INTRODUCTIONSome terms and definitionsThe biologist’s dream:Theories of Drug-Receptor InteractionLaw of mass actionThe theoretical basis for characterizing receptors using saturation radioligand assaysRADIOACTIVITY, RADIOLIGANDS, AND BINDING ASSAYSHow to separate Bound from FreeEquilibrium dialysis:Centrifugation:Filtration:Total vs. free concentrations of ligand. Ligand depletion.RadioactivitySpecific radioactivityCalculating the concentration of the radioligandRadioactive decaySelecting the radioligandWhat chemical structure: agonist vs. antagonistChoice of isotopeThe Poisson distributionCHARACTERIZATION OF A RECEPTOR USING A RADIOLIGANDAnalysis of saturation radioligand binding dataNonspecific binding (NSB)Analysis of data to determine Bmax and KDScatchard plotsDETERMINATION OF KINETIC PARAMETERSDissociation binding dataHow "off rate" experiments work?Association binding dataHow "on rate experiments" workTo determine kon if you don't know koff:COMPETITIVE BINDING DATA WITH ONE CLASS OF RECEPTORSPresent and future uses of competition binding assaysWhat is a competitive binding curve?Entering dataDecisions to make before fitting the dataWeightingConstantsInterpreting the resultsAssumptionsIC50KIShallow competitive binding curvesThe slope factor or Hill slopeThe Hill EquationCompetitive binding with two sitesComparing one- and two-site modelsAgonist binding and the ternary complexLigand, Prism and other curve fitting programsANTAGONISMCompetitive Antagonism.Schild RegressionNoncompetitive Antagonism.PARTIAL AGONISTSWhat does a partial agonist look like?Estimating the potency of partial agonistsDecreasing receptor concentrationBIOCHEMICAL AND MOLECULAR TOXICOLOGY RECEPTOR THEORY AND PRACTICE At the end of this section, you should be able to: 1. Know the fundamentals of characterizing a receptor. 2. Know the fundamentals of characterizing a drug or toxicant. 3. Be able to use small ligands properly in toxicological studies as guided by basic principles. 1 INTRODUCTION..............................................................................................................................................................1 1.1 SOME TERMS AND DEFINITIONS ................................................................................................................................1 1.2 THEORIES OF DRUG-RECEPTOR INTERACTION...........................................................................................................2 2 RADIOACTIVITY, RADIOLIGANDS, AND BINDING ASSAYS.................................................................................6 2.1 HOW TO SEPARATE BOUND FROM FREE.....................................................................................................................7 2.2 TOTAL VS. FREE CONCENTRATIONS OF LIGAND. LIGAND DEPLETION...........................................................................7 2.3 RADIOACTIVITY ......................................................................................................................................................8 2.4 SELECTING THE RADIOLIGAND .................................................................................................................................9 3 CHARACTERIZATION OF A RECEPTOR USING A RADIOLIGAND ...................................................................12 3.1 ANALYSIS OF SATURATION RADIOLIGAND BINDING DATA.........................................................................................12 4 DETERMINATION OF KINETIC PARAMETERS .....................................................................................................14 4.1 DISSOCIATION BINDING DATA ................................................................................................................................ 14 4.2 ASSOCIATION BINDING DATA ................................................................................................................................. 15 5 COMPETITIVE BINDING DATA WITH ONE CLASS OF RECEPTORS ................................................................17 5.2 SHALLOW COMPETITIVE BINDING CURVES............................................................................................................... 20 5.3 LIGAND, PRISM AND OTHER CURVE FITTING PROGRAMS........................................................................................... 25 6 ANTAGONISM ...............................................................................................................................................................25 6.1 COMPETITIVE ANTAGONISM. .................................................................................................................................25 6.2 SCHILD REGRESSION ............................................................................................................................................. 27 6.3 NONCOMPETITIVE ANTAGONISM............................................................................................................................ 28 7 PARTIAL AGONISTS ....................................................................................................................................................28 7.1 WHAT DOES A PARTIAL AGONIST LOOK LIKE?.......................................................................................................... 29 7.2 ESTIMATING THE POTENCY OF PARTIAL AGONISTS...................................................................................................30 7.3 DECREASING RECEPTOR CONCENTRATION .............................................................................................................. 31Receptor Theory and Practice: Page 1 1 INTRODUCTION Note: Gluttons for punishment can refer to the following for more in dpeth information. • LE Limbird. Cell surface receptors: A Short Course on Theory and Methods, Second Edition. Kluwer Academic Publishers, 1996. • HI Yamamura et al. Methods in Neurotransmitter Receptor Analysis, Raven Press, 1990. • T Kenakin. Pharmacologic Analysis of Drug Receptor Interaction (2nd ed.) Raven Press, 1993. 1.1 Some terms and definitions • Affinity: the “tenacity” by which a drugs binds to its receptor (Discussion: the solution of a lipid containing drug in the bilayer may be essentially irreversible: is this a high affinity event?) • Intrinsic activity (=efficacy?): the relative maximal response caused by a drug in a tissue preparation.