BIOCHEMICAL 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 31 Receptor 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 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 a drug s ability to affect a receptor and cause a biological response hence a property of a drug Discussion can a drug have negative efficacy Potency broad definition wide the ability of a drug to cause a measured biological change narrow definition wide the ability of a drug to cause a measured functional change 1 1 1 The biologist s dream From a biological perspective one might simplify drug hormone or neurotransmitter action via the following scheme Drug Receptor Drug Receptor Complex Response s Ideally we would wish to understand and then predict all of the properties of how a drug can cause a response in any tissue Unfortunately there are many factors that make achieving this goal at best difficult and at worst impossible For example events that affect the equilibrium of the drug at the receptor limited diffusion on a macro or micro scale metabolism entrapment etc can cause experimental results to deviate from theory Even more importantly the production of a stimulus often does not have a one to one correspondence to the measured response The response caused by an activated receptor can involve a variety of different mechanisms see cartoon in Figure 1 Some receptors directly effect the response of interest e g the ionotropic receptor in A Even in this case other factors including allosteric modulators cofactors etc that can influence the observed response In the case of G protein coupled receptors B the receptor may start a cascade of biochemical events due to actions at several effectors and in some cases also may be phosphorylated itself In other cases like a Receptor Theory and Practice Page 2 tyrosine kinase C the receptor may itself be modified e g phosphorylated in the process of catalyzing a reaction phosphorylation in this example Finally nuclear receptors are examples of a class D that are actually translocated as part of their normal function A B Ion R ligand ligand R R E 1 C D ligand E 2 ligand R R R R R R P P P P nucleus E 1 2 Theories of Drug Receptor Interaction There have been several major theories that have been proposed to provide a theoretical basis for understanding modeling and thereby predicting drug response Three of the most widely known of these schemes are described as follows Occupation Theory the idea that a response emanates from a receptor only when it is occupied by an appropriate ligand drug Rate Theory the idea that a response emanates from a receptor in proportion to the kinetic rate of onset and offset of drug binding to the receptor Operational Model a modified semi empirical approach that is based on occupancy theory modified to incorporate a factor relating agonist receptor complex and response All of these theories have specific strengths and appeal and all have significant failings In general rate theory is now considered to be the one of least utility Conversely since the advent of radioreceptor methods in the mid 1970 s it is now possible to measure drug receptor interactions directly making occupation theory of particular interest because receptor occupation can be measured directly for the first time As molecular tools begin to provide ways of studying several sequential molecular events operational theory or direct multi step models can certainly be applied Receptor Theory and Practice Page 3 1 2 1 Law of mass action Although there are a plethora of complexities that arise it is true that the large majority of experiments especially those using radioreceptor methods radioligand binding methods are based on very simple application of the law of mass action In the case of a drug ligand interacting with a homogeneous population of receptors this relationship can be expressed Ligand Receptor kon koff Ligand Receptor Binding occurs when ligand and receptor collide due to diffusion in the correct orientation and with enough energy The rate of association number of binding events per unit of time equals Ligand Receptor kon Once binding has occurred the ligand and receptor remain bound together for a random amount of time The rate of dissociation number of dissociation events per unit time equals ligand receptor koff The probability of dissociation is the same at every instant of time The receptor doesn t know how long it has been bound to the ligand After