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 it’ll 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….

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptorNicotinic ACh receptor

M2

receptor

Enzyme

transporter

Na+

choline

Ca2+

Voltage gated calcium channel

Action potential

Presynaptic terminal

Post synaptic terminal

Synaptic cleft

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptornAChR

M2

receptor

Na+

choline

Ca2+

Action potential

SYNTHESIS

RELEASE

RECEPTORS

INACTIVATION

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptornAChR

M2

receptor

Na+

choline

Ca2+

Action potential

SYNTHESIS

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?

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptornAChR

M2

receptor

Na+

choline

Ca2+

Action potential RELEASE

RECEPTORS

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?

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptornAChR

M2

receptor

Na+

choline

Ca2+

Action potential

RECEPTORS

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

Gated Ion

Channels

GPCR Kinase-

linked

receptor

Nuclear

Receptor

Location Membrane Membrane Membrane Intracellular

Effector Ion channel Channel or

enzyme

Enzyme Gene

Transcription

Coupling Direct G-protein Direct Via DNA

Examples Nicotinic,

GABAa

Dopamine,

cannabinoid,

adenosine,

muscarinic

GABAB

Insulin,

growth factor,

cytokine

Steroid,

thyroid

hormone

receptors

Structure Oligomeric

assembly of

subunits

surrounding

pore

Monomeric

structure of 7

transmembran

e domains

Single

transmembran

e helix linking

extracellular

receptor to

intracellular

kinase domain

Monomeric

structure with

separate

receptor and

DNA binding

domains.

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

•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.

Neuronal

nAChR

αd

β

g α

ACh

ACh

pore

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

ACh

The Receptor Pore

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

M1, M3, M5M2,M4

Potential therapeutic uses of

mAChR subtype-selective

compounds*

From: :

Nature Reviews Drug

Discovery 6, 721-733 (2007)

doi:10.1038/nrd2379

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptornAChR

M2

receptor

Na+

choline

Ca2+

Action potential

RECEPTORS

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

• β-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

• Receptor functions as an enzyme that transfersphosphate groups from ATP to tyrosine residues onintracellular target proteins.

• Tyrosine kinase receptors mediate the actions of growth factors, cytokines and certain hormones (eg insulin).

Tyrosine Kinase Receptors

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/content/full/sigtrans;2001/112/re21/F3

Proliferation pathways

• Receptor activation leads to activation of PLCγby phosphorylation.

• PLC γ-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

Fra

ctio

nal

occ

upan

cy

[L]

1

0.5

Affinity constant (Kd)

FO = [L]

Kd+[L]

If you solve the equation for Kd, you

will find that Kd=[L] when FO =0.5,

thus Kd can be defined as the

concentration of drug [L] which

occupies 50% of the existing

receptor population

LOG [L]

1

0.5

Bmax

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.

LOG [L]

1

0.5

Fra

ctio

nal

occ

upan

cy

Which drug has higher affinity?

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

% E

ffec

t

[DRUG]

100

50

C50

% E

ffec

t

LOG [DRUG]

100

50

EMAX

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 receptor’s 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.

Res

po

nse

LOG [DRUG]

Full agonist

Partial agonist

• 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

LOG [DRUG]

Res

po

nse

Po

ten

tial

LOG [DRUG]Actual Response Observed Response

Res

po

nse Tissue maximum

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…

• Bind to the receptor in a reversible manner to

compete directly with agonist binding.

– What effect will this have on an agonist response curve?

Res

ponse

LOG [AGONIST]

agonist

Reversible Competitive 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?

Res

ponse

Log [agonist]

agonist

Irreversible Antagonism

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?

Res

po

nse 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?

ACh

ACh

CHOLINE +AcCoA ACh + HSCoA

ChAT

ACh

AChE

CHOLINE + ACETATE

M1 receptornAChR

M2

receptor

Na+

choline

Ca2+

Action potential

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 (egdopamine, 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 AChEactivity 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 5HT

5HT

5HT1,2 or 4

receptor

5HT3 receptor

5HT1a

receptor

5-HTP

5HT

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.

SSRI’s

• 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.

ENZYME

P P

T

T

Ca2+

T TD

P= precursorT= transmitterD=degradation product

Axon

Cell body or dendrite

A “typical” nerve terminal

ENZYME

Action potential