3. receptors as drug targets.pdf
TRANSCRIPT
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Receptors as Drug Targets
Compiled by: Chikowe, I.
Basic Medical Sciences College of Medicine
Malawi
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Receptors Receptors: are specific areas of certain proteins and
glycoproteins that are found either embedded in cellular membranes or in the nuclei of living cells.
Cell surface/membrane receptor: receptor embedded in the cell membrane and transfers chemical information from the extracellular compartment to the intracellular compartment.
Nuclear receptor: receptor that exists in the intracellular compartment and upon activation binds to regulator regions in the DNA and modulates gene expression.
Ligand: any endogenous (messenger) or exogenous chemical agent that binds to a receptor.
Binding domain: the general region on a receptor where a ligand binds.
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Receptors and Messengers Receptors and their chemical messengers are crucial to the
communication systems of the body.
When the communication goes wrong, the body does not work normally and this can lead to ailments like:
Depression
Heart problems
Schizophrenia
Muscle fatigue and many more.
The problems could be 2 ways:
Too many messengers being released leading to overheating of target cells (metaphorically)
Too few messengers being released making the cell sluggish
So drugs can either increase messengers or block messengers. 3
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Cell
Nerve
Messenger
Signal
Receptor
Nerve
Nucleus Cell
Response
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Examples of Chemical messengers
Simple molecular neurotransmitters:
Monoamines; e.g. acetylcholine, noradrenaline, dopamine, serotonin.
Amino acids; -aminobutylic acid (GABA), glutamic acid, glycine.
Calcium ion
Complex molecular chemical messengers:
Lipids; prostaglandins, purines (adenosine or ATP).
Neuropeptides; endorphins, enkephalins
Peptide hormones; angiotensin, bradykinin
Enzymes; thrombin.
Receptors are identified by specific neurotransmitter or hormone that activates them: e.g. receptor activated by dopamine is called dopaminergic receptor; cholinergic receptor for aceytlcholine;
adrenergic receptor or adrenoceptor for adrenaline or noradrenaline. 5
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Neurotransmitters do not undergo reaction when they bind receptor. They leave receptor unchanged after passing on their message.
The binding of messenger induces change in shape which causes the opening of ion channel.
A target cell may have various receptors specific to different types of messengers.
Not all receptors activated by same chemical messenger are exactly the same throughout the body. E.g.
adrenergic receptors in lungs slightly different from adrenergic receptors in heart; due to variations in amino acid composition.
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Nerve 1
Nerve 2 Hormone
Blood
supply
Neurotransmitters
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Cell surface receptor
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Nuclear receptor
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Structure and Function of Receptors
Most receptors are proteins with various post-translational modifications like covalent attachments of carbohydrate, lipid and phosphate.
Responses to extracellular environment involve receptors that modulate cellular components which generate, amplify, coordinate and terminate post-receptor signaling via (cytoplasmic) second messengers. E.g. cyclic adenosinemonophosphate (cAMP).
These secondary messengers promote a sequence of biochemical events that result in an appropriate physiological response
Signal transduction: the mechanism by which any message carried by the ligand is translated through the receptor system into a tissue response.
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Examples of common bonding forms in drug receptor interactions (minus van der waals forces)
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Structure and Function-Mechanism
Receptors contain a binding site (hollow or cleft in the receptor surface) that is recognised by the chemical messenger
Binding of the messenger involves intermolecular bonds
Binding results in an induced fit of the receptor protein
Change in receptor shape results in a domino effect
Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell
Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound
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Illustration of Mechanism
Binding site:
A hydrophobic hollow or cleft on the receptor surface - equivalent to the active site of an enzyme
Accepts and binds a chemical messenger
Contains amino acids which bind the messenger
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Cell Membrane
Cell
Receptor
Messenger
message
Induced fit
Cell
Receptor
Messenger
Message
Cell
Messenger
Receptor
Binding site
ENZYME
Binding site
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Receptor/Messenger Binding
Binding site is nearly the correct shape for the messenger
Binding alters the shape of the receptor (induced fit)
Altered receptor shape leads to further effects - signal transduction
Bonding forces:
Ionic, H-bonding, van der Waals.
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M
M
E R R
M
E R
Signal transduction
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Example
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Receptor
Binding site
vdw
interaction
ionic
bond
H-bond
Phe
Ser
O H
Asp
CO2
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Induced fit - Binding site alters shape to maximize intermolecular bonding
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Intermolecular bonds not
optimum length for
maximum binding strength
Intermolecular bond
lengths optimised
Phe
Ser O
H
Asp
CO2 Induced
Fit
Phe
Ser
O H
Asp
CO2
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Transmembrane signaling of cell surface receptor
This is accomplished by only a few mechanisms:
Transmembrane ion channels: open or close upon binding of a ligand or upon membrane depolarization
G-protein-coupled receptors: Transmembrane receptor that stimulates a GTP-binding signal transducer protein (G-protein) which then generates intracellular 2nd messenger
Nuclear receptors: Lipid soluble ligand that crosses the cell membrane and acts on an intracellular receptor
Kinase-linked receptors: Transmembrane receptor proteins with intrinsic or associated kinase activity which is allosterically regulated by a ligand that binds to the receptors extracellular domain.
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Summary of receptors
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Ion Channels Rapidly acting (milliseconds) transmembrane ion channels:
Multi-unit complexes with central aqueous channel. Upon binding of a ligand, channel opening allows a specific ion travel down its concentration gradient.
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Control of Ion Channels
Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory.
Anionic ion channels for Cl- (e.g. GABAA) = inhibitory. 19
Cell membrane
Five glycoprotein subunits
traversing cell membrane
Messenger
Cell membrane
Receptor
Induced
fit
Gating
(ion channel
opens)
Binding
site
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Induced fit
and opening
of ion channel
ION
CHANNEL
(open)
Cell
Cell
membrane
MESSENGER
Ion
channel
Ion
channel Cell
membrane
ION
CHANNEL
(closed)
Cell
RECEPTOR
BINDING
SITE
Lock Gate
Ion
channel
Ion
channel Cell
membrane
Cell
membrane
MESSENGER
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Voltage-gated ion channels:
Gating: controlled by membrane polarization/depolarization
Not controlled by binding of ligands, rather they sense the potential difference across the cell membrane.
Selectivity: Na+, K+ or Ca+ ions
Important drug targets for local anaethetics
Intracellular ligand-gated channels:
Consist of 5 protein subunits with receptor binding site being present on one or more of the subunits
Binding of neurotransmitter to ion channel receptor causes a conformational change in protein subunits so that the second transmembrane domain of each subunit rotates to open the channel
Ca+ controlled K+ channel
ATP-sensitive K+ channel. 21
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Responsible for
neurotransmission
cardiac conduction
muscle contraction etc...
E.g: Cholinergic nicotinic receptors is an example to these type of receptors. 22
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G-Protein-coupled Receptors GPCR: Large family of receptors with a probable
common evolutionary precursor. Transmembrane protein that is serpentine in shape, crossing the lipid bilayer seven times.
The G-protein-coupled receptors are membrane-bound proteins with 7 transmembrane sections. The c-terminal chain lies within the cell and the N-terminal chain is extracellular.
They activate signal proteins called G-proteins.
Location of binding sites differs between different G-protein-coupled receptors.
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Binding of messenger leads to opening of binding site for signal protein. The latter binds and fragments, with one of the subunits departing to activate a membrane-bound enzyme.
The rhodopsin-like family of G-protein-coupled receptors includes many receptors that are targets for currently important drugs. 24
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Second messengers Essential in conducting and amplifying signals from G-protein
coupled receptors.
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DAG Ca cAMP cGMP IP3
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Illustration of G-Protein-coupled Receptors Activation
Here, receptor binds messenger leading to induced fit; opens a binding site for signal protein (G-protein) and the G-Protein is destabilised then split.
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messenger
G-protein
split
induced
fit
closed open
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G-Protein-Enzyme-linked receptors
Spans the membrane once and may form dimers.
These receptors also have cytosolic enzyme activity as an integral component of their structure.
Metabolism
Growth
Differentiation
Most common Enzyme-linked receptors are: EGF
PDGF tyrosine kinase activity
ANP
Insulin
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important functions controlled by these receptors.
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Illustration of enzyme linked receptor
In some cases: G-Protein subunit activates membrane bound enzyme; binds to allosteric binding site; induced fit results in opening of active site and Intracellular reaction catalysed.
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active site (closed)
active site (open)
Enzyme
Intracellular reaction
Enzyme
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In other cases: Protein serves dual role - receptor plus enzyme; receptor binds messenger leading to an induced fit; protein changes shape and opens active site; reaction catalysed within cell.
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closed
messenger
induced
fit
active site
open
intracellular reaction
closed
messenger
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Kinase-linked Receptors Receptors directly linked to kinase enzymes.
Messengers binding leads to opening of kinase active site, allowing a catalytic reaction to take place.
A good example of Kinase-linked receptors is tyrosine-linked receptor:
Tyrosine kinase receptors have an extracellular binding site for a chemical messenger and an intracellular enzymatic active site which catalyzes the phosphorylation of tyrosine residues in protein substrates. E.g. receptor for insulin and growth factor.
Insulin receptor is preformed heterotetrameric structure that acts as a tyrosine kinase receptor.
Growth hormone receptor dimerises on binding its ligand, then binds and activates tyrosine kinase enzymes from the cytoplasm. 30
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Intracellular Receptors
Receptor is entirely intracellular.
Ligand must have sufficient lipid solubility.
Primary targets of these ligand-receptor complexes are transcription factors.
Steroid hormones exert their effects by this receptor mechanism.
DNA RNA proteins
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Illustration
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Regulation of Receptors Receptors not only initiate regulation of physiological and
biochemical function but are themselves subject to many regulatory and homeostatic controls.
Controls include
regulation of synthesis and
degradation of the receptor by multiple mechanisms;
covalent modification,
association with other regulatory proteins, and/or
relocalization within the cell.
Modulating inputs may come from other receptors.
Receptors are always subject to feedback regulation by their own signaling outputs.
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Reduced responsivity: Chronic use of an agonist can result in the receptor-effector system becoming less responsive
eg. alpha-adrenoceptor agents used as nasal decongestants
Myasthenia gravis: decrease in number of functional acetylcholine nicotinic receptors at the neuromuscular junction.
Increased responsivity: Chronic disuse of a receptor-effector system can result in an increased responsiveness upon re-exposure to an agonist.
Denervation super sensitivity at skeletal muscle acetylcholine nicotinic receptors
Thyroid induced upregulation of cardiac beta-adrenoceptors
Prolonged use of many antagonists (pharmacological as well as functional) can result in receptor upregulation.
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Receptor upregulation
Most receptors are internalized and degraded or recycled with age and use.
Antagonists slow use-dependent internalization
Inverse agonists stabilize the receptor in the inactive state to prevent internalization.
The cell continues to produce receptors.
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Drug Designing from Ligand-Receptor
Agonists: drugs designed to mimic the natural messenger
Agonists should bind and leave quickly - number of binding interactions is important
Antagonists: drugs designed to block the natural messenger
Antagonists tend to have stronger and/or more binding interactions, resulting in a different induced fit such that the receptor is not activated 38
M
M
E R R
M
E R
Signal transduction
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Design of agonists Agonists: bind reversibly to binding site and produce same
induced fit as the natural messenger - receptor is activated
Similar IMF bonds formed as with natural messenger
Agonists often similar in structure to the natural messenger
must have the correct binding groups
binding groups must be correctly positioned
must have the correct shape and size to fit the binding site
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E
Agonist
R E
Agonist
R
Signal transduction
Agonist
R
Induced fit
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Design of agonist-Binding groups
Know the structure of the natural chemical messenger and identify the functional groups involved in the bonding with receptor.
In the hypothetical neurotransmitter shown above, important binding groups and respective interactions are: aromatic ring (van der waals), alcohol (H-bonding), ammonium ion (ionic bonds).
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van der Waals binding region H-bond
binding region Ionic binding region
Binding groups
Neurotransmitter
O O 2 C
H
Binding site
Receptor
NH2Me
OHH
-
O
N H 2 M e
H
H O
O 2
C
H
Binding site
Receptor
O
N H 2 M e
H
H O
O 2
C H
Binding site
Receptor
INDUCED FIT
Induced fit allows stronger binding interactions
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Design of Agonist
Compare Binding groups:
Identify important binding interactions in natural messenger
Agonists are designed to have functional groups capable of the same interactions
Usually require the same number of interactions
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Hypothetical neurotransmitter
H O N H 2 M e
H
H-bonding
group
van der Waals
-bonding
group
Ionic
binding
group
H 2 N N H 2 M e
H
N H M e H O H O
N H 2 M e
H H
H M e
Possible agonists with similar binding groups
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O O
2 C
H
Binding site
Receptor
O O
2 C
H
Binding site
Receptor
H C H 2 M e
H
Structure II has 2 of the 3 required binding groups - weak activity
H N H 2 M e
H
I
HCH2Me
H
II
H N H 2 M e
H
Structure I has one weak binding group - negligible activity
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Binding groups must be positioned such that they can interact with complementary binding regions at the same time
Example has three binding groups, but only two can bind simultaneously
Example will have poor activity.
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H
N H 2
M e
O H
H
O O
2 C
H
Binding site
2 Interactions only
H
N H 2 M e
H
O H
No interaction
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One enantiomer of a chiral drug normally binds more effectively than the other
Different enantiomers likely to have different biological properties. 45
O O
2 C
H
Binding site
3 interactions
O
N H 2 M e
H
H
O O
2 C
H
Binding site
2 interactions
O H
N H 2 M e
H
O N H 2 M e
H
H
O M e H 2 N
H
H
Mirror
Enantiomers of a chiral molecule
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Agonist must have correct size and shape to fit binding site
Groups preventing access are called steric shields or steric blocks. 46
O
N H
2
H
H
Me
C H 3
No Fit
O
O 2
C
H
Binding site
C H 3
Steric block
Me
Steric block
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Design of Antagonists Antagonists bind to the binding site through IMF but fail to
produce the correct induced fit - receptor is not activated
Normal messenger is blocked from binding
Level of antagonism depends on strength of antagonist binding and conc. and increasing the messenger concentration reverses antagonism.
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O N
H
H
M e
H
H
O O
2 C
H
Binding site
Perfect Fit (No change in shape)
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A binding site can have extra binding regions
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OH
O 2
C
Receptor binding site
Extra binding regions
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Antagonists can form binding interactions with extra binding regions neighboring the binding site for the natural messenger
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O
O
Asp
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HO
Extra hydrophobic
binding region
Hydrophobic
binding region
Ionic binding
region
H-bond
binding region
Hypothetical neurotransmitter
NH2Me
HO
H
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Hydrophobic
region
O
O
Asp
-
HO
Induced fit resulting from binding of the normal messenger
NH2Me
HO
H
Hydrophobic
region
O
O
Asp
HO
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NH2Me
HO
HInduced fit
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Hydrophobic
region
O
O
Asp
HO
Hydrophobic
region
HO
Initial binding
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Different induced fit resulting from extra binding interaction
NHMe
HO
H
Hydrophobic
region
O
O
Asp
HO
Different induced fit
- NHMe
HO
H
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Competitive (Reversible) Antagonists
Antagonist binds reversibly to the binding site
Intermolecular bonds involved in binding
Different induced fit means receptor is not activated
No reaction takes place on antagonist
Level of antagonism depends on strength of antagonist binding and concentration
Messenger is blocked from the binding site
Increasing the messenger concentration reverses antagonism.
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An
E R
M
An
R
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Irreversible Antagonists Non-Competitive (Irreversible) Antagonists
Antagonist binds irreversibly (covalent) to the binding site
Different induced fit means that the receptor is not activated
Messenger is blocked from the binding site
Increasing messenger conc. does not reverse antagonism
Often used to label receptors. 53
X
OH OH
X
O
Covalent Bond
Irreversible antagonism
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1
N u
N u
Receptor
Propylbenzilylcholine mustard
C l
C l
Agonist binding site
Antagonist binding site
C l
C l
HOO
O
NCl
Cl
N u
N u
Receptor
2 Irreversible binding
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Non-competitive (Reversible) Allosteric Antagonists
Antagonist binds reversibly to an allosteric binding site
IMF bonds formed between antagonist and binding site
Induced fit alters the shape of the receptor
Binding site is distorted and is not recognized by messenger
Increasing messenger concentration does not reverse antagonism. 55
ACTIVE SITE (open)
ENZYME Receptor
Allosteric binding site
Binding site
(open) ENZYME Receptor
Induced fit
Binding site unrecognisable
Antagonist
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Antagonists by the umbrella effect
Antagonist binds reversibly to a neighbouring binding site
IMF bonds formed between antagonist and binding site
Antagonist overlaps the messenger binding site
Messenger is blocked from the binding site
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Antagonist
Binding site for antagonist
Binding site for messenger
messenger
Receptor Receptor
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Partial Agonist Agents which act as agonists but produce a weaker effect.
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Partial agonist Slight shift
Partial opening of an ion channel
Receptor
O O 2 C
H
1
N H M e
O
H
H H
Receptor
O
O 2 C
2
N H M e
O
H
H
Possible explanations
Agent binds but does not produce ideal induced fit for maximum effect
Agent binds to binding site in two different modes, one where the agent acts as an agonist and one where it acts as an antagonist
Agent binds as an agonist to one receptor subtype but as an antagonist to another receptor subtype.
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Inverse Agonists Properties shared with antagonists
Bind to receptor binding sites with a different induced fit from the normal messenger
Receptor is not activated
Normal messenger is blocked from binding to binding site
Properties not shared with antagonists
Block any inherent activity related to the receptor (e.g. GABA receptor)
Inherent activity = level of activity present in the absence of a chemical messenger
Receptors are in an equilibrium between constitutionally active and inactive forms. 58
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Explanation of how drugs affect receptor equilibria A) Resting state
B) Addition of agonist
C) Addition of antagonist
D) Addition of inverse agonist
E) Addition of partial agonist
Inactive conformations Active conformation
Agonist binding site
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Desensitization Receptors become desensitized on long term exposure to
agonists
Prolonged binding of agonist leads to phosphorylation of receptor
Phosphorylated receptor changes shape and is inactivated
Dephosphorylation occurs once agonist departs
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Receptor
O O2C
1
H Ion channel
(closed)
Agonist NH3
Receptor
O
H
Agonist NH3
O2C
Induced fit alters protein shape Opens ion channel
Receptor
O
H
Agonist P
O2C
NH3
Phosphorylation alter shape Ion channel closes Desensitization
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Sensitization Receptors become sensititized on long term exposure to
antagonists
Cell synthesises more receptors to compensate for blocked receptors
Cells become more sensitive to natural messenger
Can result in tolerance and dependence
Increased doses of antagonist are required to achieve same effect (tolerance)
Cells are supersensitive to normal neurotransmitter
Causes withdrawal symptoms when antagonist withdrawn
Leads to dependence 61
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Sensitization
Antagonist
Neurotransmitter
Normal response
Receptor
synthesis
No response
Response
Stop
antagonist Excess response No response
Increase
antagonist Tolerance
Receptor
synthesis
Sensitization
Dependence
No response
No response
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OMeOH
H
H H
H
H
H2O
His 524
Glu353
Arg394
Hydrophic skeleton
Oestradiol
Phenol and alcohol of estradiol are important binding groups Binding site is spacious and hydrophobic Phenol group of estradiol is positioned in narrow slot Orientates rest of molecule Acts as agonist
Design of an antagonist for the estrogen receptor
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Action of the oestrogen receptor
Oestradiol
H12
Oestrogen receptor
Binding site
AF-2 regions
Dimerisation & exposure of AF-2 regions
Coactivator
Nuclear transcription
factor
Coactivator
DNA
Transcription
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OH
S
O
O
Raloxifene
Asp351
His 524
O
Glu353
Arg394
N
H
H
Side chain
Raloxifene is an antagonist (anticancer agent) Phenol groups mimic phenol and alcohol of estradiol Interaction with Asp-351 is important for antagonist activity Side chain prevents receptor helix H12 folding over as lid AF-2 binding region not revealed Co-activator cannot bind
Design of an antagonist for the estrogen receptor
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Anticancer agent
CH2CH3
O
Me2N
Tamoxifen as an antagonist for the estrogen receptor