Mechanism of Drug Action and Drug Targets

Receptors

Neurotransmitter receptors in the plasma membrane of postsynaptic cells fall into two broad
classes: ligand-gated ion channels and G protein - coupled receptors. Synapses containing either
type can be excitatory or inhibitory, but the two types vary greatly in the speed of their response.

Ligand-Gated Ion Channels (Fast synaptic signalling)

Binding of the neurotransmitter to the extracellular binding domain on a ligand gated ion channel
causes an immediate conformational change that opens the channel portion of the protein,
allowing ions to cross the membrane and causing the membrane potential to change within 0.1 -
2 milliseconds. Binding of ligand to excitatory receptors opens cation channels that allow passage of
both Na+ and K+ ions, leading to rapid depolarization of the postsynaptic membrane; in
contrast, binding of ligand to inhibitory receptors opens Cl channels, leading to hyperpolarization
of the postsynaptic membrane.

G Protein coupled receptors (GPCRs) (Slow synaptic transmission)

Many functions of the nervous system operate with time course of seconds or minutes; regulation
of the heart rate, for instance, requires that action of neurotransmitters extend over several
beating cycles measured in seconds. In general, the neurotransmitter receptors utilized in slow
synapses are coupled to G proteins. GPCR signalling is also common in non-neuronal cells. Binding
of the neurotransmitter leads to a conformational change in the receptor, that results in activation
of a specific G protein. The G-protein may directly binds to and activate or inhibit ion-channels. In
other cases, the receptor-activated G protein activates (or inhibits) adenylate cyclase or
phospholipase C, triggering a rise in cytosolic cAMP or Ca2+, respectively; these second
messengers in turn affect the ion conductance of a linked ion channel, or trigger kinase cascades
that result in phosphorylation/dephosphorylation of target proteins. The postsynaptic responses
induced by neurotransmitter binding to GPCR are intrinsically slower and longer lasting than those
induced by ligand-gated channels.
In this lecture, the diversity of receptors for and responses to a single kind of neurotransmitter is
illustrated by acetylcholine. Synapses in which acetylcholine is the neurotransmitter are termed
cholinergic synapses. Acetylcholine receptors that cause excitatory responses lasting only
milliseconds are called nicotinic acetylcholine receptors. They are so named because nicotine, like
acetylcholine, causes a rapid depolarization . These receptors are ligand-gated channels for Na+
and K+ ions. Other acetylcholine receptors are called muscarinic acetylcholine receptors because
muscarine (a mushroom alkaloid) causes the same response as acetylcholine. There are several
subtypes of muscarinic acetylcholine receptors present in different cell types; all are coupled to G
proteins, but they induce different responses. The M2 receptor present in heart muscle activates a
Gi protein that causes the opening of a K+ channel and thus a hyperpolarization lasting seconds
when post-synaptic. Presynaptic M2 receptors, in addition to inhibiting adenylate cylase, inhibit
the opening of voltage gated calcium channels, leading to decreased ACh release from the nerve
terminal. The M1, M3, and M5 subtypes are coupled to Gq and activate phospholipase C; the M4
subtype activates Gi and inhibits adenylate cyclase. Thus, a single neurotransmitter induces very
different responses in different nerve and muscle cells, depending on the type of receptor found
in the target cells.

How drugs act at receptors

Affinity: a measure of how tightly a drug binds to the receptor. If the drug does not bind well,
then the action of the drug will be shorter and the chance of binding will also be less. This can be
measured numerically by using the dissociation constant KD. The KD is the concentration of drug
when 50% of receptors are occupied. Thus, the higher the KD the lower the affinity of the drug
(the more drug itll take to bind to the receptors).

Potency is a measure of how much a drug is required in order to produce a particular effect. This is
defined in a concentration response curve by the EC50 - the concentration of drug required to
produce 50% of maximal response. If a drug has high potency drug only a small amount is required
to induce a large response.
Efficacy is the ability of a drug produce a response. Agonists are described as having affinity and
efficacy, partial agonists are drugs which bind with affinity, but do not produce maximal response
- even at 100% receptor occupancy, thus they are less efficacious than full agonists. Antagonists
are drugs which have no intrinsic efficacy - they can bind to the receptor (i.e. they have affinity),
but do not induce a conformational change in the receptor. They produce their effects by
preventing agonist from binding.

Enzymes

Many enzymes form useful drug targets eg acetylcholinesterase (neostigmine); cyclo-oxygenase

(asprin), angiotensin converting enzyme (captopril). Often the drug mimics the natural substrate
acting as a competitive inhibitor of the enzyme (eg captopril); in other cases the binding is non-
competitive and irreversible (eg asprin). Drugs may also act as false substrates, where the drug
molecule undergoes transformation to form an adnormal product that subverts the normal
metabolic pathway. An example of this is the anticancer drug fluorouracil, which replaces uracil as
an intermediate in purine biosynthesis, but cannot be converted into thymidylate, thus blocking
DNA synthesis and preventing cell division. Some drugs require enzymic degradation to convert
them from a prodrug into an active form. Other drugs may be converted into a toxic or reactive
metabolite. We will consider the example of acetylcholine esterase inhibitors in the lecture.

Carrier/Transporter Molecules

Transportation of polar molecules across lipid membranes often requires a carrier protein. The
carrier proteins embody a recognition site that makes them specific for a particular permeating
species, and these sites can also be targets for drugs, whose effect is generally to block the
transporter. We will consider the example of the serotonin transporter, which are the drug target
for selective serotonin reuptake inhibitors (SSRIs), for depression.

Ion Channels

Ion channels are protein molecules that span the cell membrane, and can switch between open
and closes states, allowing the controlled entry or exit of specific ions across the cell membrane in
their open state. Ion channels are highly selective for the ions that they control. The channels are
all gated i.e they have particular triggers that allow for their opening. You are already familiar
with two forms of gating - ligand gated ion channels, which are an intergral part of a receptor, and
second messenger gated ion channels, which are opened either by G-proteins directly, or
downstream effector molecules such as cAMP. The third major class of gated-ion channel are the
voltage gated ion channels, which open in response to voltage changes across cell membrane.
These channels open when the membrane becomes depolarized, and are critical to the
mechanism of membrane excitability (development of action potentials). Channel opening is
usually short lasting. Drugs may bind directly within the ion channel and prevent the flow of ions,
or may bind elsewhere on the molecule and increase or decrease the probability of the channel
opening in response to a change in voltage. Local anaesthetics are a class of drugs which block
pain pathways by blocking voltage gated sodium channels and therefore preventing the action
potential that carries the pain signal from the site of injury to the brain.

How do drugs work?

Basic Mechanism of Drug Action and
Drug Targets
Associate Professor Michelle Glass
m.glass@auckland.ac.nz
 Learning Objectives
Describe the potential drug targets within a human body.
Describe the role of receptors, enzymes, ion channels and transporters in
drug action.
Understand how drugs bind to receptors, and define the principles of
affinity, efficacy and potency and be aware of the influence of the tissue
on these properties.
Understand the concentration response curve and what information can
be gained from it.
Differentiate between inverse agonism, agonism and antagonism and
explain them using the two state model of receptor activity.
Differentiate between different types of antagonism and understand their
impact on the concentration response curve.

Mechanism of Drug Action

Most drugs produce their effects by binding to protein
molecules. Drug binding often leads to a conformational
change in the protein. Four primary drug targets are:
- Ion channels.
- Enzymes.
- Carrier molecules.
- Receptors.
The only important exception to proteins as target sites is
DNA on which a number of antitumour and antimicrobial
drugs act.
 Cell-to-cell communication

Neuroscience of psychoactive substance use and dependence. I. World Health Organization. ISBN 92 4 156235 8

Neurotransmission is a four step

process..
Neurotransmitter synthesis
Neurotransmitter release
Action on receptors
Inactivation

Drugs can act on all of these processes - we

will look at these in more detail using the
cholinergic system .
 Action potential
Enzyme

transporter
Ca2+
choline
Voltage gated calcium channel
Na+
ChAT
CHOLINE +AcCoA ACh + HSCoA

ACh

ACh
M2 Presynaptic terminal
receptor

ACh Synaptic cleft

CHOLINE + ACETATE

AChE
Post synaptic terminal

Nicotinic ACh receptor

M1 receptor

RELEASE

SYNTHESIS

RECEPTORS
M1 re
INACTIVATION
 Action potential

SYNTHESIS
Ca2+

+ HSCoA

ACh

ACh
M2
receptor
CHOLINE + ACETATE ACh

AChE

nAChR
M1 receptor

Synthesis
Two key sites of action
- Choline transporter - critical to uptake of choline,
rate limiting step in ACh synthesis.
- Choline Acetyl Transferase (ChAT) - enzyme
involved in synthesis of choline

What effect would drugs that inhibited these

proteins have on neurotransmission?
 RELEASE

choline

Na+

CHOLINE +AcCo

M2
receptor
CHOLINE + ACETATE

AChE

RECEPTORS
nAChR
M1 receptor

Release
ACh is packaged in synaptic vesicles by the
vesicular ACh transporter.
Vesicles are held in the cytoskeleton by Ca2+
sensitive vesicle membrane proteins (VAMPs).
When an action potential reaches the terminal,
voltage dependent calcium channels open and
Ca2+ rushes in triggering vesicular fusion with the
cell membrane and release of ACh into the
synapse
Sites of Drug Action? - what effect?
 Action potential

Ca2+
choline

Na+
ChAT
CHOLINE +AcCoA ACh + HSCoA

ACh

ACh

CHOLINE + ACETATE ACh

AChE

RECEPTORS
M1 re

Receptors
Proteins which specifically recognise a particular
neurotransmitter/hormone and upon binding
undergo a conformation change leading to
activation/inhibition of cell signalling.
Four main families of receptor:
- Ligand Gated Ion Channels (Ionotropic receptors)
- G-protein coupled receptors
- Tyrosine kinase/cytokine receptors
- Nuclear/Steroid Hormone Receptors
 Ligand GPCR Kinase- Nuclear
Gated Ion linked Receptor
Channels receptor
Location Membrane Membrane Membrane Intracellular

Effector Ion channel Channel or Enzyme Gene

enzyme Transcription
Coupling Direct G-protein Direct Via DNA

Examples Nicotinic, Dopamine, Insulin, Steroid,

GABAa cannabinoid, growth factor, thyroid
adenosine, cytokine hormone
muscarinic receptors
GABAB
Structure Oligomeric Monomeric Single Monomeric
assembly of structure of 7 transmembran structure with
subunits transmembran e helix linking separate
surrounding e domains extracellular receptor and
pore receptor to DNA binding
intracellular domains.
kinase domain

Ligand Gated Ion Channels

Mediate fast signal transmission at synapses (action
occurs in a fraction of a millisecond, and is equivalent to
about 107 ions per second)
All are multi-subunit complexes
All have three important properties:
- They are activated in response to specific ligands
- They conduct ions through the otherwise impermeable
cell membrane
- They select among different ions
 Nicotinic Acetylcholine Receptors
ACh
d Neuronal
nAChR
g
ACh

Subunits clustered around central receptor channel

Each subunit has 4 transmembrane domains (20 TM domains in
total)
ACh binding site is a dimer formed by 3 or more peptide loops on a
subunit and 2 loops on adjacent subunit (complementary component).
Each ACh binding site sites at the interface between one of the two
alpha-subunits and its neighbour.
Binding to both sites is needed for channel opening.

The Receptor Pore

pore

ACh

gate

The five M2 helices are sharply kinked inwards halfway through the
membrane forming a constriction.
The M2 helices are believed to snap to attention when Ach binds, opening
the channel
 The receptor pore
Ion conductivity is highly selective, and determined by
amino acids in TM2.
Most excitatory neurotransmitters (ACh, Glutamate)
cause an increase in Na+ and K+ permeability.
The inside of the cell therefore becomes more positive
(depolarised) and has an increased probability for an
action potential.
For nACh, mutation of a critical residue in TM2 changes
the ion channel from being cation selective to being
anion selective.

Ionotropic Receptors As Drug Targets

GABAA - benzodiazepines and barbiturates (sedation and
anxiolytic effects). Muscimol (hallucinogenic mushroom).
Flumazinal.
Glutamate - ketamine (aneasthetic). Major target for
neuroprotection and anti-convulsants, but to date all
compounds have shown major adverse effects
(predominantly hallucinations)
Nicotinic- nicotine, pancuronium (antagonist) used as
muscle relaxants during anaesthesia.
 Families Of Receptors
Ligand Gated Ion Channels (Ionotropic
receptors)
G-protein coupled receptors
Tyrosine kinase/cytokine receptors
Nuclear/Steroid Hormone Receptors

Key GPCR signalling pathways

M2,M4 M1, M3, M5
 Potential therapeutic uses of
mAChR subtype-selective
compounds*
From: :
Nature Reviews Drug
Discovery 6, 721-733 (2007)
doi:10.1038/nrd2379

Action potential

Ca2+
choline

Na+
ChAT
CHOLINE +AcCoA ACh + HSCoA

ACh

ACh

ACh
CHOLINE + ACETATE

AChE

RECEPTORS
nAChR
M1 receptor
 Pre-Synaptic Receptors
Presynaptic receptors are usually Gi linked.
Activation of them leads to inhibition of voltage sensitive
Ca2+ channels
This results in decreased neurotransmitter release
(feedback loop)
Because the pre-synaptic receptors are
pharmacologically distinct from the post-synaptic
receptors, specific drugs can be designed to target these
receptors.
Drugs which block presynaptic receptors can result in a
10 fold increase in neurotransmitter release.

Other drugs acting through GPCRs

-adrenoceptor - propranolol, isoprenaline
Adenosine receptors - caffeine, theophylline
Dopamine receptors - L-dopa, haloperidol,
bromocryptine
Opioid receptors - morphine, codeine
Serotonin receptors - buspirone, ondansetron, LSD
Muscarinic receptors - atropine
Cannabinoid receptors - cannabis, rimonabant, Sativex
 Families Of Receptors
Ligand Gated Ion Channels (Ionotropic
receptors)
G-protein coupled receptors
Tyrosine kinase/cytokine receptors
Nuclear/Steroid Hormone Receptors

Tyrosine Kinase Receptors

Receptor functions as an enzyme that transfers
phosphate groups from ATP to tyrosine residues on
intracellular target proteins.
Tyrosine kinase receptors mediate the actions of
growth factors, cytokines and certain hormones (eg
insulin).
 Vascular Endothelial Growth Factor
Receptors
Essential for angiogenesis during
development, pregnancy, wound healing
Also in pathophysiological conditions eg
cancer, rheumatoid arthritis, cardiovascular
disease.
Multiple receptors/multiple ligands, we will
look briefly at VEGFR2

VEGFR2
Ligand stimulated receptor dimerisation
Autophosphorylation of tyrosine residues in cytoplasmic
domain
Associates with SH2 domain proteins
Activation regulates a multitude of biological functions
- Endothelial cell survival
- Endothelial cell proliferation
- Endothelial cell migration
- NO and PGI2 production
- Increase vascular permeability
 http://stke.sciencemag.org/cgi/c 12/re21/F3

Proliferation pathways
Receptor activation leads to activation of PLC
by phosphorylation.
PLC -hydrolyses PIP2 to DAG + IP3
DAG activates PKC
PKC activation leads to activation of ERK via
Raf and MEK
ERK activation leads to increased gene
transcription

What therapies might target these

pathways?
Angiogenesis inhibitors?
Angiogenesis stimulators?
 How do drugs bind to receptors
Van der Waals forces - weak forces
Hydrogen binding - stronger
Ionic interactions - between atoms with
opposite charges, stronger than hydrogen,
weaker than covalent.
Covalent binding - essentially irreversible

Affinity Constants
If you solve the equation for Kd, you
will find that Kd=[L] when FO =0.5,
FO = [L] thus Kd can be defined as the
Kd+[L] concentration of drug [L] which
occupies 50% of the existing
receptor population
Fractional occupancy

1 1 Bmax

0.5 0.5

Affinity constant (Kd)

[L] LOG [L]

 Affinity
The higher the affinity of the drug for the receptor, the
lower the concentration at which it produces a given level of
receptor occupancy.
Fractional occupancy

0.5 Which drug has higher affinity?

LOG [L]

Affinity vs Biological Response

Concentration - response curves are not a good measure
of affinity because the relationship between receptor
occupancy and response is not strictly proportional :
- considerable amplification may exist - it may only take a
low level of receptor occupancy to cause a maximal
response in some tissues.
- Many factors downstream from the receptor binding may
interact to produce the final response.
 Biological Response
Biological response, eg a rise in blood pressure, contraction
of a strip of smooth muscle in an organ bath, or the
activation of an enzyme can be measured and plotted as a
concentration response curve.
In any system the response to a drug can be classified by its
C50 and Emax

EMAX
100 100
% Effect
% Effect

50 50
C50
[DRUG] LOG [DRUG]

Potency

C50 is used to measure the potency of an agonist.

C50 is the effective concentration of an agonist that
produces 50% of the maximal response
The more potent the agonist, the lower the C50.
Antagonist potencies are more complicated to
determine, but a similar principle holds.
 Efficacy
The ability of a drug to bind to a receptor and cause
a change in the receptors action is termed efficacy
and measured by Emax
- A drug with positive efficacy will activate a receptor to
promote cellular response - AGONISTS
- A drug with negative efficacy will bind to receptors to
decrease basal receptor activity - INVERSE AGONIST
- A drug with no efficacy will bind to the receptors but
have no effect on activity -ANTAGONIST (what would
the effect of this be?)

Agonism
Drugs the elicit the maximum tissue response are full
agonists, drugs that produce less than the maximum
response are partial agonists.

Full agonist
Response

Partial agonist

LOG [DRUG]

Partial agonists can not produce maximal response even at

100% receptor occupancy.
 Agonism
While affinity is thought to be a function of only the drug and the
receptor, the maximum response of a tissue to a drug is determined
by efficacy and tissue properties. A drug may appear to be a partial
agonist in one tissue and a full agonist in another
Response
Potential

Response
Tissue maximum

LOG [DRUG] LOG [DRUG]

Actual Response Observed Response

Antagonists

A compound that binds to but does not

activate (or inactivate) the receptor.
Antagonists have affinity but NO efficacy.
Defined by how they bind to the receptor
 Reversible Competitive Antagonism

Bind to the receptor in a reversible manner to

compete directly with agonist binding.
- What effect will this have on an agonist response
curve?
Response

agonist

LOG [AGONIST]

Irreversible Antagonism

- Drug covalently binds to the receptor. Not reversible

- Reduces the number of receptors available to the agonist.
What would this do to an agonist dose response curve?

agonist
Response

Log [agonist]
 Irreversible antagonist
Sometimes at low doses an irreversibe antagonist
looks like a reversible antagonist - what does this
tell you about the number of receptors required to
produce maximal response?
Response

agonist
agonist + increasing
Concentrations of
irreversible
antagonist
[Agonist]

In contrast to drugs that act on

receptors which can activate or
inactivate, most drugs acting on
enzymes or transporters only inhibit
- why is this?
 Action potential

Ca2+
choline

Na+
ChAT

CHOLINE +AcCoA ACh + HSCoA

ACh

ACh
M2
receptor

nAChR
M1 receptor
INACTIVATION

Inactivation
Once in the synapse neurotransmitter must be
quickly removed or chemically inactivated in
order to prevent constant stimulation of the
post-synaptic cell
There are two key mechanisms of inactivation
- Transport back into the pre-synaptic synapse (eg
dopamine, serotonin)
- Enzymic degradation (eg Acetylcholine)
Inhibitors of Acetylcholine esterase
The effect that a specific AChE inhibitor can have on the body depends
largely on the chemical properties of the molecule and the strength of the
bond it forms with AChE.
Irreversible AChE inhibitors are highly toxic -
- organophosphorus compounds or nerve gases
- form incredibly stable phosphorus bonds with AChE. Which resist
hydrolytic cleavage for up to several hours inhibiting all normal AChE
activity and leading to an excessive build-up of ACh at cholinergic
synapses.
- Lead to profuse sweating, dimmed vision, uncontrollable vomiting and
defecation, convulsions, filling of the bronchial passages with mucus,
bronchial constriction, and at last, paralysis and asphyxiation from
respiratory failure.

Acetylcholinesterase inhibitors.
Medium duration Irreversible
 Reversible AChE inhibitors
Bind to AChE for a short time.
Used in disorders characterized by a decrease in cholinergic function -
increase the duration that Ach is available ins synapse, so one molecule
can activate more receptors.
Used to treat myasthenia gravis
- an autoimmune disorder characterized by debilitating muscle weakness.
- weakness is caused by a progressive breakdown of ACh receptor sites on the
muscular endplate, making muscle movement difficult.
- Pyridostigmine bromide, (Mestinon or Regonol), is the most widely used.
Cannot pass the blood-brain barrier, so their action is limited to the
inactivation of AChE at the neuromuscular junction., so no psychotropic
effects.
Used in the treatment of Alzheimer's disease
- brain cell loss results in a progressive and significant loss of cognitive and
behavioral function.
- Drugs must readily cross the blood-brain barrier.
- Tetrohydroaminoacridine (Tacrine or Cognex), and Donepezil (Aricept)
- can help to ease some of the memory and language deficits.

Serotonin (5HT) Transporter

5HT is involved in sleep, appetite, memory, sexual
behaviour, neuroendocrine function and mood.

It is synthesized from the amino acid precursor

tryptophan, packaged in vesicles, and released into
synapses following an action potential.

Reuptake, determines the extent and duration of

receptor activation.
 Tryptophan 5-HTP 5HT

5HT1a
receptor

5HT 5HT

5HT3 receptor
5HT1,2 or 4
receptor

Serotonin Transporter
Part of a family of sodium and chloride dependent transporters.
Twelve transmembrane domains.

Na+ binds first to the carrier, followed by 5-HT (1). Cl- is needed
for transport but not for transmitter binding.
The molecules are translocated in the membrane and dissociate
(2). K+ then binds (3), and is translocated to the outside of the
membrane (4), and dissociates to complete the cycle.
 SSRIs
What does this stand for?
Can you think of an example?
What will the effect be?

Revision
On the following stylised synapse find at
least ten sites of drug action.
Describe what the likely effect of the drug is
on neurotransmission
For five give an example of a drug that acts in
this way.
 typical nerve terminal

P= precursor Action potential

T= transmitter
D=degradation product

Ca2+
P P ENZYME
T
Axon

D T T
ENZYME
Cell body or
dendrite