PROTEINS AS DRUG TARGETS:

RECEPTORS

©1
Contents
1. Structure and function of receptors (2 slides)
1.1. Chemical Messengers (2 slides)
1.2. Mechanism (2 slides)
2. The binding site
3. Messenger binding
3.1. Introduction
3.2. Bonding forces (2 slides)
4. Overall process of receptor/messenger interaction
5. Signal transduction
5.1. Control of ion channels (4 slides)
5.2. Activation of signal proteins (2 slides)
5.3. Activation of enzyme active site
6. Competitive (reversible) antagonists
7. Non competitive (irreversible) antagonists
8. Non competitive (reversible) allosteric antagonists
9. Antagonists by umbrella effect
10. Agonists
[24 slides]
©1
Receptors

©1
 1. Structure and function of receptors

• Globular proteins acting as a cell’s ‘letter

boxes’

• Located mostly in the cell membrane

• Receive messages from chemical messengers

coming from other cells

• Transmit a message into the cell leading to

a cellular effect

• Different receptors specific for different

chemical messengers

• Each cell has a range of receptors in ©

the
1
1. Structure and function of receptors

Nerve Nerve
Signal

Messenger
Receptor

Response
Nucleus
Cell Cell

©1
 1. Structure and function of receptors

Chemical Messengers

Neurotransmitters: Chemicals released from

nerve endings which travel across a nerve
synapse to bind with receptors on target cells,
such as muscle cells or another nerve. Usually
short lived and responsible for messages
between individual cells

Hormones: Chemicals released from cells or

glands and which travel some distance to bind
•with
Chemical messengers
receptors on ‘switch
targeton’cells
receptors without
throughout the
undergoing a reaction
body

©1
Neurotransmitters relay signal between a neuron and another cell

©1
Classical Hormones are produced by the glands of the
endocrine system, shown below

The major
endocrine
glands: (Male
left, female
right) 1 Pineal
gland 2
Pituitary gland
3 Thyroid gland
4 Thymus 5
Adrenal gland 6
Pancreas 7 Ovary
8 Testes

©1
1. Structure and function of receptors

Nerve 1

Blood Nerve 2
supply
Hormone

Neurotransmitters

©1
©1
 1. Structure and function of receptors
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 ©1
 1. Structure and function of receptors
Mechanism

Messenger Induced fit Messenger

Messenger

Cell
Membrane Receptor Receptor Receptor

Cell Cell Cell

message
Message

©1
2. The 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

• No reaction or catalysis takes place

Binding site
Binding site

ENZYME

©1
The Binding Site

©1
 3. Messenger binding
3.1 Introduction

Messenger
M

Induced fit

• 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 ©1
 3. Messenger binding
3.2 Bonding forces
• Ionic
• H-bonding
• van der Waals

Example: vdw
interaction

H-bond
Binding site
H ionic
O Phe
bond
Ser
CO2

Asp

Receptor

©1
 3. Substrate binding
3.2 Bonding forces
• Induced fit - Binding site alters shape to
maximise intermolecular bonding

Phe
Phe

H
O
H
O Ser
Ser CO2
CO2 Induced
Asp Fit Asp

Intermolecular Intermolecular bond

bonds not optimum lengths optimised
length for maximum
binding strength

©1
Letting Go

©1
 4. Overall process of receptor/messenger interaction

M M

RE R RE

Signal transduction
• Binding interactions must be:
- strong enough to hold the messenger
sufficiently long for signal
transduction to take place
- weak enough to allow the messenger to
depart
• Implies a fine balance
• Drug design - designing molecules with ©
1
5. Signal transduction
5.1 Control of ion channels

• Receptor protein is part of an ion channel

protein complex
• Receptor binds a messenger leading to an
induced fit
• Ion channel is opened or closed
• Ion channels are specific for specific ions
(Na+, Ca2+, Cl-, K+)
• Ions flow across cell membrane down
concentration gradient
• Polarises or depolarises nerve membranes
©1
Closed or Opened?

©1
 5. Signal transduction
5.1 Control of ion channels

Hydrophilic
tunnel

Cell
membrane

©1
Opening the Door

©1
 5. Signal transduction
5.1 Control of ion channels

Binding
Receptor site Messenger

Induced
Cell fit Cell
membrane membrane
‘Gating’
(ion channel
opens)
Five glycoprotein subunits
traversing cell membrane

ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = ex

ion channels for Cl- (e.g. GABAA) = inhibitory
©1
 5. Signal transduction
5.1 Control of ion channels:

MESSENGER

ION
CHANNEL RECEPTOR ION
(closed) BINDING CHANNEL
SITE (open) MESSENGER

Induced fit
and opening
Lock
Gate of ion channel
Cell Ion Ion Cell Cell Ion Ion Cell
membrane channel channel membrane membrane channel channel membrane

Cell Cell

Link
©1
GABAA
Receptor

©1
P2X4 Receptor Ion
Channel

©1
 5. Signal transduction
5.2 Activation of signal proteins
• Receptor binds a messenger leading to an
induced fit
• Opens a binding site for a signal protein (G-
protein)
messenger
• G-Protein binds, is destabilised then split
induced
fit

closed open

Link G-protein
Link split

©1
 5. Signal transduction
5.2 Activation of signal proteins
• G-Protein subunit activates membrane
bound enzyme
Binds to allosteric binding site
Induced fit results in opening of
active site
• Intracellular reaction catalysed

Enzyme Enzyme

active site active site

(closed) (open)

Intracellular
reaction
©1
5. Signal transduction
5.3 Activation of enzyme active site
• 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
messenger messenger
induced
fit

active site
closed closed
open

intracellular reaction ©1
Intracellular Receptors

Link

©1
Competitive Antagonists

©1
 6. Competitive (reversible) antagonists

M
An

An

RE R

• 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© 1
Irreversible Antagonists

©1
 7. Non competitive (irreversible) antagonists
X

Covalent Bond

OH OH O

Irreversible antagonism

• Antagonist binds irreversibly to the

binding site
• Different induced fit means that the
receptor is not activated
• Covalent bond is formed between the drug
and the receptor
1
• Messenger is blocked from the binding ©site
 8. Non competitive (reversible) allosteric antagonists
Binding site
Binding site
unrecognisable

Induced
ACTIVE SITE
fit
(open)
Receptor
ENZYME
(open)
Receptor
ENZYME
Allosteric
site

Antagonist

• Antagonist binds reversibly to an allosteric

site
• Intermolecular bonds formed between
antagonist and binding site
• Induced fit alters the shape of the receptor
• Binding site is distorted and is not
recognised by the messenger
© 1not
• Increasing messenger concentration does
The Umbrella Effect

©1
 9. Antagonists by umbrella effect
• Antagonist binds reversibly to a
neighbouring binding site
• Intermolecular bonds formed between
antagonist and binding site
• Antagonist overlaps with the messenger
messenger
binding site
Binding site is blocked from the binding site
• Messenger
for antagonist
Binding site
for messenger Antagonist

Receptor Receptor

©1
Agonists

©1
10. Agonists
• Agonist binds reversibly to the binding site
• Similar intermolecular bonds formed as to
natural messenger
• Induced fit alters the shape of the receptor
in the same way as the normal messenger
• Receptor is activated
• Agonists are often similar in structure to
the natural messenger
Agonist Agonist Agonist

Induced fit

RE R RE

Signal transduction
©1
 Clonidine

Dexmedetomidine ©1
http://www.uri.edu/pharmacy/animation/animation.htm

©1
 Patrick
An Introduction to Medicinal Chemistry 3/e

Chapter 6

PROTEINS AS DRUG
TARGETS:
RECEPTOR STRUCTURE &
SIGNAL TRANSDUCTION
Part 1: Sections 6.1 - 6.2

©1
Contents
Part 1: Sections 6.1 - 6.2

1. Receptor superfamilies
2. Ion channel receptors (Ligand gated ion channels)
2.1. General structure (3 slides)
2.2. Structure of protein subunits (4-TM receptor
subunits)
2.3. Detailed structure of ion channel
2.4. Gating (2 slides)

[9 slides]

©1
 1. Receptor superfamilies

RESPONSE
TIME
• ION CHANNEL RECEPTORS
msecs
• G-PROTEIN COUPLED RECEPTORS MEMBRANE
BOUND
seconds
• KINASE LINKED RECEPTORS minutes
• INTRACELLULAR RECEPTORS

©1
 2. Ion channel receptors (Ligand gated ion channels)
.1 General structure

Recept Binding site

or Messenger

INDUCED
Cell FIT Cell
membrane membrane
‘GATING’
(ion channel
opens)
Five glycoprotein subunits
traversing cell membrane

ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = ex

ion channels for Cl (e.g. GABAA) = inhibitory

©1
 2. Ion channel receptors (Ligand gated ion channels)

nsverse view (nicotinic receptor)

Binding
sites

Ion channel
 
 

Cell  

 
membrane


Two ligand binding sites

x subunits mainly on -subunits

©1
 2. Ion channel receptors (Ligand gated ion channels)

ansverse view (glycine receptor)

Binding
sites

Ion channel


 
  
Cell 
membrane 


Three ligand binding sites

xxsubunits on -subunits

©1
 2. Ion channel receptors (Ligand gated ion channels)

ucture of protein subunits (4-TM receptor subun

Neurotransmitter binding region

H2N Extracellular loop

CO2H

Cell TM1 TM2 TM3 TM4

membrane

Intracellular Variable loop

loop

4 Transmembrane (TM) regions

(hydrophobic)
©1
 2. Ion channel receptors (Ligand gated ion channels)
Detailed structure of ion channel
Protein
TM4 subunits
TM1 TM3

TM3 TM2 TM1

TM4 TM2 TM2 TM4

TM1 TM3 Transmembrane

regions
TM3 TM2 TM2 TM1

TM4 TM1 TM3 TM4

: TM2 of each protein subunit ‘lines’ the central p

©1
 2. Ion channel receptors (Ligand gated ion channels)

2.4 Gating

Neurotransmitter Induced fit ‘Domino effect’ Rotation of 2TM regions

binds at binding site of each protein subunit

Ion flow

Cell TM2 TM2

membrane

TM2 TM2

TM2 TM2
TM2 TM2
Transverse view Transverse view
TM2 TM2 TM2
of TM2 subunits of TM2 subunits
TM2
Closed
Open
©1
• Link
• Link ©1
Link ©1
 2. Ion channel receptors (Ligand gated ion channels)

2.4 Gating
• Fast response measured in msec
• Ideal for transmission between nerves
• Binding of messenger leads directly to
ion flows across cell membrane
• Ion flow = secondary effect (signal
transduction)
• Ion concentration within cell alters
• Leads to variation in cell chemistry

©1
 Patrick
An Introduction to Medicinal Chemistry 3/e

Chapter 6

PROTEINS AS DRUG
TARGETS:
RECEPTOR STRUCTURE &
SIGNAL TRANSDUCTION
Part 2: Sections 6.3 - 6.6
©1
Contents

Part 2: Sections 6.3 - 6.6

3. G-protein-coupled receptors (7-TM receptors)

3.1. Structure - Single protein with 7 transmembrane regions
3.2. Ligands
3.3. Ligand binding site - varies depending on receptor type
3.4. Bacteriorhodopsin & rhodopsin family (2 slides)
3.5. Receptor types and subtypes (2 slides)
3.6. Signal transduction pathway
a) Interaction of receptor with Gs-protein (3 slides)
b) Interaction of s with adenylate cyclase (2 slides)
c) Interaction of cyclic AMP with protein kinase A (PKA) (4 slides)
3.7. Glycogen metabolism - triggered by adrenaline in liver cells (2 slides)
3.8. GI proteins
3.9. Phosphorylation
3.10. Drugs interacting with cyclic AMP signal transduction
3.11. Signal transduction involving phospholipase C (PLC) (2 slides)
3.12. Action of diacylglycerol (2 slides)
3.13. Action of inositol triphosphate (2 slides)
3.14. Resynthesis of PIP2

[29 slides]
©1
 3. G-protein-coupled receptors (7-TM receptors)
ructure - Single protein with 7 transmembrane reg

Extracellular
loops NH2

-Terminal
N chain

Transmembrane
Membrane VII VI V IV III II I helix

G-Protein
binding region

HO2C
Variable
Intracellular loops
intracellular loop
-Terminal
C chain

©1
 3. G-protein-coupled receptors (7-TM receptors)
3.2 Ligands

• Monoamines e.g. dopamine, histamine,

noradrenaline, acetylcholine (muscarinic)
• Nucleotides
• Lipids
• Hormones
• Glutamate
• Ca++

©1
 3. G-protein-coupled receptors (7-TM receptors)
gand binding site - varies depending on receptor

Ligand

A B C D

A) Monoamines - pocket in TM helices

B) Peptide hormones - top of TM helices +

extracellular loops + N-
terminal chain
C) Hormones - extracellular loops + N-terminal
chain

D) Glutamate - N-terminal chain

©1
 3. G-protein-coupled receptors (7-TM receptors)
Bacteriorhodopsin & rhodopsin family
• Rhodopsin = visual receptor
• Many common receptors belong to this same family
• Implications for drug selectivity depending on
similarity (evolution)
• Membrane bound receptors difficult to crystallise
• X-Ray structure of bacteriorhodopsin solved -
bacterial protein similar to rhodopsin
• Bacteriorhodopsin structure used as ‘template’ for
other receptors
• Construct model receptors based on template and
amino acid sequence
• Leads to model binding sites for drug design
• Crystal structure for rhodopsin now solved©-1
 3. G-protein-coupled receptors (7-TM receptors)
Bacteriorhodopsin & rhodopsin family

Common ance stor

Monoamines
muscarinic
alpha beta

Bradykinin, Opsins, Rhodopsins

Endothelins Angiotensin.Tachykinins
Interleukin-8 Receptor
types

Receptor
2 4 5 3 1 H1 H2 1 2A 2B 2C D4 D3 D2 D1A D1B D5 3 2 1 sub-types

Muscarinic Histamine -Adrenergic Dopaminergic -Adrenergic

©1
 3. G-protein-coupled receptors (7-TM receptors)

5 Receptor types and subtypes

Reflects differences in receptors which

recognise the same ligand

Receptor Types Subtypes

Adrenergic Alpha () 1, 2A, 2B, 2C

Beta () 1 , 2 , 3

Muscarinic Nicotinic
Muscarinic 

©1
 3. G-protein-coupled receptors (7-TM receptors)

5 Receptor types and subtypes

• Receptor types and subtypes not equally

distributed amongst tissues.
• Target selectivity leads to tissue
selectivity
rt muscle - 1 adrenergic receptors
cells - 3 adrenergic receptors
nchial muscle - 1& 2 adrenergic receptors
tract - 1 2 & 2 adrenergic receptors

©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway

a) Interaction of receptor with Gs-protein

- membrane bound protein of 3 subunits (

GS-Protein
- S subunit has binding site for GDP
-GDP bound non covalently

 

GDP

©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway

a) Interaction of receptor with Gs-protein

Ligand

Ligand G-protein
Cell membrane
binding binds
Receptor
ß ß Induced  ß
 Induced 

fit for 
 fit
G-protein
GProtein GDP GTP

Binding site for G-protein opens G-Protein alters shape

GDP binding site distorte
GDP binding weakened
GDP departs

= GDP
©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway

a) Interaction of receptor with Gs-protein

 ß
 ß  ß


GTP binds 
Fragmentation

and release
Induced fit
Binding site recognises GTP G-protein alters shape
Complex destabilised

ss repeated for as long as ligand bound to receptor

l amplification - several G-proteins activated by o
nit carries message to next stage

©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway GTP
GDP s-subunit
b) Interaction of s with adenylate Adenylate
cyclase cyclase
Binding site
for s subunit GTP hydrolysed
to GDP catalysed
by s subunit

Binding
Induced P
fit ATP cyclic AMP
ATP cyclic AMP
Active site Active site Active site
(closed) (open) (closed)
Signal
s Subunit changes shape
transduction
(con) Weaker binding to enzyme
Departure of subunit
Enzyme reverts to inactive
s Subunit recombines with  dimer
state
to reform Gs protein
©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway
b) Interaction of s with adenylate cyclase

eral 100 ATP molecules converted before s-GTP deact

resents another signal amplification
lic AMP becomes next messenger (secondary messenger
lic AMP enters cell cytoplasm with message
NH2 NH2

N N
N N

N Adenylate cyclase N
O O O N N
HO P O P O P O
O O
OH OH OH
H H H H
H O H Cyclic AMP
ATP
OH OH P O OH
O OH

©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway
c) Interaction of cyclic AMP with protein
kinase A (PKA)
• Protein kinase A = serine-threonine kinase
• Activated by cyclic AMP
• Catalyses phosphorylation of serine and
threonine residues on protein substrates
• Phosphate unit provided by ATP
O O O O
H Protein H H Protein H
N C kinase A N C N C kinase A N C

H H H H
HC OH HC O
OH O
CH3 P O
CH3
P OH
Serine Threonine HO
HO O
OH

©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway
c) Interaction of cyclic AMP with protein
kinase A (PKA)

Adenylate
cyclase

ATP cyclic AMP

Activation

Protein
kinase
P
Enzyme Enzyme
(inactive) (active)

Chemical
reaction ©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway
c) Interaction of cyclic AMP with protein
kinase A (PKA)
kinase A - 4 protein subunits
- 2 regulatory subunits (R) and 2 catalytic subunits
cAMP
Ccatalytic subunit
C
R R
R R
cAMP C
binding
C
sites
catalytic subunit

Note
Cyclic AMP binds to PKA
Induced fit destabilises complex
Catalytic units released and activated©
1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction pathway
c) Interaction of cyclic AMP with protein
kinase A (PKA)

Protein Protein
+ ATP + ADP

osphorylation of other proteins and enzymes

gnal continued by phosphorylated proteins
rther signal amplification
©1
 3. G-protein-coupled receptors (7-TM receptors)
ycogen metabolism - triggered by adrenaline in live
Adrenaline
s s
-Adrenoreceptor adenylate
cyclase
cAMP

Glycogen Protein kinase A

synthase
(active) Inhibitor (inactive)
Catalytic
C subunit of
Glycogen PKA Inhibitor-P Phosphatase
synthase-P (active) (inhibited)
(inactive)
Phosphorylase Phosphorylase
kinase (inactive) kinase-P (active)

Phosphorylase b Phosphorylase a
(inactive) (active)

Glycogen Glucose-1-phosphate

©1
 3. G-protein-coupled receptors (7-TM receptors)
cogen metabolism - triggered by adrenaline in liver

Coordinated effect - activation of

glycogen metabolism
- inhibition of glycogen
synthesis

Adrenaline has different effects on

different cells -
activates fat metabolism in fat cells

©1
 3. G-protein-coupled receptors (7-TM receptors)
.8
3.8 GI proteins

s to different receptors from those used by Gs prote

anism of activation by splitting is identical
ubunit binds adenylate cyclase to inhibit it
ylate cyclase under dual control (brake/accelerator
ground activity due to constant levels of s and i
all effect depends on dominant G-Protein
nant G-protein depends on receptors activated

©1
 3. G-protein-coupled receptors (7-TM receptors)
.9 Phosphorylation
alent in activation and deactivation of enzymes
phorylation radically alters intramolecular binding
lts in altered conformations

N H3 N H3
NH3

O
O P O

O O
O H O P O
O
O O O

Active site
Active site open
closed

©1
 3. G-protein-coupled receptors (7-TM receptors)
Drugs interacting with cyclic AMP signal transducti
Cholera toxin - constant activation of
c.AMP - diahorrea

Theophylline and caffeine

- inhibit
phosphodiesterases
- phosphodiesterases responsible for
metabolising O cyclic AMP O CH 3

- cyclic
HC
3
AMP activity
H
N HCprolonged
N
3
N N

N N
O N O N

CH3 CH3

Theophylline Caffeine
©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction involving phospholipase C (PLC
roteins - interact with different receptors from GS and
it by same mechanism to give q subunit
ubunit activates or deactivates PLC (membrane bound enz
ction catalysed for as long as q bound - signal amplifi
ke and accelerator
Active site Active site
(closed) (open)
DG
   PIP2
PLC PLC PLC

IP3

Active site
GTP hydrolysis q departs (closed)
DG
 PIP2 
PLC PLC

Phosphate IP3 enzyme

deactivated
Binding weakened
©1
 3. G-protein-coupled receptors (7-TM receptors)
Signal transduction involving phospholipase C (PLC)

R R
O C C O
O O O P H
CH2 CH CH2 R R
HO O P
O PLC H HO O C O C
OH H + O O
O P O H H
CH2 CH CH2
O H O P
OH
HO O P
HO
OH
IP3 DG
O P

PIP2

sphatidylinositol diphosphate
Inositol triphosphate
Diacylglycerol
tegral part of cell membrane)
(polar and moves (remains in membrane)
into cell cytoplasm)

R= long chain hydrocarbons P = PO 3 2-

©1
 3. G-protein-coupled receptors (7-TM receptors)
2 Action of diacylglycerol
• Activates protein kinase C (PKC)
• PKC moves from cytoplasm to membrane
• Phosphorylates enzymes at Ser & Thr residues
• Activates enzymes to catalyse intracellular
reactions
• Linked to inflammation, tumour propagation, smooth
muscle activity etc
Cell membrane
DG DG
Binding
site for DG DG

PKC PKC

Active site
PKC
closed Enzyme Enzyme
(inactive)(active)

Cytoplasm Cytoplasm Cytoplasm

Chemical
reaction
PKC moves DG binds to Induced fit
to membrane DG binding site opens active site

©1
 3. G-protein-coupled receptors (7-TM receptors)
2 Action of diacylglycerol
inhibiting PKC - potential anti cancer agents
CHCO2Me

Me
CH3CH2CH2 CH CH CH CH Me
C O O Me
H H
O OH Me
MeO2C CH C O O OH O
H HO O
O H Me

H
O
Me C
OH
H

Bryostatin (from sea moss)

©1
 3. G-protein-coupled receptors (7-TM receptors)
Action of inositol triphosphate

- hydrophilic and enters cell cytoplasm

ilises Ca2+ release in cells by opening Ca2+ ion chan

activates protein kinases

tein kinases activate intracellular enzymes

l chemistry altered leading to biological effect

©1
 3. G-protein-coupled receptors (7-TM receptors)
Action of inositol triphosphate

Cell membrane

IP3
Cytoplasm

Calmodulin
Calcium
Ca++ Calmodulin Ca++
stores
Activation Activation
Protein Protein
kinase P kinase
P

Enzyme Enzyme Enzyme Enzyme

(inactive)(active) (inactive) (active)

Chemical Chemical
reaction reaction

©1
 3. G-protein-coupled receptors (7-TM receptors)
14
.14 Resynthesis of PIP2

several
steps
IP3 + DG PIP2
Inhibition

Li+ salts

Lithium salts used vs manic depression

©1
 Patrick
An Introduction to Medicinal Chemistry 3/e

Chapter 6

PROTEINS AS DRUG
TARGETS:
RECEPTOR STRUCTURE &
SIGNAL TRANSDUCTION
Part 3: Section 6.7
©1
Contents

Part 3: Section 6.7

4. Tyrosine kinase linked receptors

4.1. Structure
4.2. Reaction catalysed by tyrosine kinase
4.3. Epidermal growth factor receptor (EGF- R)
(2 slides)
4.4. Insulin receptor (tetrameric complex)
4.5. Growth hormone receptor
4.6. Signalling pathways (5 slides)

[9 slides]

©1
4. Tyrosine kinase linked receptors

• Bi-functional receptor /
enzyme

• Activated by hormones

• Over-expression can
result in cancer

©1
 4. Tyrosine kinase linked receptors
4.1 Structure

Ligand binding region

Extracellular
N H2
N-terminal
chain

Hydrophilic
Cell membrane
transmembrane
region (-helix)

Catalytic binding region

(closed in resting state)
Intracellular
C-terminal C O2 H
chain

©1
 4. Tyrosine kinase linked receptors
Reaction catalysed by tyrosine kinase

O Tyrosine O
N C kinase N C
Protein Protein Mg++ Protein Protein

OH ATP ADP O P

Tyrosine Phosphorylated
residue tyrosine
residue

©1
 4. Tyrosine kinase linked receptors
Epidermal growth factor receptor (EGF- R)

EGF

Cell Ligand binding

and dimerisation Phosphorylation
membrane

HO OH PO OP
OH OH ATP OP OP
ADP
Inactive EGF-R Induced fit
monomers opens tyrosine kinase
active sites

Binding site for EGF

EGF - protein hormone - bivalent ligand
Active site of tyrosine kinase ©1
http://www.iressa.com/iressaHCP/9898_12811_0_0_0.aspx?mid=24

©1
 4. Tyrosine kinase linked receptors
Epidermal growth factor receptor (EGF- R)

• Active site on one half of dimer catalyses

phosphorylation of Tyr residues on other
half
• Dimerisation of receptor is crucial
• Phosphorylated regions act as binding sites
for further proteins and enzymes
• Results in activation of signalling proteins
and enzymes
• Message carried into cell

©1
 4. Tyrosine kinase linked receptors
Insulin receptor (tetrameric complex)

Insulin

Phosphorylation
Cell
membrane
HO OH PO OP
ATP ADP OP
OH OH OP

Kinase active site

opened by induced fit

Insulin binding site

Kinase active site

©1
http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/moaction/surface.html

©1
 4. Tyrosine kinase linked receptors
Growth hormone receptor
Tetrameric complex constructed in presence of growth hormone
GH

GH binding
& Binding Activation and
dimerisation of kinases phosphorylation

GH receptors ATP ADP

(no kinase activity)
HO OH PO OP
kinases OH OH OP OP

HO Kinase active site

OH OH
OH opened by induced fit

Growth hormone binding site

Kinase active site ©1
 4. Tyrosine kinase linked receptors
.6 Signalling pathways

Ligand Ligand

P P
P P
P P
P P
P P

signalling protein

©1
 4. Tyrosine kinase linked receptors
.6 Signalling pathways
1-TM Receptors

Tyrosine kinase Guanylate cyclase

inherent or associated

Signalling proteins cGMP

PLC IP3 GAP Grb2 Others

kinase

IP3 DG PIP3

Ca++ PKC
©1
 4. Tyrosine kinase linked receptors
.6 Signalling pathways
Receptor
binding
site

GROWTH FACTOR RECEPTOR

Tyrosine kinase
active site
(inactive)
HO
OH
HO OH

©1
 4. Tyrosine kinase linked receptors
.6 Signalling pathways
Growth
factor

1) Binding of
growth factor Dimerisation
Phosphorylation
2) Conformational
change
HO HO HO HO PO PO
OH OH OH OH OP OP
HO HO OH HO OHHO OH PO OPPO OP
OH

OH
Binding
Grb2 Ras and
Ras GDP
Binding and OP GTP/GDP OP GTP
phosphorylation Grb2 exchange
PO PO PO PO
of Grb2 OP OP OP OP
PO OPPO OP PO OPPO OP

©1
 4. Tyrosine kinase linked receptors
.6 Signalling pathways

OP Ras
Gene transcriptio
PO PO
OP OP
PO OPPO OP

Raf (inactive) Raf (active)

Mek (inactive) Mek (active)

Map kinase (inactive)Map kinase (active)

Transcription Transcription
factor (inactive) factor (active)

©1
http://www.bioinformaticscourses.com/ISB/sp2003/1FMK/mechanism.html

©1
http://faculty.plattsburgh.edu/donald.slish/tyrosinekinase/TK1.html

©1
 Patrick
An Introduction to Medicinal Chemistry 3/e

Chapter 6

PROTEINS AS DRUG
TARGETS:
RECEPTOR STRUCTURE &
SIGNAL TRANSDUCTION
Part 5: Case Study
©1
Contents

Part 5: Case Study

6. Case Study - Inhibitors of EGF Receptor Kinase
6.1. The target (4 slides)
6.2. Testing procedures
- In vitro tests (3 slides)
- In vivo tests (2 slides)
- Selectivity tests
6.3. Lead compound – Staurosporine
6.4. Simplification of lead compound (2 slides)
6.5. X-Ray crystallographic studies (2 slides)
6.6. Synthesis of analogues
6.7. Structure Activity Relationships (SAR)
6.8. Drug metabolism (2 slides)
6.9. Further modifications (3 slides)
6.10.Modelling studies on ATP binding (4 slides)
6.11.Model binding studies on Dianilinophthalimides (4 slides)
6.12.Selectivity of action (3 slides)
6.13.Pharmacophore for EGF-receptor kinase inhibitors
6.14.Phenylaminopyrrolopyrimidines (3 slides)
6.15.Pyrazolopyrimidines
[43 slides]
©1
Case Study - Inhibitors of EGF Receptor Kina
The target - Epidermal growth factor receptor
- Dual receptor / kinase enzyme role

Extracellular Binding site

space
Receptor

cell
membrane

Cell
Kinase active site
(closed)

©1
6.1 The target Overexpression
of erbB1 gene

Excess
receptor
KINASE INHIBITOR
-
Excess sensitivity
to EGF
Potential
anticancer
Excess signal agent
from receptor

Excess cell growth

and division

Tumours
©1
6.1 The target
H H
N
Protein
N
N O O O HN Protein

N N O P O P O P O
HO O
O O O
O
H H Tyrosine
H H residue
OH OH

ATP
Mg
Tyrosine kinase

H H
N

N
N O O Protein

O P O P O HN Protein
N N

O O O
O O
O
H H P
O
H H O
OH OH
Phosphorylated
ADP tyrosine residue

©1
6.1 The target

Inhibitor Design

ble versus binding site for tyrosine region

ble versus binding site for ATP

hibitors of the ATP binding site

ign a potent but selective inhibitor versus EGF rec

and not other protein kinases.

©1
.2 Testing procedures
In vitro tests
Enzyme assay
using kinase portion of the EGF receptor produced
by recombinant DNAtechnology. Allows enzyme studies
in solution.

EGF-R
cell Recombinant
DNA
membrane

Water
Cell soluble
kinase

©1
.2 Testing procedures

In vitro tests
assay
hibitors by ability to inhibit standard enzyme catalysed reac

ATP ADP
OH O P

Angiotensin II Angiotensin II
Assay product
kinase
to test inhibition

Inhibitors

sts inhibitory activity only and not ability to cross cell me

st potent inhibitor may be inactive in vivo
©1
.2 Testing procedures
n vitro tests
Cell assays
• Use cancerous human epithelial cells which are
sensitive to EGF for growth
• Measure inhibition by measuring effect on cell growth -
blocking kinase activity blocks cell growth.
• Tests inhibitors for their ability to inhibit kinase
and to cross cell membrane
• Assumes that enzyme inhibition is responsible for
inhibition of cell growth

Checks
• Assay for tyrosine phosphorylation in cells - should
fall with inhibition
• Assay for m-RNA produced by signal transduction -
should fall with inhibition
© 1 in
• Assay fast growing mice cells which divide rapidly
6.2 Testing procedures

In vivo tests
cancerous human epithelial cells grafted onto mice
ect inhibitor into mice
ibition should inhibit tumour growth
ts for inhibitory activity + favourable pharmacokin

©1
6.2 Testing procedures

Selectivity tests

Similar in vitro and in vivo tests carried

out on serine-threonine kinases and other
tyrosine kinases

©1
3 Lead compound - Staurosporine
H
N
O

N N

O
H3C

H3C
O

NH
H3C

crobial metabolite
ghly potent kinase inhibitor but no selectivity
mpetes with ATP for ATP binding site
mplex molecule with several rings and asymmetric ce
fficult to synthesise
©1
Simplification of lead compound
H
N
O

Simplification
Remove asymmetric
N N
ring
O H
H 3C N
* * O
H 3C *
O *

NH
Simplification
H 3C Symmetry
N N H
H H
Staurosporine O
N
O

N N
H H

Arcyriaflavin A
• Symmetrical
molecule
• Active and
selective vs PKC
but not EGF-R ©1
Simplification of lead compound maleimide ring
H
N
O O

Bisindolylmaleimides
PKC selective
N N
H H

H indole ring
indole ring
O Phthalimide
N
O

Simplification
N N
H H Simplification
H
N
Aniline Aniline O O

Dianilinophthalimide (CGP
52411)
• Selective inhibitor for N N
H H
EGF receptor and not
other kinases
• Reversal of selectivity

©1
 X-Ray crystallographic studies

ferent shapes implicated in different selectivity

Arcyriaflavin Bisindolyl-maleimides
Dianilino-phthalimid

Planar Bowl shaped Propellor shaped

asymmetric
H
N
H O O
H N
N O O
O O
NH HN

N N N N
H H H H

©1
 X-Ray crystallographic studies

opeller conformation relieves steric clashes

H
N
H O O
N
O O

Steric Steric H H
clash HH HH clash Twist H
H
NH HN
NH HN
Propeller
Planar shape

©1
6 Synthesis of analogues

H 3C CH3
Diels Alder Anilines
O H 2C O Si (CH3) 3 Toluene O O
TMSCl, NEt3
O O
DMF, 100 oC MeO2C Acetic acid, 120 oC
O H 2C O Si (CH3) 3
C

C
(H3C) 3SiO OSi(CH3) 3
CO2Me

H 3C CH3
O O R
a) LiOH, MeOH O O O O N O
O NH3 or formamides
O
b) (Ac) 2O, toluene 140-150 oC

R1 R1 R1 R1 R1 R1
NR 2 2
R N NR2 R N 2
NR2
R N2

©1
 Structure Activity Relationships (SAR)
R
N
O O

R1 R1
NR2 R 2N

Activity lost if N is substituted

ine aromatic rings essential (activity lost if cycloh
or F (small groups). Activity drops for Me and lost
Activity drops if N substituted
ine N’s essential. Activity lost if replaced with S
carbonyl groups important. Activity drops for lactam
H
N
O

NH HN

©1
 Structure Activity Relationships (SAR)

ent Structure: R=R1=R2=H chosen for preclinical tria

= 0.7 M

H
O N O

NH HN

CGP 52411

©1
6.8 Drug metabolism
H
N
Excretion
O O

Glucuronylation
Glucose O Drug
HO NH HN

H
N
O O
Metabolism
(man,mouse,
rat, dog)

NH HN

CGP 52411

H
N
O O
Metabolism Glucuronylation
(monkey) Glucose O Drug O Glucose

HO NH HN OH Excretion

©1
6.8 Drug metabolism

oduce F at para position as metabolic blocker

H
N
O O

F NH HN F

Metabolic Metabolic
blocker CGP 53353 blocker

©1
9 Further modifications

a) Chain extension
H
N
O O

Chain extension NH HN
Chain extension

CGP58109

Activity drops

©1
9 Further modifications

) Ring extension / expansion

extension
ring
expansion
H HN NH HN N
N
O O
O O
remove
O
polar groups

NH HN NH HN NH HN

CGP 52411 (IC50 0.7M) CGP54690 (IC50 0.12M) CGP57198 (IC50 0.18M)
Inactive in cellular assays
Active in vitro and in vivo
due to polarity
(unable to cross cell membrane)

©1
9 Further modifications

c) Simplification

H H
N N
O O O O
Simplification

NH HN NH OH

CGP52411 CGP58522
Similar activity in enzyme assay
Inactive in cellular assay

©1
0 Modelling studies on ATP binding

• No crystal structure for EGF- receptor

available

• Make a model active site based on

structure of an analogous protein which
has been crystallised

• Cyclic AMP dependant protein kinase used

as template

©1
0 Modelling studies on ATP binding

Cyclic AMP dependant protein kinase

+ Mg + ATP + Inhibitor (bound at
substrate site)

Crystallise

Crystals

X-Ray Crystallography

Structure of protein /
inhibitor / ATP complex

Molecular modelling

Identify active site

and binding interactions
for ATP
©1
0 Modelling studies on ATP binding

• ATP bound into a cleft in the enzyme with

adenine portion buried deep close to
hydrophobic region.

• Ribose and phosphate extend outwards

towards opening of cleft

• Identify binding interactions (measure

distances between atoms of ATP and
complementary atoms in binding site to see
if they are correct distance for binding)

• Construct model ATP binding site for EGF-

receptor kinase by replacing amino acid’s
of cyclic AMP dependent protein kinase for
©1
those present in EGF receptor kinase
0 Modelling studies on ATP binding
empty H-bond interaction
Gln767 HN pocket
Thr766
H2NOC
H
N O
O
H

H O
H3C N
Leu768 H H
H3C N
O

Met769 N
N N 6 O O O
S H 1
H3C
N O P O P O P O
N
O O O
O O
H H
H H
OH OH

'ribose' pocket
a H bond acceptor
is a H-bond donor
e forms H-bonds to Glu in ribose pocket ©1
Model binding studies on Dianilinophthalimides
empty H-bond interaction
Gln767 HN pocket
Thr766
H2NOC
H
N O
O
H

H O
H3C N O
Leu768 H
H3C O N

Met769 N
S H O NH
H3C

O HN

'ribose' pocket

©1
Model binding studies on Dianilinophthalimides

• Both imide carbonyls act as H-bond acceptors

(disrupted if carbonyl reduced)
• Imide NH acts as H bond donor (disrupted if
N is substituted)
• Aniline aromatic ring fits small tight
ribose pocket
• Substitution on aromatic ring or chain
extension prevents aromatic ring fitting
pocket
• Bisindolylmaleimides form H-bond
interactions but cannot fit aromatic ring
into ribose pocket.
©1
•
Model binding studies on Dianilinophthalimides
empty
Gln767 HN pocket H-bond interaction
Thr766
H2NOC
H
N O
O
H

H O O
H3C N
Leu768 HN
H3C O H N
Met769 N NH
S H
H3C O

O
HN

'ribose' pocket

©1
Model binding studies on Dianilinophthalimides
empty
Gln767 HN pocket
Thr766
H2NOC H-bond interaction
H
N O
O
H

H O O
H3C N
Leu768 H
H3C
N
O

Met769 N O NH
S H
H3C

NH
O

'ribose' pocket

©1
2 Selectivity of action

POSERS ?

• Ribose pocket normally accepts a polar ribose

so why can it accept an aromatic ring?

• Why can’t other kinases bind

dianilinophthalimides in the same manner?

©1
 6.12 Selectivity of action

no Acids present in the ribose pocket

Hydrophobic Hydrophilic

Protein Kinase A Leu,Gly,Val,Leu Glu,Glu,Asn,Thr

Leu,Gly,Val,Leu,CysArg,Asn,Thr
EGF Receptor Kinase

©1
 6.12 Selectivity of action

e pocket is more hydrophobic in EGF-receptor kinase

an stabilise and bind to aromatic rings (S-Ar inter
empty
pocket
Gln767 HN
Thr766
H2NOC
H
N O
O
H

H O O
H3C N
Leu768
H
H3C O N

Met769
N
S H O NH
H3C

O HN
H
S

'ribose' pocket

lisation by S-Ar interaction not present in other k

to selectivity of action ©1
 Pharmacophore for EGF-receptor kinase inhibitors

O
HBD H HBD
N

HBA O NH
HBA

HN

Ar Ar
Pharmacophore

ophore allows identification of other potential inhibi

databases for structures containing same pharmacophore
ionalise activity of different structural classes of i

©1
14 Phenylaminopyrrolopyrimidines

59326 - Two possible binding modes for H-bonding

H
HBD N
Cl

HBD H N H
N HBA N
N
N
HBA
Ar
N N
H
Cl

Mode I Mode II

Only mode II tallies with pharmacophore and

explains activity and selectivity

©1
14 Phenylaminopyrrolopyrimidines

empty
empty pocket
Cl pocket O
O

H H
N N

N
N N H H
H N
N
N N
N H

CGP59326 CGP59326 H
H
S
S
Cl

'ribose' pocket
'ribose' pocket

Binding Mode I like ATP Binding mode II (favoured)

(not favoured)
Illustrates dangers in comparing structures and
assuming similar interactions (e.g. comparing
CGP59326 with ATP) ©1
14 Phenylaminopyrrolopyrimidines

HBD H
N HBD

HBA N H
N HBA
N

Ar
Ar

Cl

©1
 15 Pyrazolopyrimidines
.15
i) Lead compounds

Cl
NH2

N NH2
N

N N
N
N

H 2N N N
H

(I) EC50 0.80M (II) EC50 0.22M

• Both structures are selective EGF-receptor

kinase inhibitors
• Both structures belong to same class of
compounds
• Docking experiments reveal different binding
modes to obey pharmacophore ©1
.15 Pyrazolopyrimidines

i) Structure I

empty
Extra binding
pocket interactions
O
HBD H
N
H
H H
N

Structure
N
N
H
N
N
I HBA N
N
N
N N

S
Ar

'ribose' pocket

©1
.15 Pyrazolopyrimidines
i) Structure I

NH2 NH2

N N
N N

N N N
N

(I) EC50 0.80M (III) EC50 2.7M

©1
.15 Pyrazolopyrimidines
ii) Structure II
ot bind in same mode since no fit to ribose pocket
ds in similar mode to phenylaminopyrrolopyrimidines

empty
pocket
Cl

O
H N
N NH

N
Structure
N II
H
N
H 2N

unoccupied
'ribose' pocket

©1
.15 Pyrazolopyrimidines
Extra
Drug design on structure II H-bonding
interaction

Cl Cl Cl OH

HBD HBD
H N H N H N H N
N NH N NH N NH N NH

HBA HBA
N N
SimplificationN Extension NH
Extension N
NH
(remove extra (add aromatic
N N ring for ribose N N
functional group)
H 2N pocket)
Ar
Cl Cl

(II) (IV) (V) (VI)

EC50 0.22M EC50 0.16M EC50 0.033M EC50 0.001M
Activity increases Activity increases Activity increases
Ar fits ribose pocket

• Upper binding pocket is larger than ribose

pocket allowing greater variation of
substituents on the ‘upper’ aromatic ring © 1