From Pharmacological Mechanisms to Precision Medicine (BBS3012) 1
From Pharmacological Mechanisms to
Precision Medicine (BBS3012)
Case 4, 5, 6
Name
Institution
Course
Tutor
Date
, From Pharmacological Mechanisms to Precision Medicine (BBS3012) 2
CASE 4 – STIMULATING BETA EXPERIMENTS
LG:
1. Make graphs from the results and process the data in order to calculate the values.
Aim: we have chosen beta-2 receptor (stimulation of beta 2 receptors on the airway
smooth muscle cells results, via activation of G-proteins and effector enzymes, in
smooth muscle relaxation and therefore bronchodilation) as our target receptor for our
new compound. We want to evaluate this compound as a new bronchodilator
compound.
Materials: airway tissue (containing beta-2 adrenergic receptor) homogenate and
smooth muscle preparation, radioactive ligand (isoprenaline), filtration apparatus and
scintillation (radioactivity) counter.
Experiment 1 – saturation analysis
Aim: to determine the dissociation constant of the radioactive ligand. We incubate fixed
amounts of tissue homogenate with increasing concentrations of radioactive ligand.
After 10 minutes we filter the incubations and count radioactivity.
→ Receptor binding techniques
Receptor binding studies are possible because of the high affinity that some agonists and
antagonists have for their receptor. Consequently, at low concentrations of a drug, high
proportion is bound to the receptor compared to the proportion which binds to non-
receptor sites.
Saturation binding studies allow determination of the affinity of a receptor for a
radioligand and, more importantly, receptor densities.
In addition to binding to receptors of interest, radioligands also bind to other sites.
Binding to the receptor of interest is called specific binding, while binding to other sites
is called nonspecific binding.
, From Pharmacological Mechanisms to Precision Medicine (BBS3012) 3
• Nonspecific binding represents interaction of ligand with
membranes.
• Nonspecific binding can also be binding to receptors,
transporters or other proteins not of interest to the investigator.
Nonspecific binding is usually proportional to the concentration of radioligand.
Nonspecific binding is detected by measuring radioligand binding in the presence of a
saturating concentration of an unlabeled drug that binds to the receptor. Under those
conditions, virtually all the receptors are occupied by the unlabeled drug so the
radioligand can only bind to nonspecific sites. Subtract the nonspecific binding at a
particular concentration of radioligand from the total binding at that concentration to
calculate the specific radioligand binding to receptors.
→ Radioactivity counting in binding assays
Radioligand is a radioactively labeled drug that can associated with a receptor of
interest. Measuring the rate and extent of binding provides information on the number of
binding sites, and their affinity and pharmacological (biological) characteristics.
→ Saturation binding
We vary the concentration of radioligand and measure binding at equilibrium. The goal
is to determine the Kd (ligand concentration that binds to half the receptor sites at
equilibrium) and Bmax (maximum number of binding sites).
The Kd-value is a measure of the affinity of the ligand for its binding sites. Since the Kd
is the concentration at which the ligand binds to half its binding sites, the ligand’s
affinity for the receptor is small when its Kd is large. Likewise, ligands with a high
affinity have small Kd.
Specific binding is obtained by subtracting non-specific binding from total. Note that
nonspecific binding increases linearly with the concentration of the radioligand, while
specific binding becomes saturated at Bmax.
→ Why do we use radioactive ligand?
Isoprenaline binds to beta 2 receptors.
You can buy isoprenaline with radioactive receptor – with new compound we have
synthesized this and it is hard to make it with radioactive analogue of the compoud.
, From Pharmacological Mechanisms to Precision Medicine (BBS3012) 4
We do this experiment only once and can test lot of compounds in our second and third
experiments.
→ Slope
We take the last two points because we assume that the increase is due to non-specific
binding in our total binding curve.
Multiply the concnetration times the slope. This is because the last two points are due to
increase in non-specific binding so we multiply our concentration times the slope.
The specific binding is determined by subtracting nonspecific binding from total
binding.
Bmax = 873.08 cpm
Kd (isoprenaline) = 1.2 – 1.3 nM (50% of the receptors are occupied at this value)
Experiment 2 – competition (inhibition or displacement) assay
Aim: We want to evaluate the ability of our test compound to displace the radioactive
ligand from the beta-2 receptors.
From Pharmacological Mechanisms to
Precision Medicine (BBS3012)
Case 4, 5, 6
Name
Institution
Course
Tutor
Date
, From Pharmacological Mechanisms to Precision Medicine (BBS3012) 2
CASE 4 – STIMULATING BETA EXPERIMENTS
LG:
1. Make graphs from the results and process the data in order to calculate the values.
Aim: we have chosen beta-2 receptor (stimulation of beta 2 receptors on the airway
smooth muscle cells results, via activation of G-proteins and effector enzymes, in
smooth muscle relaxation and therefore bronchodilation) as our target receptor for our
new compound. We want to evaluate this compound as a new bronchodilator
compound.
Materials: airway tissue (containing beta-2 adrenergic receptor) homogenate and
smooth muscle preparation, radioactive ligand (isoprenaline), filtration apparatus and
scintillation (radioactivity) counter.
Experiment 1 – saturation analysis
Aim: to determine the dissociation constant of the radioactive ligand. We incubate fixed
amounts of tissue homogenate with increasing concentrations of radioactive ligand.
After 10 minutes we filter the incubations and count radioactivity.
→ Receptor binding techniques
Receptor binding studies are possible because of the high affinity that some agonists and
antagonists have for their receptor. Consequently, at low concentrations of a drug, high
proportion is bound to the receptor compared to the proportion which binds to non-
receptor sites.
Saturation binding studies allow determination of the affinity of a receptor for a
radioligand and, more importantly, receptor densities.
In addition to binding to receptors of interest, radioligands also bind to other sites.
Binding to the receptor of interest is called specific binding, while binding to other sites
is called nonspecific binding.
, From Pharmacological Mechanisms to Precision Medicine (BBS3012) 3
• Nonspecific binding represents interaction of ligand with
membranes.
• Nonspecific binding can also be binding to receptors,
transporters or other proteins not of interest to the investigator.
Nonspecific binding is usually proportional to the concentration of radioligand.
Nonspecific binding is detected by measuring radioligand binding in the presence of a
saturating concentration of an unlabeled drug that binds to the receptor. Under those
conditions, virtually all the receptors are occupied by the unlabeled drug so the
radioligand can only bind to nonspecific sites. Subtract the nonspecific binding at a
particular concentration of radioligand from the total binding at that concentration to
calculate the specific radioligand binding to receptors.
→ Radioactivity counting in binding assays
Radioligand is a radioactively labeled drug that can associated with a receptor of
interest. Measuring the rate and extent of binding provides information on the number of
binding sites, and their affinity and pharmacological (biological) characteristics.
→ Saturation binding
We vary the concentration of radioligand and measure binding at equilibrium. The goal
is to determine the Kd (ligand concentration that binds to half the receptor sites at
equilibrium) and Bmax (maximum number of binding sites).
The Kd-value is a measure of the affinity of the ligand for its binding sites. Since the Kd
is the concentration at which the ligand binds to half its binding sites, the ligand’s
affinity for the receptor is small when its Kd is large. Likewise, ligands with a high
affinity have small Kd.
Specific binding is obtained by subtracting non-specific binding from total. Note that
nonspecific binding increases linearly with the concentration of the radioligand, while
specific binding becomes saturated at Bmax.
→ Why do we use radioactive ligand?
Isoprenaline binds to beta 2 receptors.
You can buy isoprenaline with radioactive receptor – with new compound we have
synthesized this and it is hard to make it with radioactive analogue of the compoud.
, From Pharmacological Mechanisms to Precision Medicine (BBS3012) 4
We do this experiment only once and can test lot of compounds in our second and third
experiments.
→ Slope
We take the last two points because we assume that the increase is due to non-specific
binding in our total binding curve.
Multiply the concnetration times the slope. This is because the last two points are due to
increase in non-specific binding so we multiply our concentration times the slope.
The specific binding is determined by subtracting nonspecific binding from total
binding.
Bmax = 873.08 cpm
Kd (isoprenaline) = 1.2 – 1.3 nM (50% of the receptors are occupied at this value)
Experiment 2 – competition (inhibition or displacement) assay
Aim: We want to evaluate the ability of our test compound to displace the radioactive
ligand from the beta-2 receptors.
