Receptor Theory & Toxicant-Receptor InteractionsRichard B. MailmanSome examples of receptors1 E2RE1ligandβγβγαα2 IonRRligandligandnucleusRR3 ligandERR4 RRATPADPPATPADPPPPWhat 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 Definitions• Curie: 1 Ci = 2.22 x 1012 disintegrations/min (dpm)• Becquerel: 1 Bq = 60 disintegration/min – The Bq has replaced Ci in the SI system• Efficiency: the percentage of dpm that are actually captured (cpm)– What affects this?• Specific activity: how many moles of radioactive atom are on each radioactive molecule– Usually expressed in radioactivity units per unit of mole– Why is this important to a toxicologist?Radioactivity Principles• Specific activity depends ONLY 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 ÆHe– 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 InteractionsLigand + ReceptorLgand-ReceptorComplexResponse(s)Bimolecular Interactions: Foundation of Most StudiesLigand + Receptor Ligand-Receptorkonkoff[Ligand] [Receptor] k [Ligand Receptor] kon off⋅⋅=⋅⋅Rearrange that equation to define the equilibrium dissociation constant KD.[Ligand] [Receptor][Ligand Receptor]kkKoffonD⋅⋅==At equilibrium:Ligand + ReceptorLigand-ReceptorComplexResponse(s)Saturation EquationsF+KB*FDmax=BFractional occupancy[Ligand][Ligand] KD=+DDKBBKFBmax1+−=Michealis-Menten formScatchard formLinear & Semilog020 40 60 80 100FreeLinear Plot 00.20.40.60.81Bound-2 -1 0 1 2log [Free]Semi-Log Plot00.20.40.60.81BoundSaturation EquationsF+KB*FDmax=BFractional occupancy[Ligand][Ligand] KD=+DDKBBKFBmax1+−=Michealis-Menten formScatchard formCalculations from Basic Theory (I)log [competing ligand] (M)Specific Binding (%)10-910-810-710-610-510-410-3025507510091%9%100-foldCommit this to memory!!!!!Saturation Radioreceptor Assaysunbound labeled drug + unbound test drugdrug-receptorcomplex radiolabeleddrugreceptorpreparationFiltrationBetaCounter TissuePreparationCharacterizing Drug-Receptor Interactions:Saturation curves0 2 4 6 8 1012141618Radioligand Added (cpm x 1000)Amount BoundSpecific Binding! (calculated)Non-SpecificTotal Binding8006004002000Saturation EquationsF+KB*FDmax=BFractional occupancy[Ligand][Ligand] KD=+DDKBBKFBmax1+−=Michealis-Menten formScatchard formScatchard plotB(Specific Binding)B/F(Specific Binding/ Free Radioligand)Bmax-1/KDCompetition Radioreceptor Assaysunbound labeled drug + unbound test drugdrug-receptorcomplex radiolabeleddrugreceptorpreparationtestdrugFiltrationBetaCounter TissuePreparationCompetition Curvelog [ligand] (nM)01020304050607080901000.10.01 1.0 10 100Total Binding (dpm *10, e.g.)IC50TopBottomSpecific BindingNSBCalculations from Basic Theory (I)log [competing ligand] (M)Specific Binding (%)10-910-810-710-610-510-410-3025507510090%10%81 FoldCalculations from Basic Theory (II)log [competing ligand] (M)Specific Binding (%)10-910-810-710-610-510-410-3025507510091%9%100-foldCommit this to memory!!!!!Competition CurvesLog [ligand] (nM)01020304050607080901000.10.01 1.0 10 100 1000Specific Binding (%)BAConcentration (nM)01020304050607080901000.10.01 1.0 10 100 1000Specific Binding (%)ADCBSchild Analysis: Functional effects & antagonistsLog Agonist Concentration (M)00.20.40.60.81.0-10-11 -9 -8 -7 -6Response (Fraction of maximal)Control(agonist with noantagonist)+ Increasingconcentrationsof antagonist B Raw DataMore Advanced Concepts of Receptor TheorySpare receptors and “full agonists”αD1E1βγE2αRE1βγcAMP stimulation????????D1D1Full & Partial AgonistsConcentration (nM)020406080100Full agonistPartial agonist(% stimulation relative to dopamine)cAMP synthesis1 10 100 1000 10000 100000Normal Agonist F.S. DrugD2RG-proteinαβγNo activationABCDFunctionalComplex#1FunctionalComplex#2Ligand #1Typical AgonistLigand #2Functionally Selective AgonistαβγFunctional SelectivityTherapeutic consequences:Traditional Drug•TherapeuticEffect•SideEffectTherapeutic consequences:Functionally Selective Drug•TherapeuticEffect•SideEffectFunctional selectivity:JPET, January 2007tissue or organism. Besides the heuristically interesting natureof functional selectivity, there is a clear impact on drug discov-ery, because this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizingtherapeutic action. It also may be timely to revise classic con-August
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