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Receptor Theory&
Toxicant-Receptor Interactions
Richard B. Mailman
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Some examples of receptors
1
E2
R E1
ligand
β γ
β γ
α α
2Ion
R R
ligand
ligand
nucleus
R
R
3 ligand
E
R R
4
R R
ATP
ADP
P
ATP
ADP
PP
P
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What is a receptor?
• To a neuroscientist
– A protein that binds a neurotransmitter/modulator• To a cell biologist or biochemist
– A protein that binds a small molecule
– A protein that binds another protein
– A nucleic acid that binds a protein• To a toxicologist
– A macromolecule that binds a toxicant
• Etc.
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Definitions
• Affinity:
– the “tenacity” by which a ligand binds to its receptor• Intrinsic activity (= “efficacy”):
– the relative maximal response caused by a drug in a tissue preparation. A full
agonist causes a maximal effect equal to that of the endogenous ligand (or
sometimes another reference compound if the endogenous ligand is not
known); a partial agonist causes less than a maximal response.
– Intrinsic efficacy (outmoded): the property of how a ligand causes biological
responses via a single receptor (hence a property of a drug).
• Potency:
– how much of a ligand is needed to cause a measured change (usuallyfunctional).
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Radioactivity Definitions
• Curie: 1 Ci = 2.22 x 1012 disintegrations/min (dpm)
• Becquerel: 1 Bq = 60 disintegration/min– The Bq has replaced Ci in the SI system
• Efficiency: the percentage of dpm that are actually captured (cpm)
– What affects this?
• Specific activity: how many moles of radioactive atom are on each
radioactive molecule
– Usually expressed in radioactivity units per unit of mole
– Why is this important to a toxicologist?
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Radioactivity Principles
• Specific activity depends ONLY on half-life, and is totally
independent of mode or energy of decay.
• When decay occurs for all of the biologically important isotopes
(14C; 3H; 32P; 35S; 125I; etc.), the decay event changes the chemical
identity of the decaying atom, and in the process, destroys themolecule on which the atom resided.
– e.g., 3HHe
– Do NOT adjust the specific activity of your radiochemical based on decay – for
every decay, there is a loss of the parent molecule.
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Drug-Receptor Interactions
Ligand + ReceptorLgand-Receptor
Complex
Response(s)
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Bimolecular Interactions:
Foundation of Most Studies
Ligand + Receptor Ligand-Receptork on
k off
[Ligand] [Receptor] k [Ligand Receptor] kon off ⋅ ⋅ = ⋅ ⋅
Rearrange that equation to define the equilibrium dissociation constant KD.
[Ligand] [Receptor]
[Ligand Receptor]
k
kKoff
on
D
⋅
⋅= =
At equilibrium:
Ligand + ReceptorLigand-Receptor
Complex Response(s)
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Saturation Equations
F+K
B*F
D
max= B
Fractional occupancy
[Ligand]
[Ligand] K D= +
D D K
B B
K F
B max1+−=
Michealis-Menten form
Scatchard form
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Linear & Semilog
0 20 40 60 80 100
Free
Linear Plot
0
0.2
0.4
0.6
0.8
1
B o u
n d
-2 -1 0 1 2log [Free]
Semi-Log Plot
0
0.2
0.4
0.6
0.8
1
B o u n d
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Saturation Equations
F+K
B*F
D
max
= B
Fractional occupancy [Ligand][Ligand] K D
=+
D D K B B
K F B max1 +−=
Michealis-Menten form
Scatchard form
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Calculations from Basic Theory (I)
log [competing ligand] (M)
S p e c i f i c B i n d i n g
( % )
10-9 10-8 10-7 10-6 10-5 10-4 10-3
0
25
50
75
100
91%
9%
100-fold
Commit this to memory!!!!!
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Saturation Radioreceptor Assays
unbound labeled drug +
unbound test drug
drug-receptorcomplex
radiolabeled
drug
receptor
preparation
FiltrationBeta
Counter
Tissue
Preparation
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Characterizing Drug-Receptor Interactions:
Saturation curves
0 2 4 6 8 10 12 14 16 18
Radioligand Added (cpm x 1000)
A m o
u n
t B
o u n
d Specific Binding! (calculated)
Non-Specific
Total Binding
800
600
400
200
0
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Saturation Equations
F+K
B*F
D
max= B
Fractional occupancy[Ligand]
[Ligand] K D
=+
D D K
B BK F
Bmax
1+−=
Michealis-Menten form
Scatchard form
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Competition Radioreceptor Assays
unbound labeled drug +
unbound test drug
drug-receptorcomplex
radiolabeled
drug
receptor
preparation
test
drug
FiltrationBeta
Counter
Tissue
Preparation
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Competition Curve
log [ligand] (nM)
0
10
20
30
40
50
60
70
80
90
100
0.10.01 1.0 10 100
T o
t a l
B i n d
i n g
( d p
m * 1 0
, e
. g . )
IC50
Top
Bottom
Specific
Binding
NSB
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Calculations from Basic Theory (I)
log [competing ligand] (M)
S p e c i f i c B i n d i n g
( % )
10-9
10-8
10-7
10-6
10-5
10-4
10-3
0
25
50
75
100
90%
10%
81 Fold
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Calculations from Basic Theory (II)
log [competing ligand] (M)
S p e c
i f i c B i n d i n g
( % )
10-9
10-8
10-7
10-6
10-5
10-4
10-3
0
25
50
75
100
91%
9%
100-fold
Commit this to memory!!!!!
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Competition Curves
Log [ligand] (nM)
0
10
20
30
40
50
60
70
80
90
100
0.10.01 1.0 10 100 1000
S p
e c
i f i c B i
n d i n
g ( %
)
B
A
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Concentration (nM)
0
10
20
30
40
50
60
70
80
90
100
0.10.01 1.0 10 100 1000
S p
e c i f i c
B i n
d i n
g ( %
)
A DCB
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Schild Analysis: Functional effects & antagonists
Log Agonist Concentration (M)
0
0.2
0.4
0.6
0.8
1.0
-10-11 -9 -8 -7 -6
R e s p
o n
s e ( F
r a c t i o
n o
f m
a x i m
a l )
Control
(agonist with no
antagonist)
+ Increasingconcentrations
of antagonist B
Raw Data
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More Advanced Concepts of Receptor Theory
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Spare receptors and “full agonists”
αD1
E1
β γ
E2
α
RE1
β γ
cAMP stimulation
????
????
D1 D1
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Full & Partial Agonists
Concentration (nM)
0
20
40
60
80
100
Full agonist
Partial agonist
( % s t i
m u
l a t i o n
r e l a t i
v e
t o d
o p
a m
i n e
)
c A M
P s y n
t h e s i s
1 10 100 1000 10000 100000
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Normal Agonist F.S. Drug
D2R
G-protein α
βγ
No activation
A B
C D
Functional
Complex
#1
Functional
Complex
#2
Ligand #1
Typical Agonist
Ligand #2
Functionally Selective Agonist
α
βγ
Functional Selectivity
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Therapeutic consequences:
Traditional Drug
•TherapeuticEffect
•SideEffect
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Therapeutic consequences:
Functionally Selective Drug
•TherapeuticEffect
•SideEffect
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Functional selectivity:
JPET, January 2007
tissue or organism. Besides the heuristically interesting natureof functional selectivity, there is a clear impact on drug discov-ery, because this mechanism raises the possibility of selectingor designing novel ligands that differentially activate only asubset of functions of a single receptor, thereby optimizingtherapeutic action. It also may be timely to revise classic con-
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August 2007