BS2013 - Topic 1: Receptors, Agonists and Antagonists

Pharmacology

the study of how drug mechanisms interact with living systems

Receptors

Proteins that receive extracellular information (ligands) and initiate a cellular response.

Agonists

substances that bind to receptors to produce a response

2 Actions of agonists (lock and key analogy)

1. receptor binding

2. receptor activation

Occupancy

proportion of receptors occupied

Simple equation/definition for occupancy

no. of receptors occupied/total no. of receptors

Example of how occupancy can be measured directly?

Radioligand binding

Radioligand binding

use of a radioactive isotope to radiolabel an agonist for detection

Radiolabelling method

1. isolate cells

2. aliquot cells/membranes into multiple tubes or wells

3. add radiolabelled agonist to the samples across a wide range of concentrations

4. equilibrate

5. after equilibration, remove unbound drug by filtration (bound drug remains attached to cells/membranes on filter)

6. count residue to measure the radioactivity

Methods of isolating cells/membranes

• enzymatic treatment to dissociate cells

• detergent treatment and separation by centrifugation

Total binding

the binding of a ligand to all sites in the system (including both the particular receptors of biological interest and non-specific binding)

Non-specific binding

low-affinity, undesired and non-saturable binding of a ligand to components (cell membrane proteins, apparatus) of an experimental system

Specific binding

binding of a ligand to the particular receptors of biological interest

How to measure non-specific binding

• repeat radioligand binding experiment in the presence of saturating concentration of non-labelled agonist

• the radioligand can only bind to non-specific sites, either on the membrane or the filter

How to obtain the specific binding curve (specific binding to receptors of interest)

specific binding = total binding - non-specific binding

How to clearly observe the relationship between agonist binding and concentration on a graph

semi-log plots

semi-log plot

x-axis is plotted on a logarithmic scale (y-axis remains the same)

Benefit of the semi-log plot

allows the relationship between specific binding and agonist concentration to be seen more clearly

Law of Mass Action

RATE of a reversible chemical reaction is proportional to the product of the concentration of the reactants

Langmuir equation assumptions

1. Agonist-receptor interactions are at equilibrium

2. agonist-receptor binding does not reduce agonist concentration (i.e. [agonist] »»[receptor] — the agonist concentration must always be in great excess compared to the no. of receptors)

K_D

the equilibrium dissociation constant

2 parameters that describe the Langmuir equation

• the concentration of the drug (X_A)

• the equilibrium dissociation constant (K_D)

B_max

the maximum no. of receptors bound, achieved when all receptors are occupied by a specific drug (expressed in mol/mg of protein)

Relationship between K_D and affinity

reversely proportional, as the K_D increases the agonist affinity decreases

The Langmuir Equation

P_A (occupancy) = X_A/(X_A + K_D)

The Langmuir equation (experimental)

Bound = B_max x X_A/(X_A + K_D)

Receptor reserve

not all receptors need to be occupied to generate a maximal response

EC₅₀

concentration of agonist required to produce 50% of the maximal response

potency

measure of a drug's activity in terms of concentration required to produce a specific, defined effect.

How does EC₅₀ correlates to potency

lower EC₅₀ = higher potency

K_d (equilibrium dissociation constant)

molar concentration of agonist at which 50% of receptors are occupied at equilibrium

How does K_d correlate to affinity

a lower K_d signifies higher affinity (strong binding)

Advantages of signalling amplification and “receptor reserve”

• small increases in agonist concentration can maximally activate a cellular response (physiologically efficient)

• loss of receptors with age or disease does not readily lead to loss of cell/organ performance

full agonist

an agonist that can stimulate 100% of the maximal response of the tissue

partial agonist

agonists that cannot stimulate 100% of the maximal response (of a specific tissue) regardless of their concentration

E_max

maximum effect a drug can produce, regardless of dose

Efficacy (in pharmacology)

how effectively a drug can stimulate the receptor to generate a functional response

Affinity (in pharmacology)

probability of a drug binding to its receptor at any given instant

Antagonists

a drug that prevents the biological response of agonist

5 different classes of antagonism

• chemical

• pharmacokinetic

• physiological (functional)

• downstream signalling

• receptor antagonism

chemical antagonism

antagonists which combine with or chemically modify an agonist to dampen their effects

pharmacokinetic antagonism

antagonists that reduce agonist concentration within the body by altering absorption, metabolism or excretion of agonist

physiological (functional) antagonism

interaction of two agonists with opposing cellular responses in the body.

antagonism of downstream signalling

antagonists that block a step between receptor activation and functional response

receptor antagonism

When an antagonist competes with an agonist for receptor occupancy (either reversible or irreversible)

competitive receptor antagonist

a ligand which binds at the receptor but produces no response (efficacy = 0) reducing agonist-receptor occupancy, thus inhibiting agonist-receptor responses

Characteristics of a reversible competitive (surmountable) antagonist

• effects of antagonist can be reversed/washed off

• block of the antagonist can be overcome by addition of more agonist

• does not effect the maximal response of tissue to agonist

• more useful clinically as its effects can be controlled more easily (than an irreversible antagonist)

Characteristics of an irreversible competitive (insurmountable) antagonist

• antagonist forms stable often covalent, chemical bonds with the receptor

• effects cannot be reversed/washed off

• block of the antagonist cannot be overcome by further agonist addition

• time dependent

• decreases maximal response of a tissue to the agonist

The Gaddum equation

X_A/(X_A + K_A[1 +X_B/K_B])

Dose ratio

how many fold greater agonist concentration is required in the presence of an antagonist to produce the same response

Equation for dose ratio

[agonist EC50 in presence of antagonist] ÷ [agonist EC50 in absence of antagonist]

pA₂

the negative log of the molar concentration of antagonist required to produce a dose ratio of 2

How does pA₂ correlate to potency

greater pA₂ = greater potency because lower concentration of antagonist is required to shift the EC₅₀ to generate a dose ratio of 2

Schild equation

log₁₀(DR-1) = log₁₀[X_B] - log₁₀K_B

x intercept of a Schild plot represent

negative pA₂

A₂

the molar concentration of antagonist that gives a dose ratio of 2

How to work out the A₂ value from a Schild plot

inverse log of x intercept