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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