pcl471laboratory slides

PCL471 LaboratoryReceptor Pharmacology

Maurice Shen

maurice.shen@utoronto.ca

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

• 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

• 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

• 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

• 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

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

Steps of Ligand-Receptor Interactions

3. After dissociation, the ligand and receptor remainunchanged.

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

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

3. Neither the receptor nor the ligand is altered by binding.

The model ignores changes in receptor conformation

4. All bindings are reversible.

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

Non-specific Binding

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

Saturation Binding Curves

Scatchard Plot

Advantages: Requires very few data pointsEasy to interpret visually (Kd & Bmax)

Disadvantage: Inaccurate

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.

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, [3H] 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?

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

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…

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.

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

Competition Binding Curve

IC50: the concentration of cold drug that displaces 50% of specific binding of the radiolabeled drug from the receptor

Competition Binding Curve

Which drug has higher affinity to this receptor?

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.

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]

Kd1+

[D] = concentration of radiolabeled drug

Kd = dissociation constant of the radiolabeled drug

IC50 is obtained from the competition curve of the cold drug

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

GPCR Cycle

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.

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

Biphasic Nature of Agonist Binding Curve

The two Ki values are approximately 2 log units apart

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

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

Affinity Chromatography

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

Photoaffinity Labeling

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

Receptor Autoradiography

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

Positron Emission Tomography (PET)

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)

3. References

4. 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

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

• CalculationsGive 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