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Total Synthesis and Rational Design of Protein Kinase Inhibitors

Total Synthesis and Rational Design of Protein Kinase Inhibitors. Matthew Noestheden 1 st Seminar Thursday, February 16 th , 2006. Outline. What are protein kinases? Why are they important to study? Total synthesis of Wortmannin Rational design - purine scaffold

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Total Synthesis and Rational Design of Protein Kinase Inhibitors

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  1. Total Synthesis and Rational Design of Protein Kinase Inhibitors Matthew Noestheden 1st Seminar Thursday, February 16th, 2006

  2. Outline • What are protein kinases? • Why are they important to study? • Total synthesis of Wortmannin • Rational design - purine scaffold • Isoform specific kinase inhibitors

  3. What are they? • Catalyze the phosphorylation of Ser, Thr and Tyr amino acid residues

  4. What are they? OH ATP ATP ATP OPO3- OPO3- OPO3- OPO3- OPO3-

  5. ~500 in human genome1 1) Manning, et al. 2002. Science. 298(5600); 1912-1934.

  6. Importance? Neurodegenerative Disorders • Rho kinase inhibitors effective at treating animal models of Alzheimer’s • Cannot distinguish between Rho isoforms IC50 = 1 nM ATP Dimethyl-Fasudil

  7. Importance? Cancer • Constitutive activation of a variety of protein kinases has been directly linked to certain cancers CML BCR-ABL c-KIT PDGFRα PDGFRβ Imatinib (Gleevec)

  8. Studying Protein Kinases • Understanding non-disease linked protein kinase function? OH OPO3- OPO3- OPO3- OPO3- OPO3-

  9. No Effect Mild Phenotype Death Studying Protein Kinases • Genetic manipulation to affect kinase activity • Mild phenotype – redundancy & compensatory action • Developmental/viability issues Mutant lacking gene of interest Desired Phenotype

  10. No Effect Desired Phenotype Death Studying Protein Kinases • Small-molecules • Eliminate developmental issues • Limit compensatory action

  11. Small-Molecule Kinase Inhibitors Wortmannin Staurosporine Herbimycin A Purine Analogs Imatinib (Gleevec)

  12. Wortmannin • First isolated from Penicillium wortmanii in 19571 • Part of larger family of steroidal furanoids 1) Brian, et al. 1957. Trans. Brit. Mycol. Soc. 40; 365-368.

  13. Wortmannin Wortmannin

  14. Wortmannin

  15. Wortmannin Drahl, et al. 2005. Angew. Chem. Int. Ed. 44: 5788-5809.

  16. Activity • Inhibit PI3 kinase • Wortmannin inhibits 5 other human kinases IC50 = 4.2 nM

  17. Syntheses to Date: Shibasaki, et al. 1996. Tetrahedron. Lett.37(34); 6141-6144. Shibasaki, et al. 2005. Tetrahedron.61; 5057-5065. • Optically pure hydrocortisone • 1st formal total synthesis of (+)-wortmannin Shibasaki, et al. 2002. Angew. Chem. Int. Ed.41(24); 4680-4682. • (±)-wortmannin

  18. Total Synthesis of Wortmannin 1) Shibasaki, et al. 2002. Angew. Chem. Int. Ed.41(24); 4680-4682. 2) Shibasaki, et al. 2005. Tetrahedron.61; 5057-5065.

  19. Total Synthesis of Wortmannin

  20. Total Synthesis of Wortmannin

  21. Total Synthesis of Wortmannin

  22. Total Synthesis of Wortmannin

  23. Total Synthesis of Wortmannin

  24. Total Synthesis of Wortmannin

  25. Total Synthesis of Wortmannin

  26. Total Synthesis of Wortmannin (+)-Wortmannin 0.0002% yield over 53 steps

  27. Purine Analogs • Simple scaffold • Several potential sites amenable to modification Adenine Scaffold ATP

  28. Purine Analogs

  29. Purine Library Gray, et al. 1998. Science. 281(41); 533-538.

  30. Purine Library • Synthesized library to develop a more potent inhibitor of CDK21 Olomoucine IC50 = 7µM Gray, et al. 1998. Science. 281(41); 533-538.

  31. Purine Library Purvalanol A Purvalanol B Compound 52 cdc2-cyclin B CDK2-cyclin A CDK2-cyclin E CDK5-p35 Cdc28p Pho85p cdc2-cyclin B CDK2-cyclin A CDK2-cyclin E CDK5-p35 Cdc28p Pho85p Cdc2 CDK2 Gray, et al. 1998. Science. 281(41); 533-538.

  32. Summary • Total synthesis of (+)-Wortmannin • Wortmannin and purine analogs inhibit protein kinases with high affinity (low nM IC50) • Specificity limited, especially amongst closely related proteins Purine Analogs Wortmannin

  33. Specificity?

  34. Normal Kinase Mutant Kinase Specificity? • Alter kinase active-site? Pocket Generation Active Inactive 1) Shokat et al. 1998. Curr. Biol.8; 257-266. 2) Shokat et al. 1999. J. Am. Chem. Soc. 121; 627-631.

  35. Normal Kinase Mutant Kinase Isoform Specific Inhibitors1,2 • v-Src Ile338 → Gly/Ala mutant identified • “Gatekeeper” residue or 1) Shokat et al. 1998. Curr. Biol.8; 257-266. 2) Shokat et al. 1999. J. Am. Chem. Soc. 121; 627-631.

  36. Isoform Specific Inhibitors1,2 • No significant change to in vivo function • Expanded ATP active-site accepts N6-adenine analogs Mutant Kinase Mutant Kinase 1) Shokat et al. 1998. Curr. Biol.8; 257-266. 2) Shokat et al. 1999. J. Am. Chem. Soc. 121; 627-631.

  37. Gatekeeper Limitations1,2 • ID residue in nearly all eukaryotic protein kinases • ~30% are intolerant to gatekeeper mutation • ~20% contain Thr gatekeeper 1) Shokat et al. 1998. Curr. Biol.8; 257-266. 2) Shokat et al. 1999. J. Am. Chem. Soc. 121; 627-631.

  38. Rescuing Catalytic Activity • ~30% lose catalytic activity • 2nd mutation that rescues catalytic activity? 2nd Mutation Gatekeeper Mutation Active Kinase Inactive Kinase 1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.

  39. 2nd Mutation Gatekeeper Mutation Inactive Kinase Rescuing Catalytic Activity • N-terminal Asn→Thr mutation rescued activity of intolerant kinase • Analogous residue identified in 3 representative intolerant protein kinases (CDK2, MEKK1, GRK2)

  40. Rescuing Catalytic Activity • CDK2 – anti-cancer, anti-viral, cardiovascular diseases 10-fold Serial Dilutions 1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.

  41. Rescuing Catalytic Activity • MEKK1 – wound healing, cell motility and adhesion 1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.

  42. Rescuing Catalytic Activity • GRK2 – heart failure Relative Activity (%) 80% of kinome accessible! 1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.

  43. Covalent Inhibition • ~20% have smaller (Thr) gatekeepers Normal Kinase Mutant Kinase Inactive Inactive 1) Taunton et al. 2005. Science. 308; 1318-1321.

  44. Covalent Inhibition

  45. Covalent Inhibition • 2nd selectivity filter from bioinformatics analysis of 1˚ sequences Reactive Cys Gatekeeper 1) Taunton et al. 2005. Science. 308; 1318-1321.

  46. Covalent Inhibition • Pyrrolopyrimidine scaffold mimics adenine core of ATP • Install electrophile at C8 to react with Cys Adenine Pyrrolopyridine ATP mimic 1) Taunton et al. 2005. Science. 308; 1318-1321.

  47. Covalent Inhibition Occupies expanded active-site Reacts with Cys R = Kinase Acylated Enzyme α-fluoromethylketone (fmk) 1) Taunton et al. 2005. Science. 308; 1318-1321.

  48. Covalent Inhibition = fmk = biotin • Wild-type • preincubation with fmk abrogates biotin-fmk binding Normal Kinase Normal Kinase Cys Mutation • No reaction with biotin-fmk Normal Kinase Gatekeeper Mutation • No reaction with biotin-fmk Inactive Inactive fmk Biotin-fmk (1 µM) Need both selectivity filters for inhibition 1) Taunton et al. 2005. Science. 308; 1318-1321.

  49. Covalent Inhibition • Selectively targets RSK1/RSK2 in complex whole cell extract RSK1/RSK2

  50. Summary • Specificity important to Medicinal Chemistry, requisite for Cell Biology • Engineering novel protein kinases active sites • ‘Bump-Hole’ methodology • Inhibition of distinct protein isoforms with rationally designed small-molecules • >80% of kinome accessible for isoform specific study

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