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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

Total Synthesis and Rational Design of Protein Kinase Inhibitors

Matthew Noestheden

1st Seminar

Thursday, February 16th, 2006

outline
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
What are they?
  • Catalyze the phosphorylation of Ser, Thr and Tyr amino acid residues
what are they1
What are they?

OH

ATP

ATP

ATP

OPO3-

OPO3-

OPO3-

OPO3-

OPO3-

500 in human genome 1
~500 in human genome1

1) Manning, et al. 2002. Science. 298(5600); 1912-1934.

importance
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

importance1
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
Studying Protein Kinases
  • Understanding non-disease linked protein kinase function?

OH

OPO3-

OPO3-

OPO3-

OPO3-

OPO3-

studying protein kinases1

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

studying protein kinases2

No Effect

Desired Phenotype

Death

Studying Protein Kinases
  • Small-molecules
      • Eliminate developmental issues
      • Limit compensatory action
small molecule kinase inhibitors
Small-Molecule Kinase Inhibitors

Wortmannin

Staurosporine

Herbimycin A

Purine Analogs

Imatinib (Gleevec)

wortmannin
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.

wortmannin1
Wortmannin

Wortmannin

wortmannin3
Wortmannin

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

activity
Activity
  • Inhibit PI3 kinase
  • Wortmannin inhibits 5 other human kinases

IC50 = 4.2 nM

syntheses to date
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
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 wortmannin8
Total Synthesis of Wortmannin

(+)-Wortmannin

0.0002% yield over 53 steps

purine analogs
Purine Analogs
  • Simple scaffold
  • Several potential sites amenable to modification

Adenine Scaffold

ATP

purine library
Purine Library

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

purine library1
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 library2
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
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

specificity1

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.

isoform specific inhibitors 1 2

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 inhibitors 1 21
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 limitations 1 2
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
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.

rescuing catalytic activity1

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 activity2
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 activity3
Rescuing Catalytic Activity
  • MEKK1 – wound healing, cell motility and adhesion

1) Zhang et al. 2005. Nature Methods. 2(6); 435-441.

rescuing catalytic activity4
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
Covalent Inhibition
  • ~20% have smaller (Thr) gatekeepers

Normal Kinase

Mutant Kinase

Inactive

Inactive

1) Taunton et al. 2005. Science. 308; 1318-1321.

covalent inhibition2
Covalent Inhibition
  • 2nd selectivity filter from bioinformatics analysis of 1˚ sequences

Reactive Cys

Gatekeeper

1) Taunton et al. 2005. Science. 308; 1318-1321.

covalent inhibition3
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 inhibition4
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 inhibition5
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 inhibition6
Covalent Inhibition
  • Selectively targets RSK1/RSK2 in complex whole cell extract

RSK1/RSK2

summary1
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
Acknowledgements
  • John Pezacki
  • Natalie Goto
  • Bojana Rakic
  • Trevor Mischki
  • Gianni Lorello
  • Jane Hu

Pezacki Lab

importance2
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

slide54

APH(3’)-IIIa

APH(3’)-IIIaM90G/A

M90G/A

  • Error prone PCR
  • Clone into expression
  • vector

Neomycin

Kanamycin

Express in E. coli

Circular DNA with mutated

APH(3’)-IIIa

rescuing catalytic activity5
Rescuing Catalytic Activity

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. 2005. Nature Methods. 2(6); 435-441.

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

IC50 = 1.5 µM

IC50 = 0.55 µM

activity1
Activity
  • Inhibit PI3 kinase
  • Wortmannin inhibits 5 other human kinases

IC50 = 4.2 nM

IC50 = 2 nM, 0.1 nM

slide59

Normal Kinase

Normal Kinase

Normal Kinase

Inactive

Inactive

summary2
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