Transcript Brief Receptor Theory

Receptor Theory
&
Toxicant-Receptor Interactions
Richard B. Mailman
Some examples of receptors
2
ligand
1
Ion
ligand
R
E
1
a
bg
b
3
R
R
a
g E
2
ligand
4
ligand
R
R
R
R
ATP
ATP
R
R
ADP
ADP
P
nucleus
E
P
P
P
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.
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 (usually
functional).
Radioactivity Principles
• Specific activity depends 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 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.
Drug-Receptor Interactions
Ligand + Receptor
Lgand-Receptor
Complex
Response(s)
Bimolecular Interactions:
Foundation of Most Studies
Ligand + Receptor
Ligand + Receptor
Ligand-Receptor
Complex
kon
Ligand-Receptor
koff
At equilibrium:
[Ligand]  [Receptor]  k on  [Ligand  Receptor]  k off
Rearrange that equation to define the equilibrium dissociation constant KD.
[Ligand]  [Receptor] k off

 KD
[Ligand  Receptor]
k on
Response(s)
Saturation Equations
FBmax
B
KD + F
B
1
Bmax

B
F
KD
KD
[Ligand]
Fractional occupancy 
[Ligand]  K D
Linear & Semilog
Linear Plot
0.8
0.6
0.4
0.2
0
20
0
40
Free
60
80
100
Semi-Log Plot
1
0.8
Bound
Bound
1
0.6
0.4
0.2
0
-2
-1
0
log [Free]
1
2
Radioreceptor Assay (Simple)
receptor preparation
radiolabeled drug
test drug
Tissue
Preparation
drug-receptor
complex
Beta
Counter
Filtration
unbound labeled drug +
unbound test drug
Characterizing Drug-Receptor Interactions:
Saturation curves
Total Binding
800
Specific Binding! (calculated)
600
400
200
Non-Specific
0
0
2
4
6
8
10
12
Radioligand Added (cpm x 1000)
14
16
18
Specific Binding/ Free Radioligand
Scatchard plot
-1/KD
Bmax
Specific Binding
Competition Curve
100
Top
90
80
70
60
Specific
Binding
50
40
30
IC50
20
10
Bottom
NSB
0
0.01
0.1
1.0
10
log [ligand] (nM)
100
Calculations from Basic Theory (I)
Specific Binding (%)
100
90%
75
50
25
10%
0
81 Fold
10-9
10-8
10-7
10-6
10-5
log [competing ligand] (M)
10-4
10-3
Calculations from Basic Theory (II)
Commit this to memory!!!!!
Specific Binding (%)
100
91%
75
50
25
9%
0
100-fold
10-9
10-8
10-7
10-6
10-5
log [competing ligand] (M)
10-4
10-3
Competition Curves
100
90
80
70
60
A
50
40
B
30
20
10
0
0.01
0.1
1.0
10
Log [ligand] (nM)
100
1000
100
90
80
70
60
50
40
A
30
B
C
D
20
10
0
0.01
0.1
1.0
10
Concentration (nM)
100
1000
Functional effects & antagonists
+ Increasing
Raw Data
concentrations
of antagonist B
1.0
0.8
Control
(agonist with no
antagonist)
0.6
0.4
0.2
0
-11
-10
-9
-8
-7
Log Agonist Concentration (M)
-6
Spare receptors and “full agonists”
D1
E1
D1
a
D1
bg
E1
a
cAMP stimulation
????
bg
E2
????
R
Full & Partial Agonists
100
Full agonist
80
60
Partial agonist
40
20
0
1
10
100
1000
Concentration (nM)
10000
100000