Drug-Receptor Interactions: Affinity

Ligand

A molecule that binds to a specific receptor site on another molecule

Affinity

Tendency for a drug to bind to a receptor

Efficacy

Tendency for a drug, once bound, to activate a receptor

Agonists have significant efficacy, antagonists have zero efficacy

KD

Constant that defines the affinity of a drug for a receptor

Drug-receptor interaction

• Most of the interactions are reversible

• Involve the interaction of 2 molecules

Forward rate of reaction equations

k+1[A]*[R]

Reverse rate of reaction equation

k-1 [AR]

Reaction when at equilibrium equation

k+1[Aeq]*[Req] = k-1 [AeqReq]

KD= k-1 / k+1 = [Aeq]*[Req]/ [AeqReq]

Occupancy

Proportion of receptors occupied that varies with drug concentration

Measured that equilibrium

Governed by affinity

Varies between 0 (no drug present) and 1 (receptors occupied)

Occupancy equation

Occupancy = number of receptors occupied/ total number of receptors

Some experimental approaches to measure drug affinity

• Radioligand Binding Assays (RBA)

• Fluorescence Polarization Assays

• Surface Plasmon Resonance

• Isothermal Titration Calorimetry

• Computational Modelling

RBA: source of receptors & incubation conditions

Sources: tissues/ cells selected to contain recognition sites of interest (isolated membranes, slices, synaptosomes, cultured cells, purified receptors)

Incubation: to preserve integrity of both ligand and receptors. Temperature is usually low room temperature to 0

RBA: the radioligand

• must be biologically active

• Must be extremely pure chemically

• No degradation

• Labelling must achieve high specific activity to allow very low concentrations

RBA: how to solve the problem of degradation

• Store at low temps

• Avoid light

• Incorporation of antioxidant

• Free-radical scavenger in drug solution

RBA: examples of radio-labels used

3H, 125I

RBA: separating bound from free ligand

• usually done by filtration or centrifugation.

• For soluble binding: techniques like dialysis, column chromatography, precipitation.

• Major consideration: rate of dissociation of ligand-receptor complex.

Does lower affinity requires a faster or slower separation

Faster and more efficient separation

RBA: Non specific binding

• Ligands bind non-specifically to substances like plastic, filter paper, glass, etc.

• Can be reduced by anti-absorbants

• Measuring proportion of specific and non-specific binding is key element to assay

What is non-specific binding determined by

Addition of excess non-radioactive drug

Specific binding equation

Specific binding = total bound - nonspecific binding

Binding data is plotted on what type of scale

Semi-logarithmic

(Specific binding has a sigmoidal shape that plateaus)

What does the Langmuir/ Scatchard equation describe

Relationship between receptor occupancy, affinity and drug concentration

Specific binding equation in relation to Langmuir/ Scatchard equation

Specific Bound = (Bmax - Xa)/ (Xa + KD)

• measures occupancy

What is Bmax

• Binding capacity, expressed per mg protein

• direct measure of concentration of receptors present

How do you determine KD from Langmuir/ Scatchard graph

The concentration of ligand require to ‘occupy’ 50% of receptors at equilibrium

Does low KD show high or low affinity

High affinity

• Low KD —> high affinity

• High KD —> low affinity

When does Ki ≈ KD

When the inhibitor competes with the ligand for the same binding site on the receptor