Receptors as Drug Targets

Compiled by: Chikowe, I.

Basic Medical Sciences College of Medicine

Malawi

1

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.

2

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

4

Cell

Nerve

Messenger

Signal

Receptor

Nerve

Nucleus Cell

Response

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

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.

6

Nerve 1

Nerve 2 Hormone

Blood

supply

Neurotransmitters

Cell surface receptor

7

Nuclear receptor

8

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.

9

Examples of common bonding forms in drug receptor interactions (minus van der waals forces)

10

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

11

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

12

Cell Membrane

Cell

Receptor

Messenger

message

Induced fit

Cell

Receptor

Messenger

Message

Cell

Messenger

Receptor

Binding site

ENZYME

Binding site

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

Example

14

Receptor

Binding site

vdw

interaction

ionic

bond

H-bond

Phe

Ser

O H

Asp

CO2

Induced fit - Binding site alters shape to maximize intermolecular bonding

15

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

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.

16

Summary of receptors

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.

18

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

20

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

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

Responsible for

neurotransmission

cardiac conduction

muscle contraction etc...

E.g: Cholinergic nicotinic receptors is an example to these type of receptors. 22

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.

23

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

Second messengers Essential in conducting and amplifying signals from G-protein

coupled receptors.

25

DAG Ca cAMP cGMP IP3

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.

26

messenger

G-protein

split

induced

fit

closed open

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

27

important functions controlled by these receptors.

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.

.

28

active site (closed)

active site (open)

Enzyme

Intracellular reaction

Enzyme

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

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

31

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

32

Illustration

33

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.

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.

35

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.

36

37

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

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

39

E

Agonist

R E

Agonist

R

Signal transduction

Agonist

R

Induced fit

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).

40

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

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

42

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

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

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.

44

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

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

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

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.

47

O N

H

H

M e

H

H

O O

2 C

H

Binding site

Perfect Fit (No change in shape)

A binding site can have extra binding regions

48

OH

O 2

C

Receptor binding site

Extra binding regions

Antagonists can form binding interactions with extra binding regions neighboring the binding site for the natural messenger

49

O

O

Asp

-

HO

Extra hydrophobic

binding region

Hydrophobic

binding region

Ionic binding

region

H-bond

binding region

Hypothetical neurotransmitter

NH2Me

HO

H

50

Hydrophobic

region

O

O

Asp

-

HO

Induced fit resulting from binding of the normal messenger

NH2Me

HO

H

Hydrophobic

region

O

O

Asp

HO

-

NH2Me

HO

HInduced fit

51

Hydrophobic

region

O

O

Asp

HO

Hydrophobic

region

HO

Initial binding

-

Different induced fit resulting from extra binding interaction

NHMe

HO

H

Hydrophobic

region

O

O

Asp

HO

Different induced fit

- NHMe

HO

H

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.

52

An

E R

M

An

R

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

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

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

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

56

Antagonist

Binding site for antagonist

Binding site for messenger

messenger

Receptor Receptor

Partial Agonist Agents which act as agonists but produce a weaker effect.

57

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.

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

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

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

60

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

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

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

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

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

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

Anticancer agent

CH2CH3

O

Me2N

Tamoxifen as an antagonist for the estrogen receptor