Download G-Protein Coupled Receptors Past, Present, Future Outline and

G-Protein Coupled Receptors
Past, Present, Future
Michael B. Bolger, Ph.D.
USC School of Pharmacy
PSCi 664 - 2006
Outline and Objectives
• Receptors and Autonomic Physiology
– Langley (1905), Dale (1906), Ahlquist (1948)
•
•
•
•
Discovery of c-AMP (Sutherland, 1957)
Discovery of G-proteins
Radio-ligand Receptor Binding Assay
Dopamine Receptors
– SAR and associated drugs
– Anatomical location
– Links to disease
• Current Medicinal Chemistry
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1
John Newport Langley (1852 – 1925)
N
O
CH3
N
H3 C
+
N
O
Acetylcholine
Nicotine
D-Tubocurarine
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Langley J. Physiol. 33:374 (1905)
Gastrocnemius Muscle response to
Nicotine and d-Tubocurarine
20 mg Nicotine and
Stimulation of sciatic
nerve.
20 mg Nicotine
followed by 30 mg
and 20 mg or curari
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Langley Conclusions
“Since, in the normal state, both nicotine and curari abolish
the effect of nerve stimulation, but do not prevent
contraction from being obtained by direct stimulation of the
muscle or by a further adequate injection of nicotine, it may
be inferred that neither the poisons nor the nervous impulse
act directly on the contractile substance of the muscle but
on some accessory substance. Since this accessory
substance is the recipient of stimuli which it transfers to the
contractile material, we may speak of it as the receptive
substance of the muscle.”
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Langley Conclusions
“I conclude then that in all cells two constituents at least must be distinguished,
(1) substance concerned with carrying out the chief functions of the cells, such
as contraction, secretion, the formation of special metabolic products, and (2)
receptive substances especially liable to change and capable of setting the
chief substance in action. Further, that nicotine, curari, atropine, pilocarpine,
strychnine, and most other alkaloids, as well as the effective material of
internal secretions produce their effects by combining with the receptive
substance, and not by an action on axon-endings if these are present, nor by a
direct action on the chief substance (muscle, heart, etc.).”
“The varied effects produced by poisons show that the receptive substance
varies in different cells (cp. p. 408). But the effects show also that the receptive
substance connected with the cells of any one class have frequently some
common characters, and that they differ more or less from the receptive
substances connected with the nerve fibres of any other class. The
preferential action is very marked in the case of adrenalin.”
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Sir Henry Dale (Nobel Prize, 1936)
Fig. 11
Effect of 0.1 mg
Epinephrine on
jejunum and
blood pressure.
Fig. 12
Effect of vagal
stimulation on
jejunum in the
presence of 150 mg
of an ergot alkaloid
(α2-receptor
antagonist)
Dale H.H., J. Physiol. 34:163 (1906)
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Sir Henry Dale (Nobel Prize, 1936)
Dale H.H., J. Physiol. 34:163 (1906)
Right eye: Effect of intravenous 100 mg of ergot (α-antagonist).
Left eye: Effect of left carotid occlusion. Blocks the baroreceptor
reflex by the depressor nerve producing increased sympathetic
outflow from the cardiovascular control center resulting in slow
dilation as the blockade is overcome locally in left eye.
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Sir Henry Dale (Nobel Prize, 1936)
At the Nobel lecture, speaking about acetylcholine
which was isolated by his colleage Dr. Ewins in 1914.
“I was thus able to describe it as having two apparently distinct types of action.
Through what I termed its "muscarine" action, it reproduced at the periphery all
the effects of parasympathetic nerves, with a fidelity which, as I indicated, was
comparable to that with which adrenaline had been shown, some ten years
earlier, to reproduce those of true sympathetic nerves. All these peripheral
muscarine actions, these parasympathomimetic effects of acetylcholine, were
very readily abolished by atropine. When they were thus suppressed, another
type of action was revealed, which I termed the "nicotine" action, because it
closely resembled the action of that alkaloid in its intense stimulant effect on all
autonomic ganglion cells, and, as later appeared, on voluntary muscle fibres.”
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Raymond Ahlquist
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Ahlquist R., Am. J. Physiol. 153:586 (1948)
5
Raymond Ahlquist
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Ahlquist R., Am. J. Physiol. 153:586 (1948)
Raymond Ahlquist
“The original paper was rejected by the Journal of Pharmacology and
Experimental Therapeutics, was a loser in the Abel Award competition,
and finally was published in the American Journal of Physiology due to my
personal friendship with a great physiologist, W.F. Hamilton.”
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Ahlquist R., Am. J. Physiol. 153:586 (1948)
6
Earl W. Sutherland (Nobel Prize 1971)
Discovery of c-AMP
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Earl W. Sutherland (Nobel Prize 1971)
Discovery of c-AMP
Only the particulate fraction of homogenized liver cells could
restore phosphorylase activity. c-AMP was isolated from this
particulate fraction.
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http://nobelprize.org/nobel_prizes/medicine/laureates/1994/
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Martin Rodbell (1/2 Nobel Prize 1994)
Requirement for GTP in hormonal activation of adenylyl cyclase
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Schramm M, Rodbell M, JBC 250(6):2232 (1975)
8
Martin Rodbell (1/2 Nobel Prize 1994)
Requirement for GTP in hormonal activation of adenylyl cyclase
Rodbell M, et al., J.B.C., 246:1877 (1971)
Membranes, Preincubation, 36x Dilution, Final Incubation & Assay
Schramm M, Rodbell M, JBC 250(6):2232 (1975)
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Martin Rodbell (1/2 Nobel Prize 1994)
Requirement for GTP in hormonal activation of adenylyl cyclase
Membranes, Preincubation, 36x Dilution, Final Incubation & Assay
Schramm M, Rodbell M, JBC 250(6):2232 (1975)
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Alfred G?. Gilman (Nobel Prize 1994)
Northrup J. et al. PNAS 77:6516 (1980)
α = 45 kDa β = 35 kDa γ = 5-10 kDa
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Alfred G?. Gilman (Nobel Prize 1994)
Receptor
γ = 5-10 kDa
α = 45 kDa β = 35 kDa
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GPCR Cycle: Principles of Drug Action, 3rd Ed.
Pratt and Taylor
α-subunit
activates
Start Here
High
Affinity
Low
Affinity
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Gs and Gi Proteins Modulate Different Receptors
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Dopamine Analog Conformations
D. A. Williams and T.L.
Lemke, Foye’s
Priniciples of Medicinal
Chemistry, 5th edition,
Part II, Chap. 20, pg
489. (2002)
HO
+
NH2
HO
SKF 38393
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Renal Dopamine D1 Receptors
Propranolol =
β- adrenergic blocker
DA = D1 & D2 agonist
Haldol = D1/2 antagonist
ISO = β agonist
BRADY = vasodilation
X = test compound
AcCh = muscarinic and
nicotinic agonist
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Goldberg L.I., et al., Ann. Rev. Pharmacol. Toxicol. 18:57 (1978)
12
Agonists Relative Potency and Selectivity
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Antagonists Relative Potency and Selectivity
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Types of Binding Assays:
Saturation Assay
Scatchard Plot
Saturation: Each tube has
different amount of 3H-Ligand
Total Binding
3H-Ligand only
Non-specific Binding
3H-Ligand +
1 μM (+)-butaclamol
Bound = Total – Non-specific
Sibley D., J. Biol. Chem.,
257(11):6351 (1982)
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Radio-ligand Receptor Binding Assay
Prepare an assay with a radioactive
ligand and test chemical at various
concentrations in test tubes. Place
glass fibre paper in the harvester
block and lock it down. Apply a
vacuum to the machine and suck up
your assay on to the filter paper. At
this point the test tubes are washed
three or four times with a buffer from
the wash media pump, controlled
from the harvesting probe head.
After this, the vacuum is turned off,
the block is opened and the paper
(already scored) is placed in vials,
where a suitable scintillant is added.
The vials are then capped and are
ready for the radioactive count in a
scintillation counter.
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Other Binding Assay Controls
Linear: tissue vs. Bmax
Linear: dissociation kinetics
Pseudo-first Order Association Kinetics
Sibley D., J. Biol. Chem.,
257(11):6351 (1982)
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Types of Binding Assays:
Competition Assay
Competition: Each tube has
small amount of 3H-Ligand (< Kd)
Plus increasing amount of test (3 log units).
Competition Plot
Kd = 4.8 nM
Total Binding
3H-Ligand plus
Unlabeled test
Non-specific Binding
+ unlabeled +
1 μM (+)-butaclamol
3H-Ligand
Bound = Total – Non-specific
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Competition Binding Results (Kd nM)
•
•
•
•
•
•
•
•
•
•
•
•
•
Dopamine
210
D1D2 Ago.
(-) Apomorphine
4.8 D1D2 Ago.
(-) N-propylapomor.
0.25 D1D2 Ago.
(-) Epinephrine
670
α,β Ago.
(-) Serotonin
23,000
5-HT Ago.
Thorazine
28
D1D2 Ant.
cis-Flupenthixol
4.3 D1D2 Ant.
trans-Flupenthixol 1,600
D1D2 Ant.
(+) Butaclamol
1.3 D2 Ant.
(-) Butaclamol
23,000
n/a
Spiroperidol
0.32 D2/5HT Ant.
Propranolol
37,000
β1β2 Ant.
(-) Sulpiride
1,400
D2 Ant.
tran-flupenthixol
S
CF3
S
OH
N
N
CF3
cis-flupenthixol
N
OH
N
N
H3 C
H3C
OH
CH3
(+) Butaclamol
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Dopamine SAR
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Creese I., Ann. Rev. Neurosci. 6:43 (1983)
Brain Autoradiography
3H-Spiperone
D2S2-Antag.
Alone.
3H-Spiperone
+ Ketanserin
S2- antag.
Claustrum
Layer 5A
Altar CA, Science, 228:597 (1985)
3H-Spiperone
+ butaclamol
D2S2 – antagonist
Nucleus accumbens
Caudate-putamen
Layer 5A Cortex
3H-Spiperone
+ sulpiride
D2 – antagonist
Nucleus accumbens
Caudate-putamen
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Current Status of
Dopamine Receptors
Calne, N. Engl. J. Med. 329(14):1021 (1993)
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Receptor Binding Results
(+) Butaclamol
Kd = 1.3 nM
Sibley D., J. Biol. Chem., 257(11):6351 (1982)
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Receptor Binding Results
(+/-) ADTN
Kdh = 31 nM
KdL = 1800 nM
+
NH3
HO
HO
2-amino-5,6-dihydroxytetrahydronaphthalene
(A-5,6-DTN)
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Sibley D., J. Biol. Chem., 257(11):6351 (1982)
GTP Analog Structure
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Receptor Binding Results
(-) Apomorphine w/o Gpp(NH)p
Kdh = 9.3 nM
KdL = 380 nM
(-) Apomorphine w/ 100 μM Gpp(NH)p
KdL = 380 nM
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GPCR Cycle: Principles of Drug Action, 3rd Ed.
Pratt and Taylor
α-subunit
activates
Start Here
High
Affinity
Low
Affinity
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Receptor Binding Results
(+) Butaclamol
KdL = 1.8 nM
(-) Apomorphine w/o Gpp(NH)p
Kdh = 5 nM
KdL = 350 nM
(-) Apomorphine w/ 100 μM Gpp(NH)p
KdL = 360 nM
(-) Apomorphine w/ whole cells
KdL = 330 nM
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Creese I., Ann. Rev. Neurosci. 6:43 (1983)
Receptor Binding D2 receptor
Bovine Anterior Pituitary (mainly D2 receptors)
Sibley D.,
Life Sciences
31(7):637 (1982)
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Receptor Binding D1 receptor
Rat Striatum (has D1 & D2 receptors)
Sibley D.,
Life Sciences
31(7):637 (1982)
Incubations done
w/ 3 μM Domperidone
Selective D2 Antag.
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Dopamine SAR: Receptor Binding
Sibley D.,
Life Sciences
31(7):637 (1982)
SKF-38393
HO
+
NH2
HO
HO
HO
+
NH2
SKF-82526
Fenoldopam
HO
Cl
HO
HO
S
SKF-83742
N CH
3
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Brain Tissue Perfusion
Superfusion of
rat neocortex
with no drug or
SKF 38393
(D1 agonist)
3x10-6 M
3x10-7 M
3x10-8 M
No Agonist
Normalized
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Stoof J.C., Nature 294:366 (1981)
Brain Tissue Perfusion
Stoof J.C., Nature 294:366 (1981)
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Fenoldopam
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Future
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