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: (1)characterize receptors in their natural environment as well

as those transfected into cell lines; (2)study receptor dynamics and localization; (3)identify novel chemical structures that interact with receptors (4)define ligand activity and selectivity in normal and diseased

tissues.

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.

Radioligand Binding Studies

kon Association rate constant or on-rate constant

koff Dissociation rate constant or off-rate constant

Kd Equilibrium dissociation constant

Law of mass action

At Equilibrium

[Ligand] Fractional Occupancy

0 0%

1.Kd 50%

4.Kd 80%

9.Kd 90%

99.Kd 99%

The model assumes:

• All receptors are equally accessible to ligands.

• Receptors are either free or bound to ligand. It doesn't allow for more than one affinity state, or states of partial binding.

• 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 experiment

3. Identify a method for separating bound from free

4. Identify a method for distinguishing specific from nonspecific binding

Criteria for selecting a radioactive ligand

a. The specific activity of the radioligand should be high enough to detect the receptor in the tissue being studied.

b. The radioligand should have a high affinity for the receptor.

When using a filtration method to separate bound from free, it usually takes 5 to 15 seconds to filter the sample and rinse the filter. During this time less than 10% of the radioligand should dissociate from the receptor.

c. The radioligand should have a high degree of selectivity for the receptor being studied.

d. The radioligand should be chemically stable in the assay media during the binding reaction.

e. The radioligand should be pure.

Radioligand binding could be :

1)Specific

The site that we want to study is referred to as the SPECIFIC SITE.

2) Non-Specific

All other sites are called NONSPECIFIC SITES.

Examples of nonspecific binding sites

1) 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.

2) 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 bind to 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 filters used to separate bound from free.  This binding would be considered nonspecific binding. 

Binding to test tubes can sometimes be eliminated by choosing the type of test tube (glass or plastic) used to do the binding assay. 

Binding to glass fiber filters can usually be eliminated to some extent by trying different types of filters from different manufactures or coating the filter with polyethyleneamine which puts a negative charge on the filter.

SATURABLE and NON-SATURABLE binding

SATURABLE binding sites are always present ininfinite amounts. Another way of thinking of this is that if you add enough ligand all of the sites will be occupied with ligand.

NON-SATURABLE binding sites are sites that are present in essentially infinite amounts. No matter how much ligand you add, not all of the sites will be occupied with ligand. The site is 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 concentration of radioactive ligand used in a saturation curve. Under these conditions all of the receptors would be occupied by radioactive ligand.

Binding is also measured in the presence of the same concentration of radioactive ligand plus increasing concentrations of unlabeled ligand. As the concentration of unlabeled ligand increases the amount of radioactive ligand bound decreases until a plateau is reached. At this point no matter how much unlabeled ligand is added the amount of radioactive ligand bound remains constant.

This residual amount of radioligand bound represents binding to nonspecific, non-saturable sites which can't be displaced by unlabeled ligand.

The amount of inhibitor to use would be the concentration which completely blocks the saturable binding sites.In this example it would be between 10-9 to 10-8 M.

Guidelines -Appropriate assay conditions

Radioligand SelectionThe 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. In some cases, the radioligand requires special storage procedures, such as addition of antioxidants or protease inhibitors in the case of a peptide radioligand, to slow or prevent degradation.

Nonspecific bindingThe physicochemical properties of a ligand determine its level of nonspecific binding due to interactions with lipid membranes and/or filter or scintillation bead material inthe assay.Factors such as lipophilicity and aqueous solubility should be taken into consideration when selecting a ligand to radiolabel.

Buffers usedIn most cases, a homogenization / assay buffer is selected that yields the highest ratio of specific versus nonspecific binding.

Common buffers - Tris, Hepes, sodium phosphate and glycylglycine, 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 best so that the results can be compared. 

Some binding assays require the presence of special ions. For example, 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 the pH.  Generally using a physiological pH, such as 7.4,  is best so that the results are comparable to what is seen in vivo.

Optically active radioligandsIf a ligand contains a chiral center, use of the stereochemically active form is preferable.With few exceptions, most receptors differentiate between the optical isomers of compounds, with the pharmacologically-active isomer binding with higher affinity than the less-active isomer.

Additional reagents MgCl2 is used in many receptors binding assays.  Initially this was

used to help precipitate the radioligand receptor complex during centrifugation.  NaCl is often used in adrenergic receptor binding studies to convert the receptor to a form that has a lower affinity for agonists.  GTP or its non-hydrolyzeable analog (Gpp(NH)p) are often used when agonist binding is also going to be studied in subsequent competition experiments.  Gpp(NH)p converts the receptor into a form that has a low affinity for agonist

Temperature of the binding reactionMost binding assays can be conducted at room temperature. Some assays work better at 4oC than at room temperature, and sometimes the affinity of the radioligand for the receptor is higher at 4oC  than at 25oC .  It takes longer to reach steady-state when the reaction is run at 4oC   than at room temperature.

Major Types of Radio ligand Study

1.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 experiments

Experiment used to be analyzed with Scatchard plots (more accurately attributed to Rosenthal), they are sometimes called "Scatchard experiments".

The analyses depend on the assumption that you have allowed the incubation to proceed to equilibrium (few minutes to many hours, depending on the ligand, receptor, temperature, and other experimental conditions.)

The lowest concentration of radioligand will take the longest to equilibrate. When testing equilibration time, therefore, use a low concentration of radioligand (perhaps 10-20% of the KD).  

Purpose of the Saturation experiment

1)To determine the affinity or Kd of a radioligand for a

receptor Kd is the equilibrium dissociation constant. It is equal to the concentration of radioactive ligand required to occupy 50 % of the receptors.

2)The density (Bmax) of a specific receptor or receptor

subtype in a given tissueBmax is the total number of receptor sites in the tissue being studied. It occurs when the all of the receptor molecules are occupied by radioactive drug.

Basic characteristics of a saturation experiment

In a saturation experiment increasing concentrations of a radioactive ligand are allowed to bind to the receptor until steady-state conditions occur. After reaching steady state, the bound ligand is separated from the free ligand. The most widely used methods for separation of bound ligand from free ligand are filtration and centrifugation. The amount of ligand bound to the filter or trapped in the pellet is then measured. A radioactive ligand is used because the radioactivity of low concentrations of ligand can be detected in filters or membrane pellets.

Methods used to determine Kd and Bmax from a saturation 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 (the amount of radioactive ligand that is bound to the receptor) on the Y axis and FREE (the free concentration of radioactive ligand) on the X axis.

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 the binding sites

Getting an accurate estimate of Kd and Bmax from this graph by eye is difficult.   The curve is usually analyzed by nonlinear regression analysis

By fitting the data to the equation for a saturation curve.

where B is Bound           F is Free       

Rosenthal Plot

The data from the saturation experiment can be plotted with Bound/Free on the Y axis and Bound on the X axis.

Single site binding data can be analyzed by linear regression to give a straight line.

The slope of the line is -1/Kd and the X-intercept

is Bmax. This is a Rosenthal Plot.

Most scientists call it a Scatchard Plot.

Rosenthal Plot

It is easy to visually compare two sets of data on a Rosenthal plot. 

If the Kd for both sets of data are similar the

slopes will be similar. 

If the Bmax changes then the X intercept will

change. 

Two-site fit to the data can be visualized more easily with a Rosenthal Plot than with a saturation curve.  

Advantages of Rosenthal Plot

Experimental conditions that need to beconsidered in doing saturation experiments

1. Concentration of radioligand used

2. Concentration of tissue 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.

 

1. Concentration of radioligand used

2. The concentration of tissue used:

It is dependent on the number of binding sites per mg of tissue.  The tissue concentrations required in the assay will vary for the same binding site in different tissues and for different receptors in the same 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 high that more than 10% of the radioligand is bound. 

The equations used for generation of Kd and Bmax are

based on the assumption that the free concentration of radioligand does not change. 

It is generally assumed that these conditions are met if less than 10% of the radioligand is bound to the receptor. 

One way to determine quickly whether 10% of the radioligand is bound to the receptor is to look at the position 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 assume

that the free ligand concentration is not changing during the binding reaction. These conditions are generally assumed to be met if less than 10% of the radioligand is bound to the receptor at steady state 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 radioligandThe 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 to filters used in a filter assay then bound would be total bound.  (Note that total bound represents the binding of the radioligand to both specific and nonspecific sites.)  

Free = Total Added - (Specific bound + Nonspecific bound)or  Free = Total Added - Total bound

If the nonspecific binding comes predominately from the binding of the radioligand to the filters used to separate bound from free, then this nonspecific binding is not to the tissue and occurs after the reaction is complete.  In this case, only specific binding should be subtracted from total binding to give free.Free = Total added - Specific bound

Nonlinear regression analysis of the saturation curve

Saturation curves are best fit to the following equation

where B is bound and F is free.

Conditions that need to be met when doing saturation experiments

•The measured amount of bound radioligand needs to reflect the amount of radioligand bound under equilibrium conditions. That is, equilibrium conditions need to be present when bound is separated from free. The bound ligand should not dissociate from the receptor during the course of separating free from bound.

•A time course can be run at the lowest concentration of radioligand under your experimental conditions to demonstrate that equilibrium conditions are present when bound is separated from free.

Less than 10% of the radioligand should dissociate from the receptor in the process of separating bound from free. 

Less than 10% of the radioligand should be depleted from the reaction mixture.

The amount of bound radioligand needs to be determined accurately

Specific binding must be correctly defined.

Ligand should not be degraded during the course of the reaction.

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 Bmax

1 and Bmax2 are the binding site density

for sites 1 and 2

Kd1 and Kd

2 are the Kd values for site 1 and

site 2

2 Competition ExperimentsMany ligands for receptors are not available in a radioactive form. Since they are unlabeled there is no way to directly measure their affinity for the receptor.The affinity of the unlabeled ligand for the receptor can be determined indirectly by measuring its ability to compete with a radioactive ligand for the receptor. In a competition experiment various concentrations of an unlabeled ligand are allowed to compete with a fixed concentration of a radiolabeled ligand for a receptor.

As the concentration of unlabeled ligand increases, the amount of radioligand bound to the receptor decreases.

The competitive inhibitor can be either an agonist or an antagonist.

It is called a competitive inhibitor because its value is determined by measuring the ability of the unlabeled drug to compete with a radiolabeled drug for the receptor.

The Ki value for an unlabeled drug should be the

same as the Kd value obtained for the same drug

in radiolabeled form.

Analyses of the results of the competition experiment are simpler and more accurate if the radiolabeled ligand is only binding to one site.

Hormone and neurotransmitter receptors exits in conformations which have either high affinity or low affinity for the agonist.

A true antagonist has equal affinities for the high and low affinity states of the receptor. The analysis of the data is simpler if the radiolabeled ligand is an antagonist

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

Purpose of Competition Experiments

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 used

Binding 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 the affinity is high, the EC50 will be low. The affinity is usually quantified as the equilibrium dissociation constant, Ki. The Ki is the concentration of the competing ligand that will bind to half the binding sites at equilibrium, in the absence of radioligand or other competitors. If the Ki is low, the affinity of the receptor for the inhibitor is high.

•  The concentration of the radioligand. If you choose to use a higher concentration of radioligand, it will take a larger conc of unlabeled drug to compete for half the radioligand binding sites.

•  The affinity of the radioligand for the receptor (Kd). It takes more unlabeled drug to compete for a tightly bound radioligand (low Kd) than for a loosely bound radioligand (high Kd).

Two-Site Competition Experiments

Two site binding in a competition experiment under the following conditions:

If the unlabeled ligand is an agonist. Receptors have both low and high affinity states for agonist. Unless steps are taken to convert all the receptor molecules into either high or low affinity states, agonist will bind to more than one affinity state of a receptor in a competition experiment.

If the unlabeled ligand binds to different subtypes of the receptor with different affinities and there is more than one subtype in the tissue you are studying.

If the unlabeled ligand binds to more than one receptor with different affinities. This assumes the radioligand is also binding to more than one receptor.

The characteristics of a competition curve with one-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 with two-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 using Competition Studies

Methods used to identify the pharmacological profile for a specific subtype of a receptor

•To initial identify the pharmacological profile of a receptor subtype it is important to first have a tissue which selectively expresses only that receptor subtype.

•Competition studies are done to determine the affinity of a large number of drugs for the receptor. It is important to use drugs which have both a high and low affinity for the receptor. The affinity of these drugs for the receptor define the pharmacological profile for the receptor. This profile is used to determine the identify of receptor subtypes in specific tissues

Methods used to identify the receptor subtypes in an unknown 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.