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

2. Some examples of receptors 1 E 2 R E 1 ligand       2 Ion R R ligand nucleus R R 3 ligand E R R 4 R R ATP ADP P ATP ADP P P 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: –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 ( 14 C; 3 H; 32 P; 35 S; 125 I; 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., 3 H  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 Ligand + Receptor Lgand-Receptor Complex Response(s)

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

8. Saturation Equations

9. Linear & Semilog 0 20406080100 Free Linear Plot 0 0.2 0.4 0.6 0.8 1 Bound -2012 log [Free] Semi-Log Plot 0 0.2 0.4 0.6 0.8 1 Bound

10. Radioreceptor Assay (Simple) unbound labeled drug + unbound test drug drug-receptor complex radiolabeled drugreceptor preparationtest drug Filtration Beta Counter Tissue Preparation

11. Characterizing Drug-Receptor Interactions: Saturation curves 024681012141618 Radioligand Added (cpm x 1000) Amount Bound Specific Binding! (calculated) Non-Specific Total Binding 800 600 400 200 0

12. Scatchard plot Specific Binding Specific Binding/ Free Radioligand B max -1/K D

13. Competition Curve log [ligand] (nM) 0 10 20 30 40 50 60 70 80 90 100 0.10.011.010100 Total Binding (dpm *10, e.g.) IC50 Top Bottom Specific Binding NSB

14. Calculations from Basic Theory (I) log [competing ligand] (M) Specific Binding (%) 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 0 25 50 75 100 90% 10% 81 Fold

15. Calculations from Basic Theory (II) log [competing ligand] (M) Specific Binding (%) 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!!!!!

16. Competition Curves Log [ligand] (nM) 0 10 20 30 40 50 60 70 80 90 100 0.10.011.0101001000 Specific Binding (%) B A

17. Concentration (nM) 0 10 20 30 40 50 60 70 80 90 100 0.10.011.0101001000 Specific Binding (%) ADCB

18. Functional effects & antagonists Log Agonist Concentration (M) 0 0.2 0.4 0.6 0.8 1.0 -10-11-9-8-7-6 Response (Fraction of maximal) Control (agonist with no antagonist) + Increasing concentrations of antagonist B Raw Data

19. Spare receptors and “full agonists”  D1D1 E1E1   E2E2  R E1E1   cAMP stimulation ???? D1D1 D1D1

20. Full & Partial Agonists Concentration (nM) 0 20 40 60 80 100 Full agonist Partial agonist (% stimulation relative to dopamine) cAMP synthesis 110100100010000100000