Total Synthesis and Rational Design of Protein Kinase Inhibitors

Name: Total Synthesis and Rational Design of Protein Kinase Inhibitors
Uploaded: 2017-07-30T14:35:27+00:00
Duration: PTM20S48
Channel: Ami Gallagher
Description: Total Synthesis and Rational Design of Protein Kinase Inhibitors

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 Science. 298(5600);

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 BCR-ABL CML c-KIT PDGFRα PDGFRβ Imatinib (Gleevec)

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

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

Small-molecules Eliminate developmental issues Limit compensatory action No Effect Death Desired Phenotype

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 Trans. Brit. Mycol. Soc. 40;

Wortmannin Wortmannin

Wortmannin

Wortmannin Drahl, et al Angew. Chem. Int. Ed. 44:

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

Syntheses to Date: Optically pure hydrocortisone
Shibasaki, et al Tetrahedron. Lett. 37(34); Shibasaki, et al Tetrahedron. 61; Optically pure hydrocortisone 1st formal total synthesis of (+)-wortmannin Shibasaki, et al Angew. Chem. Int. Ed. 41(24); (±)-wortmannin

Total Synthesis of Wortmannin
1) Shibasaki, et al Angew. Chem. Int. Ed. 41(24); 2) Shibasaki, et al Tetrahedron. 61;

0.0002% yield over 53 steps

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

Purine Analogs

Purine Library Gray, et al Science. 281(41);

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

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 Science. 281(41);

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 Wortmannin Purine Analogs

Specificity?

Specificity? Alter kinase active-site? Normal Kinase Pocket Generation
Mutant Kinase Active Inactive 1) Shokat et al Curr. Biol. 8; 2) Shokat et al J. Am. Chem. Soc. 121;

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

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 Curr. Biol. 8; 2) Shokat et al J. Am. Chem. Soc. 121;

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 Curr. Biol. 8; 2) Shokat et al J. Am. Chem. Soc. 121;

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

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

CDK2 – anti-cancer, anti-viral, cardiovascular diseases 10-fold Serial Dilutions 1) Zhang et al Nature Methods. 2(6);

MEKK1 – wound healing, cell motility and adhesion 1) Zhang et al Nature Methods. 2(6);

GRK2 – heart failure Relative Activity (%) 80% of kinome accessible! 1) Zhang et al Nature Methods. 2(6);

Covalent Inhibition ~20% have smaller (Thr) gatekeepers Normal Kinase
Mutant Kinase Inactive Inactive 1) Taunton et al Science. 308;

Covalent Inhibition

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

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 Science. 308;

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

filters for inhibition
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 Science. 308;

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

Acknowledgements John Pezacki Pezacki Lab Natalie Goto Bojana Rakic
Trevor Mischki Gianni Lorello Jane Hu

Importance? Type II Diabetes
Insulin insensitivity increases blood glucose Glucose production via gluconeogenesis PDH regulated by PDH kinase Diabetes related ailments Pyruvate ↑ Blood Glc Inhibition PDH PDH Kinase

Circular DNA with mutated
APH(3’)-IIIa APH(3’)-IIIaM90G/A M90G/A Error prone PCR Clone into expression vector Express in E. coli Kanamycin Neomycin Circular DNA with mutated

Location Mutation Y17H R18H N-Terminal Lobe E24V E24K N87T Gatekeeper G90V G90E E101A E103A E103V S135N C-Terminal Lobe L136V L136I T163S R169C L175I G205S Y219N Applied identical methodology to 3 distantly related intolerant protein kinases Not applicable to protein kinases Nearest to gatekeeper in 1º and 3º structure Mutation to larger, wt residues rescues catalytic activity Not applicable to protein kinases 1) Zhang et al Nature Methods. 2(6);

Selectivity?

Furanoids Inhibit the pp60v-Src oncogene
Halenaquinol inhibits EGFR (IC50 = 19 µM) IC50 = 1.5 µM IC50 = 0.55 µM

Activity Inhibit PI3 kinase Wortmannin inhibits 5 other human kinases
IC50 = 4.2 nM IC50 = 2 nM, 0.1 nM

Normal Kinase Normal Kinase Normal Kinase Inactive Inactive

Summary Protein kinases important in disease states
Wortmannin and purine analogs inhibit protein kinases with high affinity (low nM IC50) Specificity limited, especially amongst closely related proteins Highly conserved active-site across kinome

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