2. What is a Receptor?
• Paul Ehrlich (1800s) : Drug might act as a “Magic Bullet”
directed at a vulnerable “Receptor”
• A component of a cell or organism that interacts with a
specific ligand and initiates a chain of biochemical events
leading to the ligand’s observed effects.
– Ligands can be exogenous (drugs) or endogenous (hormones
and neurotransmitters etc.)
• 3 main characteristics of Ligand/Receptor Interactions:
– Saturable
– Reversible Binding
– Dose-Response Relationship
3. • Agonists: Drugs that elicit biological effects that are
comparable to endogenous ligands.
– Full: Maximal Efficacy
– Partial: Sub-maximal Efficacy
– Inverse: Produces opposite biological effects as the full
agonists
• Antagonists: Drugs that bind to the receptor but do not
elicit any biological effect; Zero Efficacy.
– Reversible & Competitive: transiently bound to the same site as
Agonists
– Non-competitive: bound to a different site as Agonists
– Irreversible: covalently bind to the same site as Agonists
Clinical Utilities?
Various Types of Drugs
4. • Ligand-Gated Ion Channels: Trans-membrane channels
that allows for the influx of ions such as Na+
, Ca2+
,Cl-
.
– Neurotransmitters systems: Glutamate, GABA, Acetylcholine
• Receptor Tyrosine Kinases: Trans-membrane
polypeptides that autophosphorylate at tyrosine residues
upon activation
– Growth Factors: Insulin, Epidermal Growth Factor (EGF), Platelet-
derived Growth Factor (PDGF).
• Nuclear Hormone Receptors: Cytosolic receptors that
regulates gene transcription within a cell.
– Hormones: Glucocorticoids, Thyroid Hormone, Sex Steroids etc.
Various Types of Receptors
5. • G Protein-Coupled Receptors: 7 trans-membrane
proteins that are coupled to specific G proteins; elicit
second messenger-mediated signaling cascade upon
activation.
– Largest family of cell surface receptors, more than 50% of drugs
on the market target GPCRs.
– Wide range of ligands including hormones, neurotransmitters,
light, and olfactory stimuli.
– Downstream signaling may lead to gene transcription, protein
synthesis, protein phosphorylations
Dopamine Receptors are GPCRs
Various Types of Receptors
6. • The use of varying concentrations of a specific
radiolabelled ligand to determine receptor affinity to
the ligand (Kd) and total receptor density (Bmax) in a
membrane preparation.
• The theory of radioligand binding experiments are
based on the Laws of Mass Action, where:
Ligand + Receptor [Ligand-Receptor]
Radioligand Binding Assays
Kon
Koff
7. 1. Binding occurs when ligand and receptor collide due
to random diffusion and when the collision has the
correct orientation and sufficient energy.
The rate of association is defined by:
[ligand] x [receptor] x Kon
Steps of Ligand-Receptor Interactions
2. Once the bound complex is formed, the ligand and
receptor remain bound for a random amount of
time, determined by their relative affinity for one
another
The rate of dissociation is defined by:
[ligand-receptor] x Koff
8. Steps of Ligand-Receptor Interactions
3. After dissociation, the ligand and receptor remain
unchanged.
4. Equilibrium is reached when the rate of Ligand-Receptor
complex formation equals to the rate of dissociation.
Therefore, at equilibrium:
[ligand] x [receptor] x Kon = [ligand-receptor] x Koff
[ligand] x [receptor] Koff
[ligand-receptor] Kon
The Dissociation Constant (Kd) is defined as the concentration of
ligand that occupies half of the available receptors at equilibrium
= = Kd
9. Assumptions Made in the Model
1. All receptors are equally accessible to the ligand.
2. All receptors are either ligand-bound or ligand-free.
The model ignores partial binding
1. Neither the receptor nor the ligand is altered by
binding.
The model ignores changes in receptor conformation
1. All bindings are reversible.
10. Drug Bindings
1. Total Binding: Total radioligand bound to the cell
membrane.
2. Non-specific Binding: Determined by measuring the
radioligand binding in the presence of excess cold drug
with high affinity to the receptor of interest.
Assumes all receptors are bound by the cold drug
3. Specific Binding: The difference between Total and
Non-specific binding.
Specific binding curve is shown as an rectangular
hyperbolic function that yields Kd and Bmax
12. Drug Bindings
1. Total Binding: Total radioligand bound to the
membrane.
2. Non-specific Binding: Determined by measuring the
radioligand binding in the presence of excess cold drug
with high affinity to the receptor of interest.
Assumes all receptors are bound by the cold drug
3. Specific Binding: The difference between Total and
Non-specific binding.
Specific binding curve is shown as a rectangular
hyperbolic function that yields Kd and Bmax
15. Radioactivity Measurements
1. β particles emitted from the radioactive ligand transfer
energy to flours in the solvent.
2. Excited flours dissipate energy by emitting light, which is
detected by photomultiplier tube.
16. Today…
1. COS-7 cells transfected with either D1 or D5 cDNA;
membranes harvested by differential centrifugation
2. Two-point saturation binding of D1 and D5 dopamine
receptors using radiolabeled dopamine receptor
antagonist, [3
H] SCH-23390.
3. Radiation counts measured using scintillation counter.
Can you identify Dx and Dy based on the Kd derived
from the 2-point Scatchard plot?
17. Week 2
1. Incubation Period: 45 minutes
Unlabeled Competitive Drugs
Group 1: A
Group 2: B
Group 3: C
Group 4: D
Group 5: E
Both Dx and Dy
18. Can you identify Dx and Dy based on the
Kd derived from the 2-point Scatchard
plot?
No,
Why
?
Both D1 and D5 receptors exhibit similarly high
affinity for SCH23390. Cannot tell the difference
between Dx and Dy based on Kd.
Must employ another method to identify Dx and Dy…
19. Competition Binding
The use of competition curves:
Measures the binding of a single concentration of radioligand
in the presence of increasing concentrations of an unlabeled
drug
1. To screen for compounds that can selectively bind to a
specific receptor
2. To validate an assay:
Compete with a cold drug with known potency that
was derived from functional assays.
Confirms the cold drug’s binding affinity to the
receptor.
20. Competition Binding Curve
Total Radioligand
binding
Non-specific
binding
The cold drug compete with the
radioligand at the same site on
the receptor.
The drop of specific binding
from 90% to 10% represents an
81-fold increase in [cold drug],
which usually corresponds to 2
log units
21. Competition Binding Curve
IC50: the concentration of cold drug that displaces 50% of specific
binding of the radiolabeled drug from the receptor
23. IC50 is determined by..
1. The concentration of the radioligand:
Higher [radioligand] would require higher [cold drug] to displace
radioligand binding.
2. The affinity of the radioligand to the receptor:
Higher affinity of the radioligand would require higher [cold
drug] to displace 50% of specific binding
Therefore..
IC50 may vary depending on the experimental setups;
A universal value is needed to describe the affinity of a
drug to the receptor.
24. Inhibition Constant, Ki
Defined as the concentration of unlabeled drug that will bind to half the
receptor binding sites at equilibrium in the absence of radioligand or other
competitors.
Ki is a property of the unlabeled drug and the receptor,
it can be obtained from the Cheng-Prusoff Equation:
Ki =
IC50
[D]
Kd
1+
[D] = concentration of radiolabeled drug
Kd = dissociation constant of the radiolabeled drug
IC50 is obtained from the competition curve of the cold drug
25. Assumptions made in the Cheng-Prusoff
Equation..
1. Only a small fraction of labeled and unlabeled ligands
has bound
2. Homogenous receptor population
3. No cooperativity
Binding to one binding site does not alter the drug’s affinity to
another site.
4. Reaction has reacted equilibrium
5. Reversible binding that follows the Laws of Mass
Action
6. Kd of the radioligand is known
27. Ternary Complex Model of GPCR
G-protein coupling determines GPCR’s affinity to Agonist
binding.
G-protein coupled state: The G-protein is bound to GDP; GPCR is
in the inactive state and has high affinity to agonists.
G-protein uncoupled state: The G-protein is bound to GTP; GPCR
is activated and has low affinity to agonists.
G-protein coupling does not affect GPCR’s affinity to
Antagonist binding.
GPCR displays high affinity to antagonist binding despite the state of
G-protein coupling.
28. Competition Binding Curves of
Agonist vs. Antagonist
Agonist curve is biphasic due to the high/low affinity states, with
two IC50 and thus two Ki (Kihigh vs Kilow)
Antagonist curve is uniphasic due to single high affinity state
29. Biphasic Nature of Agonist Binding Curve
The two Ki values are approximately 2 log units apart
30. Today…
Perform competition binding experiments using the following
compounds with [3H]SCH23390 to elucidate the identity of Dx
and Dy through the rank order of potency of these compounds
Butaclamol
Dopamine
Noradrenaline
Spiperone
SCH23390
31. Other Binding Techniques
Affinity Chromatography
Separates proteins on the basis of reversible interaction between a
protein and a specific ligand that is coupled to a chromatography matrix
Used to purify receptor of interest with high selectivity
Requires a selective ligand covalently linked to chromatography matrix
Ligand-Receptor binding must be reversible
Receptor is eluted via changes in pH or ionic strength, or via competition
33. Other Binding Techniques
Photoaffinity Labeling
Direct probing of a target protein through a covalent bond which is
photochemically introduced between a ligand and its specific receptor
The ligand is linked to a photoreactive group sensitive to UV light
After binding to the receptor, UV light induces covalent linkage
The Ligand-Receptor complex can be identified by SDS-PAGE
Advantageous for Target Identification via quantification of the complex
formed; more complex will form if a ligand has high affinity vs low affinity
35. Other Binding Techniques
Receptor Autoradiography
Direct binding of a selective radioligand to its receptor in native tissue
Shows the distribution of the receptors in native tissue; ie brain slices.
Allows for quantitative analysis of the receptors
Ligand of choice determines selectivity
In theory similar to Immunohistochemistry, with the use of selective
ligands instead of antibodies
37. Other Binding Techniques
Positron Emission Tomography (PET)
Most commonly used isotope is fluorodeoxyglucose (FDG), which is
glucose with radioactive flourine attached
Imaging technique which produces a three dimensional image
or map of functional processes in the body
System detects pairs of gamma rays emitted indirectly by a
positron-emitting radioisotope
Isotope is introduced in body on a metabolically active
molecule
39. Lab Reports (double spaced)
1. Title Page
2. Body (Max 5 pages)
Introduction
Methods (can use tables)
Results (report data from your group)
Discussion (Dx, Dy, Drug A-E identification, summary, errors)
1. References
2. Graphs (must be hand-drawn)
A. 2-point Scatchard Plot (group)
B. 15-point Scatchard Plot (lab manual)
C. Competition Binding Curve (group)
Axis, Figure Legends at bottom
40. Lab Reports (double spaced)
1. Tables
A. Kd, Bmax from 2-point and 15-point Scatchard Plot for Dx and
Dy.
B. IC50 and Ki of all competitor drugs for Dx and Dy
Descriptions on top, Abbreviations
• Calculations
Give one example for each calculation, show step by step, be
specific
Specific Binding, Free, Bound/Free, Kd, Bmax, IC50, Ki
(shown as % of 10 -14
) etc.
Questions?
maurice.shen@utoronto.ca
Subject Title: PCL471
Editor's Notes
I assume the majority of you guys are in the specialist program, so most of what I’m going to talk about today will probably just be a brief review of what you already know from PCL201 and PCL302. These are the crystal structures of 3 different types of receptors, which are GABAA receptor, dopamine D3 receptor, and insulin receptor. I put them there simply to make the slides look cool.
So what is a receptor? The concept of a receptor exists all the way back in the 1800s, where this dude, Paul Ehrlich, proposed that a drug might act as a magic bullet that targets a specific receptor when he was trying to develop an anti-syphilis drug. I don’t know why he was studying syphilis but he did. Nowadays the existence of a receptor is well established, and as some of you probably know, this year’s Nobel price for chemistry was given to Robert Leftkowiz and Brian Kobilka who studied the G protein coupled receptors. A receptor is defined as a component of a cell or organism that interacts with a specific ligand and initiates a chain of biochemical events leading to the ligand’s observed effects. A ligand for a receptor can be endogenous or exogenous. For example, the endogenous ligand for the dopamine receptors would be dopamine, and an exogenous ligand would be L-DOPA. There are 3 main characteristics of ligand/receptor interactions. The binding of a ligand to its receptor is often saturable and reversible, and there exists a dose-response relationship where increasing concentration of a ligand will lead to enhanced biological effect until all the receptors are saturated.
Since we are pharmacologists, we are more interested in exogenous ligands, or drugs, as opposed to endogenous ligands. We can classify drugs into different types based on their abilities to regulate known biological processes. Agonists are rugs that elicit biological effects that are comparable to endogenous ligands. For instance, L-DOPA would be an agonist for the dopamine receptor since it mimics dopamine’s effects on motor function. An agonist is considered as a full agonist when it induces maximal biological effects, whereas a partial agonist induces sub-maximal biological effects. In contrast, an inverse agonist will produce opposite biological effects as the full agonists. For instance, if an full agonist leads to increased calcium level in the brain, an inverse agonist will lead to reduced calcium level in the brain.
Antagonists are drugs that bind to the receptor but do not elicit any biological effect. In other words, antagonists have zero efficacy. An antagonist for the dopamine receptor would be the anti-psychotic drug haloperidol. Different types of antagonists exist based on how they interact with the receptor. A reversible and competitive antagonist transiently binds to the same site as agonists. A non-competitive antagonist binds to a site on the receptor that is different from agonists, and they are also known as negative allosteric modulators. An irreversible antagonist covalently binds to the same site as agonists. Of all these types of drugs, partial agonists and antagonists have the best clinical utilities since it is easier to fine tune existing biological processes than to produce a biological effect from scratch, which might be harder to control.
As you know, many different types of receptors exist in a living organism. There are ligand-gated ion channels, which are trans-membrane channels that allows for the influx of ions such as Na+, Ca2+, Cl-. These receptors are generally found in the brain and the nervous system, and are activated by different neurotransmitters such as glutamate, GABA, and acetylcholine. Receptor tyrosine kinases are trans-membrane polypeptides that autophosphorylate at tyrosine residues upon activation. They are generally activated by growth factors such as insulin, epidermal growth factor, and platelet-derived growth factor. Nuclear hormone receptors are cytosolic receptors that regulates gene transcription within a cell, and as their name suggest, they are mainly activated by hormones such as glucocorticoids, thyroid hormone, sex steroids etc.
And finally, there are the G protein-coupled receptors. These are 7 trans-membrane proteins that are coupled to specific G proteins, and they elicit second messenger-mediated signaling cascade upon activation that may lead to gene transcription, protein synthesis, protein phosphorylations. GPCRs are the largest family of cell surface receptors and more than 50% of drugs on the market target GPCRs. They have wide range of ligands including hormones, neurotransmitters, light, and olfactory stimuli. The receptor of interest in this lab, the dopamine receptors, is a GPCR.
The technique that we will be using to study the receptor-ligand interaction in this module is called radioligand binding assay. It involves the use of varying concentrations of a specific radiolabelled ligand to determine receptor affinity to the ligand (Kd) and total receptor density (Bmax) in a membrane preparation. The theory of radioligand binding experiments are based on the Laws of Mass Action, where.
In an in vitro system, where you have your receptors transfected into a cell line, and you add your drug of interest, binding occurs when ligand and receptor collide due to random diffusion and when the collision has the correct orientation and sufficient energy, and the rate of association is defined by. Once the bound complex is formed, the ligand and receptor remain bound for a random amount of time, as determined by their relative affinity for one another. The rate of dissociation is defined by.
After dissociation, we assume the ligand and the receptor remain unchanged. Equilibrium is reached when the rate of Ligand-Receptor complex formation equals to the rate of dissociation. Therefore, at equilibrium. The Dissociation Constant (Kd) is defined as the concentration of ligand that occupies half of the available receptors at equilibrium.
There are 4 important assumptions made in the drug binding model I just described. First, we assume all receptors are equally accessible to the ligand. Second, we assume the receptor is either bound to a drug, or is in its free form. Partial binding is ignored. Third, we assume neither the receptor nor the ligand is altered by binding. Change in receptor conformation upon ligand binding is ignored. And lastly, we assume all bindings are reversible.
Since the receptors are transfected into a cell line, the drug of interest may also bind to membrane proteins other than the receptor itself. Total binding is defined as the total radioligand bound to the membrane. This is determined by measuring the total radioactivity on the cell membrane. Non-specific binding is then determined by measuring the radioligand binding in the presence of excess cold drug with high affinity to the receptor of interest. The cold drug must be different from the radiolabeled drug of interest, and it must have high enough affinity in order to specifically occupy all the receptors, rendering the radiolabeled ligand to only be able to bind non-specifically to membrane proteins.
This diagram depicts the difference between total binding and non-specific binding. The measurement of total binding is straightforward and includes specific and non-specific binding. To measure non-specific binding, all receptors are occupied by a different cold drug, and the radioactivity only comes from radiolabeled drug that is bound to other membrane proteins. An important assumption here is that the cold drug and the radiolabled drug of interest have completely different binding affinity to other membrane proteins.
We are then able to calculate specific binding by subtracting non-specific binding from total binding. Specific binding curve is shown as a rectangular hyperbolic function that yields Kd and Bmax, as shown in this figure.
Knowing how much drug is specifically bound and how much total drug was added, we are then able to plot a Scatchard plot by plotting bound drug over free drug on the y-axis, and the concentration of bound drug on the x-axis. The Scatchard plot requires very few data points, and Kd and Bmax can be easily deduced from the graph, which are the -1 over slope and the x-intercept, respectively. However, one major disadvantage of Scatchard plot is that the Kd and Bmax value may not be as accurate as the ones derived from the saturation binding curve.
Lastly, it is also important to know how the radioactivity from the radiolabeled ligand is measured, since our whole module relies on this measurement. The radioactivity is measured using the scintillation counter. The β particles emitted from the radioactive ligand transfer energy to flours in the solvent, and excited flours dissipate energy by emitting light, which is then detected by photomultiplier tube in the scintillation counter.
So for today, COS-7 cells were transfected with either D1 or D5 cDNA, and the membranes were harvested by differential centrifugation. This was already done for you. What you guys have to do today is to establish two-point saturation binding of D1 and D5 dopamine receptors using radiolabeled dopamine receptor antagonist, [3H] SCH-23390. The question to think about is whether you could identify Dx and Dy based on the Kd derived from the 2-point Scatchard plot? Why and why not? You guys will have to address this question in your lab report.
