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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 Matthew Noestheden 1st Seminar Thursday, February 16th, 2006
Outline • What are protein kinases? • Why are they important to study? • Total synthesis of Wortmannin • Rational design - purine scaffold • Isoform specific kinase inhibitors
What are they? • Catalyze the phosphorylation of Ser, Thr and Tyr amino acid residues
What are they? OH ATP ATP ATP OPO3- OPO3- OPO3- OPO3- OPO3-
~500 in human genome1 1) Manning, et al. 2002. Science. 298(5600); 1912-1934.
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
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)
Studying Protein Kinases • Understanding non-disease linked protein kinase function? OH OPO3- OPO3- OPO3- OPO3- OPO3-
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
No Effect Desired Phenotype Death Studying Protein Kinases • Small-molecules • Eliminate developmental issues • Limit compensatory action
Small-Molecule Kinase Inhibitors Wortmannin Staurosporine Herbimycin A Purine Analogs Imatinib (Gleevec)
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.
Wortmannin Wortmannin
Wortmannin Drahl, et al. 2005. Angew. Chem. Int. Ed. 44: 5788-5809.
Activity • Inhibit PI3 kinase • Wortmannin inhibits 5 other human kinases IC50 = 4.2 nM
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
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.
Total Synthesis of Wortmannin (+)-Wortmannin 0.0002% yield over 53 steps
Purine Analogs • Simple scaffold • Several potential sites amenable to modification Adenine Scaffold ATP
Purine Library Gray, et al. 1998. Science. 281(41); 533-538.
Purine Library • Synthesized library to develop a more potent inhibitor of CDK21 Olomoucine IC50 = 7µM Gray, et al. 1998. Science. 281(41); 533-538.
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.
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
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.
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.
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.
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.
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.
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)
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.
Rescuing Catalytic Activity • MEKK1 – wound healing, cell motility and adhesion 1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.
Rescuing Catalytic Activity • GRK2 – heart failure Relative Activity (%) 80% of kinome accessible! 1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.
Covalent Inhibition • ~20% have smaller (Thr) gatekeepers Normal Kinase Mutant Kinase Inactive Inactive 1) Taunton et al. 2005. Science. 308; 1318-1321.
Covalent Inhibition • 2nd selectivity filter from bioinformatics analysis of 1˚ sequences Reactive Cys Gatekeeper 1) Taunton et al. 2005. Science. 308; 1318-1321.
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.
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.
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.
Covalent Inhibition • Selectively targets RSK1/RSK2 in complex whole cell extract RSK1/RSK2
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