Receptor Theory & Toxicant-Receptor Interactions Richard B. Mailman

Some examples of receptors 1 E 2 R ligand b g a 2 Ion R ligand ligand nucleus R 3 ligand E R 4 ATP ADP 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: 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).

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 Lgand-Receptor Complex Ligand + Receptor Response(s)

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.

Saturation Equations Michealis-Menten form Scatchard form

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]

Saturation Equations Michealis-Menten form Scatchard form

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

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)

Saturation Equations Michealis-Menten form Scatchard form

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

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

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)

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)

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)

Competition Curves A B Specific Binding (%) Log [ligand] (nM) 100 90 10 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)

A B C D Specific Binding (%) Concentration (nM) 10 20 30 40 50 60 70 10 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)

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)

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

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)

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

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

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

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

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

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.