AL - Quantitative Studies I

Question 1

Q: What information do quantitative receptor studies provide? (4)

Answer 1

• Affinity – Strength of drug-receptor interactions.
• Number of binding sites – Total receptor availability for drug binding.
• Pharmacological properties – Drug efficacy, potency, and response.
• Structure-function relationships – How receptor structure influences function.

Question 2

Q: What is the receptor binding assay workflow? (5)

Answer 2

1. Incubate tissue or cells with a radiolabeled ligand.
2. Separate bound vs. free ligand using centrifugation or filtration.
3. Measure ligand binding at different concentrations to determine affinity (K_D).
4. Displace ligands using other compounds to study binding site pharmacology.
5. Introduce receptor mutations to study structure-function relationships.

Question 3

Q: What are the two key parameters in quantitative receptor binding studies? (3)

Answer 3

Affinity (Kₐ or K_D) – Ligand concentration at which 50% of receptors are occupied.
- Lower K_D → Higher affinity (stronger binding).

Receptor Density (Bmax) – Total number of binding sites available on the receptor.

Question 4

Q: How do binding curves help analyze receptor-ligand interactions? (3)

Answer 4

• Total binding (○) – Includes both specific and nonspecific binding.
• Specific binding (●) – Determined by subtracting nonspecific binding.
• Nonspecific binding (△) – Binding that occurs independent of receptor interaction, measured using excess unlabeled ligand.

Question 5

Q: Why are quantitative receptor studies important? (4)

Answer 5

• Pharmacological profiling – Identifying ligands for specific receptor subtypes.
• Receptor identification & isolation – Understanding receptor presence in tissues.
• Quantifying receptor number – Important for studying upregulation or downregulation.
• Key Limitation – Radioligand binding does not provide efficacy/function information.

Question 6

Q: What key principles underlie receptor-ligand binding analysis? (3)

Answer 6

1) Law of Mass Action – Binding follows the reversible reaction:
- Ligand (L) + Receptor (R) ⇌ Bound Complex (B).

2) Free Drug Approximation – Only a small fraction of ligand binds to receptors, so:
- Free drug concentration ≈ Total drug concentration.

3) Equilibrium Condition – At equilibrium, the rate of association = dissociation:
- K₁ (association rate) & K₋₁ (dissociation rate) determine binding affinity (K_D).

Question 7

Q: What is affinity, and how is it quantified?

Answer 7

Affinity – Strength of ligand-receptor interaction.

Measured by K_D (dissociation constant):
- K_D = k₋₁ / k₁, where:
- k₁ = association rate constant.
- k₋₁ = dissociation rate constant.
- Lower K_D → Higher affinity.

Affinity Ranges:
- Nanomolar (nM, 10⁻⁹ M) → High affinity 😊.
- Micromolar (µM, 10⁻⁶ M) → Moderate affinity.
- Millimolar (mM, 10⁻³ M) → Low affinity ☹️.

Question 8

Q: What are the units of equilibrium constants in ligand binding? (2)

Answer 8

• K_A (Association Constant) – Expressed in L/mol (inverse of K_D).
• K_D (Dissociation Constant) – Expressed in mol/L (M).

Question 9

What is the equilibrium equation for ligand-receptor binding? (3)

Answer 9

At equilibrium: k₁[L][R] = k₋₁[B]
Rearranged: k₋₁ / k₁ = K_D = ([L][R] / [B])
Receptor availability: [R] = Bmax - [B]

Question 10

Q: What is the Langmuir Binding Equation, and what does it describe?

Answer 10

Describes ligand-receptor interactions using:

B = (Bmax × [L]) / (K_D + [L])

Where:
- B = Bound ligand concentration.
- Bmax = Total receptor density (maximum binding).
- [L] = Free ligand concentration.
- K_D = Equilibrium dissociation constant (ligand concentration at which half of receptors are occupied).

Valid for simple bimolecular ligand-receptor interactions.

Question 11

Q: What is Bmax, and why is it important? (3)

Answer 11

Bmax – Total number of receptors available for binding.

Typically expressed in:
- 10⁻¹² to 10⁻¹⁵ moles/mg protein (picomolar to femtomolar levels).

Key Takeaways:
- Higher Bmax → More receptors → Greater ligand binding potential.
- Lower Bmax → Fewer receptors → May indicate receptor downregulation.
- Bmax is independent of K_D – Receptor density and affinity are distinct parameters.

Question 12

Q: How do you calculate receptor binding using real data? (4)

Answer 12

1) Subtract non-specific binding from total bound to get specific c.p.m.

2) Convert to specific d.p.m using:
- Specific c.p.m / 0.44 (counting efficiency) = Specific d.p.m.

3) Calculate ligand amount in moles:
- Specific d.p.m / (2.2 × 10¹² × 77 × 10³).
- Example: 3582/(2.2 × 10¹² × 77 × 10³) = 2.1 × 10⁻¹⁴ mol.

4) Normalize by protein amount (e.g., 0.13 mg per assay tube):
- 162 × 10⁻¹⁵ mol/mg = 162 fmol/mg.