Pharmacodynamics 1:
Affinity and Efficacy
A/Prof Angela Finch
1
Applied
Concepts
Core Concepts
PHAR2011 – Introductory Pharmacology and Toxicology Map
Pharmacodynamics
“what the drug does to the body”
Drug Properties
Pharmacokinetics
“what the body does to the drug”
Drug binds to protein target
Drug type: small molecule,
peptide/protein, biological
Absorption: dose/route
Drug selectivity
Signalling and
cellular response
Side effects and
toxicity
Physiological/
therapeutic
response
Drug safety and
pharmacovigilance
Measured effect
(affinity/potency/
efficacy)
Drug action: agonist, partial agonist,
antagonist
Distribution: drug movement and
concentration in the body
Metabolism: how the drug is degraded
Excretion: drug removal
Drug safety and pharmacovigilance
Autonomic targets to treat
disease
Neurotransmitter regulation to
treat disease
Autacoid regulation to treat
disease
Asthma
Neuromuscular Disorders
Migraine
Cardiovascular Disease
Migraine
Gastrointestinal Disorders
Overactive Bladder
Depression
Inflammation, Itch and Allergy
Neuromuscular Disorders
ADHD
Asthma
Learning Objectives
By the end of this lecture you should be able to:
1. Describe the concept of affinity and how it is measured
2. Apply concepts of affinity to interpret pharmacological data
3. Describe the concept of efficacy and how this relates to agonists and antagonists
4. Identify the features of a concentration response curve and relate them to drug
action
5. Identify full agonists and partial agonists based on pharmacological data
3
Agents cannot act without binding
“corpora non agunt, nisi fixata”
Ehrlich P. Lancet 1913; 2:445-451.
Paul Ehrlich
The Nobel Prize in
Physiology or Medicine 1908
“The combining group of the protoplasmic molecule to which the
introduced group is anchored will here after be termed receptor”
Paul Ehrlich 1909
Drug affinity is the physical interaction between the drug and receptor
Drug affinity measures how strongly a drug interacts with the receptor
1
4
Types of interactions between a receptor and a ligand
Multiple Noncovalent Bonds Confer Binding Affinity
1
Molecular Cell Biology. 4th edition., Lodish et al. W. H. Freeman; (New York:,
USA) 2000.
Molecular Biology of the Cell. 4th edition. Alberts B, et al. New York:
Garland Science; 2002.
5
How does the interactions between the ligand and receptor relate
to the affinity and specificity of a drug?
• Affinity is the strength of the interaction between a ligand and a receptor.
It is governed by:
• Shape complementarity. For example, if a molecule doesn’t fit into the binding pocket less
van der Waals interactions can occur leading to decreased affinity
• The strength and number of non-covalent interactions between the ligand and
the receptor. If more and/or stronger the bonds form, then the affinity will be higher.
• Specificity describes the case when a ligand has increased affinity for one receptor
over others.
It is governed by:
• shape complementarity
• specific interactions between the ligand and receptor
1
6
Why do we want to know the affinity of our ligands?
When we are developing drugs knowing if they have affinity for our target (and any other target) helps in
the development process. Establishing the affinity a compound has for the target allows us to compare it
to other compounds so we can develop high affinity compounds.
Ligand binding assays are easy to perform, can be automated to achieve high throughput. The data allows
for the determination of drug affinity, but also can detect allosteric interactions, the characterisation of
receptor subtypes, and estimates of level of receptor expression in a tissue/cell.
https://www.bmglabtech.com/ru/high-throughput-screening/
7
How can we quantify the binding of a ligand (drug) to a receptor?
Receptor + Ligand ⇌ R.L (complex)
kon
R + L ⇌ R.L (complex)
koff
kon units = M-1s-1 concentration dependent rate
koff units = s-1 concentration independent rate
1
8
Affinity
The strength of the reversible interaction between a drug and its receptor, as measured by the binding
dissociation constant (KD)
kon
R + L ⇌ R.L (complex)
koff
koff
kon
=
KD =
[R] x [L]
[RL]
• Bmax is the total density (concentration) of
receptors in a sample of tissue or per cell
• KD is the ligand concentration at which half of
the total number of receptors are bound to
ligand
1
KD units = M
Bmax units = sites/cell or fmol/mg protein
9
Experimentally measuring affinity: Saturation Assays
We need a method to measure the amount of ligand bound to the receptor. Common approaches
are to label the ligand with either a radioactive isotope (3H, 125I) or a fluorescent molecule.
Labelled ligand
Incubate labelled ligand
and receptor (e.g. cells,
tissue or cell membrane)
Remove unbound
labelled ligand
(e.g. filtration)
Measure signal from
the label on the ligand
Receptor in
cell membrane
Saturation binding experiments use increasing concentrations of radioligand and require
incubation until equilibrium is reached.
1
Aroaoz et al., Anal Bioanal Chem (2010) 397:1695–1704
10
Experimentally measuring affinity: KD and Bmax
Comparing the affinity of three different
radioligands for the same receptor
1, 2
Comparing the expression level of two
different receptors.
11
Don’t confuse saturation binding with enzyme kinetics
(mol/s)
Michaelis-Menten equations
• Bmax is the total density (concentration) of
receptors in a sample of tissue or per cell
• KD is the ligand concentration at which half of
the total number of receptors are bound to
ligand
1
• Vmax is the maximum rate of an enzyme catalysed
reaction
• Km is the concentration of substrates when the
reaction reaches half of Vmax
Experimentally measuring affinity: Competition binding assays
Labelling ligands is expensive, time consuming and sometimes impossible. Competition binding assays allow us to
indirectly measure the affinity of an unlabelled ligand by determining the amount of the test ligand required to
compete for receptor binding with the labelled ligand.
Competitive binding experiments use a single concentration of labelled ligand and increasing concentrations
of unlabelled ligand and require incubation until equilibrium is reached.
Decreasing binding of
labelled ligand
The affinity of the
unlabelled ligand (IC50 ) is
the concentration of drug
required for 50% inhibition
of labelled-ligand binding
unlabelled-ligand
labelled-ligand
receptor
Increasing concentration of
unlabelled ligand
1
13
Experimentally measuring affinity: Competition binding assays
Drug
IC50 (nM)
1
0.9
2
10
3
0.26
• Drug 3 has the highest affinity
• Drug 2 has the lowest affinity
1, 2
14
Experimentally measuring affinity: Ki
The IC50 is a property of the experiment, as it is dependent on the concentration of labelled ligand. We can
convert this to an absolute value for a ligand and receptor pair that is independent on the amount of labelled
ligand added by using the Cheng-Prusoff equation.
Cheng–Prusoff equation
[L] = the concentration of the labelled ligand
Kd = is the affinity of the labelled ligand for the receptor – this is determined by doing a saturation assay
Ki = equilibrium dissociation constant for binding of the unlabelled drug to the receptor
Note: Ki is a property of the receptor and unlabelled drug, while IC50 is a property of the experiment.
1
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Experimentally measuring affinity: Ki
The IC50 values for compound-X are 20 nM when 10 nM of labelled-ligand was used and 500 nM when 500 nM of
labelled-ligand was used.
500 nM
The labelled-ligand has a KD
for the receptor of 10 nM
20 nM
Cheng–Prusoff equation
Ki = 20 nM/ (1+ 10 nM/10 nM) = 10 nM
Ki = 500 nM/ (1+ 500 nM/10 nM) = 10 nM
Ki = 10 nM for compound-X under both conditions.
1, 2
16
Efficacy
The extent to which a drug can produce a response
Ligand (L) + inactive Receptor (R)
Response
LR
Occupancy governed by
AFFINITY
LR* (R* =active receptor)
Activation governed by
EFFICACY
3
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Efficacy: The extent to which a drug can produce a response
Full Agonist
Partial agonist
Antagonist
Maximum Effect
Smaller Effect
No Effect
Agonist: a compound that can bind to and cause activation of a receptor, thus mimicking the
actions of the endogenous ligand.
Antagonist: a compound that can bind to but not activate a receptor, thus blocking the actions
of the endogenous ligand.
3, 5
18
Experimentally measuring efficacy
Concentration (Dose)-response curves
Concentration-response relationships graphically describe the relationship between the
concentration of a ligand applied to cells and the resulting response.
Linear scale (hyperbolic)
4
Semi-log scale (sigmoidal)
19
Concentration response curves- Slope (Hill Slope)
slope
threshold
Response (% maximum)
maximum
A
B
Drug A slope = 1
Drug B slope = 4
Log Drug concentration (nM)
Concentration response curves typically
have a slope of 1 (Hill Slope).
A Hill slope of 1 will occur when a ligand
binds to a single binding site (receptor)
Hill slope >1 indicates positive cooperativity.
Hill slope <1 indicates negative cooperativity.
Perelson & Deeks (2011) Sci Transl Med. 3(91):91ps30
4
Experimentally measuring efficacy
Concentration response curves- Emax
Efficacy refers to the maximum response that can be achieved with a drug (maximum
usually compared to a reference ligand). Given by Emax (maximum effect or response)
Drug
Emax (%)
A
100
B
100
Drug B = Full agonist
– an agonist with maximal efficacy
compared to the reference ligand (drug A)
3, 5
21
Experimentally measuring efficacy
Concentration response curves- Emax
Efficacy refers to the maximum response that can be achieved with a drug (maximum
usually compared to a reference ligand). Given by Emax (maximum effect)
Drug
Emax (%)
A
100
B
100
C
50.4
Drug C = Partial agonist
– an agonist with less
than maximal efficacy
3, 5
22
Learning Objectives
By the end of this lecture you should be able to:
1. Describe the concept of affinity and how it is measured
2. Apply concepts of affinity to interpret pharmacological data
3. Describe the concept of efficacy and how this relates to agonists and antagonists
4. Identify the features of a concentration response curve and relate them to drug
action
5. Identify full agonists and partial agonists based on pharmacological data
23
You have tested three new drugs (A, B, & C) that activate the Class I nuclear receptor WB1. You have included hormone WB, a
known full agonist for the WB1 receptor, in your assay (data displayed in the graphs above).
(i) Rank the potency of Drugs A to C and hormone WB from highest to lowest and include the EC50 for each compound. How did
you determine the ranking? [3.5 marks]
(ii) Rank the efficacy of Drugs A to C and hormone WB from highest to lowest. How did you determine the efficacy? [1.5 mark]
(iii) What type(s) of agonist are each of the drugs? [1 mark]
(iv) Describe the structural characteristics of the family of receptors the WB1 receptor belongs to. [1.5 marks]
(v) Describe how this type of receptor signals. Include in your answer the time scale for a cellular response. [2.5 marks]
Which ONE of the following conclusions can be made based on the data above?
A. [3H] spiperone has a higher affinity for the D2 receptor than compounds 1-4
B. Compound 2 has a longer binding time at the D2 receptor than compound 3
C. Compound 4 has a higher affinity for the D2 receptor than compound 2
D. Compound 3 has higher potency at the D2 receptor than compound 1
E. Compounds 1, 2, 3 and 4 have equal efficacy at the D2 receptor