1. Receptor Theory & Toxicant-Receptor Interactions
Richard B. Mailman

2. Some examples of receptors1 E 2 R
ligand b g a 2
Ion R
ligand
ligand
nucleus R 3
ligand E R 4
ATP
ADP P

3. 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.

4. Definitions Affinity: Intrinsic activity (= “efficacy”): Potency:
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).

5. 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.

6. Drug-Receptor Interactions
Lgand-Receptor
Complex
Ligand + Receptor
Response(s)

7. Bimolecular Interactions: Foundation of Most Studies
Ligand-Receptor Complex
Ligand + Receptor
Response(s)
At equilibrium:
Rearrange that equation to define the equilibrium dissociation constant KD.

8. Saturation Equations
Michealis-Menten form
Scatchard form

9. Linear & Semilog Linear Plot Bound Semi-Log Plot Bound Free log [Free]
0.2
0.4
0.6
0.8 1
Bound 20 40 60 80
100
Free
Semi-Log Plot
0.2
0.4
0.6
0.8 1
Bound -2 -1 1 2
log [Free]

10. Saturation Equations
Michealis-Menten form
Scatchard form

11. Saturation Radioreceptor Assays
preparation
radiolabeled
drug
Tissue Preparation
drug-receptor complex
Beta Counter
Filtration
unbound labeled drug + unbound test drug

12. Characterizing Drug-Receptor Interactions: Saturation curves
Total Binding
800
600
400
200
Specific Binding! (calculated)
Amount Bound
Non-Specific 2 4 6 8 10 12 14 16 18
Radioligand Added (cpm x 1000)

13. Saturation Equations
Michealis-Menten form
Scatchard form

14. (Specific Binding/ Free Radioligand)
Scatchard plot
-1/KD
(Specific Binding/ Free Radioligand)
B/F
Bmax
B (Specific Binding)

15. Competition Radioreceptor Assays
preparation
radiolabeled
drug
test
drug
Tissue Preparation
drug-receptor complex
Beta Counter
Filtration
unbound labeled drug + unbound test drug

16. Competition Curve IC50 Total Binding (dpm *10, e.g.) log [ligand] (nM)10 20 30 40 50 60 70 80 90
100
Top
Total Binding (dpm *10, e.g.)
Specific Binding
IC50
Bottom
NSB
0.1
0.01
1.0 10
100
log [ligand] (nM)

17. Calculations from Basic Theory (I)25 50 75
100
90%
Specific Binding (%)
10%
81 Fold
10-9
10-8
10-7
10-6
10-5
10-4
10-3
log [competing ligand] (M)

18. Calculations from Basic Theory (II)
Commit this to memory!!!!! 25 50 75
100
91%
Specific Binding (%) 9%
100-fold
10-9
10-8
10-7
10-6
10-5
10-4
10-3
log [competing ligand] (M)

19. Competition Curves A B Specific Binding (%) Log [ligand] (nM) 100 9010 20 30 40 50 60 70 80 90
100 A
Specific Binding (%) B
0.1
0.01
1.0 10
100
1000
Log [ligand] (nM)

20. A B C D Specific Binding (%) Concentration (nM) 10 20 30 40 50 60 7010 20 30 40 50 60 70 80 90
100
Specific Binding (%) A B C D
0.1
0.01
1.0 10
100
1000
Concentration (nM)

21. Functional effects & antagonists
+ Increasing concentrations of antagonist B
Raw Data
0.2
0.4
0.6
0.8
1.0
Control (agonist with no antagonist)
Response (Fraction of maximal)
-10
-11 -9 -8 -7 -6
Log Agonist Concentration (M)

22. Spare receptors and “full agonists”b g E2 a R E1 b g
cAMP stimulation
????
????

23. Full & Partial Agonists
100
Full agonist 80
cAMP synthesis 60
(% stimulation relative to dopamine)
Partial agonist 40 20 1 10
100
1000
10000
100000
Concentration (nM)

24. Functionally Selective Agonist
Ligand #1
Typical Agonist
Ligand #2
Functionally Selective Agonist A B
Normal Agonist
F.S. Drug bg a bg
Functional
Complex #1
D2R a
G-protein C D
Functional
Complex #2
No activation

25. Ligand action on three pathways via a single receptor: Traditional view of “full” agonist
Therapeutic Effect 1
Side Effect 1
Side Effect 2

26. Ligand action on three pathways via a single receptor: Traditional view of “partial” agonist
Therapeutic Effect 1
Side Effect 1
Side Effect 2

27. Ligand action on three pathways via a single receptor: Traditional view of antagonist
Therapeutic Effect 1
Side Effect 1
Side Effect 2

28. Activation of three pathways via a single receptor: “Functionally selective” compound
Therapeutic Effect 1
Side Effect 1
Side Effect 2

29. Lessons of functional selectivity
Increases complexity in understanding mechanisms of toxicity.
BUT …. provides opportunities to dissociate toxicity from therapeutic effects mediated via a single receptor.
Universal to almost all targets for small molecules.