PHAR2821

Drug Discovery III

Nicotinic Acetylcholine Receptors
Semester 2 2016

Presented by
A/Prof Thomas Balle
Faculty of Pharmacy

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 ACETYLCHOLINE IN THE CNS

– Cholinergics – mediate their effect via receptors stimulated by Acetylcholine

(ACh)
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From Rang & Dale’s Pharmacology 7th Ed.
 THE CHOLINERGIC SYNAPSE

Synaptic cleft
Effector Cell

Cholinergic Neuron
ACh
Muscarinic ACh Receptor
Acetyl CoA
ACh ACh ACh Nicotinic ACh Receptors
Choline
Ser Choline Choline

Ser

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 ACETYLCHOLINE RECEPTORS

– Two types of receptors responding to acetylcholine (ACh)

(ACh)
Muscarinic acetylcholine receptors Nicotinic acetylchomine receptors

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 NACHR ARCHITECTURE

PENTAMERIC LIGAND GATED ION CHANNELS

ACh

ACh

ACh

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 Curare

Strychnos toxifera Tubocurarine

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 Curare

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 Nerve cell ACh = Acetylcholine
Curare

Muscle Contraction Paralysis

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 Slangetoxin: a-Bungarotoxin

Bungarus Multicinctus

a-Bungarotoxin

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 Snake toxin: a-Cobratoxin

a-Cobratoxin

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 POISONOUS CONE SNAILS

NH2-CC----C-------C-CONH2

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 MOVIE

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 NICOTINIC ACETYLCHOLINE RECEPTORS

Nicotine

(ACh)

Mucscle Type Receptors Neuronal Receptors

Stimuli Many different sub-groups (subtypes)

Implicated in many CNS disorders

Muscle contraction e.g. dependence
Alzheimer’s disease
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 NICOTINIC ACETYLCHOLINE RECEPTORS

NICOTINIC RECEPTORS & DISEASE

Cigarrette smoking is related to cancer and heart dissease

Inverse correlation ~ smoking and parkinson’s disease
Patients with autosomal dominant nocturnal frontal lobe epilepsy (ADFLE) who
smoke have fewer seizures
In other types of epilepsy cigarette smoking can trigger seizures
Huge over-representation of cigarette smokers in certain patient groups, e.g.
schizophrenics and ADHD patients

Mutations in a4 and b2 subunits in some epilepsies, a7 in schizophrenia, and a5 in

nicotine addiction
Mutations cause imbalance in the properties of neuronal circuits

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 NICOTINIC ACETYLCHOLINE RECEPTORS

SMOKING AND DEPRESSION: A BIDIRECTIONAL CAUSALITY

People with depression or a history of depression are 3x more likely to smoke

and become addicted than people without such background (Breslau et al., 1998)

Chronic smoking increases a person’s risk of depression as a result of

changes in neurophysiology (Markou & Kenny, 2002)

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 NICOTINIC ACETYLCHOLINE RECEPTORS

SMOKING & DEPRESSION

– At least five of the DSM-IV criteria for Nicotine Withdrawal overlap with those
of and Major Depression (Paperwalla et al., 2004)
– DSM-IV criteria for Nicotine Withdrawal:
:

1. Insomnia
2. Irritability
3. Frustration or anger
4. Anxiety
5. Difficulty concentrating
6. Restlessness
7. Decreased heart rate
8. Increased appetite/weight gain

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DSM = Diagnostic and Statistical Manual of Mental Disorders
 CHOLINERGIC NEUROTRANSMISSION

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Miwa J M et al. Physiology 2012;27:187-199
 NACHR ARCHITECTURE

AGONIST BINDING IN SUBUNIT INTERFACES

– Multiple different subunits also complicate the picture
– a1-a10, b1-b4, g, d, e – 17 different subunits!
– A wealth of unique combinations possible

Homomeric Binary Ternary Quartenary

a7 a7 α4 b2 a4 a5 a1 b1
a7 a7 b2 b2 b2 b2 g d
a7 α4 a4 α1
(a7)5 (a4)2(b2)3 (a4)2(b2)2a5 (a1)2b1gd

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Millar & Gotti, Neuropharmacol, 2009
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 nAChRs SUBTYPES HAVE DISTINCT PHARMACOLOGY

Subtype

a1bdg/e a3b4 a4b2 a7

Neuromuscular
Tissue distribution Autonomic ganglia Brain Brain
junction

Modulation of Modulation of
Neuromuscular Transmission in
Physiology neurotransmitter neurotransmitter
transmission autonomic ganglia
release release

Selective ligands D-tubocurarine () Mecamylamine () ABT-894 () SSR180711 ()

Analgesia,
Neuromuscular Cognitive
Pharmacology Anti-hypertensive cognitive
blockade enhancement
enhancement
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 NACHR ARCHITECTURE

AGONISTS BIND IN SUBUNIT INTERFACES

– From a drug discovery perspective ligand gated ion channels are
complicated
Agonists bind in the interface between two subunits – i.e. not in a pocket
deep within a protein but in the inteface between two seperate
proteins
Coordinated action from at least two agoists required simultaneously
for activation

a4 b2

b2 b2
a4

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 nACHR DRUG DISCOVERY

– It’s Like An Onion

Cholinergic Receptors

Muscarinic Nicotinic

Muscle Type Neuronal

Subtypes

a4 b2
Stoichiometries
b2 b2
a4
(α4)2(β2)3 (α4)3(β2)2

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 STOICHIOMETRIES

BINARY RECEPTORS ARE NOT JUST BINARY RECEPTORS

They exist in (at least) two different stoichiometries
The most abundant nAChR in the brain, the a4b2 nAChR, exists in two distinct
stochiometries
a4 b2

b2 b2
a4

a4 b2 Control of
stoichiometries is key
b2 b2
a4 Otherwise pharmacology
(α4)2(β2)3 (α4)3(β2)2 is a mess

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 MESSY DATA

BIOLOGICAL DATA
- Lack of correlation between binding and functional data
- Lack of in depth understanding of drivers for potency and efficacy
- Differences between different expression systems (HEK vs. XO)
- Lack of reproducibility - from day to day - and between laboratories
- Poor fits of data (low Hill coefficients, large error bars, biphasic curves)

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Tosco et al., J. Med Chem, 2009 Crestey, Jensen et al., J Med Chem, 2013
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 MESSY DATA

FUNCTIONAL POTENCIES a4b2 nAChRs

Activation profile

NS3531
100 N

N
% of nicotine response

75
NS3950
N

O N

50
Br N

O N
25
N
NS3573

-10 -9 -8 -7 -6 -5 -4
Log [C]

Interpretation of data in a structural

context requires a well-defined biological
response that ideally is or can be related to binding
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 FUNCTIONAL ASSAY – XENOPUS OOCYTES (XO)
a mRNA
More b mRNA b mRNA

More a mRNA

Time (s)
Antagonist
ACh (300 µM)
Current (nA)

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 FUNCTIONAL ASSAY – XENOPUS OOCYTES
a mRNA
b mRNA

Time (s)

Current (nA)

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 STOICHIOMETRIES

ACETYLCHOLINE In XO biphasic α4β2 responses have been

observed for numerous agonists including ACh
a4:b2
By altering α4:β2 cRNA ratios monophasic
1:1 (■)
responses can be obtained

1:10 ratio results in HS (high sensitivity) R’s ●

10:1 ratio results in LS (low sensitivity) R’s ▼

Assumption: Pure receptor populations

produce monophasic resonses
Deviations from ideal monophasic bahaviour
was asttributed to contamination with other
stochiometries

Nelson et al., Mol Pharmacol, 2003

Zhou et al., J Neurosci, 2003
Kuryatov et al., Mol Pharmacol, 2005
Moroni et al., Mol Pharmacol, 2006
Zwart et al., Eur J Pharmacol, 2006
Carbone et al., Br J Pharmacol, 2009
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Data from Moroni et al., Mol Pharmacol, 2006
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 a4b2 STOICHIOMETRIES

a4:b2 ratio a4:b2 a4:b2 b2-6-a4 & a4

1:4 (□) 20:1 100:1 1:1
Acetylcholine
4:1 (■)

The biphasic response is an

inherrent feature of LS
(a4)3(b2)2 receptors

Curve fitting with monophasic equations looks awful

Curves are better approximated by biphasic
NS3573 equations !!!
LS component is constant
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 WHY IS THE RESPONSE BIPHASIC?
40
EC50: 20 nM

% of maximal ACh
EC50_1: 6.3 nM 30
EC50-1 = 9.8 nM
% of maximal ACh

15 EC50_2: 6.3 M
EC50-2 = 7.9 µM
Frac: 0.27 20 Mutate the α4α4 site
10 to resemble the
10
α4β2 sites
5 Green  purple
0
NS3573 -11 -10 -9 -8 -7 -6 -5 -4
0 log[NS3573]
-11 -10 -9 -8 -7 -6 -5 -4
log[NS3573]
15
EC50: 23 M

% of maximal ACh
Mutate the α4β2
α4 α4 10
sites to resemble
the α4α4 sites
β2 β2 5 purple  green
α4
0
(α4)3(β2)2 -11 -10 -9 -8 -7 -6 -5 -4
log[NS3573]
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Harpsøe et al., J. Neurosci. 2011
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 AN a4a4 AGONIST BINDING SITE

EC50: 3.2 M
Same experiement using ACh 100

% of maximal ACh
EC50_1: 0.79 M 75
100
% of maximal ACh

EC50_2: 63 M
Frac: 0.15 50 Mutate the α4α4
75
site to resemble
25
50 ACh the α4β2 sites
25 0
-9 -8 -7 -6 -5 -4 -3 -2
0 log[ACh]
-9 -8 -7 -6 -5 -4 -3 -2
100
EC50: 140 M

% of maximal ACh
log[ACh]
75
Mutate the α4β2
sites to resemble
α4 α4 50
the α4α4 sites
25
β2 β2
0
α4 -9 -8 -7 -6 -5 -4 -3 -2
log[ACh]
(α4)3(β2)2

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Harpsøe et al., J. Neurosci. 2011 3
 DIFFERENTIAL EFFECTS OF a4b2 AGONISTS

SERIES OF STRUCTURALLY RELATED AGONISTS

NS3531 NS3920
Ki*= 0.72 nM Ki = 0.32 nM

NS3573 NS3950 NS3570

Ki = 0.62 nM Ki = 0.25 nM Ki = 0.80 nM

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* [3H]Cytisine Page 33

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 a4b2 AGONISTS - XO

1. NS3531 2. NS3920 3. NS3570

monophasic fits monophasic fits monophasic fits
90 90 25
80 80
a4b2 HS ab HS a4b2 HS
70 70 20
a4b2 LS a4b2 LS a4b2 LS
% I/IACh max

% I/IACh max

% I/IACh max
60 60
15
50 50
40 40
10
30 30
20 20 5
10 10
NB poor fit
0 0 0
-11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 -3
log[NS3531] \M log[NS3920] \M log[NS3570] \M

4. NS3573 5. NS3950

45
monophasic fits 90
monophasic fits Blue Curves 5
80
40
a4b2 HS a4b2 HS
35 70 2
a4b2 LS a4b2 LS
% I/IACh max

60
% I/IACh max

30
25 50 1, 4
20 40
30
15 3
10 20

5 10 NB reasonable fit
NB horrible fit 0
0
-10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4

log[NS3573] \M log[NS3950] \M

ab HS: (a4)2(b2)3

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 SAR AT THE a4b2 INTERFACE

HALOGEN BONDING EXPLAINS HIGHER EFFICACY OF COMPDS 2 & 5

(a4)2(b2)3
5

1, 4

Rohde et al, JBC, 2012

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 SAR AT THE a4b2 INTERFACE

INTER-SUBUNIT BRIDGE FORMATION GOVERNS EFFICACY

– Ligand-based

Onium
group C
N+
Aryl Centroid
or Carbonyl C

X Hydrogen
bond
Sheridan et al., J Med Chem, 1986 acceptor
Figure from Glennon & Dukat, Bioorg Med Chem Lett, 2004
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 MODULATION

α4α4 SITE CAN BE MODULATED BY MUTATIONS

EC50_1: 0.79 M EC50: 3.2 M
100 100
GABA +
% of maximal ACh

EC50_2: 63 M

% of maximal ACh
Frac: 0.15 Diazepa
75
a4b2 75
a4
m b2
3M

50 4:1 50 4:1
GABA
25 25

0 0
-9 -8 -7 -6 -5 -4 -3 -2 -9 -8 -7 -6 -5 -4 -3 -2
log[ACh] log[ACh]

α4 α4

β2 β2
α4

(α4)3(β2)2

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Lindquist & Birnir, J. Neurochem. 2006 3
 MODULATION

BENZODIAZEPINE-LIKE MODULATION IN THE a4b2 RECEPTOR

No effect
NS9283 Timmermann et al. (2012) Br J Pharmacol 167, 164-82

ACh
A C h + N S 9 2 8 3 ( 3 1 .6 µ M )

1 .0

0 .8

ACh
E C 5 0 : 0 .1 1 µ M EC 50: 93µ M
0 .6

I/I 1 m M
0 .4

0 .2
E C 5 0 : 1 .1 µ M
NS9283 is a novel selective modulator that 0 .0
-1 0 -9 -8 -7 -6 -5 -4 -3 -2

activates only 3a:2b a4b2 receptors lo g [A C h ] \M

Analgesic effects by augmentation of the effect of a4b2 agonists

Procognitive
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effects Page 40
Timmermann et al. Br J Pharmacol, 2012
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 ALLOSTERIC MODULATION

STOICHIOMETRY SELECTIVITY INSPIRED SEARCH FOR BINDING SITE

Based on homology model from Harpsøe et al.

Based on PDB:2BG9 from Unwin
(2011) J Neurosci 31, 10759-66
(2005) J Mol Biol 346, 967-89
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Olsen et al., JBC, 2013 4
 ALLOSTERIC MODULATION

NO EFFECT ON a4 TRIPPLE MUTANT

NS9283
H142V, Q150F, T152L

3a :2 b
600
a 4 + b2
\% ACh

400 3m
a4 + b2
I/I 1 0 µ M

200

0
-8 -7 -6 -5 -4
lo g [N S 9 2 8 3 ] \M

Based on homology model from Harpsøe et al.

Based on PDB:2BG9 from Unwin
(2011) J Neurosci 31, 10759-66
(2005) J Mol Biol 346, 967-89
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Olsen et al., JBC, 2013 4
 NS9283 CO-CRYSTAL STRUCTURE WITH LS-AChBP

~ 60 M

Co-crystal structure with Ls-AChBP @2.7Å

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Olsen et al., JBC, 2014 PDB: 4NZB
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 NS9283 CO-CRYSTAL STRUCTURE

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Olsen et al., submitted
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 NS9283 CO-CRYSTAL STRUCTURE

HOMOLOGY MODEL a4-a4 INTERFACE

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Olsen et al., submitted
4
 NS9283 CO-CRYSTAL STRUC

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Olsen et al., submitted
4
 NS9283 CO-CRYSTAL STRUCTURE

ANOTHER NIFTY DETAIL

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Olsen et al., submitted
4
 NS9283 CO-CRYSTAL STRUCTURE

QM CALCULATION SUPPORT FAVOURABLE INTERACTION WITH W182

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Olsen et al., submitted (B3LYP/CC-PVTZ++//B3LYP/6-31G*) 4
 CO-CRYSTAL STRUCTURE - SUMMARY

– X-ray structure, mutational data and homology model collectively support a

binding mode of NS9283 overlapping that of ACh.
– NS9283 forms contacts to residues known to mediate agonist efficacy and
potency.
– NS9283 forms a bridge across the subunit interface as we have suggested for
agonists
– NS9283 has favorable contacts to the functionally important W182

– HOW COME NS9283 IS A MODULATOR AND NOT AN AGONIST?

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 MODULATOR THAT MECHANISTICALLY ACTS AS AN AGONIST

WELL – IT’S NOT –

(a4)3(b2)2 (a4)3(b23M)2

NS9283 activates receptors with more than one NS9283-compatible

binding site -
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Olsen et al., submitted
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 CAN OTHER RECEPTORS BE MODULATED THROUGH NON-
CANONICAL INTERFACES?

ACh
A C h + N S 9 2 8 3 ( 3 1 .6 µ M )

100
EC50: 3.2 M
GABA +

% of maximal ACh
1 .0

Diazepa
a4
m b2
0 .8
75 3M
ACh

E C 5 0 : 0 .1 1 µ M EC 50: 93µ M
0 .6

4:1
I/I 1 m M

50
0 .4 GABA
0 .2
E C 5 0 : 1 .1 µ M 25
0 .0
-1 0 -9 -8 -7 -6 -5 -4 -3 -2
0
lo g [A C h ] \M
-9 -8 -7 -6 -5 -4 -3 -2
log[ACh]
NS9283 site

Agonist site α4 α4

β2 β2
α4

(α4)3(β2)2 Agonist site

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Lindquist & Birnir, J. Neurochem. 2006 5
 CURRENT nAChR RESEARCH PROJECTs

Faculty of Pharmacy - Mental Health Research Theme

– Antidepressants and Smoking Status
– Better treatment of memory impairment in ADHD (a4b2 modulators targeting
the a4a4 binding site)
– Diagnostics – Ligands for positron emission tomography
– a6-containing nAChRs – Memory impairment and parkinsons’s disease
– a4b2 superagonists for treatment of neurophatic pain

Expressions of intrest regarding PhD projects are always welcome

thomas.balle@sydney.edu.au

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