Topic 10 Drugs of the Nervous
System
Ch 19,20 Patrick
Part VI- Nervous system -Corey
Contents
Part 1: Cholinergics & anticholinesterases
1.
2.
3.
4.
Nerve Transmission
Neurotransmitter
Transmission
Cholinergic receptors
4.1.
Nicotinic receptor
4.2.
Muscarinic receptor - G Protein coupled receptor
5. Cholinergic agonists
5.1.
Acetylcholine as an agonist
5.2.
Nicotine and muscarine as cholinergic agonists
5.3.
Requirements for cholinergic agonists
6. SAR for acetylcholine
7. Binding site (muscarinic)
8. Active conformation of acetylcholine
9. Instability of acetylcholine
10. Design of cholinergic agonists
11. Uses of cholinergic agonists
CHOLINERGIC
NERVOUS
SYSTEM
1. Nerve Transmission
Peripheral nervous system
CNS
Brain
Peripheral nerves
Muscle
Heart
Gastrointestinal
tract
(GIT)
Spinal cord
1. Nerve Transmission
Peripheral nervous system
Skeletal
muscle
CNS
(Somatic)
Ach
(N)
CNS
(Autonomic)
Synapse
Ach (N)
NA
Sympathetic
Adrenaline
Parasympathetic
Ach
(N)
Adrenal
medulla
AUTONOMIC
Synapse
Ach
(N)
Ach
(M)
Smooth muscle
Cardiac muscle
1. Nerve Transmission
Synapses
100-500A
Receptors
New signal
Nerve impulse
Nerve
Nerve
Vesicles containing
neurotransmitters
Release of
neurotransmitters
Receptor binding
and new signal
2. Neurotransmitter
Acetylcholine (Ach)
O
+
C
H 3C
Acetyl
NMe 3
O
Choline
3. Transmission process
Signal in nerve 1
...
Nerve 1
Signal
...
.
Nerve 2
...
Acetylcholinesterase enzyme
Acetylcholine
Vesicle
Cholinergic receptor
3. Transmission process
Vesicles fuse with membrane and release Ach
Nerve 1
Signal
Nerve 2
3. Transmission process
Nerve 2
3. Transmission process
•
•
•
•
Receptor binds Ach
Induced fit triggers 2o message
Triggers firing of nerve 2
Ach undergoes no reaction
2o Message
Nerve 2
3. Transmission process
•
•
•
Ach departs receptor
Receptor reverts to resting state
Ach binds to acetylcholinesterase
Nerve 2
3. Transmission process
Ach hydrolysed
by acetylcholinesterase
O
O
C
H 3C
O
Acetylcholine
HO
C
NMe3
H 3C
Acetic acid
OH
+
NMe3
Choline
Nerve 2
3. Transmission process
Choline binds to carrier protein
Choline
Nerve 1
Carrier protein for choline
Nerve 2
3. Transmission process
Choline transported into nerve
Nerve 1
Nerve 2
3. Transmission process
Ach resynthesised
Nerve 2
Nerve 1
E 1 = Choline acetyltransferase
O
O
C
H 3C
SCoA
+
C
E1
HO
CH2 CH2 NMe3
Choline
H 3C
O
Acetylcholine
NMe3
3. Transmission process
Ach repackaged in vesicles
Nerve 1
Nerve 2
4. Cholinergic receptors
Receptor types
• Not all cholinergic receptors are identical
• Two types of cholinergic receptor - nicotinic and muscarinic
• Named after natural products showing receptor selectivity
HO
NMe
Me
N
Nicotine
O
CH2NMe3
L-(+)-Muscarine
Activates cholinergic
Activates cholinergic
receptors at nerve synapses
receptors on smooth
and on skeletal muscle
muscle and cardiac muscle
Acetylcholine is natural messenger for both receptor types
Peripheral nervous system
Skeletal
Muscle
CNS
(Somatic)
SOMATIC
Ach
(N)
CNS
(Autonomic)
Synapse
Ach (N)
NA
Sympathetic
Adrenaline
Parasympathetic
Ach
(N)
Adrenal
medulla
AUTONOMIC
Synapse
Ach
(N)
Ach
(M)
Smooth Muscle
Cardiac Muscle
4.1 Nicotinic receptor
Control of Cationic Ion Channel:
Receptor
Binding
site
Cell
membrane
Five glycoprotein subunits
traversing cell membrane
Messenger
Induced
fit
‘Gating’
(ion channel
opens)
Cell
membrane
4.1 Nicotinic receptor
The binding sites
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
4.2 Muscarinic receptor - G Protein coupled receptor
Activation of a signal protein
• Receptor binds messenger leading to an induced fit
• Opens a binding site for a signal protein (G-protein)
messenger
induced
fit
closed
open
G-protein
bound
G-protein
split
4.2 Muscarinic receptor - G Protein coupled receptor
Activation of membrane bound enzyme
• G-Protein is split and subunit activates a membrane bound
enzyme
• Subunit binds to an allosteric binding site on enzyme
• Induced fit results in opening of an active site
• Intracellular reaction is catalysed
Enzyme
subunit
active site
(closed)
Enzyme
active site
(open)
Intracellular
reaction
5. Cholinergic agonists
5.1 Acetylcholine as an agonist
Advantages
• Natural messenger
• Easily synthesized
O
HO
+
NMe 3
AcO
NMe3
NMe3
Ac2O
Disadvantages
• Easily hydrolysed in stomach (acid catalysed hydrolysis)
• Easily hydrolysed in blood (esterases)
• No selectivity between receptor types
• No selectivity between different target organs
5. Cholinergic agonists
5.2 Nicotine and muscarine as cholinergic agonists
Advantages
• More stable than Ach
• Selective for main cholinergic receptor types
• Selective for different organs
Disadvantages
• Activate receptors for other chemical messengers
• Side effects
5. Cholinergic agonists
5.3 Requirements for cholinergic agonists
•
Stability to stomach acids and esterases
•
Selectivity for cholinergic receptors
•
Selectivity between muscarinic and nicotinic receptors
•
Knowledge of binding site
•
SAR for acetylcholine
6. SAR for acetylcholine
Ethylene
bridge
O
Me
O
Acetoxy
NMe3
4 o Nitrogen
Quaternary nitrogen is essential
O
H3C
O
O
CMe3
Bad for activity
H3C
O
NMe2
6. SAR for acetylcholine
Ethylene
bridge
O
Me
O
Acetoxy
•
•
NMe3
4 o Nitrogen
Distance from quaternary nitrogen to ester is important
Ethylene bridge must be retained
O
O
H 3C
O
NMe3
H 3C
Bad for activity
O
NMe3
6. SAR for acetylcholine
Ethylene
bridge
O
Me
O
NMe3
4 o Nitrogen
Acetoxy
Ester is important
H 3C
O
NMe3
H 3C
Bad for activity
NMe3
6. SAR for acetylcholine
Ethylene
bridge
O
Me
O
NMe3
4 o Nitrogen
Acetoxy
Minimum of two methyl groups on quaternary nitrogen
O
O
H 3C
Et
Et
O
N
Et
H 3C
O
Me
Me
Et
Bad for activity
N
Active
6. SAR for acetylcholine
Ethylene
bridge
O
Me
NMe3
O
4 o Nitrogen
Acetoxy
Methyl group of acetoxy group cannot be extended
O
H 3C
O
NMe3
Bad for activity
6. SAR for acetylcholine
Ethylene
bridge
O
Me
Acetoxy
O
NMe3
4 o Nitrogen
Conclusions:
• Tight fit between Ach and binding site
• Methyl groups fit into small hydrophobic pockets
• Ester interacting by H-bonding
• Quaternary nitrogen interacting by ionic bonding
7. Binding site (muscarinic)
hydrophobic
pocket
CH3
Trp-307
Asp311
CH3
CO2
N
O
O
H
O
H
N
Asn-617
CH3
hydrophobic
pockets
CH3
Trp-616 Trp-613
hydrophobic
pocket
7. Binding site (muscarinic)
Trp-307
vdw
CH3
CH3
Ionic bond
CO2
N
O
Asp311
CH3
vdw
O
H-bonds
H
O
H
N
Asn-617
CH3
Trp-616 Trp-613
vdw
7. Binding site (muscarinic)
•
•
Possible induced dipole dipole interaction between quaternary
nitrogen and hydrophobic aromatic rings in binding site
N+ induces dipole in aromatic rings
δ+
δ-
R
NMe3
δ+
δ-
8. Active conformation of acetylcholine
O
H
H
Me
C
Me
Me
N
O
Me
H
H
•
Several freely rotatable single bonds
•
Large number of possible conformations
•
Active conformation does not necessarily equal the most
stable conformation
8. Active conformation of acetylcholine
Rigid Analogues of acetylcholine
HO
Me
O
CH2NMe3
Me
NMe3
O
CH2NMe3
Me
O
H
MUSCARINE
•
•
•
H
O
O
Rotatable bonds ‘locked’ within ring
Restricts number of possible conformations
Defines separation of ester and N
O
4.4A
O
5.9A
N
N
Muscarinic
receptor
Nicotinic
receptor
9. Instability of acetylcholine
Me3N
!" O
!+ C
H 3C
O
•
Neighbouring group participation
•
Increases electrophilicity of carbonyl group
•
Increases sensitivity to nucleophiles
10. Design of cholinergic agonists
Requirements
•
Correct size
•
Correct pharmacophore - ester and quaternary nitrogen
•
Increased stability to acid and esterases
•
Increased selectivity
10. Design of cholinergic agonists
Use of steric shields
Rationale
•
Shields protect ester from nucleophiles and enzymes
•
Shield size is important
•
Must be large enough to hinder hydrolysis
•
Must be small enough to fit binding site
10. Design of cholinergic agonists
hinders binding to esterases
and provides a shield to
nucleophilic attack
Me
O
Methacholine
Me
O
NMe3
*
asymmetric centre
Properties
• Three times more stable than acetylcholine
• Increasing the shield size increases stability but decreases
activity
• Selective for muscarinic receptors over nicotinic receptors
• S-enantiomer is more active than the R-enantiomer
• Stereochemistry matches muscarine
• Not used clinically
HO
Me
O
O
MUSCARINE
CH2NMe3
Me
O
Me
O
(S)
CH2NMe3
H
Me
H
O
(R)
CH2NMe3
Me
10. Design of cholinergic agonists
Use of electronic factors
•
•
Replace ester with urethane
Stabilises the carbonyl group
H 2N
O
O
C
C
H 2N
O
=
!+
H 2N
C
!"
10. Design of cholinergic agonists
O
C
H2N
O
NMe3
Carbachol
Properties
• Resistant to hydrolysis
• Long lasting
• NH2 and CH3 are equal sizes. Both fit the hydrophobic pocket
• NH2 = bio-isostere
• Muscarinic activity = nicotinic activity
• Used topically for glaucoma
10. Design of cholinergic agonists
Steric + Electronic factors
O
Me
C
H2N
O
*
NMe3
Bethanechol
Properties
•
Very stable
•
Orally active
•
Selective for the muscarinic receptor
•
Used to stimulate GI tract and urinary bladder after surgery
10. Design of cholinergic agonists
Nicotinic selective agonist
O
C
Me
O
*
Me
NMe3
* asymmetric centre
11. Uses of cholinergic agonists
Nicotinic selective agonists
Treatment of myasthenia gravis
- lack of acetylcholine at skeletal muscle causing weakness
Muscarinic selective agonists
•
Treatment of glaucoma
•
Switching on GIT and urinary tract after surgery
•
Treatment of certain heart defects. Decreases heart muscle
activity and decreases heart rate
Peripheral nervous system
Skeletal
Muscle
CNS
(Somatic)
SOMATIC
Ach
(N)
CNS
(Autonomic)
Synapse
Ach (N)
NA
Sympathetic
Adrenaline
Parasympathetic
Ach
(N)
Adrenal
medulla
AUTONOMIC
Synapse
Ach
(N)
Ach
(M)
Smooth Muscle
Cardiac Muscle
Contents
Part 1: Cholinergics & anticholinesterases
1.
2.
3.
4.
Nerve Transmission
Neurotransmitter
Transmission process
Cholinergic receptors
4.1.
Nicotinic receptor
4.2.
Muscarinic receptor - G Protein coupled receptor
5. Cholinergic agonists
5.1.
Acetylcholine as an agonist
5.2.
Nicotine and muscarine as cholinergic agonists
5.3.
Requirements for cholinergic agonists
6. SAR for acetlcholine
7. Binding site (muscarinic)
8. Active conformation of acetylcholine
9. Instability of acetylcholine
10. Design of cholinergic agonists
11. Uses of cholinergic agonists
CHOLINERGIC
NERVOUS
SYSTEM
1. Nerve Transmission
Peripheral nervous system
CNS
Brain
Peripheral nerves
Muscle
Heart
Gastrointestinal
tract
(GIT)
Spinal cord
1. Nerve Transmission
Peripheral nervous system
Skeletal
muscle
CNS
(Somatic)
Ach
(N)
CNS
(Autonomic)
Synapse
Ach (N)
NA
Sympathetic
Adrenaline
Parasympathetic
Ach
(N)
Adrenal
medulla
AUTONOMIC
Synapse
Ach
(N)
Ach
(M)
Smooth muscle
Cardiac muscle
1. Nerve Transmission
Synapses
100-500A
Receptors
New signal
Nerve impulse
Nerve
Nerve
Vesicles containing
neurotransmitters
Release of
neurotransmitters
Receptor binding
and new signal
Contents
Part 2: Cholinergics & anticholinesterases
12. Cholinergic Antagonists (Muscarinic receptor)
12.1.
Atropine
12.2.
Hyoscine (scopolamine)
12.3.
Comparison of atropine with acetylcholine
12.4.
Analogues of atropine
12.5.
Simplified Analogues
12.6.
SAR for Antagonists
12.7.
Binding Site for Antagonists
13. Cholinergic Antagonists (Nicotinic receptor)
13.1.
Curare
13.2.
Binding
13.3.
Analogues of tubocurarine
12. Cholinergic Antagonists (Muscarinic receptor)
•
•
•
Drugs which bind to cholinergic receptor but do not activate it
Prevent acetylcholine from binding
Opposite clinical effect to agonists - lower activity of
acetylcholine
Postsynaptic
nerve
Postsynaptic
nerve
Ach
Ach
Antagonist
Ach
12. Cholinergic Antagonists (Muscarinic receptor)
Clinical Effects
• Decrease of saliva and gastric secretions
• Relaxation of smooth muscle
• Decrease in motility of GIT and urinary tract
• Dilation of pupils
Uses
• Shutting down digestion for surgery
• Ophthalmic examinations
• Relief of peptic ulcers
• Treatment of Parkinson’s Disease
• Anticholinesterase poisoning
• Motion sickness
12. Cholinergic Antagonists (Muscarinic receptor)
Me
12.1 Atropine
N
easily racemised
H
CH2 OH
O
CH
C
*
O
•
•
•
•
•
Racemic form of hyoscyamine
Source - roots of belladonna (1831) (deadly nightshade)
Used as a poison
Used as a medicine
decreases GIT motility
antidote for anticholinesterase poisoning
dilation of eye pupils
CNS side effects - hallucinations
12. Cholinergic Antagonists (Muscarinic receptor)
12.2 Hyoscine (scopolamine)
Me
N
H
O
CH2 OH
H
H
O
C
CH
*
O
•
•
•
Source - thorn apple-Datura-jimsonweed
Medical use - treatment of motion sickness
CNS effects, hallucinations
12. Cholinergic Antagonists (Muscarinic receptor)
12.3 Comparison of atropine with acetylcholine
Me
N NMe3
CH2
CH
H2
O
O
C
O
•
•
•
•
•
•
CH2 OH
CH3
CH
C
O
Relative positions of ester and nitrogen similar in both molecules
Nitrogen in atropine is ionised
Amine and ester are important binding groups (ionic + H-bonds)
Aromatic ring of atropine is an extra binding group (vdW)
Atropine binds with a different induced fit - no activation
Atropine binds more strongly than acetylcholine
12. Cholinergic Antagonists (Muscarinic receptor)
12.4 Analogues of atropine
Br
CH3
CH(CH3) 2
H 3C
H 3C
N
NO3
N
H
H
CH2 OH
CH2 OH
O
C
CH
O
Ipratropium
(bronchodilator & anti-asthmatic)
•
•
•
O
C
CH
O
Atropine methonitrate
(lowers GIT motility)
Analogues are fully ionized
Analogues unable to cross the blood brain barrier
No CNS side effects
12. Cholinergic Antagonists (Muscarinic receptor)
12.5 Simplified Analogues
Pharmacophore = ester + basic amine + aromatic ring
Et
Me
N
Et
Me
CH
CH2 CH2
CH2
CH2 OH
O
CH
C
O
HO
C
CH2 CH2N(Et)3
Br
O
C
CH2CH2 N
Me
CH
Me
Me
O
Cl
O
Amprotropine
Tridihexethyl bromide
Propantheline chloride
12. Cholinergic Antagonists (Muscarinic receptor)
12.5 Simplified Analogues
Me
N
Me2N
N
H
O
CH2CH3
N
CH
O
O
CH
OH
CH
O
CH2OH
Tropicamide
(opthalmics)
Benztropine
(Parkinsons disease)
Cyclopentolate
(opthalmics)
O
HN
N
N
C
N
C
CH2
CH
Benzhexol
(Parkinsons disease)
N
N
Me
O
Pirenzepine
(anti-ulcer)
12. Cholinergic Antagonists (Muscarinic receptor)
12.6 SAR for Antagonists
R'
O
CH2
R 2N
CH2
R' = Aromatic or
Heteroaromatic
CH
C
R'
O
Important features
• Tertiary amine (ionised) or a quaternary nitrogen
• Aromatic ring
• Ester
• N-Alkyl groups (R) can be larger than methyl (unlike agonists)
• Large branched acyl group
• R’ = aromatic or heteroaromatic ring
• Branching of aromatic/heteroaromatic rings is important
12. Cholinergic Antagonists (Muscarinic receptor)
12.6 SAR for Antagonists
Me
Me
CH
O
O
C
CH2CH2 N
O
Me
CH
C
Me
Me
CH2
O
Cl
Active
Inactive
O
CH2 CH2NR2
12. Cholinergic Antagonists (Muscarinic receptor)
12.6 SAR for Antagonists vs. Agonists
SAR for Antagonists
SAR for Agonists
Tertiary amine (ionized)
or quaternary nitrogen
Aromatic ring
Ester
N-Alkyl groups (R) can be
larger than methyl
R’ = aromatic or heteroaromatic
Branching of Ar rings important
Quaternary nitrogen
Aromatic ring
Ester
N-Alkyl groups = methyl
R’ = H
12. Cholinergic Antagonists (Muscarinic receptor)
12.7 Binding Site for Antagonists
van der Waals
binding regions
for antagonists
Acetylcholine
binding site
RECEPTOR SURFACE
12. Cholinergic Antagonists (Muscarinic receptor)
12.7 Binding Site for Antagonists
O
Me
O
C
O
O
CH2CH2
Me
CH
N
Me
C
Me
CH
Me
O
CH2
Cl
CH2
NMeR2
CO2
O
H 2N
Asn
13. Cholinergic Antagonists (Nicotinic receptor)
13.1 Curare
• Extract from curare plant- Strychnos toxifera
• Used for poison arrows
• Causes paralysis (blocks acetylcholine signals to muscles)
• Active principle = tubocurarine
MeO
Me
N
HO
Me
H
O
Me
H
H
CH2
CH2
O
N
OH
OMe
Tubocurarine
13. Cholinergic Antagonists (Nicotinic receptor)
Pharmacophore
• Two quaternary centres at specific separation (1.15nm)
• Different mechanism of action from atropine based antagonists
• Different binding interactions
Clinical uses
• Neuromuscular blocker for surgical operations
• Permits lower and safer levels of general anaesthetic
• Tubocurarine used (previously) as neuromuscular blocker
but side effects
13. Cholinergic Antagonists (Nicotinic receptor)
13.2 Binding
protein complex
(5 subunits)
diameter=8nm
S
N
8nm
N
9-10nm
a) Receptor dimer
Probably not bridging Ach sites
b) Interaction with tubocurarine
N
N
Tubocurarine
Acetylcholine binding site
13. Cholinergic Antagonists (Nicotinic receptor)
13.3 Analogues of tubocurarine
Me3N(CH2) 10NMe3
Me3NCH2CH2
O
Decamethonium
•
•
•
Long lasting
Long recovery times
Side effects on heart
O
O
C
C
CH2 CH2
O
CH2 CH2NMe3
Suxamethonium
•
•
•
•
Esters incorporated
Shorter lifetime (5 min)
Fast onset and short duration
Side effects at autonomic ganglia
13. Cholinergic Antagonists (Nicotinic receptor)
13.3 Analogues of tubocurarine
O
Me
Pancuronium (R=Me)
Vecuronium (R=H)
Me
Me
H
O
O
•
•
•
•
•
Me
N
H
N
O
H
H
Me
Steroid acts as a spacer for the quaternary centres (1.09nm)
Acyl groups are added to introduce the Ach skeleton
Faster onset then tubocurarine but slower than suxamethonium
Longer duration of action than suxamethonium (45 min)
No effect on blood pressure and fewer side effects
13. Cholinergic Antagonists (Nicotinic receptor)
13.3 Analogues of tubocurarine
MeO
Me
N
MeO
CH2
Atracurium
OMe
OMe
•
•
•
•
•
•
•
CH2
O
O
C
C
O
(CH2)5
O
OMe
H
CH2
CH2
N
OMe
MeO
OMe
Design based on tubocurarine and suxamethonium
Lacks cardiac side effects
Rapidly broken down in blood both chemically and metabolically
Avoids patient variation in metabolic enzymes
Lifetime is 30 minutes
Administered as an i.v. drip
Self destruct system limits lifetime
13. Cholinergic Antagonists (Nicotinic receptor)
13.3 Analogues of tubocurarine
Me
N
Ph
CH2
R
CH
H
-H
C
Me
N
R
H2C
O
O
Ph
ACTIVE
Atracurium stable at acid pH
Hofmann elimination at blood pH (7.4)
CH C
INACTIVE
13. Cholinergic Antagonists (Nicotinic receptor)
13.3 Analogues of tubocurarine
Mivacurium
MeO
Me
N
O
MeO
OMe
O
O
N
OMe
O
H3C
OMe
MeO
OMe
OMe
•
•
Faster onset (2 min)
Shorter duration (15 min)
Contents
Part 3: Cholinergics & anticholinesterases
14. Acetylcholinesterase
14.1.
Role
14.2.
Hydrolysis reaction catalysed
14.3.
Effect of inhibition
14.4.
Structure of enzyme complex
14.5.
Active site - binding interactions
14.6.
Active site - Mechanism of catalysis
15. Anticholinesterases
15.1.
Physostigmine
15.2.
Mechanism of action
15.3.
Physostigmine analogues
15.4.
Organophosphates
15.5.
Anticholinesterases as ‘Smart Drugs’
14. Acetylcholinesterase
14.1 Role
• Hydrolysis and deactivation of acetylcholine
• Prevents acetylcholine reactivating receptor
...
Nerve 1
Signal
...
Nerve 2
...
Acetylcholinesterase enzyme
14. Acetylcholinesterase
14.2 Hydrolysis reaction catalysed
O
O
C
CH3
O
CH3
Acetylcholine
active
HO
C
NMe3
OH
+
NMe3
Choline
Acetic acid
inactive
14. Acetylcholinesterase
Enzyme inhibitor
(Anticholinesterase)
14.3 Effect of inhibition
2o Message
Ach
•
•
•
•
Inhibitor blocks acetylcholinesterase
Ach is unable to bind
Ach returns to receptor and reactivates
it
Enzyme inhibitor has the same effect as
a cholinergic agonist
Nerve 2
14. Acetylcholinesterase
14.4 Structure of enzyme complex
S
S
Enzyme
S
S
S
S
Enzyme
S
S
S
collagen
S
S
S
S
S
S
S
S
S
Enzyme
14. Acetylcholinesterase
14.5 Active site - binding interactions
Ester binding region
Anionic binding region
Serine
OH
Aspartate
Histidine
N
hydrophobic
pockets
Ionic
Me
N
C
N
O
:
vdw
:O :
CH2
CH2
O
O
H-bond
H
CH3
O
vdwMe
Me
Tyrosine
•
•
•
Anionic binding region similar to cholinergic receptor site
Binding and induced fit strains Ach and weakens bonds
Molecule positioned for reaction with His and Ser
14. Acetylcholinesterase
14.6 Active site - Mechanism of catalysis
O
C
O
CH2 CH2 NMe 3
:
:O
:
CH3
:N
H
NH
:O :
CH3
C
R
O
NH
:N
O
H
Serine
(Nucleophile)
Histidine
(Base)
Histidine
(Base catalyst)
:
CH3
C
O
:
: O:
:O :
R
O
H N
NH
CH3
C
OR
O
Histidine
Acid catalyst
:N
NH
H
Histidine
14. Acetylcholinesterase
14.6 Active site - Mechanism of catalysis
H2O
ROH
:
:O :
CH3
O
C
OR
O
:N
NH
CH3
C
N
O
H
Histidine
Histidine
:
C
H
O
::
O
_
: O:
O
CH3
NH
NH
:N
CH3
C
O
OH
H
NH
:N
H
Histidine
Histidine
Basic catalyst
14. Acetylcholinesterase
14.6 Active site - Mechanism of catalysis
:
_
:
:O :
:O :
CH3
C
CH3
OH
O
H
:N
NH
C
OH
:O :
H
N
Histidine
Basic catalyst
Histidine
(Acid catalyst)
:
_
:O :
CH3
NH
C
O
OH
:N
O
NH
CH3
C
OH
H
Histidine
OH
:N
NH
14. Acetylcholinesterase
•
Serine and water are poor nucleophiles
•
Mechanism is aided by histidine acting as a basic catalyst
•
Choline and serine are poor leaving groups
•
Leaving groups are aided by histidine acting as an acid catalyst
•
Very efficient - 100 x 106 faster than uncatalysed hydrolysis
•
Acetylcholine hydrolysed within 100 µsecs of reaching active
site
•
An aspartate residue is also involved in the mechanism
14. Acetylcholinesterase
The catalytic triad
•
An aspartate residue interacts with the imidazole ring of
histidine to orient and activate it
:
:
H
:O:
N
:
:O
H
Serine
(Nucleophile)
:N
O
Histidine
(Base)
Aspartate
15. Anticholinesterases
•
Inhibitors of acetylcholinesterase enzyme
•
Block hydrolysis of acetylcholine
•
Acetylcholine is able to reactivate cholinergic receptor
•
Same effect as a cholinergic agonist
15. Anticholinesterases
15.1 Physostigmine
O
C O
Me
N
H
Urethane
or
Carbamate
Me
Pyrrolidine N
N
N
Me
Me
http://www.floradelaterre.com/index.php?id=19
• Natural product from the African calabar (ordeal) beanPhysostigma
• Carbamate is essential (equivalent to ester of Ach)
• Aromatic ring is important
• Pyrrolidine N is important (ionized at blood pH)
• Pyrrolidine N is equivalent to the quaternary nitrogen of Ach
: :
15.2 Mechanism of action
O
MeNH
H
H
O
:N
NH
MeNH
O Ar
C O
:N
Ar
NH
:O :
:
C
O
Physostigmine
:O :
H
Ar
N
O
NH
MeNH
C
:O :
:
C O
:
MeNH
: :
O
:N
O Ar
H
NH
15.2 Mechanism of action
-ArOH
O
MeNH
C
O Ar
O
NH
MeNH
H
Stable carbamoyl
intermediate
O
Hydrolysis
very slow
H
:N
:N
C
:
:O :
:N
NH
Rate of hydrolysis slower by 40 x 106
O
NH
15.2 Mechanism of action
O
O
N
O
C
N
::
H
C
O
:
H
:
Me
Stable carbamoyl
intermediate
Me
Carbonyl group
'deactivated'
15.3 Physostigmine analogues
Me
O
C
Me
O
CH
NMe2
N
H
•
•
•
•
O
Me
C
O
NMe3
N
Me
(ionised at blood pH)
Miotine
Neostigmine
Simplified analogue
Susceptible to hydrolysis
Crosses BBB as free base
CNS side effects
•
•
•
•
•
Fully ionized
Cannot cross BBB
No CNS side effects
More stable to
hydrolysis
Extra N-methyl group
increases stability
15.4 Organophosphates
a) Nerve gases
iPrO
O
iPrO
P
iPrO
P
F
Dyflos
(Diisopropyl fluorophosphonate)
•
•
•
•
O
Me
F
Sarin
Agents developed in World War 2
Agents irreversibly inhibit acetylcholinesterase
Permanent activation of cholinergic receptors by Ach
Results in death
15.4 Organophosphates
b) Mechanism of action
-H
Serine
O
Serine
H
O
iPrO
iPrO
O
P
iPrO
•
•
F
Irreversible phosphorylation
P-O bond very stable
P
iPrO
STABLE
O
F-
15.4 Organophosphates
c) Medicinal organophosphate
O
Me3N
CH2
CH2
S
P
OEt
Ecothiopate
OEt
•
•
•
•
Used to treat glaucoma
Topical application
Quaternary N is added to improve binding interactions
Results in better selectivity and lower, safer doses
15.4 Organophosphates
d) Organophosphates as insecticides
EtO
S
MeO
P
EtO
S
CO2Et
P
O
NO2
MeO
S
CH
CH2 CO2Et
Parathion
•
•
•
Malathion
Relatively harmless to mammals
Agents act as prodrugs in insects
Metabolised by insects to produce a toxic metabolite
15.4 Organophosphates
d) Organophosphates as insecticides
MAMMALS
EtO
EtO
S
O
P
P
EtO
INSECTS
O
NO2
PARATHION
(Inactive Prodrug)
Insect
Oxidative
desulphurisation
EtO
O
NO2
Active drug
Mammalian
Metabolism
EtO
S
Phosphorylates enzyme
P
EtO
OH
INACTIVE
& excreted
DEATH
15.4 Organophosphates
e) Design of Organophosphate Antidotes
Strategy
• Strong nucleophile required to cleave strong P-O bond
• Find suitable nucleophile capable of cleaving phosphate esters
• Water is too weak as a nucleophile
• Hydoxylamine is a stronger nucleophile
O
O
NH 2 OH +
Hydroxylamine
•
•
O P OR
RO P OR
OR
H2 N
+
ROH
OR
Hydroxylamine is too toxic for clinical use
Increase selectivity by increasing binding interactions with active
site
15.4 Organophosphates
e) Design of Organophosphate Antidotes
Pralidoxime
N
CH3
•
•
•
•
CH N
OH
Quaternary N is added to bind to the anionic region
Side chain is designed to place the hydroxylamine moiety in
the correct position relative to phosphorylated serine
Pralidoxime 1 million times more effective than
hydroxylamine
Cannot act in CNS due to charge - cannot cross bbb
15.4 Organophosphates
e) Design of Organophosphate Antidotes
O
N
CH N
N
H
O
Me
Me
O
CH N
O
P
OR
OR
OR
P
CO2
H
Active Site (Blocked)
O
OR
CO2
OH
SER
SER
Active Site (Free)
15.4 Organophosphates
e) Design of Organophosphate Antidotes
H
H
ProPAM
NOH
N
CH3
•
•
•
Prodrug for pralidoxime
Passes through BBB as free base
Oxidised in CNS to pralidoxime
15.5 Anticholinesterases as ‘Smart Drugs’
•
Act in CNS
•
Must cross blood brain barrier
•
Used to treat memory loss in Alzheimers disease
•
Alzheimers causes deterioration of cholinergic receptors in
brain
•
Smart drugs inhibit Ach hydrolysis to increase activity at
remaining receptors
NMe2
15.5 Anticholinesterases as ‘Smart Drugs’ Me
Et
CH
O
N
C
Me
O
Cl
O
MeO
NH2
CH2
Rivastigmine (Exelon)
(analogue of physostigmine)
N
H
MeO
Donepezil
H
N
N
Tacrine (Cognex)
Toxic side effects
N
O
P
MeO
MeO
HO
Anabaseine
(ants and marine worms)
CCl3
OH
Metrifonate
(organophosphate)
O
MeO
S
N
N
N
O
Me
Galanthamine
(daffodil and snowdrop bulbs
N
Me
Xanomeline
Me