measuring receptor affinity: practical

Question 1

What do we need to measure to find the affinity of a drug for a target

Answer 1

KD= k-1(rate of unbinding) / k+1 (rate of binding) = [Aeq]*[Req]/ [AeqReq] eq= equilibrium
-For a given drug concentration [A], the Occupancy [AR]/[R]
-by measuring the occupancy you can find KD= which is affinity

Question 2

what can we determine if we measure occupancy at equilibrium

Answer 2

-affinity
-higher the conc of drug= higher occupancy
-size of response although clearly related to occupancy (by agonist) it will also depend on efficacy

Question 3

what are the Experimental approaches used to measure drug affinity

Answer 3

-Radioligand Binding Assays
=principle: A radiolabeled drug is incubated with receptor-containing tissue or cells. The amount of radiolabel bound to the receptors is measured.
-Fluorescence Polarization Assays (FPAs)
=principle: A fluorescently labeled drug is used. When it binds to a receptor, its rotational freedom decreases, leading to increased polarization of emitted fluorescence.
-Surface Plasmon Resonance (SPR)
=Principle: Measures changes in the refractive index of a surface when molecules bind to it.
-Isothermal Titration Calorimetry (ITC)
=Principle: Measures the heat released or absorbed during a binding interaction.
-Computational Modelling
=Principle: Uses computational methods to predict drug-receptor interactions based on molecular structures.

Question 4

A and D of Radioligand binding assay

Answer 4

-Advantages: Sensitive, can be used with a wide range of receptors.
-Disadvantages: Requires radioactive materials, may not be suitable for all receptors.

Question 5

A and D of Fluorescence Polarization Assays (FPAs)

Answer 5

-Advantages: Non-radioactive, high-throughput, can be used for both small and large molecules.
-Disadvantages: Requires a fluorescently labeled drug, may be less sensitive than radioligand binding assays.

Question 6

A and D of surface plasmon resonance (SPR)

Answer 6

-Advantages: Real-time measurement, can be used for both small and large molecules, label-free.
-Disadvantages: Requires specialized equipment, may be less sensitive for low-affinity interactions

Question 7

A and D of Isothermal Titration Calorimetry (ITC)

Answer 7

-Advantages: Provides thermodynamic parameters (enthalpy, entropy), can be used for both small and large molecules.
-Disadvantages: Requires specialized equipment, may be less sensitive for low-affinity interactions.

Question 8

A and D of Computational Modelling

Answer 8

-Advantages: Can provide insights into binding mechanisms, can be used to screen large libraries of compounds.
-Disadvantages: Requires computational expertise, may not always accurately predict experimental results.

Question 9

what are the key steps of this practical

Answer 9

1) source of receptor and incubation conditions
2)the radioligand
3) choice of radio labels
4) separating bound from free
5) non-specific binding