; Wiget, P; Kamat, I; Anslyn, E; Robertus, J. J. ACS Med Chem Lett2012, 3, 588-591

  15. Peptide-conjugated pterins IC50= 20 µM IC50= 15 µM Saito R, Pruet JM, Manzano LA, Jasheway K, Monzingo AF, Wiget PA, Kamat I, Anslyn EV, Robertus JD. J Med Chem. 2013, 56(1), 320-329

  16. IC50= 35µM IC50≈ 30µM IC50= 35µM Saito R, Pruet JM, Manzano LA, Jasheway K, Monzingo AF, Wiget PA, Kamat I, Anslyn EV, Robertus JD. J Med Chem. 2013, 56(1), 320-329

  17. RTA overview • Pterins are a reliable core for designing RTA inhibitors • 7-substituted pterins show more promise than 6-isomer • 7-carboxy pterin (7CP) IC50 = 230µM • Improved route towards 7CP via regiospecific acyl radical insertion • Initial amide screen directed future synthesis • Furans • Order of magnitude improvement with expanded furans • DBU greatly improves synthesis of new pterin amides • Triazoles give rise to more potent inhibitors • Ethylene diamine / glycine linkers • Peptide-conjugated pterins • Edge-to-face interaction • “Decarboxy” peptide isosters

  18. Proposed Research New pterin chemistry for B12-independent Methionine Synthase inhibition

  19. What is Methionine Synthase? • Net reaction • Biological importance • Folate cycle • Only enzyme to regenerate tetrahydrofolate (THF) from 5-Me-THF • Shortage can cause megaloblastic anemia • Nucleotide biosynthesis • Important for rapidly dividing cells • S-adenosyl-methionine • Methyltransferase reactions for DNA, proteins, etc

  20. Two classes of Methionine Synthase • Cobalamin-dependant • Found in mammals, other animals & bacteria • 140,000 kDa • 5-Me-THF transfers methyl to cobalamin (B12) which then transfers to Hcy • Folate and Hcy binding sites separated by ~50Å • Cobalamin-independent • Found in plants, fungi & bacteria • Smaller (~80 kDa) • Folate and Hcy sites in close proximity • Methyl transferred directly to Zn-bound Hcy

  21. Why is B12-independent Methionine Synthase an attractive target? • Humans only use cobalamin-dependent MetSyn • Inhibitors specifically tailored to B12-independent enzyme may serve as anti-fungal • Candida albicans • Simple assay • Fluorescent sensing of homocysteine

  22. Selective anti-fungal treatment via B12-independentMetSyn inhibitors • Target folate and Hcy binding sites simultaneously • Sites too separated in human form of MetSyn

  23. New pterin chemistry aided by DBU • Baylis-Hillman reaction • Mechanism

  24. New pterin chemistry aided by DBU • Baylis-Hillmanon pterin aldehyde • Baylis-Hillman/amidation cascade reaction

  25. Inhibitor design

  26. Summary of proposed research • Strong history of exploring pterins • Insights and expertise in their chemistry • Undergraduates will have opportunity to learn about targeted drug design • Optimization of Baylis-Hillman reaction • Construction of novel inhibitor libraries • Optimizing Baylis-Hillman/Thiol-addition/amidation cascade • Fluorescent detection of Hcy • Developing broad spectrum anti-fungals • Highly beneficial for immuno-compromised patients

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