1 – Receptor Pharmacology
First-in-class drug discovery approaches
1. Target-based
With a hypothesis already, you already know which protein to target and follow with in vitro/in
vivo screening assays.
2. Systems-based
Looking into a system without a specific hypothesis (random) and monitors phenotypic changes
in vitro/in vivo
Phenotypic screening
- Add chemicals to a model system and see the effects
- High content (screening a lot at once)
- High throughput (a lot of results in a short period of time)
Chemocentric approach
Using known molecules (e.g. salicylic acid for aspirin) as a starting molecule
3. Structure-based
Performing screening in silico, using a computer. We need the crystal structure of the target
protein and the library to perform digital binding and biophysical assays.
Types of target for drug action
1. Receptors
Testing with agonist/antagonist → Interferes with cell-cell communication and intracellular
cascade(e.g. ion channel opening/closing, enzyme activation/inhibition, DNA transcription)
2. Ion channels
Particularly in nerve cells using blockers or modulators to manipulate channel opening
probability
3. Enzymes
Inhibitor → inhibition of normal reaction
False substrate → abnormal metabolite production
Prodrug (inactive drug that needs to be cleaved into their active form) → drug activation
4. Transporters
Testing with inhibitors to block transport or testing with false substrates leading to an
accumulation of abnormal compounds.
Types of drug receptors
1. Receptor-operated channels
When a ligand binds to the receptor, the ion channel opens, hyperpolarization/depolarisation
may occur and lead to cellular effects.
Timescale: milliseconds
2. G protein-coupled receptors
When a ligand binds, small G proteins are released that can open ion channels and stimulate
second messengers (cAMP) → Ca2+ release, protein phosphorylation → cellular effects
Timescale: seconds
,3. Enzyme receptors
It is called a receptor because it is in the membrane but it has enzymatic activities that catalyze
intracellular pathways (e.g. tyrosine kinases). Binding of a ligand outside the cell causes an
enzymatic reaction inside the cell (e.g. protein phosphorylation).
Timescale: hours
4. DNA-coupled receptors
This receptor is found in the nucleus so the ligand has to be lipophilic. When a ligand binds, gene
transcription is activated → protein synthesis → cellular effects.
Timescale: hours to days
Technologies in target discovery
1. Genomics
In genomics, the genes of a healthy person are compared with the genes for a sick person to find
the mutations responsible.
2. Proteomics
This method is difficult as it is hard to find the whole diseased proteomics compared to a healthy
person through challenging MS.
3. Metabolomics
Easier to analyze by using MS
4. Untargeted metabolomics
This method tries to generate a global metabolomic profile of a sample, thus ‘untargeted’. By
using LC/MS on the sample, they make a fingerprint profile of all metabolites in a diseased cell vs
healthy cells and compare them by looking at the peaks.
Drug-receptor interaction
[𝐷] + [𝑅] ↔ [𝐷𝑅]
k1: association constant
k2: dissociation constant
𝑘2 [𝐷][𝑅]
𝐾 = 𝑘1 = [𝐷𝑅] = equilibrium dissociation constant
[𝐷𝑅] [𝐷] 1
[𝑅𝑡]
= [𝐷] + 𝐾
= 1+(𝐾/[𝐷])
[𝑅] = [𝑅𝑡] − [𝐷𝑅]
Rt= total receptors
[𝐷𝑅]
When = 0.5, it means that 50% of the binding sites are occupied.
[𝑅𝑡]
𝐾 = [𝐷] = binding affinity. The higher the affinity, the lower the K value.
Radioactive labels can be used to quantify drug-receptor binding affinity/rate. It can take the shape
of a hyperbolic or semilog (S-shaped) curve.
Determination of specific binding
In a separate experiment, the membrane is incubated with
a high concentration of ligand that is not radioactively
,labeled. A lot is given such that all specific binding is occupied by non-radioactive molecules. The
radioactively labeled compound is then added so it binds to non-specific sites. A straight line will be
obtained because it is a non-saturable phenomenon. Non-specific binding occurs when the receptors
are saturated such that they occupy spaces in between the receptors.
Thus, 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 = 𝑇𝑜𝑡𝑎𝑙 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 − 𝑛𝑜𝑛𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑏𝑖𝑛𝑑𝑖𝑛𝑔
Concentration–effect relationship
[𝐸𝐴] [𝐴]
𝑛
[𝐸𝐴𝑚𝑎𝑥]
= 𝑛 𝑛
[𝐴] +𝐾𝐴
[A] = concentration of ligand
n = hill slope
Potency of a drug is defined as the drug concentration where 50% of the maximum effect is reached.
Highest potency is when the lowest concentration of drug is needed for maximum effect.
Affinity of a drug is defined as the drug concentration where 50% of the maximum binding is
reached.
Efficacy of a drug is defined as the maximum effect you can reach with a compound.
Types of agonism
- Full agonist: the compound likes to bind to the active state, hence it pulls equilibrium to the
active state.
- Partial agonist: The receptor is active by itself even in the absence of ligands and the compound
cannot fully pull the equilibrium
- Neutral agonist: has equal affinities to both resting and activated state, thus show no effects
- Inverse agonist: has more affinity to the resting state, thus reduce basal activity
, Receptor desensitization
Refers to the decreased responsiveness that occurs with repeated or chronic exposure to agonist. It
can occur by a number of mechanisms:
- The receptor is uncoupled from its signaling cascade, such that activation will lead to an
attenuated intracellular effect
- Some drugs bind relatively strongly to a receptor and switch it on, but then subsequently block
the receptor after a certain period of time. The mechanism of how this takes place is not clear,
but it is believed that prolonged binding of the agonist to the receptor results in the
phosphorylation of hydroxyl or phenolic groups in the receptor. This causes the receptor to
alter shape to an inactive conformation despite the binding site being occupied
- The receptor/drug complex may be removed completely from the cell membrane by
endocytosis
- Cell reducing its synthesis of the receptor protein
Types of antagonism
- Competitive antagonist
This type of antagonism results in a right parallel shift of the curve (as in more agonists will be
needed to overcome the effect). You can overcome the effects by increasing the concentration of
the agonist. In a Schild’s plot, the linear slope is ~1.
- Uncompetitive/Allosteric antagonist: does not bind to the ‘native’ substrate binding site but
deforms the receptor such that no reaction occurs between the receptor and the ‘native’
substrate. You cannot overcome it by increasing concentration. In a Schild’s plot, the linear slope
is not ~1 and in a dose-response curve leads to the decrease in maximum effect.
Receptor sensitization
Can occur when an antagonist is bound for a long period of time. The cell synthesizes more receptors
to counter the antagonistic effect. This could lead to tolerance (increased doses of the antagonist are
required to achieve the same effect) and/or dependence (withdrawal symptoms occur upon acute
stopping of the antagonist).
Gaddum’s equation
First-in-class drug discovery approaches
1. Target-based
With a hypothesis already, you already know which protein to target and follow with in vitro/in
vivo screening assays.
2. Systems-based
Looking into a system without a specific hypothesis (random) and monitors phenotypic changes
in vitro/in vivo
Phenotypic screening
- Add chemicals to a model system and see the effects
- High content (screening a lot at once)
- High throughput (a lot of results in a short period of time)
Chemocentric approach
Using known molecules (e.g. salicylic acid for aspirin) as a starting molecule
3. Structure-based
Performing screening in silico, using a computer. We need the crystal structure of the target
protein and the library to perform digital binding and biophysical assays.
Types of target for drug action
1. Receptors
Testing with agonist/antagonist → Interferes with cell-cell communication and intracellular
cascade(e.g. ion channel opening/closing, enzyme activation/inhibition, DNA transcription)
2. Ion channels
Particularly in nerve cells using blockers or modulators to manipulate channel opening
probability
3. Enzymes
Inhibitor → inhibition of normal reaction
False substrate → abnormal metabolite production
Prodrug (inactive drug that needs to be cleaved into their active form) → drug activation
4. Transporters
Testing with inhibitors to block transport or testing with false substrates leading to an
accumulation of abnormal compounds.
Types of drug receptors
1. Receptor-operated channels
When a ligand binds to the receptor, the ion channel opens, hyperpolarization/depolarisation
may occur and lead to cellular effects.
Timescale: milliseconds
2. G protein-coupled receptors
When a ligand binds, small G proteins are released that can open ion channels and stimulate
second messengers (cAMP) → Ca2+ release, protein phosphorylation → cellular effects
Timescale: seconds
,3. Enzyme receptors
It is called a receptor because it is in the membrane but it has enzymatic activities that catalyze
intracellular pathways (e.g. tyrosine kinases). Binding of a ligand outside the cell causes an
enzymatic reaction inside the cell (e.g. protein phosphorylation).
Timescale: hours
4. DNA-coupled receptors
This receptor is found in the nucleus so the ligand has to be lipophilic. When a ligand binds, gene
transcription is activated → protein synthesis → cellular effects.
Timescale: hours to days
Technologies in target discovery
1. Genomics
In genomics, the genes of a healthy person are compared with the genes for a sick person to find
the mutations responsible.
2. Proteomics
This method is difficult as it is hard to find the whole diseased proteomics compared to a healthy
person through challenging MS.
3. Metabolomics
Easier to analyze by using MS
4. Untargeted metabolomics
This method tries to generate a global metabolomic profile of a sample, thus ‘untargeted’. By
using LC/MS on the sample, they make a fingerprint profile of all metabolites in a diseased cell vs
healthy cells and compare them by looking at the peaks.
Drug-receptor interaction
[𝐷] + [𝑅] ↔ [𝐷𝑅]
k1: association constant
k2: dissociation constant
𝑘2 [𝐷][𝑅]
𝐾 = 𝑘1 = [𝐷𝑅] = equilibrium dissociation constant
[𝐷𝑅] [𝐷] 1
[𝑅𝑡]
= [𝐷] + 𝐾
= 1+(𝐾/[𝐷])
[𝑅] = [𝑅𝑡] − [𝐷𝑅]
Rt= total receptors
[𝐷𝑅]
When = 0.5, it means that 50% of the binding sites are occupied.
[𝑅𝑡]
𝐾 = [𝐷] = binding affinity. The higher the affinity, the lower the K value.
Radioactive labels can be used to quantify drug-receptor binding affinity/rate. It can take the shape
of a hyperbolic or semilog (S-shaped) curve.
Determination of specific binding
In a separate experiment, the membrane is incubated with
a high concentration of ligand that is not radioactively
,labeled. A lot is given such that all specific binding is occupied by non-radioactive molecules. The
radioactively labeled compound is then added so it binds to non-specific sites. A straight line will be
obtained because it is a non-saturable phenomenon. Non-specific binding occurs when the receptors
are saturated such that they occupy spaces in between the receptors.
Thus, 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 = 𝑇𝑜𝑡𝑎𝑙 𝑏𝑖𝑛𝑑𝑖𝑛𝑔 − 𝑛𝑜𝑛𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑏𝑖𝑛𝑑𝑖𝑛𝑔
Concentration–effect relationship
[𝐸𝐴] [𝐴]
𝑛
[𝐸𝐴𝑚𝑎𝑥]
= 𝑛 𝑛
[𝐴] +𝐾𝐴
[A] = concentration of ligand
n = hill slope
Potency of a drug is defined as the drug concentration where 50% of the maximum effect is reached.
Highest potency is when the lowest concentration of drug is needed for maximum effect.
Affinity of a drug is defined as the drug concentration where 50% of the maximum binding is
reached.
Efficacy of a drug is defined as the maximum effect you can reach with a compound.
Types of agonism
- Full agonist: the compound likes to bind to the active state, hence it pulls equilibrium to the
active state.
- Partial agonist: The receptor is active by itself even in the absence of ligands and the compound
cannot fully pull the equilibrium
- Neutral agonist: has equal affinities to both resting and activated state, thus show no effects
- Inverse agonist: has more affinity to the resting state, thus reduce basal activity
, Receptor desensitization
Refers to the decreased responsiveness that occurs with repeated or chronic exposure to agonist. It
can occur by a number of mechanisms:
- The receptor is uncoupled from its signaling cascade, such that activation will lead to an
attenuated intracellular effect
- Some drugs bind relatively strongly to a receptor and switch it on, but then subsequently block
the receptor after a certain period of time. The mechanism of how this takes place is not clear,
but it is believed that prolonged binding of the agonist to the receptor results in the
phosphorylation of hydroxyl or phenolic groups in the receptor. This causes the receptor to
alter shape to an inactive conformation despite the binding site being occupied
- The receptor/drug complex may be removed completely from the cell membrane by
endocytosis
- Cell reducing its synthesis of the receptor protein
Types of antagonism
- Competitive antagonist
This type of antagonism results in a right parallel shift of the curve (as in more agonists will be
needed to overcome the effect). You can overcome the effects by increasing the concentration of
the agonist. In a Schild’s plot, the linear slope is ~1.
- Uncompetitive/Allosteric antagonist: does not bind to the ‘native’ substrate binding site but
deforms the receptor such that no reaction occurs between the receptor and the ‘native’
substrate. You cannot overcome it by increasing concentration. In a Schild’s plot, the linear slope
is not ~1 and in a dose-response curve leads to the decrease in maximum effect.
Receptor sensitization
Can occur when an antagonist is bound for a long period of time. The cell synthesizes more receptors
to counter the antagonistic effect. This could lead to tolerance (increased doses of the antagonist are
required to achieve the same effect) and/or dependence (withdrawal symptoms occur upon acute
stopping of the antagonist).
Gaddum’s equation
