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8/3/2019 Tox142 - Receptor Theory - 2006
http://slidepdf.com/reader/full/tox142-receptor-theory-2006 1/20
Receptor Theory
&
Toxicant-Receptor Interactions
Richard B. Mailman
8/3/2019 Tox142 - Receptor Theory - 2006
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Some examples of receptors
1
E2
R E1
ligand
F K
FK
E E
2 Ion
R R
ligand
ligand
nucleus
R
R
3 ligand
E
R R
4
R R
ATP
ADP
P
ATP
ADP
PP
P
8/3/2019 Tox142 - Receptor Theory - 2006
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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.
8/3/2019 Tox142 - Receptor Theory - 2006
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Definitions
Aff inity:
± the ³tenacity´ by which a ligand binds to its receptor
Intrinsic activity (= ³efficacy´):
± 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 eff icacy (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 (usually
functional).
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Radioactivity Principles
S pecific activity depends on half-life, and is totally independent of
mode or energy of decay.
W
hen 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 the
molecule on which the atom resided.
± e.g., 3H He
± Do NOT adjust the specific activity of your radiochemical based on decay ± for every decay, there is a loss of the parent molecule.
8/3/2019 Tox142 - Receptor Theory - 2006
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Drug-Receptor Interactions
Ligand + Receptor Lgand-Receptor
Complex
Response(s)
8/3/2019 Tox142 - Receptor Theory - 2006
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Bimolecular Interactions:
Foundation of Most Studies
Ligand + Receptor Ligand-Receptork on
[Ligand] [Receptor] k [Ligand Receptor] k on off !
Rearrange that equation to define the equilibrium dissociation constant K D.
[Ligand] [Receptor]
[Ligand Receptor]
k
k K
off
on
D
! !
At equilibrium:
Ligand + Receptor Ligand-Receptor
ComplexResponse(s)
8/3/2019 Tox142 - Receptor Theory - 2006
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Saturation Equations
D D K
B B
K F
B
B
max
D
max
1
F+K
FB
!
!
Fractional occupancy [Ligand][Ligand] K D
!
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Linear & Semilog
0 20 40 60 80 100Free
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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Radioreceptor Assay (Simple)
unbound labeled drug +
unbound test drug
drug-receptor
complex
radiolabeled drugreceptor preparation test drug
FiltrationBeta
Counter
Tissue
Preparation
8/3/2019 Tox142 - Receptor Theory - 2006
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Characterizing Drug-Receptor Interactions:
Saturation curves
0 2 4 6 8 10 12 14 16 18
RadioligandAdded (cpm x 1000)
S pecific Binding! (calculated)
Non-Specif ic
Total Binding
800
600
400
200
0
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Scatchard plot
S pecificBinding
S p e c
i f i c B i n d i n g / F r e e R a d i o l i g a n d
Bmax
-1/KD
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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
IC50
Top
Bottom
Specif ic
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
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
A DCB
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Functional effects & antagonists
Log Agonist Concentration (M)
0
0.2
0.4
0.6
0.8
1.0
-10-11 -9 -8 -7 -6
Control
(agonist with no
antagonist)
+ Increasing
concentrations
of antagonist B
Raw Data
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S pare receptors and ³full agonists´
ED1
E1
F K
E2
ER
E1
F K
cAMP stimulation????
????
D1 D1
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Full & Partial Agonists
Concentration (nM)
0
20
40
60
80
100
Full agonist
Partial agonist
1 10 100 1000 10000 100000