Understanding Receptors

Dermot Cox BSc, PhD

Receptors
It is critically important that a cell can
respond to the changing demands that are
put on it. To facilitate this it is necessary for
the cell to detect changes in the environment
and to direct the cell to respond to those
changes. A key component of this is the
hormone-receptor system. The body signals
cells of a need to alter their function through
the production of hormones or
neurotransmitters. Cells have receptors that
detect these changes in hormone levels and
initiate the response. There are four families
of receptors based on their structure:
ligand-gated ion channels, G-proteincoupled receptors, tyrosine kinase receptors
and intracellular receptors.

Hormone

Hormone

Hormone

Hormone

Receptors and smoke detectors

One of the most familiar sensors is the smoke
detector. The smoke detector (receptor)
specifically detects smoke (hormone) and
generates a variety of signals (second
messengers) including sound (alarm) and light.
These in turn trigger responses such as
evacuation of the building, sprinklers turning on
and fire tenders being summoned.
The adrenaline receptor is a typical example of a
receptor. It is a sensor that specifically detects the
hormone adrenaline. This then triggers the
production of the second messenger cyclic
adenosine monophosphate (cAMP). This in turn
triggers a variety of responses such as an
increase in heart rate, an increase in glucose
production and changes in vascular tone

Receptor

milliSeconds Seconds Minutes Hours/days

There are three stages in the functioning of

the signal transduction pathway. The Airst is
binding of the hormone to the receptor. This
then initiates the signal transduction
pathway and each of the four receptor
families has its own characteristic pathway.
Ultimately this leads to a cellular response.
Receptors do not change the function of a
cell they simply make it do it better/faster or
reduce its ability to function. For Aine control
of cell activity it is necessary for signals to
be terminated. In the absence of an
additional signal this allows the cell to
return to its basal/resting state.

Hormone

2nd messenger

Response

G-Protein-Coupled Receptors
(GPCR)
These are monomeric membrane proteins
with a molecular weight (MW) 35-70 kDa. A
characteristic feature is that they pass
through the membrane 7 times and thus are
also known as 7 transmembrane domain
receptors. There are at least 500 different
GPCRs including light, taste, smell and
neurotransmitters.

As their name implies these receptors are

linked to G-proteins, which are enzymes that
convert GTP to
GDP, i.e., they are
GTPases. They
exist in a resting
state when GDP
is bound. The
actions of a
Guanine
nucleotide
Exchange Factor
(GEF) replaces
the GDP with
GTP activating
the G-protein and triggering down-stream
signalling. Then a GTPase-Accelerating
Protein (GAP) enhances the GTPase activity

of the G-protein causing it to convert the

GTP to GDP and thus become inactive.
There are two families of G-proteins -
monomeric (or small) and trimeric. GPCR
are coupled to the trimeric G-proteins.
These are composed of 3 subunits - , and
with the GTPase activity residing in the G
subunit. The GEF for the trimeric G-proteins
are activated GPCR. Thus, hormone binds to
the GPCR on the cell surface and activates it
which in turn binds to the G-protein and

activates it. The activated G-protein binds to

and activates an effector enzyme which
triggers down-stream signalling. At this

point the GAP protein binds activating the

GTPase activity which converts the GTP to
GDP and thus inactivating the G-protein. A
common GAP for GPCRs is a protein called
Regulator of G-protein Signalling (RGS).
There are two effector enzymes used in
GPCR signalling - adenylate cyclase and
phospholipase.

G-protein subunits. The subunit is the key

subunit in the signalling process as it has the
GTPase activity as well as directly activating
the effector enzymes. There are four families
of submit: s, i, q and some
miscellaneous subunits such as 12. The
other subunits exist as a dimer. While
they have no enzymatic activity they can
independently regulate signalling. There are
6 different -subunits and 11 different subunits. One role for the -subunit is to
act as a Guanine nucleotide Dissociation
Inhibitor (GDI) which acts to prevent the
GTP being replaced by GTP. In other words
when is bound to it keeps the subunit
in the resting state.
Gs. This subunit acts to activate adenylate
cyclase and thus will increase cAMP levels in
the cell.
Adenylate Cyclase
Adenylate cyclase is an enzyme that
converts ATP into cyclic AMP (cAMP)
(see fig). cAMP is an important regulator
of activity in many cells. There are at
least 9 different isoforms of adenylate
cyclase.
Signal termination is mediated by
phosphodiesterase which breaks down
cAMP converting it into AMP which is
inactive.

Kinases & Phosphatases

Kinases are enzymes that phosphorylate proteins
and phosphatases are enzymes that dephosphorylate proteins. Phosphorylation status is a
very important regulator of enzyme activity.
Protein kinase A: PKA is activated by cAMP
Protein kinase C: PKC is directly activated by DAG

Gi. This subunit acts to inhibit adenylate

cyclase and thus will prevent an increase in
cAMP levels.

cAMP act activates protein kinase A. This in

turns phosphorylates a number of enzymes
that subsequently become active and are
responsible for the response to hormones
such as adrenaline.
Gq. This subunit acts to activate
phospholipase C (PLC). PLC hydrolyses
phosphoinositide from the membrane
producing diacylglycerol (DAG) and inositol
triphosphate (IP3).
DAG is the lipid component of
phosphoinositide and activates PKC. Its
actions are terminated by conversion to
phosphatidic acid.

IP3 is the water soluble part of

phosphoinositide and activates an IP3
receptor on the endoplasmic reticulum. The
is an ion channel and causes an inAlux of Ca2+
into the cytosol leading to the activation of
calmodulin and other Ca-dependent
enzymes. The signal is terminated by
conversion of IP3 to IP2.

As there are at least 4 different -subunits, a

variety of -subunits and at least two
different effector systems all linked to a
range of different receptors it is not
surprising that there are so many different
GPCRs and that they are the dominant drug
targets.
Signal termination. As well as using enzymes
to metabolise the signalling molecules such
as adenylate cyclase, GPCR also use other
termination mechanisms. One of these
involves receptor deactivation typically
through phosphorylation. Another process is
receptor internalisation where the receptor
is internalised in vesicles. The internalised
receptor is either degraded or recycled back
to the membrane for re-use.

Enzyme-Linked Receptors
These are membrane-bound proteins with
an extracellular hormone recognition
domain and an intracellular domain has
region with associated enzyme activity.
Many growth factors and hormones bind to
a family of receptors where the enzyme
activity is that of a tyrosine kinase. The
response time for enzyme-linked receptors
is slower than ion channels and G-proteincoupled receptors and will minutes to hours.

These newly formed phosphorylated

tyrosines are recognised by the SH2 domain
of an adapter protein such as Grb2. The
corresponding SH3 domain of Grb2 binds to
a signalling protein such as SoS. This in turn
recruits the small G-protein Ras and
activates it (converts it to the GTP-bound
form). Ras then commences a chain of
signalling event by phosphorylating Raf,
which in turn phosphorylates MEK which in
turn phosphorylates ERK. Ultimately this
results in altering gene transcription.

Enzyme-Linked Receptors
While tyrosine kinase activity is the main
enzyme activity of these receptors there are
other enzymes involved.
Atrial Naturetic Peptide (ANP) is
associated with guanylate cyclase activity
Tumour Necrosis Factor (TNF) receptor
has no inherent enzyme activity but
recruits these enzymes using adapters

Tyrosine kinase linked receptors

Tyrosine kinase is an enzyme that
phosphorylates tyrosine residues. A typical
tyrosine kinase-linked receptor is the
Epidermal Growth Factor (EGF) receptor.
After binding of EGF to its receptor,
dimerisation occurs. This dimerisation is
critical as it allows the kinase domain of one
receptor to phosphorylate the tyrosines of
the other receptor.

Ras
Ras is a small G-protein and thus is a GTPase that is
active when GTP is bound and inactive when GDP is
bound. As Ras is important in signalling by tyrosine
kinase receptor, many of which are growth factor
receptors, it plays an important role in cell growth.
Mutations in Ras that make it constitutively active
(permanently switched on) will over-stimulate cell
growth and thus is an oncogene that is associated
with many cancers.
SOS (son of sevenless) is a family of guanine
nucleotide exchange factors (GEF) and acts to
activate Ras.

Adapters
A characteristic of enzymelinked receptors is that they
cannot directly interact with
signalling molecules - they
use adapter proteins to
mediate the interaction.
Adapters do not participate
in signalling they just
facilitate the interaction
between signalling proteins.
Grb2 is a typical adapter.
One side has an SH2
domain which binds tyrosine phosphates. The other
side has an SH3 domain which can bind to a specific
proline sequence in proteins.

Ion Channel Receptors

rapid response usually in the millisecond

range.

Ion channels are transmembrane proteins

that allow the passage of speciAic ions. There
are three major types - voltage-gated,
ligand-gated and stress-activated.

Voltage-gated ion channels are regulated by

alteration in the membrane potential and
play an important role in nerve conduction.
Ligand-gated ion channels are regulated by
binding of hormone/neurotransmitter.
There are 4 families of ligand-gated ion
channels - P2X receptor-like, Nicotinic
receptor-like, Glutamate receptor-like and
the GluR0 receptor.

Within the receptor families the ion

channels are selective for either positive or
negative ions. While there are structural
differences between the different families
they all work on the same general principle.
We will use the Nicotinic-receptor-like
family as an example of an ion channel as it
is the most important ligand-gated ion
channel family. Ion channels deliver a very

Structure. Typically ion channels are

multimeric transmembrane proteins. There
are potentially 4 different subunits that are
used to form ion channels - , , and .
Each subunit passes through the membrane
4 times with both N and C termini being
extracellular. Usually 5 or more subunits are
involved. The subunits are clustered
together in a
circular pattern
(as seen from
above). This
creates a pore in
the centre of the
complex. The
charge within the
pore determines
which ion can
pass through and
the size of the pore also adds ion selectivity.
The -subunit contains the hormone
binding site and if there are 2 subunits
then two molecules of neurotransmitter can
bind. Neurotransmitter binding causes an
opening of the receptor (activation) which
allows the ion to pass through. When the
neurotransmitter leaves the receptor the
channel closes preventing ions from passing
through. The channel then goes through a
phase where it no longer responds to
binding of neurotransmitter (desensitised
receptor) but ultimately returns to the

resting state ready to respond to

neurotransmitter again.
Cholinergic receptors. Acetylcholine
receptors can be classiAied according to the
signalling system that they use. Some bind
muscarine (a component of some
mushrooms) and these receptors are Gprotein coupled. The others bind nicotine
and these are ion channels. The nicotinic
receptors can be further divided into those
that are found in skeletal muscle and
neuromuscular junction and those that are
found in ganglia. The skeletal muscle
nicotinic receptors are formed from 2, 1,
1 and 1 (see Aig) subunits while the
ganglionic receptors are formed from 8
and 4. These are positive ion (sodium)
channels and the increase in positive charge
within the cell leads to activation.
Acetylcholine Receptors
Nicotinic Acethlycholine Receptors
[nAChR]
Channel-linked Receptors

Muscarinic Acetylcholine Receptors

[mAChR]
G-protein coupled receptors

Skeletal Muscle
Neuromuscular junction

Ganglionic
CNS

Pentamer
2, , ,

Various permutations
8 x ; 4 x

GABA receptors. GABA (-aminobutyric acid)

is a neurotransmitter that binds to a
negative ion channel (Cl- ion). The inAlux of
negative ions inhibits cellular function and
these receptors are inhibitory receptors in
the CNS. Its structure is 2, 2 and 1. The
GABA receptor is also the receptor for a
number of drugs such as benzodiazepines,
barbiturates and ethanol. Typically these
drugs act to modulate the actions of GABA.

GPCR-linked ion channels. Some GPCRs

directly open an ion channel. This is a slower
response to a ligand-gated ion channel.

Intracellular Receptors
The fourth family of receptors are unique as
they are found in the cytoplasm rather than
on the cell membrane. Thus, they only
respond to lipophilic hormones such as
steroids (e.g. glucocorticoids such as
cortisol) as the hormone needs to pass
through the cell membrane to get access to
the receptor. Drug binding to the receptor
forms a drug-receptor complex. This
complex can pass through the cytoplasm
and enter into the nucleus where it binds to
the DNA at a site known as a response
element. This then alters gene expression -
either increasing gene expression or
decreasing gene expression.

Heat shock proteins play multiple role with

the glucocorticoid receptor. Thus, Hsp70
binds to the receptor causing a
conformational change that causes the
ligand to dissociate, i.e. it inactivates the
receptor. On the other hand Hsp90 binds to
the receptor causing a re-folding of the
receptor that facilitates ligand binding and
receptor activation.
Heat Shock Proteins (Hsp)
Heat shock proteins are a family of
proteins that are dramatically
upregulated after cells are exposed to
heat and act to chaperone other proteins.
They act to help proteins fold, to re-fold
mis-folded proteins and to guide proteins
to the correct location. They are named
according to their molecular weight (eg
Hsp70 is a family of 70 kDa Hsp.

The receptor is retained in a resting state in

the cytoplasm by the binding of heat-shock
proteins (HSPs). This complex of HSPs
prevent the receptor from entering the
nucleus. When a glucocorticoid such as
cortisol enters the cytoplasm it binds to the
ligand-binding domain on the receptor. This
results in a conformational change that
displaces the HSPs. The cortisol-receptor
complex dimerises and enters into the
nucleus where it binds to the glucocorticoid
response element (GRE). The result is
inhibition of expression of pro-inAlammatory
cytokines. Ultimately this will lead to a
reduction in inAlammation.

Complexity of Receptor Signalling

Cell signalling can be very complex and often
multiple pathways are activated. The Aigure
below shows an overview of the insulin
receptor signalling pathway and clearly
illustrates its complexity. However if you
examine it closely you can see that this is a
tyrosine kinase receptor as it is a dimerised
receptor. You can also see that it binds Grb2,
which recruits SOS and in turn activates Ras.
This then leads to activation of the Raf-MEKErk pathway. This however, is only one small
part of the signalling pathway.

The reason for complexity in signalling is

signal ampliAication. Each step tends to
activate an enzyme. Each enzyme can
activate many substrate enzymes which in
turn can activate many more enzymes. This
leads to a massive signal ampliAication.

There is also a rationale for multiple

receptor types. Ion channels generate a very
rapid response, within milliseconds, while
the GPCR take seconds to generate a
response. Tyrosine kinase-linked
and intracellular linked receptors
taking much longer. Some Tyrosine
kinase-linked receptor response can
happen within minutes but
responses that involve alterations to
gene transcription can take hours/
days to become effective.

Many receptors activate more than

one signalling pathway. For instance
with GPCR we see that the -subunit
triggered a different pathway to the
-subunit. In the example below we
see that with GABA receptors (GPCR
subtype) the -subunit inhibits
adenylate cyclase while opens ion
channels. Similarly there are
multiple pathways with tyrosine
kinase receptors (see Aig).

In summary there are four families of

receptors with different signalling
pathways which allows for multiple
response with different response times.
Cells have more than one receptor on their
surface some are inhibitory and others are
excitatory. The ultimate effect on the cell is
the sum of all of the inhibitory and
stimulatory signals.

Receptors as Drug Targets

Drugs do not act unless bound
Paul Ehrlich, 1913

Drug targets
Drug targets are the molecular entities that
drugs interact with. Virtually every drug
binds to a protein typically a receptor
although some bind to enzymes. The main
drug targets are GPCRs (27%), cytosolic
receptors (13%) and ligand-gated ion
channels (8%).

Enzymes as drug targets

While receptors are the main drug target there are a
number of important drugs that target enzymes such
as drugs that inhibit cholesterol synthesis, coagulation
or bacterial growth.
Some drugs target the enzymes of the receptor
signalling cascade. The best example of this is aspirin
which inhibits cyclooxygenase an important signalling
molecule in platelets and immune cells.
The principles used to describe the interaction
between drugs and receptors are similar to those that
describe the integration of drugs with enzymes.

In the case of many receptors there may be

multiple targets. So if we take the example of
a tyrosine kinase receptor we can see that
drugs can either bind to the growth factor or
bind to the
receptor to
prevent binding
of growth factor
to the receptor.
Drugs can also
bind to the
dimerisation site
preventing
signalling rather
than growth
factor binding.
Alternatively
drugs can inhibit
some of the
signalling enzymes such as the tyrosine
kinase activity or any of the other kinases
involved in the signalling cascades.

Any compound that speciAically binds to a

receptor is classed as a ligand. Hormones,
neurotransmitters and their synthetic
analogues all speciAically bind to receptors
and trigger a response. They are known as
agonists. Some of the synthetic analogues
also bind but they are never as effective as
their natural counterpart and are known as
partial agonists.
Receptor characteristics
Paul Ehrlich described the key
characteristics of receptors:
They have structural and steric
specificity
They are expressed in specific tissues
They are saturable and have a finite
number
They have a high affinity for their
endogenous ligand (hormone)
Binding of hormone triggers a
biochemical event

Speci=icity. Receptors have a high level of

speciAicity for their natural hormone. For
instance adrenaline (epinephrine) binds to
adrenoreceptors which trigger such things
as an increase in heart rate. On the other
hand dopamine which only differs by the
addition of a methyl and hydroxy group
binds to dopamine receptors instead which
regulate reward
and control
movement in
the brain. This
speciAicity is
due to the 3D
structure of the
receptor.
Typically this
3D structure creates a binding pocket that
has a speciAic size, shape and charge. The
Aigure shows the 3D structure of the
histamine H1 receptor. This 3D structure
produces a binding pocket with a very
speciAic shape, size and charge. The yellow
balls show doxepin (H1 antagonist) sitting

Saturability. Any cell or tissue has a Aixed

number of receptors thus at some
concentration there is enough agonist
present to engage with all of the receptors.
This creates the concept of a maximum
response i.e, the response that is generated
when all of the receptors are occupied by
agonist is the maximum response. On the
other hand a partial agonist delivers its
maximum response when it occupies all of
the receptors but this is less than that
obtained with a full agonist.
The dose-response relationship. If we reduce
the concentration of agonist below the
concentration that causes a maximum effect
we should get a reduced response. The
bigger the reduction in agonist
100
80
% Response

Agonists

in the binding
pocket preventing
histamine from
binding. Receptors
also have a speciAic
distribution. Thus,
angiotensin
receptors are found
on vascular smooth
muscle but not any
other type of smooth
Nature 475, 6570, 2011
muscle and they are
found on kidney
epithelial cells but not intestinal epithelial
cells.

60
40

EC50

20
0

100

200

300

400

500

[Agonist]

concentration the bigger the reduction in

response. This is known as the dose-

response relationship. If we plot this on a

graph it generates a hyperbolic curve (see
Aig). As we can see in the graph as we
increase adrenaline concentration the hart
rate increases however, at a certain
concentration we reach a maximum
response and no matter how much we
increase the agonist concentration there is
no further increase in response, i.e, the
graph has reached a plateau and this type of
graph is known as a hyperbolic curve.
EC50. When we try to compare the potency of
different drugs a useful measure is the
concentration of drug that causes a
maximum response - the lower the
concentration the more potent the drug.
However, when you look at the hyperbolic
curve you will see that it is very difAicult to
see when it reaches a maximum. At the
lower response (e.g 20%) level a small
change in concentration causes a big change
in response while at the high response level
(80%) a very large change in concentration
is needed to alter the response. So it is easier
to choose a a concentration of drug that is
below the maximum (probably even below
80%). While we could probably choose any
response we typically choose a 50%
response (you will see later that there is
good reason to choose 50%). The
concentration of agonist that causes a 50%
response is known as the effective
concentration 50% (EC50). This is the
parameter that we use to compare drug
potency.

Binding studies. All of the early

pharmacology studies measured the
response of tissue to the presence of drug.
However, the development of radioactive
isotopes allowed for the synthesis of drugs
that were radioactive. When these radio

labelled drugs were added to tissue it was

possible to measure drug binding (counts
per minute per mg) rather than response.
When we do this we Aind that the binding of
drug to tissue mirrors the response of tissue

% Response

Log-dose response curves. The hyperbolic

dose-response curve is difAicult to work with
as most activity happens in a very narrow
dose range. To deal with this problem we
use the
100
log doseresponse
80
curve.
60
When
40
using a
EC50
log scale
20
it is
0
1
10
100 1000
possible
log [Agonist]
to plot a

wide range of data on the axis. This log doseresponse curve is a sigmoid curve and it is a
more useful representation of the
relationship. One clear difference is that the
hyperbolic curve suggests that there is no
response when there is no agonist present
but that
the
presence
of any tiny
amount of
agonist
creates a
tiny
response.
The log
doseresponse curve suggests that there is a
threshold dose below which there is no
response, i.e, at very low concentrations of
drug there is no response. Another
advantage of the sigmoid curve is that the
EC50 is very easy to measure.

to drug. Both are hyperbolic curves. In the

case of a binding study we do not use EC50 as

we do not measure a response. Instead we
measure kd which is effectively the
concentration of drug that produces 50%
binding.

Further rearrangement of this equation

gives the equation below. Kd is the ratio of
the rates of drug leaving the receptor to
binding to receptor and is known as the
dissociation constant. Its reciprocal 1/kd is
the association constant. As molar
concentrations are used the units for kd are
Moles (M2/M) and for the association
constant it is M-1.

k2
[D].[R]
= Kd =
k1
[DR]

Quantitative drug-receptor interactions

Drug (D) + Receptor (R)

k2

DR

Effect (E)

k1

The above equation simply describes the

drug-receptor interaction. It makes a
number of assumptions: 1) drug binds to
receptor in a reversible manner 2) all
receptors are equally accessible to drug 3)
the effect is proportional to the number of
occupied receptors. K1 is the rate at which
drug binds to receptor and k2 is the rate at
which drug leaves the receptor.

[D]+[R]

k1

[DR]

k2
Above is this written in a more
mathematical form where DR is the drugreceptor complex and [] indicates the
concentration of the substance. This can be
re-arranged and written as a proper
equation below.

[ D].[R].k1 = [ DR].k 2

The total number of receptors (Rt) is the

sum of free receptors and drug-bound
receptors. Thus [Rt]=[R] + [DR] and
therefore [R]= [Rt]-[DR]. If we replace [R] in
the equation above we get:

KD =

[ D].(Rt [ DR]) [ D].Rt [ D].[DR]

=
[ DR]
[ DR]

After rearrangement we get:

[ D ].Rt
[ DR ] =
K D + [ D]
which can be further rearranged to:

[ DR]
[ D]
=
Rt
K D + [ D]

[DR ]
[D ]
=
[RT ] [D ]+ K d

E
Emax

In the above equation if kd = [D](i.e., if the

free drug concentration is the same as the
dissociation constant) the equation becomes
[D]/2[D] = 0.5. The the fraction of occupied
receptors ([DR]/[Rt]) is then 50% and kd is
equivalent to 50% binding. Equally the
response (E/Emax) will also be 50% and thus
kd is equivalent to EC50.

Potency. Potency is a measure of drug

activity and is expressed as the amount of
drug necessary to achieve a given effect.
Typically we use the 50% response as the
Aixed response and compare the EC50s for
the drugs (see Aig). In this case isoprenaline
(A) is the most potent with an EC50 of 20 M,
adrenaline (B) has an EC50 of 80 M and the
least potent is noradrenaline (C) with an
EC50 of 300 M. Potency is related to the
afAinity of a drug for its receptor and
determined from the dose-response curves.

100
80

% Response

If we assume that the response is directly

proportional to the number of receptors
bound then the percentage of drug bound
receptors should be equal to the percent
maximum response. So the the following is
true:

60

Bound/Free

0.03
0.025
0.02
0.015
0.01
0.005
0
0

10

15

Bound

performed by hand so prior to the use of

computers, data was transformed to create a
a linear plot. This is often done in
biochemistry with enzyme kinetic data
(Lineweaver-Burke plot).
With drug-receptor binding data we use the
Scatchard plot which plots [DR]/[D] vs [DR].
This line intercepts the X-axis at Bmax (i.e., [Rt])
and the slope is 1/kd. This has the advantage
of being easy to do and provides a clear
visualisation of the data. However, it is error
prone due to the transformation and violation
of a key assumption of linear regression (the X
and Y-axes should be independent of each
other).

20
0
1

Scatchard Plot
While the sigmoid and hyperbolic curves are
routinely used to analyse dose-response data
this can only be done with computers and
curve fitting software to perform non-linear
regression. Linear regression can however be

40

10

100
1000
log [Agonist]uM

10000

Ef=icacy (intrinsic activity). As we have seen

above some agonist generate a maximum
response that is less than that of the natural
hormone these are the partial agonists. To
accommodate the existence of partial
agonists it is necessary to modify the
traditional dose-response relationship. In
the equation below is the additional factor
to accommodate partial agonists and is
known as efAicacy or intrinsic activity. If =1
the drug is a full agonist and this is the
normal equation. When <1 the response at
any level of receptor occupant will be
reduced. So if =0.6 the maximum response
would be 60% of the expected maximum.

Emax

[DR ]

[RT ]

In the Aigure drug A and B are full agonists

but drug A is more potent than B. Drug C is a
partial agonist with an intrinsic activity of
0.4 with a potency between A and B. While it
is more potent than Drug B, at high
concentrations B will generate a greater
response than C while at low concentrations
C will generate the greater concentrations.

Varenicline
The health consequences of smoking are well
established but nicotine is very addictive and
thus it is very difficult to quit smoking. This is
because nicotine binds to neutrons in the
brain and releases dopamine which
stimulates the reward centres. One approach
to dealing with smoking is to use a nicotine
patch. This delivers nicotine through the skin
which stimulates the release of dopamine.
One problem is that smokers are still
addicted to nicotine and they often continue
to smoke.
Varenicline is a nicotine partial agonist with
high affinity for the receptor. This drug binds
to the receptor and acts as a weak agonist
preventing the need to smoke. Smokers can
continue to smoke as the nicotine will have no
effect since all the receptors are blocked by
varenicline. However, with no reward being
generated from smoking smokers soon quit.
Typically smokers stop the drug after 12 weeks

Spare receptors. Sometimes the doseresponse curve from the binding study does
not match that from a functional study.
Typically the EC50 for binding is higher than
that for response. This means that a 100%
response is possible with less than 100%
receptor occupancy. So it is possible to have
100% response with 80% receptor
occupancy. This means that 20% of the
receptors are spare. This can affect drug
dosing as less drug is required for a given
response (based on binding studies). The
reason for this is that a higher number of
receptors allows for an increased sensitivity
at low agonist concentrations without
altering the maximum response.

Antagonists

Antagonists
Chemical antagonism occurs when a
substance is neutralised through a
chemical interaction with another
Pharmacokinetic antagonism occurs
when one drug inhibits the absorption
or metabolism of another drug
Physiological antagonism occurs when
two drugs produce opposing
physiological effects
Pharmacological antagonism occurs
when two drugs compete for the same
receptor or enzyme

We have seen that the intrinsic activity of a

full agonist is 1 but that it is possible for this
to be less than 1 in the case of a partial
agonist. However, as continues to decrease
the agonist-like activity of the drug also
decreases. A drug with = 0 has no intrinsic
activity and is known as an antagonist. The
presence of an antagonist cannot be
detected with a dose-response curve as it
generates no response. However, it can be
detected by its effect on the dose-response
curve of an agonist or in a binding study.

pocket but will also leave the binding

pocket. Once it leaves it can then bind to the
same receptor or a different one. If there is
an antagonism present then some of the
receptors will bind antagonist rather than
agonist. This is a matter of probability: if the
agonist and antagonist are present at the
same concentration and have the same kd for
the receptor then 50% of the receptors will
be occupied by antagonist and 50% by
agonist and thus there will be a 50%
reduction in response. If there is a 10:1 ratio
of agonist to antagonist then 10% of
receptors will be occupied by antagonist and
the remaining receptors by agonist with a
reduction in response of 10%. Thus, as we
increase the agonist concentration we can
overcome the effect of antagonist. The Aigure
shows the effect of a competitive antagonist
on the dose-response curve for an agonist.
As you can see the curve looks the same but
it is shifted to the right the higher the
antagonist concentration the further to the
right that the curve is shifted.

Non-competitive antagonists
These are insurmountable as adding more
agonist has no effect. The Aigure shows the
effect of a non-competitive antagonist on the
dose-response curve for an agonist. As the
There are two types of antagonist
surmountable and insurmountable.
Surmountable antagonists can be overcome
by adding in more agonist. These are known
as competitive antagonists. Insurmountable
antagonists on the other hand are not
affected by increasing the agonist
concentration. These are either irreversible
antagonists or non-competitive antagonists.
Competitive antagonists
When an agonist binds to a receptor it does
so reversible, i.e., it can enter the binding

antagonist concentration increases from B

to D the curve plateau decreases instead of

the curve shifting to the right. These
antagonists are also known as allosteric
inhibitors. Generally they act by binding to a
different site on the receptor where they
cause a conformational change that alters
the conformation of the receptor closing the
binding pocket. Since the agonist and
antagonist dont bind to the same site there
is no potential for competition. There is also
the potential for an allosteric activator, i.e., a
drug that binds to a distinct site from the
hormone and the subsequent conformation
change increases binding of the agonist.
Irreversible antagonists
These have a similar effect to that of the
non-competitive antagonist but their
mechanism of action is different. They bind
to the hormone-binding site like a
competitive antagonist but as they bind
irreversibly there is no potential for
competition.

Irreversible antagonists
Clopidogrel is an irreversible
antagonist. It is metabolised to a
chemically reactive intermediate that
binds to the ADP receptor on platelets
(P2Y12). Once bound it chemically
crosslinks to the receptor and remains
there for the life of the platelet. At one
stage it was the second biggest selling
drug in the world.

Comparing antagonists
When we compare antagonist potencies we
can use kd value as with agonists. The kd
value will also allow us to compare the
afAinity of an agonist and antagonist for the
receptor. We do not use the EC50 as there is
no effect with antagonists. The equivalent is
the inhibitory concentration 50% (IC50)
which is the concentration of antagonist that
reduces the response to agonist by 50%.

Inverse agonists.
The intrinsic activity of a drug is not
restricted to being positive and can go
negative thus it can have a value of -11.
When is negative the drug is an inverse
agonist. These drugs have an opposite effect
to the agonist and as they have an effect they
cannot be considered to be antagonists.
They occur where there is a signiAicant basal

activity in the system. So, if we are

measuring the effect of an agonist by
measuring the levels of a 2nd messenger
then a full agonist will increase the 2nd
messenger levels to a maximum possible, a
partial agonist increases them to a lesser
extent, an antagonist has no effect on them,
a partial inverse agonist will reduce the
levels and a full inverse agonist will reduce
them to zero.

seen in the Aigure below the majority of

drugs on the market have afAinities between
100 and 10 nM.

Quantal Dose-Response Curves

The Aigure below summarises the

mechanism of action of the different types of
antagonist and the effect that they have on
the dose response curve.

When it comes to discovering a new drug

companies typically like to go for the most
potent drug possible, however, potency is no
guarantee of success and lack of potency
does not necessarily mean that the drug is
not effective. Typically a company will want
a drug with a sub-micromolar kd. As can be

The dose-response curves that we have seen

above all deal with continuous data. This is
Aine for studies such as those that monitor
changes in blood pressure for instance.
However, we often have to use quantal data,
i.e., data that
is all-ornothing. This
is
particularly
common in
clinical
studies. For
instance if
we want to
investigate
the effects of
a new
antibiotic
drug. We
would look at the effect of different doses of
antibiotic and determine if the infection is
cleared or not. This is an all-or-nothing
response - patients either have the infection
or dont have the infection. We would plot
these data in the same way as for other
dose-response curves. In this case we would
plot the dose/log-dose of drug on the X-axis
and the percentage of patients that are
infection-free on the Y-axis. In this case the
parameter that we measure is the Effective
dose 50% (ED50) that is the dose that cured
50% of patients. There are many examples
of quantal data such as presence or absence

Therapeutic Index

of seizure, death, pregnancy etc. The ED50

will depend on the therapeutic effect being
investigated. So if a sedative is being tested
then ED50 for inducing sleep (when used as a
sleeping tablet) will be less than that for
deep sleep (used to perform a procedure
under sedation). So it is important to
identify the effect when citing the ED50.

An important area for quantal doseresponse curves is toxicity studies. We can

do a quantal dose-response curve for a toxic
effect (e.g., death, organ failure etc). Fig X
shows the dose-response curves for a
therapeutic effect and for a toxic effect.
These allow us to calculate the ED50 and the
TD50 (toxic dose 50%). It is obvious that we
prefer a drug to have a TD50 that is greater
than the ED50 and that the bigger the
difference the better. Of particular interest
is the Therapeutic Index which is TD50/ED50.
So the bigger the TI the safer the drug is.
This is illustrated in the Fig below where the
TI is a measure of how far apart drugs are.

TI is more important than absolute

toxicity level as it is a measure of the
likelihood of being exposed to a toxic
dose. Thus, Botox is widely used and
not necessarily by a medic yet
botulinum toxin is one of the most
toxic substances known. However,
you would need to inject yourself with
so many syringes of Botox to kill
yourself that makes it safe.

When an Antagonist isnt an

Antagonist
When we study the effect of an antagonist a
lot depends on what we look at. For instance
if a receptor has two associated signalling
pathways that are triggered by the agonist
we can get strange results if we only look at
one. So if a drug activates only one pathway
and not the other what is it? If we only study
one pathway we would conclude that it is
either a full agonist or an antagonist. If we
study both what do we call it? Is it a partial
agonist (maybe, although it has full agonist
properties on one pathway)?
Textured antagonism. Competitive
antagonists may bind to the receptor in such
a way that one pathway is activated while
the other isnt. On the other hand an
allosteric antagonist may alter the
conformation of the receptor that prevents
one pathway from being activated. Textured
antagonism refers to the concept that there
are nuances to antagonism similar to that
with agonism (partial agonism).
Non-linear response. The normal sequence of
events is for an agonist to bind to a receptor
and activate the signalling pathway. The
receptor then becomes deactivated and
internalised. What about a drug that binds
to a receptor but triggers receptor
internalisation without activating signalling.
Is it an antagonist as it prevents a response?
Is it an agonist as it triggers receptor
internalisation?

different GPIIb/IIIa molecules and if these

are on separate platelets it leads to the
formation of platelet aggregates. When these
aggregates form in the coronary blood
vessels the result is a heart attack. As a
result GPIIb/IIIa was an important drug
target for cardiovascular disease.

The screening system used the ability to

inhibit platelet aggregation as the main
criterion for going into clinical trials. What
was not appreciated was that GPIIb/IIIa was
a true receptor and when it binds Aibrinogen
a platelet activating signal was generated
which increases clot formation. The OPUS
study was a large clinical trial of the oral
GPIIb/IIIa antagonist orboAiban. Not only
did this drug not inhibit clot formation it
actually increased the rate of heart attacks.
SibraAiban, another oral GPIIb/IIIa
antagonist had a similar increase in
mortality. In both cases the companies
thought that the drug was an antagonist but
further work showed that it also behaved
like an agonist.
Tamoxifen. Tamoxifen is an inhibitor of
oestrogen receptors in the breast and is
used to treat oestrogen-dependent breast
cancer. Unfortunately one of its side effects
is that it causes uterine cell proliferation.

Orbo=iban. GPIIb/IIIa is the Aibrinogen

receptor on there surface of platelets. As
Aibrinogen is a dimer it can bind to two

This is because it is an agonist on uterine

oestrogen receptors and an antagonist on
breast oestrogen receptors. Thus, it is
known as a selective oestrogen receptor
modiAier (SERM). This led to the
development of fulvestrant which is a pure
oestrogen receptor antagonist.
Fingolimod. This drug binds to S1P1
receptors on T-cells. Rather than activating
signalling it triggers receptor
internalisation. Thus, the cells no longer
respond to S1P1 as they have no receptor on
their surface. So it functions as an antagonist
even though it has agonist-like properties.

About the author

Dermot Cox BSc, PG Dip (Ed), PhD obtained a

degree in Pharmacology from University
College Dublin and a PhD in immunology
from Dublin City University. He led a drug
discovery project in Fujisawa
Pharmaceutical Company, Osaka, Japan for
6-years. He currently lectures in
pharmacology in Royal College of Surgeons
in Ireland where is research is in the area of
drug discovery.