Patrick
An Introduction to Medicinal Chemistry 3/e
Chapter 6
PROTEINS AS DRUG
TARGETS:
RECEPTOR STRUCTURE &
SIGNAL TRANSDUCTION
Part 2: Sections 6.3 - 6.6
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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 (2 slides)
3.5. Receptor types and subtypes (2 slides)
3.6. Signal transduction pathway
a)
Interaction of receptor with Gs-protein (3 slides)
b)
Interaction of s with adenylate cyclase (2 slides)
c)
Interaction of cyclic AMP with protein kinase A (PKA) (4 slides)
3.7. Glycogen metabolism - triggered by adrenaline in liver cells (2 slides)
3.8. GI proteins
3.9. Phosphorylation
3.10.
Drugs interacting with cyclic AMP signal transduction
3.11.Signal transduction involving phospholipase C (PLC) (2 slides)
3.12.
Action of diacylglycerol (2 slides)
3.13.
Action of inositol triphosphate (2 slides)
3.14.
Resynthesis of PIP2
[29 slides]
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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
Transmembrane
helix
G-Protein
binding region
HO2C
C -Terminal chain
Variable
intracellular loop
Intracellular loops
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3. G-protein-coupled receptors (7-TM receptors)
3.2 Ligands
• Monoamines e.g. dopamine, histamine, noradrenaline,
acetylcholine (muscarinic)
• Nucleotides
• Lipids
• Hormones
• Glutamate
• Ca++
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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
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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
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3. G-protein-coupled receptors (7-TM receptors)
3.4 Bacteriorhodopsin & rhodopsin family
Common ance stor
Monoamines
muscarinic
beta
alpha
Opsins, Rhodopsins
Bradyk inin,
Endothelins Angiote nsin.Tachykinins
Inte rleuk in-8
2
4
5
Mu scarin ic
Rec eptor
types
3
1
H1 H2
Histamine
1 2A 2B 2C
D4 D3 D2 D1A D1B D5
-Adre ne rgic
Dopamine rgic
3
2
Rec eptor
sub-types
1
-Adre ne rgic
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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
Subtypes
Adrenergic
Alpha ()
Beta ()
1, 2A, 2B, 2C
 1,  2,  3
Cholinergic
Nicotinic
Muscarinic
M1-M5
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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
- 1 adrenergic receptors
Fat cells
- 3 adrenergic receptors
Bronchial muscle
- 1& 2 adrenergic receptors
GI-tract
- 1 2 & 2 adrenergic receptors
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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 (, , g)
- S subunit has binding site for GDP
-GDP bound non covalently

g

GDP
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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
g

Ligand
binding
Induced
fit
G-protein
binds
ß
g

Induced
fit for
G-protein

G Protein
GDP
Binding site for G-protein opens
= GDP
ß
g
GTP
G-Protein alters shape
GDP binding site distorted
GDP binding weakened
GDP departs
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3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
ß
g

GTP binds
Binding site recognises GTP
g
ß
g

Fragmentation
and release
ß

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
• s Subunit carries message to next stage
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3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
b) Interaction of s with adenylate cyclase
Binding site
for s subunit
GTP
GDP
s-subunit
Adenylate cyclase
GTP hydrolysed
to GDP catalysed
by s subunit
Binding
Induced
fit
Active site
(closed)
P
ATP
cyclic AMP
Active site
(open)
Signal
transduction
(con)
s Subunit recombines with ,g dimer
to reform Gs protein
ATP
cyclic AMP
Active site
(closed)
s Subunit changes shape
Weaker binding to enzyme
Departure of subunit
Enzyme reverts to inactive
state
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3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
b) Interaction of s with adenylate cyclase
•
•
•
•
Several 100 ATP molecules converted before s-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
HO P O P O P
OH
OH
N
N
O
Adenylate cyclase
H
O
H
H
O
H
OH
N
N
O
OH
ATP
N
N
P
OH
O
H
H
Cyclic AMP
O
OH
OH
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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
H
OH
CH3
Serine
P
HO
O
O
O
Threonine
Protein
kinase A
H
N
C
HC
H
O
CH3
HO
P
O
OH
OH
Link
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Kinase:
Any of various enzymes that
catalyze the transfer of a phosphate group from
a donor, such as ADP or ATP, to an acceptor.
Phosphatase: Any of numerous enzymes that
catalyze the hydrolysis of esters of phosphoric
acid and are important in the absorption and
metabolism of carbohydrates, nucleotides, and
phospholipids and in the calcification of bone.
Phosphorylase: An enzyme that catalyzes the
production of glucose phosphate from glycogen
and inorganic phosphate.
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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
Link
Enzyme
(inactive)
Enzyme
(active)
Chemical
reaction
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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
Link
R
R
C
R
C
catalytic subunit
Note Cyclic AMP binds to PKA
Induced fit destabilises complex
Catalytic units released and activated
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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
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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
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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
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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
•
I subunit binds adenylate cyclase to inhibit it
• Adenylate cyclase under dual control (brake/accelerator)
• Background activity due to constant levels of s and i
• Overall effect depends on dominant G-Protein
• Dominant G-protein depends on receptors activated
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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
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3. G-protein-coupled receptors (7-TM receptors)
3.10 Drugs interacting with cyclic AMP signal transduction
Cholera toxin - constant activation of c.AMP - diahorrea
Theophylline and caffeine
- inhibit phosphodiesterases
- phosphodiesterases responsible for metabolising
cyclic AMP
- cyclic AMP activity prolonged
O
O
H3C
H
N
H3C
N
CH3
N
N
N
N
O
N
CH3
Theophylline
O
N
CH3
Caffeine
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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 q subunit
q Subunit activates or deactivates PLC (membrane bound enzyme)
Reaction catalysed for as long as q bound - signal amplification
Brake and accelerator
Active site
(open)
Active site
(closed)


PLC
DG

PLC
PLC
PIP2
IP3
GTP hydrolysis

Phosphate
DG
PLC
PIP2
IP3
Binding weakened
Active site
(closed)
q departs

PLC
enzyme
deactivated
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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
PLC
O
O P
O P
HO
O
H
O
H HO
OH H
H
+
H
O P
R
R
O C
O C
O
O
CH2
CH
OH
O P
HO
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)
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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
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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)
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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
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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
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3. G-protein-coupled receptors (7-TM receptors)
3.14 Resynthesis of PIP2
IP3 + DG
several
steps
PIP2
Inhibition
Li+ salts
Lithium salts used vs manic depression
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