by Lee Eun Jin

MOLECULAR MECHANISM OF ACTION OF DRUGS

Drugs produce effects in the body mainly in the following ways:

1. by acting on receptors 2. by inhibiting carriers (molecules that

transport one or more ions or molecules across the plasma membrane)

3. by modulating or blocking ion channels4. by inhibiting enzymes.

TARGETS FOR DRUG ACTION

PROTEIN TARGETS• RECEPTORS • ION CHANNELS • ENZYMES • CARRIER MOLECULES (TRANSPORTERS). NON-PROTEIN TARGETS-Binding, neutralising, osmosis etc.

RECEPTORS

ION CHANNELS

ENZYMES

CARRIER MOLECULES (TRANSPORTERS).

ACTIONS OF DRUGS NOT MEDIATED BY ANY OF THESE

• Therapeutic neutralization of gastric acid by a base (antacid).

• Drugs: Mannitol -increasing the osmolarity of various body fluids and causing changes in the distribution of water to promote diuresis, catharsis, expansion of circulating volume in the vascular compartment, or reduction of cerebral edema

• Cholesterol-binding agents,(cholestyramine resin) to decrease dietary cholesterol absorption.

RECEPTORS

RECEPTORS

• The term receptor: any cellular macromolecule to which a drug binds to initiate its effects.

• Receptors are protein molecules in or on cells whose function is to interact with the body’s endogenous chemical messengers (hormones, neurotransmitters, the chemical mediators of the immune system, etc.) and thus initiate cellular responses.

• They enable the responses of the body’s cells to be coordinated • A molecule which binds (attaches) to a receptor is called a LIGAND ; - a

peptide, or other small molecule, such as a neuorotransmitter, hormone, chemical/ drug or a toxin.

• A class of cellular macromolecules (cellular proteins) that are concerned specifically and directly with chemical signaling between and within cells.

• Combination of a hormone, neurotransmitter, or intracellular messenger with its receptor(s) results in a change in cellular activity.

• A receptor functions: recognize the particular molecules that activate (act as receptors for endogenous regulatory ligands)+ Message propagation (alter cell function)

RECEPTOR• Macromolecules that bind mediator

substances and transduce this binding into an effect, i.e., a change in cell function.

• The component of a cell or organism that interacts with a drug and initiates the chain of biochemical events leading to the drug's observed effects.

• Isolation and characterization -the molecular basis of drug action.

RECEPTORS• Ligand binding and message propagation (i.e., signalling)• Functional domains within the receptor: a ligand-binding domain

and an effector domain. • The regulatory actions of a receptor : Directly on its cellular

target(s), effect or protein(s), or may be conveyed by intermediary cellular signaling molecules : Transducers.

• The receptor, its cellular target, and any intermediary molecules : Receptor–effector system or signal-transduction pathway

• An enzyme or transport protein that creates,moves, or degrades a small metabolite (e.g., a cyclic nucleotide or inositol trisphosphate) or ion (e.g., Ca2+) : Second messenger. (Neuromediator)

Eg; cAMP.IP3, DAG, PDE etc• Second messengers : diffuse in the proximity of their binding sites

and convey information to a variety of targets, which can respond simultaneously to the output of a single receptor binding a single agonist molecule.

• Receptor–transducer–effector–signal termination complexes—established via protein–lipid and protein–protein interactions.

• Receptors and their associated effector and transducer proteins also act as integrators of information as they coordinate signals from multiple ligands with each other and with the metabolic activities of the cell.

• An important property of physiological receptors : Excellent targets for drugs- they act catalytically and hence are biochemical signal amplifiers. The catalytic nature of receptors is obvious when the receptor itself is an enzyme

• A single agonist molecule binds to a receptor that is an ion channel, hundreds of thousands to millions of ions flow through the channel every second.

• Similarly, a single steroid hormone molecule binds to its receptor and initiates the transcription of many copies of specific mRNAs, which, in turn, can give rise to multiple copies of a single protein.

• Drugs that bind to physiological receptors and mimic the regulatory effects of the endogenous signaling compounds are termed AGONISTS

(Affinity and efficacy: 1)• Agents those bind to receptors without regulatory

effect, but their binding, blocks the binding of the endogenous agonist.:

ANTAGONISTS (Affinity1, efficacy 0) • Agents that are only partly as effective as agonists no

matter the dose employed are: PARTIAL AGONISTS ( Affinity 1, Efficacy <1)• Agents that stabilize the receptor in its inactive

conformation are termed INVERSE AGONISTS ( Affinity 0; Efficacy -1 ) • The strength of the reversible interaction between a

drug and its receptor, as measured by their dissociation constant, is defined as the affinity of one for the other.

MOLECULAR STRUCTURE OF RECEPTORS : FAMILIES

• The molecular organisation : Four receptor families• Individual receptors show considerable sequence

variation in particular regions• Lengths of the main intracellular and extracellular

domains- vary from one to another within the same family

• The overall structural patterns and associated signal transduction pathways are very consistent.

RECEPTOR HETEROGENEITY AND SUBTYPES

• Receptors within a given family : Molecular varieties, or subtypes, with similar architecture; differences in their sequences, pharmacological properties.

• Distinct subtypes occur in different regions/organs, and these differ from the receptors in other organ Eg: Ach- Nicotinic

• The sequence variation that accounts for receptor diversity arises at the genomic level, i.e. different genes give rise to distinct receptor subtypes.

• A single gene can give rise to more than one receptor isoform.• After translation from genomic DNA, the mRNA contains non-

coding regions that are excised splicing before the message is translated into protein.

• Splicing : result in inclusion/deletion of one/more of the mRNA coding regions, giving rise to long or short forms of the protein.

(eg: GPCR)

receptors

• Physiological receptors• Agonist – primary agonist -allosteric agonist -partial agonist• Antagonist – syntopic -allosteric

-chemical -functional

RECEPTOR HETEROGENEITY AND SUBTYPES

• Molecular heterogeneity : feature of receptors- functional proteins in general.

• New receptor subtypes and isoforms : options for therapy

• Pharmacological viewpoint: To understand individual drugs action, effects; Molecular pharmacology.

TYPES/FAMILIES OF RECEPTORS

Based on molecular structure and the nature of the linkage (the transduction mechanism)

Ligand-gated ion channels (Ionotropic) Nicotinic acetylcholine receptor, GABA A receptor G-protein-coupled receptors

(GPCRs)/Metabotropic Muscarinic acetylcholine receptor, adrenoceptors Kinase( Tyrosine)-linked and related receptors Insulin, growth factors, cytokine receptors Nuclear/ Cytosol receptors steroids, thyroid hormones, gonadal steroids,vit D

MAJOR RECEPTOR FAMILIES

Transmembrane signaling mechanisms. A. Lignad binds to the extracellular domain of a ligand-gated channel. B. Ligand binds to a domain of the serpentine receptor, which is coupled to G protein. C. Ligand binds to the extracellular domain of a receptor that activates a kinase enzyme.D. Lipid-soluble ligand diffuses across the membrane to interact with its intracellular receptor.

Ion Channel Linked • The molecules responsible for transduction are

ions (e.g., Na+ or Ca2+) that are normally found outside of cells.

• Binding of a ligand to the receptor results in an opening of a gate through the plasma membrane (hyperpolaristaion/depolaristaion) that allows entrance of the ions (both gate and receptor are proteins, likely one in the same protein).

• The increased ion concentration in the cytoplasm propagates – signal transduction – results in a direct stimulation of a response.

Structure of the nicotinic acetylcholine receptor (a typical ligand-gated ion channel)

• The five receptor subunits (α2, β, γ, δ) : a cluster surrounding a central transmembrane pore

• Contain negatively charged aminoacids , which makes the pore cation selective.

• Two acetylcholine binding sites in the extracellular portion of the receptor, at the interface between the α and the adjoining subunits.

• On ACh binding: kinked α helices straighten out or swing out of the way, thus opening the channel pore.

Typical ligand-gated ion channel receptor

Ligand-gated Ion Channel Receptor• The receptor complex consists of five subunits, each

of which contains four transmembrane domains. • Simultaneous binding of two acetylcholine (ACh)

molecules to the two α-subunits results in opening of the ion channel, with entry of Na+ (and exit of some K+), membrane depolarization, and triggering of an action potential

• The ganglionic N-cholinoceptors apparently consist only of α and β subunits (α2β2). :

• GABAA subtypeGlutamate and glycine

GPCR

GPCR• Typically "serpentine," with seven transmembrane spanning

domains, the third one of which is coupled to the G-protein effector mechanism.

• The signaling molecule binds to the G-protein coupled receptor

• This causes a change in the receptor so it is able to bind to an inactive G protein.

• This causes a GTP to replace a GDP which activates a G protein.

• Receptor systems coupled via GTP-binding proteins (G-proteins) to adenylyl cyclase,(converts ATP to a second messenger cAMP,) that promotes protein phosphorylation by activating protein kinase A.

• The protein kinase A serves to phosphorylate a set of tissue-specific substrate enzymes, thereby affecting their activity.

G-PROTEIN-COUPLED RECEPTORS

• The seven α-helical membrane-spanning domains probably form a circle around a central pocket that carries the attachment sites for the mediator substance.

• Binding of the mediator molecule or of a structurally related agonist molecule induces a change in the conformation of the receptor protein, enabling the latter to interact with a G-protein (= guanyl nucleotide-binding protein).

• G-proteins lie at the inner leaf of the plasmalemma and consist of three subunits designated α, β, and γ.

• There are various G-proteins that differ mainly with regard to their α-unit. Association with the receptor activates the G-protein, leading in turn to activation of another protein (enzyme, ion channel).

• A large number of mediator substances act via G-protein-coupled receptors

G-protein coupled receptors triggers an increase (or, less often, a decrease) in the activity of adenylyl cyclase.

G-Protein coupled effector system

1. Adenylate cyclase-cAMP system

2. Phospholipase-C-inositol phosphate system

3. Ion channels

Adenylate cyclase-cAMP system

Phospholipase-C-inositol phosphate system

Three main variants of GPCRs

1. Gs: Stimulation of Adenyl cyclase

2. Gi: Inibition of Adenyl cyclase

3. Gq: Controls phospholipase-C activity

Kinase-linked Receptors

• These receptors are directly linked to:

1. Tyrosine kinase (e.g. receptors for insulin and various growth factors)

Or2. Guanylate cyclase (e.g. receptors for atrial

natriuretic peptide)

Receptors That Function as Transmembrane Enzymes Tyrosine kinase linked receptors

• Cell-surface receptors, Membrane-spanning macromolecules • Bind a large variety of watersoluble ligands, including amines,

amino acids, lipids, peptides, and proteins. • The ligand-binding domain is connected to the cytoplasmic

domain by a single transmembrane helix. • In receptors with intrinsic enzymatic activity, the cytoplasmic

domain contains a conserved protein tyrosine kinase (PTK) core and additional regulatory sequences that are subjected to autophosphorylation and phosphorylation by heterologous protein kinases

• Binding of the ligand causes confirmational changes so that the kinase domains become activated, ultimately leading to phosphorylation of tissue-specific substrate proteins.

• It initiates a unique cellular response for each phosphorylated tyrosine.

Tyrosine kinase receptor

2.kinase linked receptorsKINASE LINKED RECEPTORS

Receptors linked to gene transcription Intracellular Cytosol/ Nulcear Receptors

• Binding of hormones or drugs to receptors releases regulatory proteins that permit of the hormone-receptor complex.

• Such complexes interact with response elements on nuclear DNA to modify gene expression.

• Eg: drugs interacting with glucocorticoid receptors lead to gene expression of proteins that inhibit the production of inflammatory mediators.

• Pharmacologic responses elicited via modification of gene expression are usually slower in onset but longer in duration than other drugs.

Mechanism of intracellular receptors (e.g. nuclear receptors).

3.Nuclear receptors

Down-regulation of Receptors

• Prolonged exposure to high concentration of agonist causes a reduction in the number receptors available for activation.

• This results due to endocytosis or internalisation of the receptors from the cell surface

Up-regulation of Receptors

• Prolonged occupation of receptors by a blocker leads to an increase in the number of receptors with subsequent increase in receptor sensitivity.

• This is due to externalisation of the receptors from inside of the cell surface.

Spare Receptors

• A drug can produce the maximal response when even less than 100% of the receptors are occupied. The remaining unoccupied receptors are just serving as receptor reserve are called spare receptors

SPARE RECEPTORS

• The production of a maximal tissue response when only a fraction of the total number of receptors are occupied

• Eg: Acetylcholine analogues in isolated tissues, capable of eliciting maximal responses at very low occupancies, often less than 1%.

• The mechanism linking the response to receptor occupancy has a substantial reserve capacity. Such system-said to possess spare receptors, or a receptor reserve.

• Common with drugs : smooth muscle contraction; less for : RESPONSES-secretion, smooth muscle relaxation or cardiac stimulation: the effect is more nearly proportional to receptor occupancy.

• Do not imply any functional subdivision of the receptor pool, • This surplus of receptors over the number actually needed might seem a

wasteful biological arrangement. It means, however, that a given number of agonist-receptor complexes, corresponding to a given level of biological response, can be reached with a lower concentration of hormone or neurotransmitter than would be the case if fewer receptors were provided..

RECEPTORS AND DISEASE

• Molecular pharmacology: revealed a number of disease states directly linked to receptor malfunction.

The principal mechanisms:• Autoantibodies directed against receptor proteins – Eg: Myasthenia gravis , - autoantibodies that inactivate

nicotinic acetylcholine receptors. Autoantibodies can also mimic the effects of agonists, as in many cases of thyroid hypersecretion, caused by activation of thyrotropin receptors

• Mutations in genes encoding receptors and proteins involved in signal transduction. – Mutations of genes encoding GPCRs:

hypoparathyroidism, cancers

Receptor Related Diseases

• Myasthenia Gravis:– Antibodies against the cholinergic nicotinic receptors

at motor end plate.

• Insulin Resistant Diabetes

• Testicular feminisation

• Familial Hypercholesterolaemia

ION CHANNELS

Some drugs produce their actions by directly interacting with ion channels. These ion channels transport ions across the plasma membrane. They are not receptors and should be distinguished from ion channels that function as ionotropic receptors

Voltage-Operated Channels

• VOC’s like ROC’s are ion channels that are gated only by voltage.

• While ROC’s assume only 2 states: Open or Close; VOC’s also assumes a third state called ‘refractory’ (inactivated) state.

Refractory State

• In this state the channel is unable to ‘open’ (or reactivate) for a certain period of time even when the membrane potential returns to a voltage that normally opens or activates the channel.

• State Dependent Binding

ION CHANNELS AS TARGETS FOR DRUG ACTION

CARRIERS AS TARGETS FOR DRUG ACTION.

• Membrane transport proteins (Transmemebrane Proteins) are two main types:

• ATP-powered ion pumps• Transporters • Both are transmembrane proteins. , termed

‘carriers

CARRIERS AS TARGETS FOR DRUG ACTION.

ATP-powered ion pumps• The three principal ion pumps are the sodium pump (the Na+/K+

ATPase), the calcium pump, and the Na+/H+ pump in the gastric parietal cell, which is the target for the proton pump inhibitor omeprazole.

• Sodium pump. - important in maintaining cellular osmotic balance and cell volume and in maintaining the membrane potential.

• In many cells (e.g. in the myocardium, the nephron) it is the primary mechanism for transporting Na+ out of the cell

• The K+ concentration is 140 mmol/l inside cells and 5 mmol/l outside. For each molecule of ATP hydrolysed, the sodium pump pumps 3Na+ out of the cell and 2K+ in against their chemical gradients

Carrier molecules (transporters) • The main transporters involved in drug action are symporters and

antiporters (exchangers)

CARRIERS AS TARGETS FOR DRUG ACTION.

Carrier molecules (transporters) • Symporters These use the electrochemical gradient of one ion (usually

Na+) to carry another ion (or molecule or several ions) across a cell membrane. Drugs can modify this action by occupying a binding site (e.g. the action of furosemide (frusemide) on the Na+/K+/2Cl– symport in the nephron (

• Similarly, thiazide diuretics bind to and inhibit the Na+/Cl– symporter in the distal tubule.

• Antiporters These use the electrochemical gradient of one ion (usually Na+) to drive another ion (or molecule) across the membrane in the opposite direction. An important example is the Ca2+ exchanger, which exchanges 3Na+ for 1Ca2+

• This calcium exchanger should be distinguished from the ATPdriven calcium pump and the ligand-gated and voltage-gated Ca2+ channels .

• The calcium exchanger is crucial in the maintenance of the Ca2+ concentration in blood vessel smooth muscle and cardiac muscle

• OtherEg: uptake carrier in the noradrenergic varicosity, which transports noradrenaline into the cell

CARRIER MOLECULES(TRANSPORTERS) AS TARGETS FOR DRUG ACTION

CARRIER MOLECULES(TRANSPORTERS) AS TARGETS FOR DRUG ACTION

ENZYMES Drugs can produce effects on enzyme reactions by substrate competition or by reversibly or irreversibly modifying the enzyme

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