Transcript ATP binding site Protein Kinase Inhibitors

ANTICANCER AGENTS
PROTEIN KINASE INHIBITORS
Chapter 21
Protein Kinases
•Enzymes that catalyse phosphorylation reactions on protein substrates
•500-2000 estimated protein kinases in a cell
•Protein kinases are present in the cytoplasm
•Protein kinase receptors - dual role as receptor and enzyme
•Overexpression can result in cancer
•Tyrosine kinases, serine-threonine kinases and histidine kinases
•ATP used as enzyme cofactor - phosphorylating agent
H
N
N
1
6
N
H
H
N
N
O
O
O
O P
O P
O P
O
O
O
N
O
N
1
6
N
H
N
N
O
H
O
H
H
OH
H
OH
O
O P
O
O
H
H
H
OH
H
OH
O
O
O P
O P
O
O
O
Protein Kinases
Tyrosine kinases
O
O
H H
N
Protein
H H
N
Protein
Protein
Protein
O
ATP
OH
ADP
P
O
OH
OH
Protein Kinases
Serine-threonine kinases
O
O
H H
N
H H
N
Protein
Protein
Protein
Protein
O
OH
ATP
ADP
P
Serine
O
OH
OH
O
H H
N
O
H H
N
Protein
Protein
Protein
Protein
H3 C
OH
Threonine
ATP
ADP
H3C
O
P
O
OH
OH
Protein Kinases
Active Site
•Contains the binding site for the protein substrate
•Contains the binding site for the ATP cofactor
•Clinically useful inhibitors target the ATP binding site
•ATP binding site is similar but not identical for all protein kinases
•Allows selectivity of inhibitor action
1. Protein Kinases
ATP binding site
Gln-767
H2NOC
Hydrophobic
pocket
H
N
HC
Leu-768 3
H3C
O
H
O
N
HBD
H
N
HBA
Met-769
S
N
H3C
H
H
Cleft
N
N
N
N
O
O
H
H
H
OH
H
OH
Ribose pocket
O
O
O
O P
O P
O P
O
O
O
O
Gln-767
H2NOC
Protein Kinases
ATP binding site
HC
Leu-768 3
H3C
O
Met-769
S
Hydrophobic
pocket
H
N
H
O
N
HBD
H
N
H3C
N
HBA
H
H
Cleft
N
N
N
N
O
O
O
O
O P
O P
O P
O
O
O
O
H
H
H
OH
H
OH
Ribose pocket
•Purine base is buried deep into the binding site
•Purine forms two hydrogen bonding interactions to the binding site
•Ribose sugar binds to a ‘ribose binding pocket’
•Triphosphate chain lies along a cleft towards the enzyme surface
•Triphosphate interacts with two metal ions and amino acids
•Specificity surface is an area of unoccupied binding site
•An empty hydrophobic pocket lies opposite the ribose binding pocket
•The gatekeeper residue is an amino acid situated at the entrance to the
hydrophobic pocket
•The size of the gatekeeper residue is important in drug design
•The nature of amino acids in the binding pockets is important to drug design
O
Protein Kinase Inhibitors
Notes
•Type I inhibitors act on the active conformation of the enzyme
•Type I inhibitors bind to the ATP binding site and block access to ATP
•Type II inhibitors act on the inactive conformation of the enzyme
•Type II inhibitors bind to the enzyme and stabilise the inactive conformation
•Type II inhibitors are likely to be more selective
Type I inhibitors
Gefitinib, erlotinib, SU11248 and seliciclib
Type II inhibitors
Imatinib, lapatinib, sorafenib and vatalanib
Gefitinib (Iressa)
F
Aniline
HN
Cl
4
6
O
7
OMe
O
N
N3
Morpholine
1
N
Quinazoline
Notes
•Developed by Astra Zeneca
•Inhibits the kinase active site of the epidermal growth factor receptor
•The EGF-receptor is a tyrosine kinase receptor
•Gefitinib is a 4-anilinoquinazoline structure
Gefitinib (Iressa)
Lead compound
Secondary
amine
HN
4
CH3
6
Small lipophilic group
OMe
N
Electron-donating
substituents
N
7
OMe
I; IC50 5 nM
Notes
•The secondary amine, electron-donating substituents and small lipophilic group
are all important for activity
•Useful in vitro activity
•Lower in vivo activity due to rapid metabolism
•Metabolised by cytochrome P450 enzymes
Gefitinib (Iressa)
Metabolism of the lead compound
OH
HN
4
CH3
6
Oxidation
N
7
I; IC50 5 nM
OMe
HN
HN
OMe
OMe
N
OH
Cytochrome
P450 enzymes
N
+
CH3
OMe
N
N
II
OMe
N
OMe
III
Notes
•Methyl group and para-position of aromatic ring are susceptible positions
•Blocking metabolism should improve the half life of the drug
Gefitinib (Iressa)
Drug design
F
HN
4
CH3
6
HN
OMe
N
Cl
OMe
N
N
7
I; IC50 5 nM
OMe
N
OMe
IV: IC50 9 nM
Notes
•Fluoro-substituent blocks para-hydroxylation of the aromatic ring
•Fluorine is similar in size to hydrogen and has no steric effect
•Methyl group is replaced by a chloro substituent
•Chlorine and methyl group have similar sizes and lipophilicities
•Chlorine acts as a bio-isotere for the methyl group
•Chlorine is resistant to oxidation
•Compound is less active in vitro, but more active in vivo
Gefitinib (Iressa)
Drug design
F
F
Morpholine
HN
4
CH3
6
HN
OMe
N
Cl
HN
Cl
4
OMe
6
N3
N
7
I; IC50 5 nM
OMe
N
OMe
IV: IC50 9 nM
N
O
N
Spacer
1
N
O
7
OMe
Gefitinib
Notes
•Morpholine ring increases water solubility
•Morpholine nitrogen allows generation of water soluble amine salts
•Spacer allows morpholine to protrude out of the active site
•Remains solvated when the drug is bound
•Avoids a desolvation penalty
Ionisable
Gefitinib (Iressa)
Binding interactions
•Identified by a molecular modelling experiment
•Gefitinib is docked with a model binding site
•Binds to the ATP binding site
•Aniline ring occupies the normally vacant hydrophobic pocket opposite the ribose
binding pocket
•Quinazoline binds to the same region as the purine ring of ATP
F
Thr-830
OH2
Hydrophobic
pocket
H
N
HBA
N
O
Cl
O
N
HBA
Met-769
OMe
N
Cleft
3. Gefitinib (Iressa)
Synthesis of gefitinib and analogues
O
O
OMe
Methionine
HN
Phenol
OH
HN
MeSO3H
N
Pyridine
Protecting group
OAc
HN
Ac2O
OMe
N
OMe
N
OMe
Aniline substituent
NHAr
Cl
OAc
SOCl2
O
N
OAc
ArNH2
N
N
N
OMe
Ar
HN
O
O
N
N
OMe
MeOH
OH
N
NH4OH
OMe
R2N(CH2)nBr
NHAr
N
N
OMe
4. Lapatinib and Etlotinib
F
O
Aniline ring
Aniline ring
NH
Cl
NH
4
O
N
O
N
N
OMe
HN
OMe
SO2Me
Quinazoline ring
Lapatinib
Notes
•4-Anilinoquinazoline structures - compare gefitinib
•EGF-receptor kinase inhibitors
N
O
Quinazoline ring
Erlotinib (Tarceva)
IC50 2 nM
5. PKI 166
Me
NH
4
Pyrrole
N
OH
Pyrimidine
N
HBA
N
H
HBD
Notes
•Pyrrolopyrimidine structure
•EGF-receptor kinase inhibitor
•Different binding mode from ATP or anilinoquinazolines
5. PKI 166
Comparison of binding interactions
•ATP and EGF-receptor kinase inhibitors all contain a pyrimidine ring
•Different binding modes are possible
Me
F
NH
PKI 166
N
N
HBA
O
HBA N
OH
N
H
HBD
N
HBA
HBD
H
N
H
N
N
OMe
ATP
N
HBA N
O
Cl
4
N
Gefitinib
H
N
O
H
H
H
OH
H
OH
O
O
O
O P
O P
O P
O
O
O
O
6. Imatinib (Glivec or Gleevec)
N
Me
H
N
N
N
N
O
H
N
N
Me
Notes
•First protein kinase inhibitor to reach the market
•Selective inhibitor for a hybrid tyrosine kinase (Bcr-Abl)
•Bcr-Abl is active in certain tumour cells
6. Imatinib (Glivec or Gleevec)
Lead compound
Pyrimidine
H Anilino substituent
N
N
N
I
•Phenylaminopyrimidine structure
•Identified by random screening of compound libraries
•Originally identified as a PKC inhibitor
•PKC is a serine-threonine kinase
6. Imatinib (Glivec or Gleevec)
Drug design
Pyridine
N
N
H
N
N
N
N
I
H
N
3' N
II
H
N
N
N
Amide
IV
(IC 50 5 M)
N
H
Increased inhibition of PKC
Inhibits tyrosine
kinases as well
O
6. Imatinib (Glivec or Gleevec)
Drug design
Conformational
blocker
N
N
Me
H
N
N
H
N
N
O
H
H
Imatinib
N
N
O
CGP 53716
(IC 50 0.1 M)
Piperazine
N
Spacer
N
Me
•Piperazine increases activity,
selectivity and water solubility
•Spacer inserted to avoid
aniline structure
H
N
N
N
N
N
N
Me
•Increased activity vs
tyrosine kinases
•No activity against
serine-threonine kinases
IV
(IC 50 5 M)
N
H
O
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Identified from a crystal structure of an inhibitor-Abl kinase complex
•Amide serves as an ‘anchoring group’ and orientates the molecule
•Amide binds to Glu and Asp
•Glu and Asp are important to the catalytic mechanism
Hydrophobic region
Selectivity region 2
Glu
Hydrophobic pocket
Selectivity region 1
H
O
N
O
H
N
N
O
N
MeS
N
O
N
H
H
H
O
Met
N
H
O
Thr
Gatekeeper
residue
Me
O
N
O2C
Asp
N
Me
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Other interactions determine target selectivity
•A hydrogen bond to the gatekeeper Thr is essential to activity
•N-Alkylation eliminates activity
Hydrophobic region
Selectivity region 2
Glu
Hydrophobic pocket
Selectivity region 1
H
O
N
O
H
N
N
O
N
MeS
N
O
N
H
H
H
O
Met
N
H
O
Thr
Gatekeeper
residue
Me
O
N
O2C
Asp
N
Me
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Molecular modelling studies suggest that the piperazinyl group interacts with a
glutamate residue
•Imatinib inhibits protein kinases containing this glutamate residue (Abl, c-Kit
and PDGF-R)
Hydrophobic region
Selectivity region 2
Glu
Hydrophobic pocket
Selectivity region 1
H
O
N
O
N
O
N
MeS
O
N
H
H
H
O
Met
N
N
H
O
Thr
Gatekeeper
residue
Me
N
Me
Piperazinyl
group
H
N
Ionic
bond
O
N
O2C
Asp
Glu
6. Imatinib (Glivec or Gleevec)
Binding interactions
•Conformational blocker aids selectivity
•Binds to a hydrophobic pocket that is not accessible if a larger gatekeeper
residue was present
Hydrophobic region
Selectivity region 2
Glu
Hydrophobic pocket
Selectivity region 1
H
O
N
O
H
N
N
O
N
MeS
N
O
N
H
H
H
O
Met
N
H
O
Thr
Gatekeeper
residue
Me
O
N
O2C
Conformational
blocker
Asp
N
Me
6. Imatinib (Glivec or Gleevec)
Drug resistance
•Mutation of the gatekeeper residue to isoleucine introduces resistance (T315I
mutation)
•Isoleucine unable to form an important hydrogen bond to the amine
Hydrophobic region
Selectivity region 2
Glu
Hydrophobic pocket
Selectivity region 1
H
O
N
O
H
N
N
O
N
MeS
N
O
N
H
H
H
O
Met
N
H
O
Thr
Gatekeeper
residue
Me
O
N
O2C
Asp
Mutation to Isoleucine
N
Me
6. Imatinib (Glivec or Gleevec)
Synthesis of imatinib and analogues
N
N
N
O
HC(OEt)2NMe2
Me
O
Phenylguanidine
derivative
H
N
N
N
Me
II
I
NO2
NMe2
N
N
Me
Me
H
N
N
H2
Pd/C
Reduction
ArCOCl
Acylation
N
NH2
III
H
N
N
N
N
O
H
Amide
Ar
7. Second Generation Bcr-Abl inhibitors
F3C
Glu-286
N
N
H
N
N
Me
N
N
O
Asp-381
N
Met-318
H
Me
Thr-315
Nilotinib
Me
N
Thr-315
N
Me
N
N
H
N
N
S
O
H
Met-318
Cl
Dasatinib; BMS-354825
N
OH
7. Second Generation Bcr-Abl inhibitors
Cl
Cl
HN
OMe
MeO
N
O
CN
N
MeN
Bosutinib
Notes
•Inhibits two protein kinase targets (Abl and Src)
•Currently in clinical trials
•Less likely to fall prey to drug resistance
7. Second Generation Bcr-Abl inhibitors
Me
O
HN
NH2
CO2H
MeO
O
O
OMe
S
MeO
N
N
H
MeO
N
GNF-2
OMe
ON012380
Notes
Notes
•Allosteric inhibitor of Bcr-Abl
•Does not bind to ATP binding site
•Stabilises inactive form of the enzyme
•Binds to an autoregulatory cleft
•Potential agent for treating leukaemia
•Binds to the protein substrate site
•Currently under study
8. Inhibitors of cyclin-dependent kinases
Cyclin-dependent kinases
•CDKs are involved in control of the cell cycle and are overexpressed in many cancer cells
•Serine-threonine kinases
•Activated by cyclins
•Inhibited by cyclin-dependent kinase inhibitors
•Synthetic inhibitors bind to the ATP binding site
8. Inhibitors of cyclin-dependent kinases
HBD HBA
OH
O
Benzopyran
Cl
HO
O
OH
Piperidine
N
Phenyl
ring
Me
Flavopiridol
•Benzopyran binds to the adenine binding region
•Piperidine binds to the region occupied by the first phosphate of ATP
•Phenyl lies over the ribose binding pocket
•Undergoing clinical trials
8. Inhibitors of cyclin-dependent kinases
HBD HBA
OH
O
H
N
R
O
7
Benzopyran
Cl
HO
HN
O
N
OH
Piperidine
N
Me
Flavopiridol
N
Me
O
Phenyl
ring
Me
N
N
HO
N
H
MeO
N
N
Me
NHMe
Staurosporine; R=H
7-Hydroxystaurosporin; R=OH
7-Hydroxystaurosporin is
undergoing clinical trials
Me
R-Roscovitine
(seliciclib)
Shows selectivity for CDK2
Undergoing clinical trials
9. Kinase Inhibitors of FGF-R and VEGF-R
FGF-R and VEGF-R
•FGF-R = fibroblast growth factor receptor
•VEGF-R = vascular endothelial growth factor receptor
•Associated with angiogenesis
•Inhibitors bind to the ATP binding site
•Currently undergoing clinical trials
9. Kinase Inhibitors of FGF-R and VEGF-R
Cl
Me
Anilino
substituent
R
Pyrrole
Oxindole
N
H
Me
O HBA
HN
N
N
Phthalazine
N
H HBD
Pyridine
SU 5416, R=H
SU 6668, R=CH2CH2CO2H
N
PTK 787 / ZK 222584
•SU 5416 in clinical trials for
treatment of colorectal
cancer
•Oxindole binds to same
region as adenine of ATP
Phase III clinical trials in
2006
10. Multi-tyrosine receptor kinase inhibitors
Notes
•Designed to be selective against a range of tyrosine receptor kinases implicated in
tumours
•Drug resistance unlikely to occur for all kinase targets
•Equivalent of combination therapy (poly-pharmacology)
•Sometimes called ‘dirty drugs’
•Promising agents against tumours that are driven by several abnormalities
10. Multi-tyrosine receptor kinase inhibitors
H
H
N
N
H
N
N
O
H3C
O
Cl
O
Me
CF3
NEt2
O
N
H
Sorafenib IC50 12 nM
N
H
F
Me
NH
O
N
N
H
Sunitinib
N
N
Cl
Vatalanib
Notes
•Sorafenib approved as a VEGF-R kinase inhibitor
•Sunitinib approved in 2006 - inhibits VEGF-R, PDGF-R and KIT receptor kinases
•Vatalanib undergoing clinical trials
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
•Lead compound found by high throughput screening
•200 000 compounds tested
•Tested against recombinant Raf-1 kinase
MeO2C
Urea
H
N
H
N
S
O
H
Lead compound;
IC50 17 M
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib - variation of substituents
MeO2C
H
N
MeO2C
H
N
S
H
N
MeO2C
H
N
S
O
H
N
S
O
H
Lead compound
IC50 17 M
H
N
O
Me
II; IC50 1.7 M
Notes
•Methyl substituent is optimum for activity
•10-fold increase in activity
•Phenoxy group is bad for activity
O
III; Poor activity
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib - variation of rings
MeO2C
H
N
Isoxazole
H
N
N
H
N
H
N
O
S
O
O
H
Lead compound
IC50 17 M
VI; Poor activity
Notes
•Variation of rings also carried out systematically
•Isoxazole ring is not good for activity
•Conventional medicinal chemistry strategies fail to achieve further improvement
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
MeO2C
Isoxazole
H
N
H
N
N
H
N
H
N
O
S
O
O
O
H
Phenoxy
group
Lead compound
IC50 17 M
IV; IC50 1.1 M
Notes
•Parallel synthesis - 1000 analogues synthesised with all possible
combinations of rings and substituents
•Structure IV has slightly increased activity - contradicts results from
conventional studies
•Isoxazole ring and phenoxy substituent are good for activity when combined
in the same structure - synergistic effect
•Structure IV taken as new lead compound
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
MeO2C
Isoxazole
H
N
H
N
N
H
N
H
N
O
S
O
O
O
H
Phenoxy
group
IV; IC50 1.1 M
Lead compound
IC50 17 M
N
H
N
Pyridine
H
N
N
O
O
O
V; IC50 0.23 M
•Ring variation
•5-fold increase in activity
•Increase in aqueous
solubility and cLogP
10. Multi-tyrosine receptor kinase inhibitors
Design of sorafenib
MeO2C
Isoxazole
H
N
H
N
N
H
N
H
N
O
S
O
O
O
H
Phenoxy
group
IV; IC50 1.1 M
Lead compound
IC50 17 M
N
H
N
Ring
variation H
N
Pyridine
H
N
N
O
O
Substituent
variation
N
H
N
O
O
N
O
Cl
Substituent CF
3
variation
V; IC50 0.23 M
H
CH3
O
Sorafenib IC50 12 nM
1000-fold increase in activity
10. Multi-tyrosine receptor kinase inhibitors
Sorafenib - binding interactions
HBD
H
H
N
O
HBA
Cl
HBD
H
HBA
N
N
N
O
CF3
Notes
•Urea functional group acts as a binding anchor
•Hydrogen bonds are formed to catalytic Asp and Glu
•Binding orientates the molecule
•Positions each half into two selectivity regions
CH3
O
(compare imatinib)
11. Inhibitors of heat shock protein 90
Notes
•HSP 90 is a kinase protein and acts as a molecular chaperone
•Important to survival of cells - inhibition likely to lead to cell death
•HSP 90 interacts selectively with many of the proteins implicated in tumours
•Targeting HSP 90 may be effective against tumour cells resistant against other
drugs
•Resistant cells contain mutated proteins - rely more on HSP 90 during the
folding process
•Resistant cells likely to be more vulnerable to inhibitors of HSP 90
11. Inhibitors of heat shock protein 90
Notes
Inhibitors bind to the ATP binding site
Lead compound - geldanamycin
Me
O
Quinone
O
HN
Urethane O
OMe
Me
H2N
OH
O
OMe
O
Me
OMe
Me
•Natural product
•Potent inhibitor
•Urethane group is crucial to activity
•Binds to region occupied by adenine
•Poor solubility
•Reactive quinone moiety
11. Inhibitors of heat shock protein 90
Geldanamycin analogues
Me
Me
O
O
HN
O
OMe
Me
OH
O
O
N
H
OMe
Me
H2N
N
H
O
Me
O
O
O
H2N
HN
OMe
NMe
OH
O
Me
Me
OMe
Me
Alvespimycin
Tanespimycin
Me
O
HN
OH
HO
N
H
OMe
O
Me
H2N
OH
O
Me
OMe
IPI 504
Me