Molecular Aspects of Drug ActionMolecular Aspects of Drug Action Receptors:Receptors: Sensing elements in the membranes that Sensing elements in the membranes that

mediate communication between cells and mediate communication between cells and coordinate the function of all the different coordinate the function of all the different cells in the body.cells in the body.

Messengers are the hormones, transmitters, Messengers are the hormones, transmitters, local hormones, peptides, Nitric local hormones, peptides, Nitric oxide………….oxide………….

Many drugs act as agonists or antagonists on Many drugs act as agonists or antagonists on receptors for known endogenous mediators.receptors for known endogenous mediators.

ReceptorsReceptors Isolation of receptors: Electric organs of Isolation of receptors: Electric organs of

many fish consist of tissues that contain many fish consist of tissues that contain acetylcholine receptors; Torpedo fish and acetylcholine receptors; Torpedo fish and electric eelselectric eels

Venoms of snakes of the cobra family Venoms of snakes of the cobra family contains polypeptides that bind with very contains polypeptides that bind with very high specificity to nicotinic receptors.high specificity to nicotinic receptors.

αα-toxins, can be labeled and used to assay -toxins, can be labeled and used to assay the content of tissues of receptors.the content of tissues of receptors.

αα-bungarotoxin (Bangarus multicinctus)-bungarotoxin (Bangarus multicinctus)

ReceptorsReceptorsTreatment of receptors with non-ionic Treatment of receptors with non-ionic

detergents to solubilize the detergents to solubilize the membranes and then isolate them and membranes and then isolate them and then purify and amino acid sequence is then purify and amino acid sequence is determined.determined.

Cloning of receptors: cloned DNA Cloning of receptors: cloned DNA encoding individual receptors. encoding individual receptors.

Receptor subtypes were isolated using Receptor subtypes were isolated using this technique.this technique.

Ion ChannelsIon Channels Ligand-gated ion channels: open only when Ligand-gated ion channels: open only when

the receptor is occupied by an agonist.the receptor is occupied by an agonist. Voltage-gated channels: are very important, Voltage-gated channels: are very important,

being stimulated by changes in the being stimulated by changes in the membrane potentialmembrane potential

Drugs can interact either with the receptor Drugs can interact either with the receptor site of the ligand gated channels or with site of the ligand gated channels or with other parts of the channel molecule.other parts of the channel molecule.

Interaction can be indirect through G-Interaction can be indirect through G-proteinsproteins

Direct interaction with the channel protein.Direct interaction with the channel protein.

Ion ChannelsIon ChannelsLocal anesthetics: act by a simple Local anesthetics: act by a simple

mechanism of plugging the channel mechanism of plugging the channel physically and prevent the sodium ion physically and prevent the sodium ion from passing through the channel.from passing through the channel.

Drugs: dihydropyridines, inhibit the Drugs: dihydropyridines, inhibit the opening of L-type calcium channelsopening of L-type calcium channels

Benzodiazepines: bind to a site near Benzodiazepines: bind to a site near the GABA-receptors (chloride channel, the GABA-receptors (chloride channel, which is a ligand gated ion channel).which is a ligand gated ion channel).

EnzymesEnzymes Enzymes are targets for many drugsEnzymes are targets for many drugs Reversible agents e.g Neostigmine, Reversible agents e.g Neostigmine,

allopurinol.allopurinol. Irreversible agents e.g Aspirin, Irreversible agents e.g Aspirin,

Phenoxybenzamine. Phenoxybenzamine. False substrates: drug molecules undergo False substrates: drug molecules undergo

chemical transformation to form an chemical transformation to form an abnormal product that fools the normal abnormal product that fools the normal metabolic pathway. Fluorouracil, which metabolic pathway. Fluorouracil, which replaces uracil as an intermediate in replaces uracil as an intermediate in purine synthesis, thus blocking DNA purine synthesis, thus blocking DNA synthesis.synthesis.

Carrier moleculesCarrier molecules Transport of ions and organic molecules Transport of ions and organic molecules

across cell membranes.across cell membranes. It requires a carrier protein, since the It requires a carrier protein, since the

molecules are too polar (lipid insoluble). molecules are too polar (lipid insoluble). Choline carrier, Noradrenaline uptake-1, Choline carrier, Noradrenaline uptake-1, Noradrenaline vesicular uptake, Renal Noradrenaline vesicular uptake, Renal tubule weak acid carrier, Natubule weak acid carrier, Na++/K/K++ClCl-- co- co-transporter in Loop of Henle, Na+/K+ transporter in Loop of Henle, Na+/K+ pump in cardiac muscle, Proton pump in pump in cardiac muscle, Proton pump in parietal cells.parietal cells.

Types of ReceptorTypes of ReceptorReceptors produce many different Receptors produce many different

types of cellular events.types of cellular events.Very rapid actions: synaptic Very rapid actions: synaptic

neurotransmission; in millisecondsneurotransmission; in millisecondsAch.Catecholamines: in secondsAch.Catecholamines: in secondsPeptides: take longerPeptides: take longerThyroid and steroids hormones: Thyroid and steroids hormones:

hours or days.hours or days.

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 –binding receptor site, in the extracellular domain.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-amino-3-

hydroxy-5-methyl-4-isoxazolepropionic acid), hydroxy-5-methyl-4-isoxazolepropionic acid), NMDA( N-methyl-D-aspartate receptor) and kinate NMDA( N-methyl-D-aspartate receptor) and kinate receptorsreceptors

5-HT5-HT33 receptors receptors

Ligand-gated receptorsLigand-gated receptorsEach subunit has a mass of 40-58 kDa Each 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 interface binding sites, each lying at the interface between one of the two between one of the two αα-subunits and its -subunits and its neighbour.neighbour.

Ach has to bind to both sites in order to Ach has to bind to both sites in order to activate the receptor. activate the receptor. ( (

Gating MechanismGating Mechanism Fastest synaptic events are mediated by Fastest synaptic events are mediated by

this mechanism, found mainly in this mechanism, found mainly in postsynaptic membranes of nerves or postsynaptic membranes of nerves or muscles.muscles.

In most cases they increase the membrane In most cases they increase the membrane permeability to Napermeability to Na++ and K+. and K+.

This leads to depolarization and may be This leads to depolarization and may be action potential.action potential.

Coupling between the receptor and the Coupling between the receptor and the ionic channel is DIRECT.ionic channel is DIRECT.

Read about, Katz et al (72) & Patch-clamp Read about, Katz et al (72) & Patch-clamp technique.technique.

G-PROTEIN-COUPLED G-PROTEIN-COUPLED RECEPTORSRECEPTORS Most abundant type of receptorsMost abundant type of receptors

Muscarinic, adrenoceptors, dopamine, 5-Muscarinic, adrenoceptors, dopamine, 5-HT, Opiate, receptors for peptides, purine HT, Opiate, receptors for peptides, purine receptors, chemoreceptors for olfaction receptors, chemoreceptors for olfaction and pheromone detection receptors(is a and pheromone detection receptors(is a secreted or excretedsecreted or excretedchemical factor that  factor that triggers a social response in members of triggers a social response in members of the same the same species).).

Quantitative pharmacological studies with Quantitative pharmacological studies with agonists and antagonist have revealed agonists and antagonist have revealed many subtypes.many subtypes.

Most neurotransmitters can interact Most neurotransmitters can interact with both GPCRs and with ligand-with both GPCRs and with ligand-gated receptors. Peptides do not do gated receptors. Peptides do not do the same.the same.

Peptides act either on GPCRs or on Peptides act either on GPCRs or on Kinase-linked receptors.Kinase-linked receptors.

Ligands that act on nuclear receptors Ligands that act on nuclear receptors also show high selectivity.also show high selectivity.

Families of GPCRsFamilies of GPCRsA-family: rhodopsin(purplish-red ,light-A-family: rhodopsin(purplish-red ,light-

sensitive pigment found in the retina of sensitive pigment found in the retina of human eye); amine neurotransmitters, human eye); amine neurotransmitters, many polypeptides, purines, many polypeptides, purines, prostanoids, cannabinoidsprostanoids, cannabinoids

B-family: Secretin/glucagon family; B-family: Secretin/glucagon family; glucagon, secretin, calcitoninglucagon, secretin, calcitonin

C-family: metabotropic family; C-family: metabotropic family; glutamate receptors, GABAglutamate receptors, GABABB receptors, receptors, calcium sensing receptors.calcium sensing receptors.

D-family: pheromones.D-family: pheromones.

G-ProteinsG-Proteins GPCRs control many different aspects of cell GPCRs control many different aspects of cell

functionsfunctions The link between the membrane receptor The link between the membrane receptor

and the first stage of signal transduction and the first stage of signal transduction cascade is established through the G-protein.cascade is established through the G-protein.

It is a membrane protein.It is a membrane protein. G-proteins represent the middle G-proteins represent the middle

management between the receptor and the management between the receptor and the effector enzymes or ion channels.effector enzymes 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 and and γγ..

G-proteinsG-proteins The The αα-subunit possessing GTPase activity.-subunit possessing GTPase activity. When an agonist bind to a receptor the When an agonist bind to a receptor the αα--

subunit dissociates and is then free to subunit dissociates and is then free to activate an ion channel or a membrane activate an ion channel or a membrane enzyme. In some cases the enzyme. In some cases the βγβγ-subunits -subunits may activate the receptors.may activate the receptors.

Activation of the effector is terminated Activation of the effector is terminated when the bound GTP molecule is when the bound GTP molecule is hydrolysed, which allows the hydrolysed, which allows the αα-subunit to -subunit to recombine with recombine with βγβγ..

Variants of G-proteinVariants of G-proteinThe question now is; how is specificity The question now is; how is specificity

achieved so that each kind of receptor achieved so that each kind of receptor produces a distinct pattern of cellular produces a distinct pattern of cellular responses? Ach and NA?responses? Ach and NA?

The main reason is the molecular The main reason is the molecular variation within the G-protein family!!!!variation within the G-protein family!!!!

20, 20, αα-subunits subtypes, 6 of -subunits subtypes, 6 of ββ-subunits -subunits and 12 of and 12 of γγ-subunits of G-proteins, -subunits of G-proteins, giving 1500 variants of the trimer. giving 1500 variants of the trimer.

Classes of G-proteinsClasses of G-proteinsThree classes of G-proteins: GThree classes of G-proteins: Gss, G, Gii and and

GGq. q. Which show selectivity with respect Which show selectivity with respect to receptor and effector cell.to receptor and effector cell.

The The αα-subunit differ in structure.-subunit differ in structure.Example of this difference is the Example of this difference is the

action of cholera and pertussis toxins.action of cholera and pertussis toxins.Cholera toxin acts on Gs and pertussis Cholera toxin acts on Gs and pertussis

toxin acts on Gi.toxin acts on Gi.

Targets for G-proteinsTargets for G-proteins Adenylate cyclase produces cAMPAdenylate cyclase produces cAMP Phospholipase C, produces inositol Phospholipase C, produces inositol

phosphate and diacylglycerol.phosphate and diacylglycerol. Ion channels: calcium and potassium.Ion channels: calcium and potassium. Adenylate cyclase system was discovered Adenylate cyclase system was discovered

by Sutherland in 1965by Sutherland in 1965 Synthesized from ATP and inactivated by Synthesized from ATP and inactivated by

hydrolysis to 5-AMP by phosphodiesterase hydrolysis to 5-AMP by phosphodiesterase family of enzymes.family of enzymes.

Adenylate cycalse/cAMP Adenylate cycalse/cAMP systemsystem

cAMP regulates cellular functions: energy cAMP regulates cellular functions: energy metabolism, cell division, cell metabolism, cell division, cell differentiation, ion channels and differentiation, ion channels and contractile proteins in smooth muscle.contractile proteins in smooth muscle.

Common mechanism; activation of protein Common mechanism; activation of protein kinases by cAMP. kinases by cAMP.

Protein kinases catalyse the Protein kinases catalyse the phosphorylation of serine and threonine phosphorylation of serine and threonine residues using ATP as a source for residues using ATP as a source for phosphate groups.phosphate groups.

Phosphorylation can either stimulate or Phosphorylation can either stimulate or inhibit target enzymes or ion channels.inhibit target enzymes or ion channels.

Adenylate cyclase/cAMP Adenylate cyclase/cAMP systemsystem

Increased activity of voltage-activated Increased activity of voltage-activated calcium channels in heart muscle cells.calcium channels in heart muscle cells.

Phosphorylation of these channels Phosphorylation of these channels increases influx of calcium during increases influx of calcium during action potential, leading to increased action potential, leading to increased force of contraction.force of contraction.

In smooth muscle, inactivation of In smooth muscle, inactivation of mAMP leads to muscle relaxation.mAMP leads to muscle relaxation.

Receptors linked to Gi-Receptors linked to Gi-proteinsproteins

Muscarinic receptors; MMuscarinic receptors; M22 in the heart in the heart αα22-adrenoceptors in smooth muscle.-adrenoceptors in smooth muscle. Opioid receptorsOpioid receptors cAMP can be stimulated directly by other cAMP can be stimulated directly by other

agents like forskolin and fluoride ions.agents like forskolin and fluoride ions. Phosphodiesterase inhibitors: caffeine, Phosphodiesterase inhibitors: caffeine,

Theophylline .Theophylline .

Phospholipase C/PO4 systemPhospholipase C/PO4 system Catalyse the formation of intracellular Catalyse the formation of intracellular

messengers; IPmessengers; IP33 and DAG from membrane and DAG from membrane phospholipids.phospholipids.

IP3 acts to increase free cytosolic CaIP3 acts to increase free cytosolic Ca2+2+ by by releasing Careleasing Ca2+2+ from intracellular from intracellular components.components.

Free CaFree Ca2+2+ initiates muscle contraction, initiates muscle contraction, secretions, enzyme activation and secretions, enzyme activation and membrane hyperpolarization.membrane hyperpolarization.

DAG activates protein kinase , which DAG activates protein kinase , which controls many cellular functions.controls many cellular functions.

Other functions of G-proteinsOther functions of G-proteinsThey also control phospholipase A and They also control phospholipase A and

thus the formation of arachidonic acid thus the formation of arachidonic acid and eicosanoids (polyunsaturated and eicosanoids (polyunsaturated fatty acids found in fish).fatty acids found in fish).

Ion channels like, potassium and Ion channels like, potassium and calciumcalcium

channels, thus affecting membrane channels, thus affecting membrane excitability, transmitter release, excitability, transmitter release, contractility secretions ……………contractility secretions ……………

Kinase-linked receptorsKinase-linked receptors Receptor tyrosine kinasesReceptor tyrosine kinases Different from GPCRs and ligand-gated Different 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; Barbacid insulin and leptin (Read: Ihle 1995; Barbacid 1996 and Snaar-Jakleska 1999). 1996 and Snaar-Jakleska 1999).

Guanylate-cyclase-linked receptors for atrial Guanylate-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.

Kinase-linked receptors ..2Kinase-linked receptors ..2 The receptor has very large extracellular The receptor has very large extracellular

domain (ligand binding site) and domain (ligand binding site) and intracellular domain (effector). intracellular domain (effector).

Generally signal transduction involves Generally signal transduction involves dimerisation of receptors, followed by dimerisation of receptors, followed by autophosphorylation of tyrosine residues. autophosphorylation of tyrosine residues.

Phosphotyrosine residues act as acceptors Phosphotyrosine residues act as acceptors for intracellular proteins.for intracellular proteins.

Involved in cell growth and differentiation Involved in cell growth and differentiation and indirectly regulate gene transcriptionand indirectly regulate gene transcription

Kinase-linked ……………. 3Kinase-linked ……………. 3 After binding of the intracellular protein After binding of the intracellular protein

(SH2 domain), to the phosphorylated (SH2 domain), to the phosphorylated receptor, a cascade of reactions take place receptor, a cascade of reactions take place known as kinase cascade which lead to known as kinase cascade which lead to phospholipid breakdown (IP3 formation) phospholipid breakdown (IP3 formation) and Ca2+ release or gene transcription.and Ca2+ release or gene transcription.

All these pathways have opened a new All these pathways have opened a new target area for new drugs in inflammatory target area for new drugs in inflammatory and immunological diseases.and immunological diseases.

Recent breakthrough: use of Imatinib in Recent breakthrough: use of Imatinib in chronic myeloid leukaemia. This drug chronic myeloid leukaemia. This drug inhibits a specific tyrosine kinase involved inhibits a specific tyrosine kinase involved in the pathogenesis of the disease.in the pathogenesis of the disease.

Nuclear ReceptorsNuclear Receptors Regulate DNA transcription.Regulate DNA transcription. Steroid and thyroid hormones act by Steroid and thyroid hormones act by

stimulating transcription of selected genes stimulating transcription of selected genes leading to synthesis of particular proteins leading to synthesis of particular proteins production of cellular effects. Most production of cellular effects. Most receptors are located in the nucleus all receptors are located in the nucleus all ligands are lipophilic.ligands are lipophilic.

Glucocorticoids inhibit transcription of the Glucocorticoids inhibit transcription of the gene for cyclooxygenase-2 (COX-2).gene for cyclooxygenase-2 (COX-2).

Mineralocorticoids stimulate the Mineralocorticoids stimulate the production of various proteins involved in production of various proteins involved in renal tubular function.renal tubular function.

Nuclear receptorsNuclear receptors Ligands include: steroid hormones, thyroid Ligands include: steroid hormones, thyroid

hormones, Vit D & retinoic acid and lipid hormones, Vit D & retinoic acid and lipid lowering (clofibrate) and antidiabetic lowering (clofibrate) and antidiabetic drugs (troglitazone).drugs (troglitazone).

Receptors are intracellular proteins, so Receptors are intracellular proteins, so ligands must first enter cells.ligands must first enter cells.

One of these nuclear receptors is One of these nuclear receptors is responsible for the increased expression of responsible for the increased expression of drug-metabolising enzymes induced by drug-metabolising enzymes induced by many therapeutic agents.many therapeutic agents.

Other types of Ion channelsOther types of Ion channels Ion channels are characterized by:Ion channels are characterized by: Selectivity for particular ion species, which Selectivity for particular ion species, which

depends on the size of the pore and depends on the size of the pore and nature of its lining.nature of its lining.

Their gating properties, the mechanism Their gating properties, the mechanism that controls the transition between open that controls the transition between open and closed states of the channeland closed states 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--..

Ion channels …………2Ion channels …………2Voltage-gated channels: open when Voltage-gated channels: open when

membrane is depolarized. Short-lived membrane is depolarized. Short-lived even if depolarization is maintained.even if depolarization is maintained.

Ligand gated channels: activated by Ligand gated channels: activated by ligands. ligands.

Some ligand gated channels in the Some ligand gated channels in the plasma membrane respond to plasma membrane respond to intracellular rather than extracellular intracellular rather than extracellular signals.signals.

Ion channels …………….. 3Ion channels …………….. 3Examples: Ca2+-activated potassium Examples: Ca2+-activated potassium

channels, occur in most cells, leading channels, occur in most cells, leading to hyperpolarization when Ca2+ to hyperpolarization when Ca2+ levels increase.levels increase.

ATP-sensitive potassium channels, ATP-sensitive potassium channels, occur in many nerve and muscle occur in many nerve and muscle cells and insulin secreting cells.cells and insulin secreting cells.

Vanilloid receptor, contain the Vanilloid receptor, contain the binding site for capsaicin.binding site for capsaicin.

Ion channels …. 4Ion channels …. 4 Calcium release channels: endoplasmic or Calcium release channels: endoplasmic or

sarcoplasmic reticulum.sarcoplasmic reticulum. IP3 and raynodine receptors. They control IP3 and raynodine receptors. They control

the release of Ca2+ from intracellular the release of Ca2+ from intracellular stores.stores.

Store-operated calcium channels: respond Store-operated calcium channels: respond to when the intracellular stores of Ca2+ are to when the intracellular stores of Ca2+ are low, they operate to replenish these stores.low, they operate to replenish these stores.

Triggle 1999, Clare 2000.Triggle 1999, Clare 2000.