Receptor Theory & Toxicant-Receptor Interactions Richard B. Mailman

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

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 ( 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.

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

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)

Saturation Equations

Linear & Semilog Free Linear Plot Bound log [Free] Semi-Log Plot Bound

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

Characterizing Drug-Receptor Interactions: Saturation curves Radioligand Added (cpm x 1000) Amount Bound Specific Binding! (calculated) Non-Specific Total Binding

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

Competition Curve log [ligand] (nM) Total Binding (dpm *10, e.g.) IC50 Top Bottom Specific Binding NSB

Calculations from Basic Theory (I) log [competing ligand] (M) Specific Binding (%) % 10% 81 Fold

Calculations from Basic Theory (II) log [competing ligand] (M) Specific Binding (%) % 9% 100-fold Commit this to memory!!!!!

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

Concentration (nM) Specific Binding (%) ADCB

Functional effects & antagonists Log Agonist Concentration (M) Response (Fraction of maximal) Control (agonist with no antagonist) + Increasing concentrations of antagonist B Raw Data

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

Full & Partial Agonists Concentration (nM) Full agonist Partial agonist (% stimulation relative to dopamine) cAMP synthesis