A Study aid on the Pharmaco-molecular aspect of Drug Action

1. Molecular Aspects of Drug ActionMolecular Aspects of Drug Action
 Receptors:Receptors:
 Sensing elements in the membranes thatSensing elements in the membranes that
mediate communication between cells andmediate communication between cells and
coordinate the function of all the different cells incoordinate the function of all the different cells in
the body.the body.
 Messengers are the hormones, transmitters,Messengers are the hormones, transmitters,
local hormones, peptides, Nitric oxide………….local hormones, peptides, Nitric oxide………….
 Many drugs act as agonists or antagonists onMany drugs act as agonists or antagonists on
receptors for known endogenous mediators.receptors for known endogenous mediators.

2. ReceptorsReceptors
 Isolation of receptors: Electric organs of manyIsolation of receptors: Electric organs of many
fish consist of tissues that contain acetylcholinefish consist of tissues that contain acetylcholine
receptors; Torpedo fish and electric eelsreceptors; Torpedo fish and electric eels
 Venoms of snakes of the cobra family containsVenoms of snakes of the cobra family contains
polypeptides that bind with very high specificitypolypeptides that bind with very high specificity
to nicotinic receptors.to nicotinic receptors.
 αα-toxins, can be labeled and used to assay the-toxins, can be labeled and used to assay the
content of tissues of receptors.content of tissues of receptors.
 αα-bungarotoxin (Bangarus multicinctus)-bungarotoxin (Bangarus multicinctus)

3. ReceptorsReceptors
Treatment of receptors with non-ionicTreatment of receptors with non-ionic
detergents to solubilize the membranesdetergents to solubilize the membranes
and then isolate them and then purify andand then isolate them and then purify and
amino acid sequence is determined.amino acid sequence is determined.
Cloning of receptors: cloned DNACloning of receptors: cloned DNA
encoding individual receptors.encoding individual receptors.
Receptor subtypes were isolated usingReceptor subtypes were isolated using
this technique.this technique.

4. Ion ChannelsIon Channels
 Ligand-gated ion channels: open only when theLigand-gated ion channels: open only when the
receptor is occupied by an agonist.receptor is occupied by an agonist.
 Voltage-gated channels: are very important,Voltage-gated channels: are very important,
being stimulated by changes in the membranebeing stimulated by changes in the membrane
potentialpotential
 Drugs can interact either with the receptor siteDrugs can interact either with the receptor site
of the ligand gated channels or with other partsof the ligand gated channels or with other parts
of the channel molecule.of the channel molecule.
 Interaction can be indirect through G-proteinsInteraction can be indirect through G-proteins
 Direct interaction with the channel protein.Direct interaction with the channel protein.

5. Ion ChannelsIon Channels
Local anesthetics: act by a simpleLocal anesthetics: act by a simple
mechanism of plugging the channelmechanism of plugging the channel
physically and prevent the sodium ionphysically and prevent the sodium ion
from passing through the channel.from passing through the channel.
Drugs: dihydropyridines, inhibit theDrugs: dihydropyridines, inhibit the
opening of L-type calcium channelsopening of L-type calcium channels
Benzodiazepines: bind to a site near theBenzodiazepines: bind to a site near the
GABA-receptors (chloride channel, whichGABA-receptors (chloride channel, which
is a ligand gated ion channel).is a ligand gated ion channel).

6. EnzymesEnzymes
 Enzymes are targets for many drugsEnzymes are targets for many drugs
 Reversible agents e.g Neostigmine, allopurinol.Reversible agents e.g Neostigmine, allopurinol.
 Irreversible agents e.g Aspirin,Irreversible agents e.g Aspirin,
Phenoxybenzamine.Phenoxybenzamine.
 False substrates: drug molecules undergoFalse substrates: drug molecules undergo
chemical transformation to form an abnormalchemical transformation to form an abnormal
product that fools the normal metabolic pathway.product that fools the normal metabolic pathway.
Fluorouracil, which replaces uracil as anFluorouracil, which replaces uracil as an
intermediate in purine synthesis, thus blockingintermediate in purine synthesis, thus blocking
DNA synthesis.DNA synthesis.

7. Carrier moleculesCarrier molecules
 Transport of ions and organic molecules acrossTransport of ions and organic molecules across
cell membranes.cell membranes.
 It requires a carrier protein, since the moleculesIt requires a carrier protein, since the molecules
are too polar (lipid insoluble). Choline carrier,are too polar (lipid insoluble). Choline carrier,
Noradrenaline uptake-1, Noradrenaline vesicularNoradrenaline uptake-1, Noradrenaline vesicular
uptake, Renal tubule weak acid carrier, Nauptake, Renal tubule weak acid carrier, Na++
/K/K++
ClCl--
co-transporter in Loop of Henle, Na+/K+ pump inco-transporter in Loop of Henle, Na+/K+ pump in
cardiac muscle, Proton pump in parietal cells.cardiac muscle, Proton pump in parietal cells.

8. Types of ReceptorTypes of Receptor
Receptors produce many different types ofReceptors produce many different types of
cellular events.cellular events.
Very rapid actions: synapticVery rapid actions: synaptic
neurotransmission; in millisecondsneurotransmission; in milliseconds
Ach.Catecholamines: in secondsAch.Catecholamines: in seconds
Peptides: take longerPeptides: take longer
Thyroid and steroids hormones: hours orThyroid and steroids hormones: hours or
days.days.

9. Ligand-gated ion channelsLigand-gated ion channels
 They are similar to ion channels but with a ligand –They are similar to ion channels but with a ligand –
binding receptor site, in the extracellular domain.binding receptor site, in the extracellular domain.
 Nicotinic acetylcholine receptors.Nicotinic acetylcholine receptors.
Consist of four subunits,Consist of four subunits, αα,, ββ,, γγ,, δδ..
 GABAGABAAA receptor, Glutamate AMPA(receptor, Glutamate AMPA(α-α-amino-3-hydroxy-5-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid), NMDA( N-methyl-D-methyl-4-isoxazolepropionic acid), NMDA( N-methyl-D-
aspartate receptor) and kinate receptorsaspartate receptor) and kinate receptors
 5-HT5-HT33 receptorsreceptors

10. Ligand-gated receptorsLigand-gated receptors
Each subunit has a mass of 40-58 kDaEach subunit has a mass of 40-58 kDa
(unified atomic mass unit ).(unified atomic mass unit ).
The structure has two acetylcholine-The structure has two acetylcholine-
binding sites, each lying at the interfacebinding sites, each lying at the interface
between one of the twobetween one of the two αα-subunits and its-subunits and its
neighbour.neighbour.
Ach has to bind to both sites in order toAch has to bind to both sites in order to
activate the receptor.activate the receptor.
((

11. Gating MechanismGating Mechanism
 Fastest synaptic events are mediated by thisFastest synaptic events are mediated by this
mechanism, found mainly in postsynapticmechanism, found mainly in postsynaptic
membranes of nerves or muscles.membranes of nerves or muscles.
 In most cases they increase the membraneIn most cases they increase the membrane
permeability to Napermeability to Na++
and K+.and K+.
 This leads to depolarization and may be actionThis leads to depolarization and may be action
potential.potential.
 Coupling between the receptor and the ionicCoupling between the receptor and the ionic
channel is DIRECT.channel is DIRECT.
 Read about, Katz et al (72) & Patch-clampRead about, Katz et al (72) & Patch-clamp
technique.technique.

12. G-PROTEIN-COUPLEDG-PROTEIN-COUPLED
RECEPTORSRECEPTORS Most abundant type of receptorsMost abundant type of receptors
 Muscarinic, adrenoceptors, dopamine, 5-HT,Muscarinic, adrenoceptors, dopamine, 5-HT,
Opiate, receptors for peptides, purine receptors,Opiate, receptors for peptides, purine receptors,
chemoreceptors for olfaction and pheromonechemoreceptors for olfaction and pheromone
detection receptors(is a secreted or excreteddetection receptors(is a secreted or excreted
chemicalchemical factor that triggers a social response in factor that triggers a social response in
members of the same members of the same speciesspecies).).
 Quantitative pharmacological studies withQuantitative pharmacological studies with
agonists and antagonist have revealed manyagonists and antagonist have revealed many
subtypes.subtypes.

13. Most neurotransmitters can interact withMost neurotransmitters can interact with
both GPCRs and with ligand-gatedboth GPCRs and with ligand-gated
receptors. Peptides do not do the same.receptors. Peptides do not do the same.
Peptides act either on GPCRs or onPeptides act either on GPCRs or on
Kinase-linked receptors.Kinase-linked receptors.
Ligands that act on nuclear receptors alsoLigands that act on nuclear receptors also
show high selectivity.show high selectivity.

14. Families of GPCRsFamilies of GPCRs
A-family: rhodopsin(purplish-red ,light-A-family: rhodopsin(purplish-red ,light-
sensitive pigment found in the retina ofsensitive pigment found in the retina of
human eye); amine neurotransmitters,human eye); amine neurotransmitters,
many polypeptides, purines, prostanoids,many polypeptides, purines, prostanoids,
cannabinoidscannabinoids
B-family: Secretin/glucagon family;B-family: Secretin/glucagon family;
glucagon, secretin, calcitoninglucagon, secretin, calcitonin
C-family: metabotropic family; glutamateC-family: metabotropic family; glutamate
receptors, GABAreceptors, GABABB receptors, calciumreceptors, calcium
sensing receptors.sensing receptors.
D-family: pheromones.D-family: pheromones.

15. G-ProteinsG-Proteins
 GPCRs control many different aspects of cellGPCRs control many different aspects of cell
functionsfunctions
 The link between the membrane receptor andThe link between the membrane receptor and
the first stage of signal transduction cascade isthe first stage of signal transduction cascade is
established through the G-protein.established through the G-protein.
 It is a membrane protein.It is a membrane protein.
 G-proteins represent the middle managementG-proteins represent the middle management
between the receptor and the effector enzymesbetween the receptor and the effector enzymes
or ion channels.or ion channels.
 G = GTP and GDP; GuanineG = GTP and GDP; Guanine
 G-proteins consist of 3 subunits,G-proteins consist of 3 subunits, αα,, ββ andand γγ..

16. G-proteinsG-proteins
 TheThe αα-subunit possessing GTPase activity.-subunit possessing GTPase activity.
 When an agonist bind to a receptor theWhen an agonist bind to a receptor the αα--
subunit dissociates and is then free to activatesubunit dissociates and is then free to activate
an ion channel or a membrane enzyme. In somean ion channel or a membrane enzyme. In some
cases thecases the βγβγ-subunits may activate the-subunits may activate the
receptors.receptors.
 Activation of the effector is terminated when theActivation of the effector is terminated when the
bound GTP molecule is hydrolysed, whichbound GTP molecule is hydrolysed, which
allows theallows the αα-subunit to recombine with-subunit to recombine with βγβγ..

17. Variants of G-proteinVariants of G-protein
The question now is; how is specificityThe question now is; how is specificity
achieved so that each kind of receptorachieved so that each kind of receptor
produces a distinct pattern of cellularproduces a distinct pattern of cellular
responses? Ach and NA?responses? Ach and NA?
The main reason is the molecular variationThe main reason is the molecular variation
within the G-protein family!!!!within the G-protein family!!!!
20,20, αα-subunits subtypes, 6 of-subunits subtypes, 6 of ββ-subunits-subunits
and 12 ofand 12 of γγ-subunits of G-proteins, giving-subunits of G-proteins, giving
1500 variants of the trimer.1500 variants of the trimer.

18. Classes of G-proteinsClasses of G-proteins
Three classes of G-proteins: GThree classes of G-proteins: Gss , G, Gii and Gand Gq.q.
Which show selectivity with respect toWhich show selectivity with respect to
receptor and effector cell.receptor and effector cell.
TheThe αα-subunit differ in structure.-subunit differ in structure.
Example of this difference is the action ofExample of this difference is the action of
cholera and pertussis toxins.cholera and pertussis toxins.
Cholera toxin acts on Gs and pertussisCholera toxin acts on Gs and pertussis
toxin acts on Gi.toxin acts on Gi.

19. Targets for G-proteinsTargets for G-proteins
 Adenylate cyclase produces cAMPAdenylate cyclase produces cAMP
 Phospholipase C, produces inositol phosphatePhospholipase C, produces inositol phosphate
and diacylglycerol.and diacylglycerol.
 Ion channels: calcium and potassium.Ion channels: calcium and potassium.
 Adenylate cyclase system was discovered byAdenylate cyclase system was discovered by
Sutherland in 1965Sutherland in 1965
 Synthesized from ATP and inactivated bySynthesized from ATP and inactivated by
hydrolysis to 5-AMP by phosphodiesterasehydrolysis to 5-AMP by phosphodiesterase
family of enzymes.family of enzymes.

20. Adenylate cycalse/cAMP systemAdenylate cycalse/cAMP system
 cAMP regulates cellular functions: energycAMP regulates cellular functions: energy
metabolism, cell division, cell differentiation, ionmetabolism, cell division, cell differentiation, ion
channels and contractile proteins in smoothchannels and contractile proteins in smooth
muscle.muscle.
 Common mechanism; activation of proteinCommon mechanism; activation of protein
kinases by cAMP.kinases by cAMP.
 Protein kinases catalyse the phosphorylation ofProtein kinases catalyse the phosphorylation of
serine and threonine residues using ATP as aserine and threonine residues using ATP as a
source for phosphate groups.source for phosphate groups.
 Phosphorylation can either stimulate or inhibitPhosphorylation can either stimulate or inhibit
target enzymes or ion channels.target enzymes or ion channels.

21. Adenylate cyclase/cAMP systemAdenylate cyclase/cAMP system
Increased activity of voltage-activatedIncreased activity of voltage-activated
calcium channels in heart muscle cells.calcium channels in heart muscle cells.
Phosphorylation of these channelsPhosphorylation of these channels
increases influx of calcium during actionincreases influx of calcium during action
potential, leading to increased force ofpotential, leading to increased force of
contraction.contraction.
In smooth muscle, inactivation of mAMPIn smooth muscle, inactivation of mAMP
leads to muscle relaxation.leads to muscle relaxation.

22. Receptors linked to Gi-proteinsReceptors linked to Gi-proteins
 Muscarinic receptors; MMuscarinic receptors; M22 in the heartin the heart
 αα22 -adrenoceptors in smooth muscle.-adrenoceptors in smooth muscle.
 Opioid receptorsOpioid receptors
 cAMP can be stimulated directly by other agentscAMP can be stimulated directly by other agents
like forskolin and fluoride ions.like forskolin and fluoride ions.
 Phosphodiesterase inhibitors: caffeine,Phosphodiesterase inhibitors: caffeine,
Theophylline .Theophylline .

23. Phospholipase C/PO4 systemPhospholipase C/PO4 system
 Catalyse the formation of intracellularCatalyse the formation of intracellular
messengers; IPmessengers; IP33 and DAG from membraneand DAG from membrane
phospholipids.phospholipids.
 IP3 acts to increase free cytosolic CaIP3 acts to increase free cytosolic Ca2+2+
byby
releasing Careleasing Ca2+2+
from intracellular components.from intracellular components.
 Free CaFree Ca2+2+
initiates muscle contraction,initiates muscle contraction,
secretions, enzyme activation and membranesecretions, enzyme activation and membrane
hyperpolarization.hyperpolarization.
 DAG activates protein kinase , which controlsDAG activates protein kinase , which controls
many cellular functions.many cellular functions.

24. Other functions of G-proteinsOther functions of G-proteins
They also control phospholipase A andThey also control phospholipase A and
thus the formation of arachidonic acid andthus the formation of arachidonic acid and
eicosanoids (polyunsaturated fatty acidseicosanoids (polyunsaturated fatty acids
found in fish).found in fish).
Ion channels like, potassium and calciumIon channels like, potassium and calcium
channels, thus affecting membranechannels, thus affecting membrane
excitability, transmitter release,excitability, transmitter release,
contractility secretions ……………contractility secretions ……………

25. Kinase-linked receptorsKinase-linked receptors
 Receptor tyrosine kinasesReceptor tyrosine kinases
 Different from GPCRs and ligand-gatedDifferent from GPCRs and ligand-gated
receptors.receptors.
 Mediate action of growth factors, cytokines,Mediate action of growth factors, cytokines,
insulin and leptin (Read: Ihle 1995; Barbacidinsulin and leptin (Read: Ihle 1995; Barbacid
1996 and Snaar-Jakleska 1999).1996 and Snaar-Jakleska 1999).
 Guanylate-cyclase-linked receptors for atrialGuanylate-cyclase-linked receptors for atrial
natriuretic peptide is included in this group.natriuretic peptide is included in this group.
Since they have similar structure.Since they have similar structure.

26. Kinase-linked receptors ..2Kinase-linked receptors ..2
 The receptor has very large extracellular domainThe receptor has very large extracellular domain
(ligand binding site) and intracellular domain(ligand binding site) and intracellular domain
(effector).(effector).
 Generally signal transduction involvesGenerally signal transduction involves
dimerisation of receptors, followed bydimerisation of receptors, followed by
autophosphorylation of tyrosine residues.autophosphorylation of tyrosine residues.
 Phosphotyrosine residues act as acceptors forPhosphotyrosine residues act as acceptors for
intracellular proteins.intracellular proteins.
 Involved in cell growth and differentiation andInvolved in cell growth and differentiation and
indirectly regulate gene transcriptionindirectly regulate gene transcription

27. Kinase-linked ……………. 3Kinase-linked ……………. 3
 After binding of the intracellular protein (SH2After binding of the intracellular protein (SH2
domain), to the phosphorylated receptor, adomain), to the phosphorylated receptor, a
cascade of reactions take place known ascascade of reactions take place known as
kinase cascade which lead to phospholipidkinase cascade which lead to phospholipid
breakdown (IP3 formation) and Ca2+ release orbreakdown (IP3 formation) and Ca2+ release or
gene transcription.gene transcription.
 All these pathways have opened a new targetAll these pathways have opened a new target
area for new drugs in inflammatory andarea for new drugs in inflammatory and
immunological diseases.immunological diseases.
 Recent breakthrough: use of Imatinib in chronicRecent breakthrough: use of Imatinib in chronic
myeloid leukaemia. This drug inhibits a specificmyeloid leukaemia. This drug inhibits a specific
tyrosine kinase involved in the pathogenesis oftyrosine kinase involved in the pathogenesis of
the disease.the disease.

28. Nuclear ReceptorsNuclear Receptors
 Regulate DNA transcription.Regulate DNA transcription.
 Steroid and thyroid hormones act by stimulatingSteroid and thyroid hormones act by stimulating
transcription of selected genes leading totranscription of selected genes leading to
synthesis of particular proteins production ofsynthesis of particular proteins production of
cellular effects. Most receptors are located in thecellular effects. Most receptors are located in the
nucleus all ligands are lipophilic.nucleus all ligands are lipophilic.
 Glucocorticoids inhibit transcription of the geneGlucocorticoids inhibit transcription of the gene
for cyclooxygenase-2 (COX-2).for cyclooxygenase-2 (COX-2).
 Mineralocorticoids stimulate the production ofMineralocorticoids stimulate the production of
various proteins involved in renal tubularvarious proteins involved in renal tubular
function.function.

29. Nuclear receptorsNuclear receptors
 Ligands include: steroid hormones, thyroidLigands include: steroid hormones, thyroid
hormones, Vit D & retinoic acid and lipidhormones, Vit D & retinoic acid and lipid
lowering (clofibrate) and antidiabetic drugslowering (clofibrate) and antidiabetic drugs
(troglitazone).(troglitazone).
 Receptors are intracellular proteins, so ligandsReceptors are intracellular proteins, so ligands
must first enter cells.must first enter cells.
 One of these nuclear receptors is responsible forOne of these nuclear receptors is responsible for
the increased expression of drug-metabolisingthe increased expression of drug-metabolising
enzymes induced by many therapeutic agents.enzymes induced by many therapeutic agents.

30. Other types of Ion channelsOther types of Ion channels
 Ion channels are characterized by:Ion channels are characterized by:
 Selectivity for particular ion species, whichSelectivity for particular ion species, which
depends on the size of the pore and nature of itsdepends on the size of the pore and nature of its
lining.lining.
 Their gating properties, the mechanism thatTheir gating properties, the mechanism that
controls the transition between open and closedcontrols the transition between open and closed
states of the channelstates of the channel
 Their molecular architecture.Their molecular architecture.
 Cation -selective channels: Ca2+, Na+, K+Cation -selective channels: Ca2+, Na+, K+
 Anion selective: ClAnion selective: Cl--
..

31. Ion channels …………2Ion channels …………2
Voltage-gated channels: open whenVoltage-gated channels: open when
membrane is depolarized. Short-livedmembrane is depolarized. Short-lived
even if depolarization is maintained.even if depolarization is maintained.
Ligand gated channels: activated byLigand gated channels: activated by
ligands.ligands.
Some ligand gated channels in the plasmaSome ligand gated channels in the plasma
membrane respond to intracellular rathermembrane respond to intracellular rather
than extracellular signals.than extracellular signals.

32. Ion channels …………….. 3Ion channels …………….. 3
Examples: Ca2+-activated potassiumExamples: Ca2+-activated potassium
channels, occur in most cells, leading tochannels, occur in most cells, leading to
hyperpolarization when Ca2+ levelshyperpolarization when Ca2+ levels
increase.increase.
ATP-sensitive potassium channels, occurATP-sensitive potassium channels, occur
in many nerve and muscle cells andin many nerve and muscle cells and
insulin secreting cells.insulin secreting cells.
Vanilloid receptor, contain the binding siteVanilloid receptor, contain the binding site
for capsaicin.for capsaicin.

33. Ion channels …. 4Ion channels …. 4
 Calcium release channels: endoplasmic orCalcium release channels: endoplasmic or
sarcoplasmic reticulum.sarcoplasmic reticulum.
 IP3 and raynodine receptors. They control theIP3 and raynodine receptors. They control the
release of Ca2+ from intracellular stores.release of Ca2+ from intracellular stores.
 Store-operated calcium channels: respond toStore-operated calcium channels: respond to
when the intracellular stores of Ca2+ are low,when the intracellular stores of Ca2+ are low,
they operate to replenish these stores.they operate to replenish these stores.
 Triggle 1999, Clare 2000.Triggle 1999, Clare 2000.