Download Topic 4 Proteins as Drug Targets

Topic 4 Proteins as Drug
Targets
Receptors-Chapters 5 and 6
Patrick and Corey 78-80
Contents
1.
Structure and function of receptors
1.1.
Chemical Messengers
1.2.
Mechanism
2.
3.
The binding site
Messenger binding
3.1.
Introduction
3.2.
Bonding forces
Overall process of receptor/messenger interaction
Signal transduction
5.1.
Control of ion channels
4.
5.
6.
7.
8.
9.
10.
5.2.
Activation of signal proteins
5.3.
Activation of enzyme active site
Competitive (reversible) antagonists
Non competitive (irreversible) antagonists
Non competitive (reversible) allosteric antagonists
Antagonists by umbrella effect
Agonists
1.
Structure and function of receptors
•
Globular proteins acting as a cell’s ‘letter boxes’
•
Located mostly in the cell membrane
•
Receive messages from chemical messengers coming from other
cells
•
Transmit a message into the cell leading to a cellular effect
•
Different receptors specific for different chemical messengers
•
Each cell has a range of receptors in the cell membrane making it
responsive to different chemical messengers
1.
Structure and function of receptors
Nerve
Nerve
Signal
Messenger
Receptor
Response
Nucleus
Cell
Cell
1.
Structure and function of receptors
Chemical Messengers
Neurotransmitters: Chemicals released from nerve endings which
travel across a nerve synapse to bind with receptors on target cells,
such as muscle cells or another nerve. Usually short lived and
responsible for messages between individual cells
Hormones: Chemicals released from cells or glands and which travel
some distance to bind with receptors on target cells throughout the
body
•
Chemical messengers ‘switch on’ receptors without
undergoing a reaction
1.
Structure and function of receptors
Nerve 1
Blood
supply
Nerve 2
Hormone
Neurotransmitters
1.
Structure and function of receptors
Mechanism
•
Receptors contain a binding site (hollow or cleft in the receptor
surface) that is recognised by the chemical messenger
•
Binding of the messenger involves intermolecular bonds
•
Binding results in an induced fit of the receptor protein
•
Change in receptor shape results in a ‘domino’ effect
•
Domino effect is known as Signal Transduction, leading to a
chemical signal being received inside the cell
•
Chemical messenger does not enter the cell. It departs the
receptor unchanged and is not permanently bound
1.
Structure and function of receptors
Mechanism
Induced fit
Messenger
Messenger
Messenger
Cell
Membrane
Receptor
Receptor
Cell
Cell
Receptor
Cell
message
Message
2. The binding site
•
A hydrophobic hollow or cleft on the receptor surface - equivalent
to the active site of an enzyme
•
Accepts and binds a chemical messenger
•
Contains amino acids which bind the messenger
•
No reaction or catalysis takes place
Binding site
Binding site
ENZYME
3. Messenger binding
3.1 Introduction
Messenger
M
Induced fit
•
Binding site is nearly the correct shape for the messenger
•
Binding alters the shape of the receptor (induced fit)
•
Altered receptor shape leads to further effects - signal
transduction
3. Messenger binding
3.2 Bonding forces
•
•
•
Ionic
H-bonding
van der Waals
Example:
vdw
interaction
H-bond
Binding site
O
Ser
H
ionic
bond
CO2
Asp
Receptor
Phe
3. Substrate binding
3.2 Bonding forces
•
Induced fit - Binding site alters shape to maximise intermolecular
bonding
Phe
Phe
O
O
H
Ser
Ser
CO2
Asp
Intermolecular bonds not
optimum length for
maximum binding strength
Induced
Fit
H
CO2
Asp
Intermolecular bond
lengths optimised
4. Overall process of receptor/messenger interaction
M
M
M
RE
R
RE
Signal transduction
•
•
•
Binding interactions must be:
- strong enough to hold the messenger sufficiently long for signal
transduction to take place
- weak enough to allow the messenger to depart
Implies a fine balance
Drug design - designing molecules with stronger binding interactions
results in drugs that block the binding site - antagonists
5. Signal transduction
5.1 Control of ion channels
•
Receptor protein is part of an ion channel protein complex
•
Receptor binds a messenger leading to an induced fit
•
Ion channel is opened or closed
•
Ion channels are specific for specific ions (Na+, Ca2+, Cl-, K+)
•
Ions flow across cell membrane down concentration gradient
•
Polarises or depolarises nerve membranes
•
Activates or deactivates enzyme catalysed reactions within cell
5. Signal transduction
5.1 Control of ion channels
Hydrophilic
tunnel
Cell
membrane
5. Signal transduction
5.1 Control of ion channels
Receptor
Binding
site
Cell
membrane
Five glycoprotein subunits
traversing cell membrane
Messenger
Induced
fit
Cell
membrane
‘Gating’
(ion channel
opens)
Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory
Anionic ion channels for Cl- (e.g. GABAA) = inhibitory
5. Signal transduction
5.1 Control of ion channels:
MESSENGER
ION
CHANNEL
(closed)
Cell
membrane
Cell
Ion
channel
RECEPTOR
BINDING
SITE
Lock
Gate
Ion
channel
ION
CHANNEL
(open)
MESSENGER
Induced fit
and opening
of ion channel
Cell
membrane
Cell
membrane
Cell
Ion
channel
Ion
channel
Cell
membrane
5. Signal transduction
5.2 Activation of signal proteins
• Receptor binds a messenger leading to an induced fit
• Opens a binding site for a signal protein (G-protein)
• G-Protein binds, is destabilised then split
messenger
induced
fit
closed
open
G-protein
split
5. Signal transduction
5.2 Activation of signal proteins
• G-Protein subunit activates membrane bound enzyme
Binds to allosteric binding site
Induced fit results in opening of active site
• Intracellular reaction catalysed
Enzyme
Enzyme
active site
(closed)
active site
(open)
Intracellular
reaction
5. Signal transduction
5.3 Activation of enzyme active site
• Protein serves dual role - receptor plus enzyme
• Receptor binds messenger leading to an induced fit
• Protein changes shape and opens active site
• Reaction catalysed within cell
messenger
messenger
induced
fit
closed
active site
open
intracellular reaction
closed
6. Competitive (reversible) antagonists
M
An
An
RE
•
•
•
•
•
•
•
R
Antagonist binds reversibly to the binding site
Intermolecular bonds involved in binding
Different induced fit means receptor is not activated
No reaction takes place on antagonist
Level of antagonism depends on strength of antagonist
binding and concentration
Messenger is blocked from the binding site
Increasing the messenger concentration reverses antagonism
7. Non competitive (irreversible) antagonists
X
Covalent Bond
X
OH
OH
O
Irreversible antagonism
•
•
•
•
•
Antagonist binds irreversibly to the binding site
Different induced fit means that the receptor is not activated
Covalent bond is formed between the drug and the receptor
Messenger is blocked from the binding site
Increasing messenger concentration does not reverse
antagonism
8. Non competitive (reversible) allosteric antagonists
Binding site
unrecognisable
Binding site
ACTIVE SITE
(open)
Receptor
ENZYME
Allosteric
site
Induced
fit
(open)
Receptor
ENZYME
Antagonist
•
•
•
•
•
Antagonist binds reversibly to an allosteric site
Intermolecular bonds formed between antagonist and binding
site
Induced fit alters the shape of the receptor
Binding site is distorted and is not recognised by the messenger
Increasing messenger concentration does not reverse
antagonism
9. Antagonists by umbrella effect
•
•
•
•
Antagonist binds reversibly to a neighbouring binding site
Intermolecular bonds formed between antagonist and
binding site
Antagonist overlaps with the messenger binding site
Messenger is blocked from the binding site
messenger
Binding site
for antagonist
Binding site
for messenger
Receptor
Antagonist
Receptor
10. Agonists
•
•
•
•
•
Agonist binds reversibly to the binding site
Similar intermolecular bonds formed as to natural messenger
Induced fit alters the shape of the receptor in the same way as
the normal messenger
Receptor is activated
Agonists are often similar in structure to the natural
messenger
Agonist
Agonist
Agonist
Induced fit
RE
R
RE
Signal transduction
Contents
Part 1: Sections 6.1 - 6.2
1. Receptor superfamilies
2. Ion channel receptors (Ligand gated ion channels)
2.1. General structure
2.2. Structure of protein subunits (4-TM receptor
subunits)
2.3. Detailed structure of ion channel
2.4. Gating
1. Receptor superfamilies
RESPONSE
TIME
• ION CHANNEL RECEPTORS
• G-PROTEIN COUPLED RECEPTORS
• KINASE LINKED RECEPTORS
• INTRACELLULAR RECEPTORS
msecs
MEMBRANE
BOUND
seconds
minutes
2. Ion channel receptors (Ligand gated ion channels)
2.1 General structure
Receptor
Binding site
Messenger
Cell
membrane
INDUCED
FIT
‘GATING’
(ion channel
opens)
Cell
membrane
Five glycoprotein subunits
traversing cell membrane
Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory
Anionic ion channels for Cl− (e.g. GABAA) = inhibitory
2. Ion channel receptors (Ligand gated ion channels)
Transverse view (nicotinic receptor)
Binding
sites
Ion channel
α
β
Cell
membrane
δ
α
α
γ
γ
α
β
δ
2xα, β, γ, δ subunits
Two ligand binding sites
mainly on α-subunits
2. Ion channel receptors (Ligand gated ion channels)
Transverse view (glycine receptor)
Binding
sites
Ion channel
α
α
β
α
Cell
membrane
β
β
α
α
α
β
3xα, 2x
2x β subunits
Three ligand binding sites
on α-subunits
2. Ion channel receptors (Ligand gated ion channels)
2.2 Structure of protein subunits (4-TM receptor subunits)
Neurotransmitter binding region
Extracellular loop
H2N
CO2H
Cell
membrane
TM1
Intracellular
loop
TM2
TM3
TM4
Variable loop
4 Transmembrane (TM) regions
(hydrophobic)
2. Ion channel receptors (Ligand gated ion channels)
2.3 Detailed structure of ion channel
Protein
subunits
TM4
TM1
TM2
TM3
TM1
TM2
TM2
TM4
TM3
TM3
TM1
TM3
TM4
TM4
TM2
TM2
TM1
TM1
TM3
TM4
Transmembrane
regions
Note: TM2 of each protein subunit ‘lines’ the central pore
2. Ion channel receptors (Ligand gated ion channels)
2.4 Gating
Neurotransmitter
binds
Induced fit
at binding site
‘Domino effect’
Rotation of 2TM regions
of each protein subunit
Ion flow
TM2
Cell
membrane
TM2
TM2
TM2
TM2
TM2
TM2
TM2
Transverse view
of TM2 subunits
TM2
TM2
TM2
TM2
Closed
Open
Transverse view
of TM2 subunits
2. Ion channel receptors (Ligand gated ion channels)
2.4 Gating
• Fast response measured in msec
• Ideal for transmission between nerves
• Binding of messenger leads directly to ion flows across
cell membrane
• Ion flow = secondary effect (signal transduction)
• Ion concentration within cell alters
• Leads to variation in cell chemistry
Contents
Part 2: Sections 6.3 - 6.6
3.
G-protein-coupled receptors (7-TM receptors)
3.1. Structure - Single protein with 7 transmembrane regions
3.2. Ligands
3.3. Ligand binding site - varies depending on receptor type
3.4. Bacteriorhodopsin & rhodopsin family
3.5. Receptor types and subtypes
3.6. Signal transduction pathway
a)
Interaction of receptor with Gs-protein
b)
Interaction of αs with adenylate cyclase
c)
Interaction of cyclic AMP with protein kinase A (PKA)
3.7. Glycogen metabolism - triggered by adrenaline in liver cells
3.8. GI proteins
3.9. Phosphorylation
3.10.
Drugs interacting with cyclic AMP signal transduction
3.11.
Signal transduction involving phospholipase C (PLC)
3.12.
Action of diacylglycerol
3.13.
Action of inositol triphosphate
3.14.
Resynthesis of PIP2
3. G-protein-coupled receptors (7-TM receptors)
3.1 Structure - Single protein with 7 transmembrane regions
Extracellular
loops
NH2
N -Terminal chain
Membrane
VII
VI
V
IV
III
II
I
G-Protein
binding region
HO2 C
C -Terminal chain
Variable
intracellular loop
Intracellular loops
Transmembrane
helix
3. G-protein-coupled receptors (7-TM receptors)
3.2 Ligands
• Monoamines e.g. dopamine, histamine, noradrenaline,
acetylcholine (muscarinic)
• Nucleotides
• Lipids
• Hormones
• Glutamate
• Ca++
3. G-protein-coupled receptors (7-TM receptors)
3.3 Ligand binding site - varies depending on receptor type
Ligand
A
B
C
D
A) Monoamines - pocket in TM helices
B) Peptide hormones - top of TM helices + extracellular loops
+ N-terminal chain
C) Hormones - extracellular loops + N-terminal chain
D) Glutamate - N-terminal chain
3. G-protein-coupled receptors (7-TM receptors)
3.4 Bacteriorhodopsin & rhodopsin family
• Rhodopsin = visual receptor
• Many common receptors belong to this same family
• Implications for drug selectivity depending on similarity (evolution)
• Membrane bound receptors difficult to crystallise
• X-Ray structure of bacteriorhodopsin solved - bacterial protein similar
to rhodopsin
• Bacteriorhodopsin structure used as ‘template’ for other receptors
• Construct model receptors based on template and amino acid sequence
• Leads to model binding sites for drug design
• Crystal structure for rhodopsin now solved - better template
3. G-protein-coupled receptors (7-TM receptors)
3.4 Bacteriorhodopsin & rhodopsin family
Common ance stor
Monoamines
muscarinic
beta
alpha
Opsins, Rhodopsins
Bradykinin,
Endothelins Angiotensin.Tachyki nins
Interleukin-8
2
4
5
Muscarinic
Receptor
types
3
1
H1 H2
Histamine
1 2A 2B 2C
D4 D3 D2 D1A D1B D5
!-Adrenergic
Dopaminergic
3
2
1
"-Adrenergic
Receptor
sub-types
3. G-protein-coupled receptors (7-TM receptors)
3.5 Receptor types and subtypes
Reflects differences in receptors which recognise the same ligand
Receptor
Types
Adrenergic
Alpha (α)
Beta (β)
Acetylcholine
Nicotinic
Muscarinic
Subtypes
α1, α2A, α2B, α2C
β1, β2, β3
Μ1−Μ5
3. G-protein-coupled receptors (7-TM receptors)
3.5 Receptor types and subtypes
• Receptor types and subtypes not equally distributed amongst
tissues.
• Target selectivity leads to tissue selectivity
Heart muscle
- β1 adrenergic receptors
Fat cells
- β3 adrenergic receptors
Bronchial muscle
- α1& β2 adrenergic receptors
GI-tract
- α1 α2 & β2 adrenergic receptors
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
GS-Protein - membrane bound protein of 3 subunits (α, β, γ)
- αS subunit has binding site for GDP
-GDP bound non covalently
β
γ
α
GDP
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
Ligand
Cell membrane
Receptor
ß
γ
α
Ligand
binding
Induced
fit
G-protein
binds
ß
γ
α
G Protein
ß
γ
α
GDP
Binding site for G-protein opens
= GDP
Induced
fit for
G-protein
GTP
G-Protein alters shape
GDP binding site distorted
GDP binding weakened
GDP departs
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
ß
γ
α
GTP binds
Binding site recognises GTP
γ
ß
γ
α
Fragmentation
and release
ß
α
Induced fit
G-protein alters shape
Complex destabilised
• Process repeated for as long as ligand bound to receptor
• Signal amplification - several G-proteins activated by one ligand
• αs Subunit carries message to next stage
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
GTP
GDP
b) Interaction of αs with adenylate cyclase
Binding site
for αs subunit
αs-subunit
Adenylate cyclase
GTP hydrolysed
to GDP catalysed
by αs subunit
Binding
Induced
fit
Active site
(closed)
P
ATP cyclic AMP
Active site
(open)
Signal
transduction
(con)
αs Subunit recombines with β,γ dimer
to reform Gs protein
ATP cyclic AMP
Active site
(closed)
αs Subunit changes shape
Weaker binding to enzyme
Departure of subunit
Enzyme reverts to inactive
state
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
b) Interaction of αs with adenylate cyclase
•
•
•
•
Several-100 ATP molecules converted before αs-GTP deactivated
Represents another signal amplification
Cyclic AMP becomes next messenger (secondary messenger)
Cyclic AMP enters cell cytoplasm with message
NH2
NH2
N
N
O
O
O
N
N
HO P O P O P O
OH OH
OH
Adenylate cyclase
O
H
H
O
H
OH
N
N
O
H
ATP
N
N
P
OH
O
H
H
Cyclic AMP
O
OH
OH
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA)
•
•
•
Protein kinase A = serine-threonine kinase
Activated by cyclic AMP
Catalyses phosphorylation of serine and threonine residues on
protein substrates
• Phosphate unit provided by ATP
O
O
H
N
C
Protein
kinase A
H
N
H
N
C
H
H
OH
O
C
HC
Serine
O
OH
H
OH
CH3
P
HO
O
O
Threonine
Protein
kinase A
H
N
C
HC
H
O
CH3
HO
P
O
OH
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA)
Adenylate
cyclase
ATP cyclic AMP
Activation
Protein
kinase
P
Enzyme
(inactive)
Enzyme
(active)
Chemical
reaction
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA)
Protein kinase A - 4 protein subunits
- 2 regulatory subunits (R) and 2 catalytic subunits (C)
cAMP
C
catalytic subunit
C
R
cAMP
binding
sites
R
R
R
C
C
catalytic subunit
Note Cyclic AMP binds to PKA
Induced fit destabilises complex
Catalytic units released and activated
3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA)
C
P
Protein
+ ATP
Protein
+ ADP
Phosphorylation of other proteins and enzymes
Signal continued by phosphorylated proteins
Further signal amplification
3. G-protein-coupled receptors (7-TM receptors)
3.7 Glycogen metabolism - triggered by adrenaline in liver cells
Adrenaline
!s
!s
!-Adrenoreceptor
adenylate
cyclase
cAMP
Glycogen
synthase
(active)
Protein kinase A
Inhibitor (inactive)
Catalytic
C subunit of
PKA
Glycogen
synthase-P
(inactive)
Phosphorylase
kinase (inactive)
Inhibitor-P
(active)
Phosphatase
(inhibited)
Phosphorylase
kinase-P (active)
Phosphorylase b
(inactive)
Phosphorylase a
(active)
Glycogen
Glucose-1-phosphate
3. G-protein-coupled receptors (7-TM receptors)
3.7 Glycogen metabolism - triggered by adrenaline in liver cells
Coordinated effect - activation of glycogen metabolism
- inhibition of glycogen synthesis
Adrenaline has different effects on different cells
- activates fat metabolism in fat cells
3. G-protein-coupled receptors (7-TM receptors)
3.8 GI proteins
• Binds to different receptors from those used by Gs protein
• Mechanism of activation by splitting is identical
• αI subunit binds adenylate cyclase to inhibit it
• Adenylate cyclase under dual control (brake/accelerator)
• Background activity due to constant levels of αs and αi
• Overall effect depends on dominant G-Protein
• Dominant G-protein depends on receptors activated
3. G-protein-coupled receptors (7-TM receptors)
3.9 Phosphorylation
•
•
•
Prevalent in activation and deactivation of enzymes
Phosphorylation radically alters intramolecular binding
Results in altered conformations
NH3
NH3
NH3
O
O
O
H
O
Active site
closed
P
O
O
O
O
O
P
O
O
O
Active site
open
3. G-protein-coupled receptors (7-TM receptors)
3.10 Drugs interacting with cyclic AMP signal transduction
Cholera toxin - constant activation of cAMP - diarrhea
Theophylline and caffeine
- inhibit phosphodiesterases
- phosphodiesterases responsible for metabolising
cyclic AMP
- cyclic AMP activity prolonged
O
O
H3C
H
N
H 3C
N
CH3
N
N
N
N
O
N
CH3
Theophylline
O
N
CH3
Caffeine
3. G-protein-coupled receptors (7-TM receptors)
3.11 Signal transduction involving phospholipase C (PLC)
•
•
•
•
•
Gq proteins - interact with different receptors from GS and GI
Split by same mechanism to give αq subunit
αq Subunit activates or deactivates PLC (membrane bound enzyme)
Reaction catalysed for as long as αq bound - signal amplification
Brake and accelerator
Active site
(open)
Active site
(closed)
α
α
PLC
DG
α
PLC
PLC
PIP2
IP3
GTP hydrolysis
DG
α
Phosphate
PLC
PIP2
IP3
Binding weakened
Active site
(closed)
αq departs
α
PLC
enzyme
deactivated
3. G-protein-coupled receptors (7-TM receptors)
3.11 Signal transduction involving phospholipase C (PLC)
R
O C
R
C O
O
O
CH2
CH
O P H
CH2
HO
PLC
O
O P
O
H
O
O P
H HO
OH H
H
HO
H
+
O P
R
R
O C
O C
O
O
CH2
CH
OH
O P
HO
OH
IP3
CH2
DG
O P
PIP2
Phosphatidylinositol diphosphate
(integral part of cell membrane)
R= long chain hydrocarbons
Inositol triphosphate
(polar and moves
into cell cytoplasm)
P
= PO3 2-
Diacylglycerol
(remains in membrane)
3. G-protein-coupled receptors (7-TM receptors)
3.12 Action of diacylglycerol
•
•
•
•
•
Activates protein kinase C (PKC)
PKC moves from cytoplasm to membrane
Phosphorylates enzymes at Ser & Thr residues
Activates enzymes to catalyse intracellular reactions
Linked to inflammation, tumour propagation, smooth muscle activity etc
Cell membrane
DG
Binding
site for DG
DG
DG
PKC
Active site
closed
PKC
Cytoplasm
PKC moves
to membrane
Cytoplasm
DG binds to
DG binding site
PKC
Enzyme
(inactive)
Cytoplasm
Enzyme
(active)
Chemical
reaction
Induced fit
opens active site
3. G-protein-coupled receptors (7-TM receptors)
3.12 Action of diacylglycerol
Drugs inhibiting PKC - potential anti cancer agents
CHCO2Me
CH3CH2CH2
CH
CH
CH
Me
CH
C
Me
O
O
H
O
MeO2C
CH
Me
H
OH
C
Me
O
O
H
OH
O
O
HO
O
H
Me
H
Me
C
O
OH
H
Bryostatin (from sea moss)
3. G-protein-coupled receptors (7-TM receptors)
3.13 Action of inositol triphosphate
• IP3 - hydrophilic and enters cell cytoplasm
• Mobilises Ca2+ release in cells by opening Ca2+ ion channels
• Ca2+ activates protein kinases
• Protein kinases activate intracellular enzymes
• Cell chemistry altered leading to biological effect
3. G-protein-coupled receptors (7-TM receptors)
3.13 Action of inositol triphosphate
Cell membrane
IP3
Cytoplasm
Calmodulin
Calcium
stores
Calmodulin
Ca++
Activation
Protein
kinase
Enzyme
(inactive)
Ca++
Activation
P
Enzyme
(active)
Chemical
reaction
Protein
kinase
Enzyme
(inactive)
P
Enzyme
(active)
Chemical
reaction
3. G-protein-coupled receptors (7-TM receptors)
3.14 Resynthesis of PIP2
several
steps
IP3 + DG
PIP2
Inhibition
Li+ salts
Lithium salts used vs manic depression
Contents
Part 3: Section 6.7
4. Tyrosine kinase linked receptors
4.1.
Structure
4.2.
Reaction catalysed by tyrosine kinase
4.3.
Epidermal growth factor receptor (EGF- R)
4.4.
4.5.
4.6.
Insulin receptor (tetrameric complex)
Growth hormone receptor
Signalling pathways
4. Tyrosine kinase linked receptors
• Bi-functional receptor / enzyme
• Activated by hormones
• Over-expression can result in
cancer
4. Tyrosine kinase linked receptors
4.1 Structure
Extracellular
N-terminal
chain
Ligand binding region
NH2
Hydrophilic
transmembrane
region (α-helix)
Cell membrane
Catalytic binding region
(closed in resting state)
Intracellular
C-terminal
chain
C O2 H
4. Tyrosine kinase linked receptors
4.2 Reaction catalysed by tyrosine kinase
Tyrosine
kinase
Mg++
O
Protein
N
C
Protein
OH
Tyrosine
residue
ATP
ADP
O
Protein
N
C
Protein
O
Phosphorylated
tyrosine
residue
P
4. Tyrosine kinase linked receptors
4.3 Epidermal growth factor receptor (EGF- R)
EGF
Ligand binding
and dimerisation
Cell
membrane
Phosphorylation
OH
HO
OH
Inactive EGF-R
monomers
OH
OP
PO
ATP
Induced fit
opens tyrosine kinase
active sites
Binding site for EGF
EGF - protein hormone - bivalent ligand
Active site of tyrosine kinase
ADP
OP
OP
4. Tyrosine kinase linked receptors
4.3 Epidermal growth factor receptor (EGF- R)
• Active site on one half of dimer catalyses phosphorylation of
Tyr residues on other half
• Dimerisation of receptor is crucial
• Phosphorylated regions act as binding sites for further
proteins and enzymes
• Results in activation of signalling proteins and enzymes
• Message carried into cell
4. Tyrosine kinase linked receptors
4.4 Insulin receptor (tetrameric complex)
Insulin
Phosphorylation
Cell
membrane
HO
OH
OH
OH
ATP
Kinase active site
opened by induced fit
Insulin binding site
Kinase active site
ADP
PO
OP
OP
OP
4. Tyrosine kinase linked receptors
4.5 Growth hormone receptor
Tetrameric complex constructed in presence of growth hormone
GH
GH binding
&
dimerisation
Binding
of kinases
GH receptors
(no kinase activity)
Activation and
phosphorylation
ATP
HO
kinases
OH
HO
OH
OH
OH
OH
OH
ADP
PO
OP
Kinase active site
opened by induced fit
http://en.wikipedia.org/wiki/Cytokine_receptor
http://www.ebi.ac.uk/interpro/potm/2004_4/Page2.htm
Growth hormone binding site
Kinase active site(Janus, JAK kinase)
OP
OP
Tales from the drug development trenchesTucson-John Kozarich,Ligand Pharmaceuticals
Thrombocyte,i.e. platelet
Eltrombopag,PROMACTA
Binds to DIFFERENT site than thrombopoetin
with Zn 2+ .
http://www.ligand.com/collaborations.php#Leading
Tales from the drug development
trenches-Tucson
http://en.wikipedia.org/wiki/Cytokine_receptor
TPO and EPO receptors (cytokine type, also growth hormone)
connected to Janus kinase (JAK) family of tyrosine kinases
4. Tyrosine kinase linked receptors
4.6 Signalling pathways
Ligand
P
P
P
P
P
P
Ligand
P
P
P
P
signalling protein
4. Tyrosine kinase linked receptors
4.6 Signalling pathways
1-TM Receptors
Tyrosine kinase
inherent or associated
Guanylate cyclase
Signalling proteins
PLCγ
IP3
DG
Ca++ PKC
IP3
kinase
PIP3
GAP
cGMP
Grb2
Others
4. Tyrosine kinase linked receptors
4.6 Signalling pathways
Receptor
binding
site
GROWTH FACTOR RECEPTOR
Tyrosine kinase
active site
(inactive)
HO
OH
HO
OH
4. Tyrosine kinase linked receptors
4.6 Signalling pathways
Growth
factor
1) Binding of
growth factor
Dimerisation
Phosphorylation
2) Conformational
change
HO
HO
HO
OH
OH
HO
OH
HO
HO
OH
OHHO
PO
OH
OH
PO
PO
OP
OPPO
Binding
Ras and
Grb2
Binding and
phosphorylation
of Grb2
HO
OH
OH
OP
PO
PO
PO
OP
OPPO
Grb2
OP
OP
GTP/GDP
exchange
OP
PO
PO
PO
OP
OPPO
OP
OP
Ras
GDP
GTP
OP
OP
4. Tyrosine kinase linked receptors
4.6 Signalling pathways
Gene transcription
OP Ras
PO
PO
PO
OP
OPPO
OP
OP
Raf (inactive)
Raf (active)
Mek (inactive)
Mek (active)
Map kinase (inactive)
Map kinase (active)
Transcription
factor (inactive)
Transcription
factor (active)
Contents
Part 4: Section 6.8
5. Intracellular receptors
5.1.
Structure
5.2.
Mechanism
5.3.
Oestrogen receptor
5. Intracellular receptors
• Chemical messengers must cross cell membrane
• Chemical messengers must be hydrophobic
• Example - steroids and steroid receptors
5. Intracellular receptors
5.1 Structure
CO2H
Steroid
binding region
Zinc
DNA binding region
(‘zinc fingers’)
H2 N
Zinc fingers contain Cys residues (SH)
Allow S-Zn interactions
5. Intracellular receptors
5.2 Mechanism
Receptor
Co-activator
protein
DNA
Messenger
Receptor-ligand
complex
Dimerisation
Cell
membrane
1. Messenger crosses membrane
5. Complex binds to DNA
2. Binds to receptor
3. Receptor dimerisation
4. Binds co-activator protein
6. Transcription switched on or off
7. Protein synthesis activated or inhibited
5. Intracellular receptors
5.3 Oestrogen receptor
Binding
site
H12
AF-2
regions
Coactivator
Coactivator
Oestradiol
DNA
Oestrogen
receptor
Dimerisation &
exposure of
AF-2 regions
Nuclear
transcription
factor
Transcription
5. Intracellular receptors
5.3 Oestrogen receptor
His 524
Me OH
H
H
Glu353
H
H
O
Hydrophic skeleton
H2O
Arg394
Oestradiol
•
•
•
•
•
H
Phenol and alcohol of oestradiol are important binding groups
Binding site is spacious and hydrophobic
Phenol group of oestradiol positioned in narrow slot
Orientates rest of molecule
Acts as agonist
5. Intracellular receptors
5.3 Oestrogen receptor
Asp351
N
H
Side
chain
His 524
O
Glu353
O
OH
H
O
S
Arg394
Raloxifene
•
•
•
•
•
•
Raloxifene is an antagonist (anticancer agent)
Phenol groups mimic phenol and alcohol of oestradiol
Interaction with Asp351 is important for antagonist
activity
Side chain prevents receptor helix H12 folding over as lid
AF-2 binding region not revealed
Co-activator cannot bind
5. Intracellular receptors
5.3 Oestrogen receptor
O
Me2N
CH2CH3
Tamoxifen (Nolvadex)
- anticancer agent which targets oestrogen receptor
Contents
Case Study-LATER
6. Case Study - Inhibitors of EGF Receptor Kinase
6.1. The target
6.2. Testing procedures
- In vitro tests
- In vivo tests
- Selectivity tests
6.3. Lead compound – Staurosporine
6.4. Simplification of lead compound
6.5. X-Ray crystallographic studies
6.6. Synthesis of analogues
6.7. Structure Activity Relationships (SAR)
6.8. Drug metabolism
6.9. Further modifications
6.10.Modelling studies on ATP binding
6.11.Model binding studies on Dianilinophthalimides
6.12.Selectivity of action
6.13.Pharmacophore for EGF-receptor kinase inhibitors
6.14.Phenylaminopyrrolopyrimidines
6.15.Pyrazolopyrimidines
6. Case Study - Inhibitors of EGF Receptor Kinase
6.1 The target
- Epidermal growth factor receptor
- Dual receptor / kinase enzyme role
Extracellular
space
Receptor
Binding site
cell
membrane
Cell
Kinase active site
(closed)
6.1 The target
Overexpression
of erbB1 gene
Excess
receptor
-
KINASE INHIBITOR
Excess sensitivity
to EGF
Excess signal
from receptor
Excess cell growth
and division
Tumours
Potential
anticancer
agent
6.1 The target
H
N
H
Protein
N
N
O
O
N
N
H
O
O
O
H
P
O
P
O
O
O
N
N
HO
O
Tyrosine
residue
H
OH OH
Mg
Tyrosine kinase
H
N
N
O
H
ATP
H
P
Protein
HN
O
O
O
N
O
O
H
H
P
H
H
OH OH
ADP
Protein
O
O
P
Protein
HN
O
O
O
O
O
P
O
O
Phosphorylated
tyrosine residue
6.1 The target
Inhibitor Design
Possible versus binding site for tyrosine region
Possible versus binding site for ATP
Inhibitors of the ATP binding site
Aims:
To design a potent but selective inhibitor versus EGF receptor
kinase and not other protein kinases.
6.2 Testing procedures
In vitro tests
Enzyme assay
using kinase portion of the EGF receptor produced by recombinant
DNAtechnology. Allows enzyme studies in solution.
EGF-R
cell
membrane
Cell
Recombinant
DNA
Water
soluble
kinase
6.2 Testing procedures
In vitro tests
Enzyme assay
Test inhibitors by ability to inhibit standard enzyme catalysed reaction
ATP
ADP
OH
OP
Angiotensin II
Angiotensin II
kinase
Assay product
to test inhibition
Inhibitors
•
•
Tests inhibitory activity only and not ability to cross cell membrane
Most potent inhibitor may be inactive in vivo
6.2 Testing procedures
In vitro tests
Cell assays
•
•
•
•
Use cancerous human epithelial cells which are sensitive to EGF for growth
Measure inhibition by measuring effect on cell growth - blocking kinase
activity blocks cell growth.
Tests inhibitors for their ability to inhibit kinase and to cross cell membrane
Assumes that enzyme inhibition is responsible for inhibition of cell growth
Checks
•
•
•
Assay for tyrosine phosphorylation in cells - should fall with inhibition
Assay for m-RNA produced by signal transduction - should fall with
inhibition
Assay fast growing mice cells which divide rapidly in presence of EGF
6.2 Testing procedures
In vivo tests
• Use cancerous human epithelial cells grafted onto mice
• Inject inhibitor into mice
• Inhibition should inhibit tumour growth
• Tests for inhibitory activity + favourable pharmacokinetics
6.2 Testing procedures
Selectivity tests
Similar in vitro and in vivo tests carried out on serinethreonine kinases and other tyrosine kinases
6.3 Lead compound - Staurosporine
H
N
O
N
N
O
H3C
H3C
O
NH
H3C
•
•
•
•
•
Microbial metabolite
Highly potent kinase inhibitor but no selectivity
Competes with ATP for ATP binding site
Complex molecule with several rings and asymmetric centres
Difficult to synthesise
6.4 Simplification of lead compound
H
N
O
N
N
H 3C
H 3C
O
Simplification
Remove asymmetric
ring
H
N
O
*
*
*
O
*
Simplification
Symmetry
NH
H 3C
Staurosporine
N
H
N
H
H
N
O
N
H
Arcyriaflavin A
• Symmetrical molecule
• Active and selective vs
PKC but not EGF-R
O
N
H
6.4 Simplification of lead compound
maleimide ring
H
N
O
O
Bisindolylmaleimides
PKC selective
N
H
N
H
H
N
O
O
indole ring
Phthalimide
indole ring
Simplification
N
H
Aniline
N
H
Simplification
Aniline
Dianilinophthalimide (CGP 52411)
• Selective inhibitor for EGF
receptor and not other kinases
• Reversal of selectivity
H
N
O
N
H
O
N
H
6.5 X-Ray crystallographic studies
Different shapes implicated in different selectivity
Arcyriaflavin
Planar
O
H
N
Bisindolyl-maleimides
Bowl shaped
O
O
H
N
Dianilino-phthalimides
Propellor shaped
asymmetric
O
N
H
N
H
O
O
NH
N
H
H
N
N
H
HN
6.5 X-Ray crystallographic studies
Propeller conformation relieves steric clashes
Steric
clash
O
H
N
O
H
N
O
HH
HH
Steric
clash
H
Twist
H
H
H
NH
NH
O
HN
Planar
HN
Propeller
shape
6.6 Synthesis of analogues
O
H 2C
TMSCl, NEt3
DMF,
O
100 oC
H 2C
O
O
H 3C
Diels Alder
Toluene
Si (CH3) 3
O
CH3
Anilines
O
O
O
(H3C) 3SiO
OSi(CH3) 3
Acetic acid, 120 oC
MeO 2C
Si (CH3) 3
C
C
CO2Me
H 3C
O
CH3
O
O
a) LiOH, MeOH
O
O
O
O
NH3 or formamides
R1
NR2
R 2N
O
140-150 oC
b) (Ac)2O, toluene
R1
O
R
N
R1
R1
NR2
R 2N
R1
R1
NR2
R 2N
6.7 Structure Activity Relationships (SAR)
O
R
N
O
R1
R1
NR2 R 2N
•
•
•
•
•
•
R=H
Activity lost if N is substituted
Aniline aromatic rings essential (activity lost if cyclohexane)
R1=H or F (small groups). Activity drops for Me and lost for Et
R2=H Activity drops if N substituted
Aniline N’s essential. Activity lost if replaced with S
Both carbonyl groups important. Activity drops for lactam
H
N
NH
O
HN
6.7 Structure Activity Relationships (SAR)
Parent Structure: R=R1=R2=H chosen for preclinical trials
IC50 = 0.7 µM
H
N
O
NH
O
HN
CGP 52411
6.8 Drug metabolism
Excretion
H
N
O
O
Glucuronylation
HO
H
N
O
NH
Glucose
O
Glucose
O
Drug
HN
O
Metabolism
(man,mouse,
rat, dog)
NH
HN
CGP 52411
H
N
O
Metabolism
(monkey)
HO
NH
O
HN
Glucuronylation
OH
Drug O Glucose
Excretion
6.8 Drug metabolism
Introduce F at para position as metabolic blocker
H
N
O
F
Metabolic
blocker
NH
O
HN
CGP 53353
F
Metabolic
blocker
6.9 Further modifications
a) Chain extension
H
N
O
Chain extension
NH
O
HN
CGP58109
Activity drops
Chain extension
6.9 Further modifications
b) Ring extension / expansion
extension
ring
expansion
H
N
O
NH
HN
O
HN
CGP 52411 (IC50 0.7µM)
NH
O
O
NH
remove
polar groups
HN
CGP54690 (IC50 0.12µM)
Inactive in cellular assays
due to polarity
(unable to cross cell membrane)
HN
N
O
NH
HN
CGP57198 (IC50 0.18µM)
Active in vitro and in vivo
6.9 Further modifications
c) Simplification
H
N
O
NH
O
HN
CGP52411
Simplification
H
N
O
NH
O
OH
CGP58522
Similar activity in enzyme assay
Inactive in cellular assay
6.10 Modelling studies on ATP binding
• No crystal structure for EGF- receptor available
• Make a model active site based on structure of an
analogous protein which has been crystallised
• Cyclic AMP dependant protein kinase used as template
6.10 Modelling studies on ATP binding
Cyclic AMP dependant protein kinase
+ Mg + ATP + Inhibitor (bound at
substrate site)
Crystallise
Crystals
X-Ray Crystallography
Structure of protein /
inhibitor / ATP complex
Molecular modelling
Identify active site
and binding interactions
for ATP
6.10 Modelling studies on ATP binding
• ATP bound into a cleft in the enzyme with adenine portion
buried deep close to hydrophobic region.
• Ribose and phosphate extend outwards towards opening of
cleft
• Identify binding interactions (measure distances between
atoms of ATP and complementary atoms in binding site to
see if they are correct distance for binding)
• Construct model ATP binding site for EGF-receptor kinase
by replacing amino acid’s of cyclic AMP dependent protein
kinase for those present in EGF receptor kinase
6.10 Modelling studies on ATP binding
Gln767
HN
H-bond interactions
empty
pocket
Thr766
H2NOC
H
N
O
O
Leu768
H3C
Met769
S
H
H3C
H
O
N
H
H
N
O
N
H3C
H
N
1
6
N
N
O
O
N
O
O
O
P
O
O
O
O
H
1N
P
O
H
H
OH
H
OH
is a H bond acceptor
6-NH2 is a H-bond donor
Ribose forms H-bonds to Glu in ribose pocket
'ribose' pocket
P
O
O
6.11 Model binding studies on Dianilinophthalimides
Gln767
HN
empty
pocket
Thr766
H-bond interaction
H2NOC
H
N
O
Leu768
H3C
Met769
S
H
H3C
O
H
O
N
O
H
N
O
N
H
NH
O
H3C
O
HN
'ribose' pocket
6.11 Model binding studies on Dianilinophthalimides
• Both imide carbonyls act as H-bond acceptors (disrupted if
carbonyl reduced)
• Imide NH acts as H bond donor (disrupted if N is substituted)
• Aniline aromatic ring fits small tight ribose pocket
• Substitution on aromatic ring or chain extension prevents
aromatic ring fitting pocket
• Bisindolylmaleimides form H-bond interactions but cannot fit
aromatic ring into ribose pocket.
• Implies ribose pocket interaction is crucial for selectivity
6.11 Model binding studies on Dianilinophthalimides
Gln767
HN
empty
pocket
Thr766
H-bond interaction
H2NOC
H
N
O
O
Leu768
H3C
Met769
S
H
H3C
H
O
N
O
HN
H
O
N
H3C
O
H
N
NH
O
HN
'ribose' pocket
6.11 Model binding studies on Dianilinophthalimides
Gln767
HN
empty
pocket
Thr766
H-bond interaction
H2NOC
H
N
O
O
Leu768
H3C
Met769
S
H
H3C
H
O
O
N
H
N
O
N
H
NH
O
H3C
NH
O
'ribose' pocket
6.12 Selectivity of action
POSERS ?
• Ribose pocket normally accepts a polar ribose so why can it
accept an aromatic ring?
• Why can’t other kinases bind dianilinophthalimides in the same
manner?
6.12 Selectivity of action
Amino Acids present in the ribose pocket
Hydrophobic
Protein Kinase A
EGF Receptor Kinase
Hydrophilic
Leu,Gly,Val,Leu
Glu,Glu,Asn,Thr
Leu,Gly,Val,Leu,Cys
Arg,Asn,Thr
6.12 Selectivity of action
• Ribose pocket is more hydrophobic in EGF-receptor kinase
• Cys can stabilise and bind to aromatic rings (S-Ar interaction)
Gln767
HN
empty
pocket
Thr766
H2NOC
H
N
O
H
H3C
Leu768
H3C
Met769
S
O
H
O
N
O
H
N
O
N
H
NH
O
H3C
O
HN
H
S
'ribose' pocket
• Stabilisation by S-Ar interaction not present in other kinases
• Leads to selectivity of action
6.13 Pharmacophore for EGF-receptor kinase inhibitors
O
HBD
HBD
H
N
HBA
NH
O
HBA
HN
Ar
•
•
•
Pharmacophore
Ar
Pharmacophore allows identification of other potential inhibitors
Search databases for structures containing same pharmacophore
Can rationalise activity of different structural classes of inhibitor
6.14 Phenylaminopyrrolopyrimidines
CGP 59326 - Two possible binding modes for H-bonding
Cl
HBD
H
HBD
HBA
N
H
N
H
N
N
N
HBA
N
Ar
N
N
H
Cl
Mode I
Mode II
Only mode II tallies with pharmacophore and explains activity
and selectivity
6.14 Phenylaminopyrrolopyrimidines
empty
pocket
Cl
O
O
H
H
N
N
H
empty
pocket
N
N
N
CGP59326
H
N
N
N
H
N
H
N
H
CGP59326
H
S
S
Cl
'ribose' pocket
Binding Mode I like ATP
(not favoured)
'ribose' pocket
Binding mode II (favoured)
Illustrates dangers in comparing structures and assuming similar
interactions (e.g. comparing CGP59326 with ATP)
6.14 Phenylaminopyrrolopyrimidines
HBD
H
HBD
N
HBA
H
N
HBA
N
N
Ar
Ar
Cl
6.15 Pyrazolopyrimidines
i) Lead compounds
Cl
NH2
N
NH2
N
N
N
N
N
H 2N
(I) EC50 0.80µM
•
•
•
N
N
H
(II) EC50 0.22µM
Both structures are selective EGF-receptor kinase inhibitors
Both structures belong to same class of compounds
Docking experiments reveal different binding modes to obey
pharmacophore
6.15 Pyrazolopyrimidines
ii) Structure I
empty
pocket
O
H
Extra binding
interactions
HBD
H
HBA
N
H
N
H
N
N
H
Structure
I
N
N
N
N
N
N
N
H
S
'ribose' pocket
Ar
6.15 Pyrazolopyrimidines
ii) Structure I
NH2
NH2
N
N
N
N
N
N
(I) EC50 0.80µM
N
N
(III) EC50 2.7µM
6.15 Pyrazolopyrimidines
iii) Structure II
• Cannot bind in same mode since no fit to ribose pocket
• Binds in similar mode to phenylaminopyrrolopyrimidines
empty
pocket
Cl
O
H
N
N
N
NH
Structure
II
N
H
N
H 2N
H
S
unoccupied
'ribose' pocket
6.15 Pyrazolopyrimidines
Extra
H-bonding
interaction
iv) Drug design on structure II
Cl
Cl
Cl
OH
HBD
HBD
H
N
N
H
N
NH
H
N
N
NH
HBA
N
H
N
NH
N
NH
HBA
N
Simplification
N
H 2N
(II)
EC50 0.22µM
(remove extra
functional group)
Extension
N
N
(add aromatic
ring for ribose
pocket)
N
NH
N
N
NH
N
Ar
(IV)
EC50 0.16µM
Activity increases
Extension
Cl
Cl
(V)
EC50 0.033µM
Activity increases
Ar fits ribose pocket
(VI)
EC50 0.001µM
Activity increases
• Upper binding pocket is larger than ribose pocket allowing greater
variation of substituents on the ‘upper’ aromatic ring