Radioligand Binding Studies A radioligand is a radioactively labeled drug that can associate with a receptor, transporter, enzyme, or any site of interest. Measuring the rate and extent of binding provides information on the number of binding sites, and their affinity and accessibility for various drugs.Radioligand binding can be used to:(2)characterize receptors in their natural environment as well as those transfected into cell lines;(3)study receptor dynamics and localization;(4)identify novel chemical structures that interact with receptors(5)define ligand activity and selectivity in normal and diseased tissues.
Radioligand Binding Studies Receptors exist in very small concentrations in tissues. The most common method for detecting the receptors in membrane preparations, tissue slices or in the purified form is to use a radioactive drug which has a high affinity and high degree of selectivity Incubate tissue with radioactive drug under the appropriate experimental conditions, the radioactive drug (D) will bind to the receptor (R) to form a drug- receptor complex (RD). The amount of drug-receptor complex (RD) can be measured because it is now radioactive.
Law of mass actionAt Equilibrium kon Association rate constant or on-rate constant koff Dissociation rate constant or off-rate constant Kd Equilibrium dissociation constant
The model assumes:• All receptors are equally accessible to ligands.• Receptors are either free or bound to ligand. It doesntallow for more than one affinity state, or states of partialbinding.• Binding does not alter the ligand or receptor.• Binding is reversible.
Factors to consider in designing receptor binding experiments.1. Identify an appropriate radioactive ligand to use for the experiment.2. Tissue preparation to be used in the experiment3. Identify a method for separating bound from free4. Identify a method for distinguishing specific from nonspecific binding
Criteria for selecting a radioactive liganda. The specific activity of the radioligand should be highenough to detect the receptor in the tissue beingstudied.b. The radioligand should have a high affinity for thereceptor.When using a filtration method to separate bound fromfree, it usually takes 5 to 15 seconds to filter the sampleand rinse the filter. During this time less than 10% of theradioligand should dissociate from the receptor.
c. The radioligand should have a high degree ofselectivity for the receptor being studied.d. The radioligand should be chemically stable in theassay media during the binding reaction.e. The radioligand should be pure.
Radioligand binding could be :3)SpecificThe site that we want to study is referred to asthe SPECIFIC SITE.2) Non-SpecificAll other sites are called NONSPECIFIC SITES.
Examples of nonspecific binding sites1) Other receptors from the same class: [3H] rauwolscine binds to alpha-2A, alpha-2B, and alpha-2C subtypes with similar affinities. If all alpha-2 adrenoceptors are to be studied without differentiating between subtypes then binding of [3H] rauwolscine to all alpha-2 adrenoceptors would be considered specific binding. If only study alpha-2A subtype, then radioligand bound to the alpha-2A subtype would be considered specific binding and binding to the other subtypes would be considered nonspecific binding.• Other receptors from a different class: [3H] rauwolscine binding to serotonin receptors as well as to alpha-2 adrenoceptors.
3)•Binding to tissue protein: The radioligand may bindto tissue protein or become trapped in the lipid membrane.•Binding to test tube or glass fiber filters:Many radioligands bind to test tubes and glass fiber filtersused to separate bound from free. This binding would beconsidered nonspecific binding.Binding to test tubes can sometimes be eliminated bychoosing the type of test tube (glass or plastic) used to dothe binding assay.Binding to glass fiber filters can usually be eliminated tosome extent by trying different types of filters fromdifferent manufactures or coating the filter withpolyethyleneamine which puts a negative charge on thefilter.
SATURABLE and NON-SATURABLE bindingSATURABLE binding sites are always present ininfinite amounts. Another way of thinking of this is that if youadd enough ligand all of the sites will be occupied with ligand.NON-SATURABLE binding sites are sites that are present inessentially infinite amounts. No matter how much ligand youadd, not all of the sites will be occupied with ligand. The siteis non-saturable.
Examples of saturable sites: • Specific binding sites are always saturable • All Receptors • Test tubes and glass fiber filters may have saturable, nonspecific binding sites as well as non-saturable binding sites.Examples of non-saturable sites: • Low affinity tissue binding sites • Binding to test tubes and glass fiber filters may be non- saturable
Binding is measured in the presence of the highest concentrationof radioactive ligand used in a saturation curve. Under theseconditions all of the receptors would be occupied by radioactive ligand.Binding is also measured in the presence of the sameconcentration of radioactive ligand plus increasing concentrationsof unlabeled ligand. As the concentration of unlabeled ligandincreases the amount of radioactive ligand bound decreases until aplateau is reached. At this point no matter how much unlabeled ligandis added the amount of radioactive ligand bound remains constant.This residual amount of radioligand bound represents binding tononspecific, non-saturable sites which cant be displaced by unlabeledligand.The amount of inhibitor to use would be the concentration whichcompletely blocks the saturable binding sites.In this example it would be between 10-9 to 10-8 M.
Guidelines -Appropriate assay conditionsRadioligand Selection The selection of the radioligand is based on its stability, specific activity, and pharmacological selectivity. In general, • Antagonists tend to bind to receptors with much higher affinity than agonists. • Additionally, agonists induce conformational changes in receptor-effector complexes that can cause ligand-receptor complexes to exist in multiple states with different binding characteristics. • Another advantage of antagonist radioligands is that they do not activate the receptor which, in the case of binding with metabolically active cells, can result in the desensitization, and reduction in affinity, of the site.
StabilityRadioligand purity must be established periodically. Insome cases, the radioligand requires special storageprocedures, such as addition of antioxidants or proteaseinhibitors in the case of a peptide radioligand, to slow orprevent degradation.Nonspecific bindingThe physicochemical properties of a ligand determine itslevel of nonspecific binding due to interactions with lipidmembranes and/or filter or scintillation bead material inthe assay.Factors such as lipophilicity and aqueous solubility shouldbe taken into consideration when selecting a ligand toradiolabel.
Buffers usedIn most cases, a homogenization / assay buffer is selected thatyields the highest ratio of specific versus nonspecific binding.Common buffers - Tris, Hepes, sodium phosphate andglycylglycine, Krebs, Ringer, or Hanks’ balanced salt solution(HBSS).Buffers can affect the binding of radioligand to the receptor,using the same buffer as for assays measuring response is bestso that the results can be compared.Some binding assays require the presence of special ions. Forexample, opioid receptor binding is modulated by sodium,GABAA receptor binding by chloride, and the N-methyl-D-aspartate (NMDA) glutamate receptor by magnesium.
pH of the assay bufferThe affinity of a ligand for a receptor generally varies with thepH. Generally using a physiological pH, such as 7.4, is best sothat the results are comparable to what is seen in vivo.Optically active radioligandsIf a ligand contains a chiral center, use of the stereochemicallyactive form is preferable.With few exceptions, most receptors differentiate between theoptical isomers of compounds,with the pharmacologically-active isomer binding with higheraffinity than the less-active isomer.
Additional reagentsMgCl2 is used in many receptors binding assays. Initially this wasused to help precipitate the radioligand receptor complex duringcentrifugation.NaCl is often used in adrenergic receptor binding studies to convertthe receptor to a form that has a lower affinity for agonists. GTP or its non-hydrolyzeable analog (Gpp(NH)p) are often usedwhen agonist binding is also going to be studied in subsequentcompetition experiments. Gpp(NH)p converts the receptor into aform that has a low affinity for agonistTemperature of the binding reactionMost binding assays can be conducted at room temperature.Some assays work better at 4oC than at room temperature, andsometimes the affinity of the radioligand for the receptor is higher at4oC than at 25oC . It takes longer to reach steady-state when thereaction is run at 4oC than at room temperature.
Major Types of Radio ligand Study• Saturation binding experiments measure equilibrium binding of various concentrations of the radioligand. Analyze the relationship between binding and ligand concentration to determine the number of sites, Bmax, and the ligand affinity, Kd.2. Competitive binding experiments measure equilibrium binding of a single concentration of radioligand at various concentrations of an unlabeled competitor. Analyze these data to learn the affinity of the receptor for the competitor.3. Kinetics experiments measure binding at various times to determine the rate constants for radioligand association and dissociation.
1. Saturation binding experimentsExperiment used to be analyzed with Scatchard plots(more accurately attributed to Rosenthal), they aresometimes called "Scatchard experiments".The analyses depend on the assumption that you haveallowed the incubation to proceed to equilibrium (fewminutes to many hours, depending on the ligand, receptor,temperature, and other experimental conditions.)The lowest concentration of radioligand will take the longestto equilibrate. When testing equilibration time, therefore, usea low concentration of radioligand (perhaps 10-20% ofthe KD).
Purpose of the Saturation experiment1)To determine the affinity or Kd of a radioligand for areceptor Kd is the equilibrium dissociation constant. It is equal to theconcentration of radioactive ligand required to occupy 50 % ofthe receptors.2)The density (Bmax) of a specific receptor or receptorsubtype in a given tissueBmax is the total number of receptor sites in the tissue beingstudied. It occurs when the all of the receptor molecules areoccupied by radioactive drug.
Basic characteristics of a saturation experimentIn a saturation experiment increasing concentrations of aradioactive ligand are allowed to bind to the receptor untilsteady-state conditions occur.After reaching steady state, the bound ligand is separatedfrom the free ligand. The most widely used methods forseparation of bound ligand from free ligand are filtration andcentrifugation.The amount of ligand bound to the filter or trapped in the pelletis then measured. A radioactive ligand is used because theradioactivity of low concentrations of ligand can be detected infilters or membrane pellets.
Methods used to determine Kd and Bmax from asaturation experiment 1. Saturation curve 2. Rosenthal plot (commonly referred to as a Scatchard plot) These experiments are called saturation experiments because at the higher radioligand concentrations all of the receptor molecules are occupied (saturated) by radioactive ligand.
Results of the saturation experiment can be plotted with BOUND (theamount of radioactive ligand that is bound to the receptor) on the Yaxis and FREE (the free concentration of radioactive ligand) on the Xaxis.The resulting graph is a hyperbola and is called a saturation curve.Bmax is the density of the receptor in the tissue being studied.Kd is the concentration of ligand required to occupy 50% of thebinding sites
where B is Bound F is FreeBy fitting the data to the equation for a saturationcurve.Getting an accurate estimate of Kd and Bmax from thisgraph by eye is difficult. The curve is usually analyzedby nonlinear regression analysis
Rosenthal PlotThe data from the saturation experiment can beplotted with Bound/Free on the Y axis andBound on the X axis.Single site binding data can be analyzed bylinear regression to give a straight line.The slope of the line is -1/Kd and the X-interceptis Bmax. This is a Rosenthal Plot.Most scientists call it a Scatchard Plot.
Advantages of Rosenthal PlotIt is easy to visually compare two sets of data ona Rosenthal plot.If the Kd for both sets of data are similar theslopes will be similar.If the Bmax changes then the X intercept willchange.Two-site fit to the data can be visualized moreeasily with a Rosenthal Plot than with asaturation curve.
Experimental conditions that need to beconsidered in doing saturation experiments5. Concentration of radioligand used2. Concentration of tissue used
1. Concentration of radioligand used At least six radioligand concentrations equally spaced on either side of the Kd should be chosen if the radioactive ligand is bound to a single site. The lowest concentration should be approximately 1/10 of the Kd. The highest concentration should be approximately 10 times the Kd value. Ninety-one percent of the receptors are occupied by radioactive ligand concentrations which are 10 times the Kd. Six serial 2:5 dilutions of the radioligand are then made from the highest concentration. If the ligand is binding to more than one site, using 15 to 25 points to clearly define both binding sites may be necessary.
2. The concentration of tissue used:It is dependent on the number of binding sites permg of tissue. The tissue concentrations requiredin the assay will vary for the same binding site indifferent tissues and for different receptors in thesame tissue.
Factors deciding tissue (protein) concentration• It is helpful if the tissue concentration is high enough so that at least 1000 cpm are bound when all of the receptors are occupied with radioactive ligand. This allows for reasonable accuracy in counting the radioactivity bound to the tissue at the lowest concentrations of radioligand.• It is important that enough tissue be present so that a measurable amount of the radioligand is bound at the lowest radioligand concentration.
•However, the tissue concentration not be so highthat more than 10% of the radioligand is bound.The equations used for generation of Kd and Bmax arebased on the assumption that the free concentrationof radioligand does not change.It is generally assumed that these conditions are metif less than 10% of the radioligand is bound to thereceptor.One way to determine quickly whether 10% of theradioligand is bound to the receptor is to look at theposition of Y-intercept in a Rosenthal plot.
Several factors can be determined from a Rosenthal plot•The ratio of free to bound ligand that should be less than 10%.Derivation of the equations for determination of Kd and Bmax assumethat the free ligand concentration is not changing during the bindingreaction. These conditions are generally assumed to be met if lessthan 10% of the radioligand is bound to the receptor at steadystate conditions.•Kd can be estimated as the inverse slope of the line.•Bmax can be estimated from the X-intercept.
Calculating specific boundby subtracting nonspecific bound from total bound. Calculating free concentration of radioligand The total concentration of radioligand is the amount added to the assay tubes. This concentration is usually determined by directly adding to scintillation vials, the same amount of the radioligand as added to assay tubes . If a filter assay is used to separate bound from free, then the filter is also placed in the scintillation vial, and the radioligand is placed directly on the filter. When ligand is bound to the tissue, the free concentration of radioligand decreases. The free concentration of ligand would be the total amount of radioligand added to the assay tube minus the amount bound. Free = Total Added - Bound
If most of the nonspecific binding is to tissue and not tofilters used in a filter assay then bound would be totalbound. (Note that total bound represents the binding of theradioligand to both specific and nonspecific sites.)Free = Total Added - (Specific bound + Nonspecific bound)orFree = Total Added - Total boundIf the nonspecific binding comes predominately from thebinding of the radioligand to the filters used to separatebound from free, then this nonspecific binding is not to thetissue and occurs after the reaction is complete. In thiscase, only specific binding should be subtracted from totalbinding to give free.Free = Total added - Specific bound
Nonlinear regression analysis of thesaturation curveSaturation curves are best fit to the following equation where B is bound and F is free.
Conditions that need to be met when doingsaturation experiments•The measured amount of bound radioligand needs to reflectthe amount of radioligand bound under equilibrium conditions.That is, equilibrium conditions need to be present when boundis separated from free. The bound ligand should not dissociatefrom the receptor during the course of separating free frombound.•A time course can be run at the lowest concentration ofradioligand under your experimental conditions to demonstratethat equilibrium conditions are present when bound is separatedfrom free.
Less than 10% of the radioligand should dissociate fromthe receptor in the process of separating bound from free.Less than 10% of the radioligand should be depletedfrom the reaction mixture.The amount of bound radioligand needs to bedetermined accuratelySpecific binding must be correctly defined.Ligand should not be degraded during the course of thereaction.
Two-Site Saturation Experiments Under the following conditions: When more than one subtype of the receptor is present. When the radioligand has a high affinity for more than one type of receptor. When the radioactive ligand is an agonist. For example: The G-protein receptors exist in at least two conformations, one with a high affinity for agonist and one with a low affinity for agonist. Unless a reagent, such as Gpp (NH)p, is used to convert all of the receptor molecules to the low affinity form, two binding sites may be detected in the saturation experiments using a radiolabeled agonist
The equation for a two-site fit for a saturation plot is: where Bmax1 and Bmax2 are the binding site density for sites 1 and 2 Kd1 and Kd2 are the Kd values for site 1 and site 2
2 Competition ExperimentsMany ligands for receptors are not available in a radioactiveform. Since they are unlabeled there is no way to directlymeasure their affinity for the receptor.The affinity of the unlabeled ligand for the receptor can bedetermined indirectly by measuring its ability to competewith a radioactive ligand for the receptor.In a competition experiment various concentrations of anunlabeled ligand are allowed to compete with a fixedconcentration of a radiolabeled ligand for a receptor.As the concentration of unlabeled ligand increases, theamount of radioligand bound to the receptor decreases.
The competitive inhibitor can be either an agonistor an antagonist.It is called a competitive inhibitor because itsvalue is determined by measuring the ability ofthe unlabeled drug to compete with a radiolabeleddrug for the receptor.The Ki value for an unlabeled drug should be thesame as the Kd value obtained for the same drugin radiolabeled form.
Analyses of the results of the competition experimentare simpler and more accurate if the radiolabeledligand is only binding to one site.Hormone and neurotransmitter receptors exits inconformations which have either high affinity or lowaffinity for the agonist.A true antagonist has equal affinities for the high andlow affinity states of the receptor.The analysis of the data is simpler if the radiolabeledligand is an antagonist
Purpose of Competition Experiments Competition experiments can be used to determine the affinity of the unlabeled ligand for the receptor The affinity of an agonist for a receptor. The pharmacological characteristics of subtypes of a particular receptor. The classification of receptor subtypes in a tissue
Experimental Conditions for Competition StudiesThe radiolabeled ligand should have a high affinity for the receptor being studied.The concentration of radiolabeled ligand used for the competition studies should be between 0.75 and 1.0 times the Kd value for the ligand determined using the same experimental conditions as is planned for use in the competition studies.It is helpful to have at least 10 concentrations of unlabeled ligand for one-site competition studies. There should always be a tube which does not contain any drug but contains the same volume of diluents as used to deliver the unlabeled drug. Increments of 1 and 3 are often usedBinding needs to reach steady-state conditions when doing either saturation or competition experiments
The value of the EC50:• The affinity of the receptor for the competing drug. If theaffinity is high, the EC50 will be low. The affinity is usuallyquantified as the equilibrium dissociation constant, Ki.The Ki is the concentration of the competing ligand that will bind tohalf the binding sites at equilibrium, in the absence of radioligandor other competitors. If the Ki is low, the affinity of the receptor forthe inhibitor is high.• The concentration of the radioligand. If you choose to use ahigher concentration of radioligand, it will take a larger conc ofunlabeled drug to compete for half the radioligand binding sites.• The affinity of the radioligand for the receptor (Kd). It takesmore unlabeled drug to compete for a tightly bound radioligand(low Kd) than for a loosely bound radioligand (high Kd).
Two-Site Competition ExperimentsTwo site binding in a competition experiment under thefollowing conditions:•If the unlabeled ligand is an agonist. Receptors haveboth low and high affinity states for agonist. Unless steps aretaken to convert all the receptor molecules into either high orlow affinity states, agonist will bind to more than one affinitystate of a receptor in a competition experiment.•If the unlabeled ligand binds to different subtypes ofthe receptor with different affinities and there is morethan one subtype in the tissue you are studying.•If the unlabeled ligand binds to more than one receptorwith different affinities. This assumes the radioligand isalso binding to more than one receptor.
The characteristics of a competition curve withone-site binding are a sigmoid curve with: • a slope of 1.0. • there is a 81 fold difference in the concentration between 90% specific bound and 10% specific bound.
The characteristics of a competition curve withtwo-site binding is a sigmoid curve with: • a slope less than 1.0 • or evidence of two slopes separated by a plateau.
Determination of Receptor Subtype usingCompetition StudiesMethods used to identify the pharmacological profile for aspecific subtype of a receptor•To initial identify the pharmacological profile of a receptorsubtype it is important to first have a tissue which selectivelyexpresses only that receptor subtype.•Competition studies are done to determine the affinity of alarge number of drugs for the receptor. It is important to usedrugs which have both a high and low affinity for the receptor.The affinity of these drugs for the receptor define thepharmacological profile for the receptor. This profile is used todetermine the identify of receptor subtypes in specific tissues
Methods used to identify the receptor subtypes in anunknown tissue• First, the pharmacological profiles for the known receptor subtypes need to be determined. It is helpful to identify drugs which have a high affinity for each of the subtypes of the receptor.• The pharmacological profile for the unknown tissue needs to be determined. In doing this profile, choose drugs which have high affinities for each of the known subtypes. Drugs which have similar affinities for multiple subtypes are not as effective.• Compare the pharmacological profile for the unknown tissue with the pharmacological profile of the known subtypes. So for example, suppose a drug has a high affinity for subtype A and low affinity for subtype B and C and it also has a high affinity for the unknown receptor in the tissue you are studying. This would suggest that the subtype in the unknown tissue is subtype A.