SHC4016 / SMC4009

Chemical Therapeutics

Lecture 19
Signal transduction and cyclic AMP
Learning outcomes

• Understand role of receptors in signaling and

regulation of cellular chemistry
• Appreciate relevance of receptors and signaling
pathways to disease states
• Describe therapeutic strategies based on
interactions with ligand, receptor and signaling
enzymes.

SHC4031 Dr. Duncan Gill Slide 2

Receptors and signalling

• Multi-cellular organisms (eg. humans) contain

specialized cells forming tissues & organs with
specific functions.
• A large number of physiological activities must be
coordinated between cells.
• This is a achieved by signaling using chemical
messengers: neurotransmitters and hormones.
• Chemical messengers act at receptors.
• Most (not all) receptors are located on the outside of
the cell.

SHC4031 Dr. Duncan Gill Slide 3

Signal tranduction
• Signal transduction describes the mechanisms by which physical
or chemical signals are transmitted through a cell.
• The most common transduction mechanism is protein
phosphorylation.
• Receptors are the proteins responsible for detecting stimuli, such
as binding of a chemical ligand.
• The changes cause upon ligand binding to a receptor lead to a
cascade of biochemical events termed a signaling pathway.
• Signalling pathways can interact with one another, giving
networks, which coordinate metabolic responses.
• Examples of biochemical events caused by signal transduction
include transcription & translation of genes, and post-translational
protein modification.
• These mechanisms control cell growth, proliferation & metabolism.
SHC4031 Dr. Duncan Gill Slide 4
Relevance to chemical therapeutics

• Conditions such as hypertension, excessive

inflammatory response (allergies; asthma); gastric
reflux; blood clotting etc. may be managed by
targeting appropriate receptor.
• Some receptors are over-expressed in cancer.
• Some signal transduction enzymes are mutated in
certain cancers.
• Three therapeutic strategies:
– intervene at ligand (monoclonal antibody);
– intervene at receptor (agonists and antagonists);
– intervene in signalling pathway (kinase inhibitors).

SHC4031 Dr. Duncan Gill Slide 5

The hallmarks of cancer
There are six hallmarks that
distinguish cancerous from
normal cells:
• Self-sufficiency in growth
signals
•Insensitivity to anti-growth
signals
•Tissue invasion and metastasis
•Limitless replicative potential
•Sustained angiogenesis
•Evasion of apoptosis
Signal transduction involved in
all these processes: potential
strategy for therapeutic
Hanahan, Weinberg, Cell 2000, 100, 57
intervention
SHC4031 Dr. Duncan Gill Slide 6
 Intervention at ligand: inhibition of VEGF by bevacizumab
• Bevacizumab (Avastin) is a monoclonal antibody that selectively
binds and inhibits vascular endothelial growth factor (VEGF)
• Used to treat colorectal and lung cancer.

https://www.avastin-hcp.com/about-avastin/proposed-moa.html
SHC4031 Dr. Duncan Gill Slide 7
Complexity of cellular signal transduction pathways

SHC4031 Dr. Duncan Gill Slide 8

 Types of receptor

There are four main types (‘superfamilies’) of receptor:

• Ion channel receptors (involved in
neurotransmission)
• G-protein coupled receptors (60% of
transmembrane receptors; many biological
processes, including sight, taste, smell)
• Kinase-linked receptors (cell growth regulation)
• Intracellular receptors (gene expression, metabolic
regulation)

SHC4031 Dr. Duncan Gill Slide 9

Receptors: structure and function

• Globular proteins mostly located

in cell membrane.
• Chemical messenger binds to
receptor on outside of cell.
• Transmits message into cell
leading to cellular effect.
• Receptor is only activated by
specific messenger molecule:
the ligand.
• Cells have a range of different
receptors.

SHC4031 Dr. Duncan Gill Slide 10

Receptor mechanism

Messenger Induced fit Messenger

Messenger

Cell
Membrane Receptor Receptor Receptor

Cell Cell Cell

message
Message

SHC4031 Dr. Duncan Gill Slide 11

Ligand binding

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

SHC4031 Dr. Duncan Gill Slide 12

Overall process

M M

RE RE
R

Signal transduction
•Binding interactions must be strong enough to hold the messenger
sufficiently long for signal transduction to take place
• Interactions must be weak enough to allow the messenger to
depart
• Implies a fine balance
• Designing molecules with stronger binding interactions can result
in drugs that block the binding site – agonists and antagonists
SHC4031 Dr. Duncan Gill Slide 13
Types of receptor
Type Functions Membrane Ligands Therapeutic Drugs
bound? significance

Ion channel Neuro- ✓ Acetylcholine Neurology Amlodipine

transmission Diabetes Diazepam
Repaglinide

G-protein Many roles ✓ Prostaglandin Various Various

coupled s
receptor

Kinase-linked Cellular ✓ Growth Oncology Gefitinib

receptor growth factors & Erlotinib
regulation hormones Lapatinib

Intramolecular Gene ✗ Steroidal Inflammatory Cortico-

receptor expression hormones Response steroids
Oncology Tamoxifen
SHC4031 Dr. Duncan Gill Slide 14
Chemical messengers

• A neurotransmitter is a chemical released from the

end of a neuron, which travels across a synapse to
bind with a receptor on a target cell, such as a
muscle cell or another neuron.
• Usually short-lived and responsible for messages
between cells.
• A hormone is a chemical released from a cell or
gland which travels some distance to bind with
receptors on target cells throughout the body.
• Chemical messengers ‘switch on’ receptors without
undergoing a chemical reaction themselves.

SHC4031 Dr. Duncan Gill Slide 15

Neurotransmitters

SHC4031 Dr. Duncan Gill Slide 16

Types of hormone

• Amines (melatonin, thyroxine, histamine)

• Peptide (enkephalin, oxytocin)
• Protein (insulin, growth hormone)
• Eicosanoid (prostaglandin)
• Steroid (testosterone; estradiol)

SHC4031 Dr. Duncan Gill Slide 17

Drugs targeting G-protein coupled receptors

• 34% of all FDA-approved drugs have a GPCR target.

• Around 20% of agents in clinical trials have a novel
GPCR target.

SHC4031 Dr. Duncan Gill Slide 18

How does a GPCR transmit a signal into the cell?
• Binding of a ligand to the GPCR results in conformational change.
• Allows binding of a specific G-protein to receptor-ligand complex.
• Several types of G-protein (Gs, Gi, Gq) specific to GPCR.
• G-proteins are membrane-bound proteins situated on inner surface
of cell membrane, made up of three sub-units: a, b, g
• The a-subunit binds guanine diphosphate (GDP) in unbound G-
protein.
• On binding to receptor, conformational change results in release of
GDP & binding of GTP.
• Further conformational change results in splitting of a– subunit from
bg–subunit.
• Both a– and bg–subunits act as secondary messengers.
• Receptor-ligand complex activates several G-proteins before ligand
departs: signal amplification.
SHC4031 Dr. Duncan Gill Slide 19
 Signal transduction involving G-protein coupled receptors
G-protein coupled Ligand
receptor (GPCR) binds

Receptor
unoccupied

GTP–GDP
G-protein complex switch

Cellular
α-Protein
events dissociates
SHC4031 Dr. Duncan Gill Slide 20
 Signal transduction: GS protein

• Receptors binding GS protein include:

 b1-Adrenoceptor (beta-blockers, eg. propanolol)
 b2-Adrenoceptor (salbutamol)
– Histamine H2 (cimetidine, ranitidine)
• GS protein made up of three subunits a, b, g.
 a-subunit activates adenylate cyclase pathway.
 bg–subunit also has a role in regulating adenylate
cyclase and other pathways.

SHC4031 Dr. Duncan Gill Slide 21

Signal transduction: GS protein
The GS protein is membrane bound
• consists of 3 subunits ( , )  
•  S subunit has binding site for GDP 
• GDP bound non covalently GDP
When an extracellular ligand binds to the receptor, there is a conformational
change in that receptor
• opens binding site for GS protein

Ligand
cannot
cross
membrane
Cell membrane Ligand
Receptor binding
ß ß
 Induced 
 fit 

GS Protein Binding site for G-

= GDP protein opens
SHC4031 Dr. Duncan Gill Slide 22
Signal transduction: GS protein

The G protein then binds to the GPCR

• G-Protein alters conformation
- GDP binding site distorted
- GDP binding weakened
- GDP departs

G-protein
binds
γ ß Induced γ ß

α
fit for α
G-protein
GDP GTP
Binding site for G-
protein opens

SHC4031 Dr. Duncan Gill Slide 23

Signal transduction: GS protein

GTP binds
γ ß γ ß

α α

Induced fit
Binding site recognises GTP G-protein alters shape
Complex destabilised

Fragmentation
and release
of α-subunit γ ß α

Process repeated for as long as ligand bound to receptor

• known as signal amplification
• several G-proteins activated by one ligand
 s Subunit carries message to next stage

SHC4031 Dr. Duncan Gill Slide 24

Signal transduction
Signal involving
transduction GS protein:
involving adenylate
GS protein: cyclase
adenylate cyclase
Binding site
for αs subunit
Binding
Induced Active site
fit open

Active site closed ATP cyclic AMP

GTP hydrolysed to GDP

catalysed by αs subunit

P
Active site closed
ATP cyclic AMP

Receptor
 ß

SHC4031 Dr. Duncan Gill Slide 25

 Signal transduction involving GS protein: adenylate cyclase
Hundreds of ATP molecules converted before  s-GTP deactivated
• signal amplification
• cyclic AMP is synthesised in the process
- cAMP becomes next signal
➡ secondary messenger
- Cyclic AMP enters cell cytoplasm with message

Adenylate
cyclase

ATP cAMP

SHC4031 Dr. Duncan Gill Slide 26

Secondary messengers: cAMP
cAMP is an important secondary messenger in a range of biological processes
• regulation of glycogen, sugar, and lipid metabolism
Protein kinase A is a serine-threonine kinase
• activated by cyclic AMP
PKA catalyses phosphorylation of serine and threonine residues on protein
substrates
The phosphate unit comes from ATP

SHC4031 Dr. Duncan Gill Slide 27

cAMP as secondary messenger: protein kinase A

Adenylate
cyclase

ATP cyclic AMP

Activation

Protein
Kinase A
P

Enzyme Enzyme
OFF ON REACTION

SHC4031 Dr. Duncan Gill Slide 28

Protein kinase A controls glycogen metabolism

Adrenaline
as as
b-Adrenoreceptor adenylate
cyclase Inhibits conversion
cAMP of phosphorylase a
Glycogen Protein kinase A to phosphorylase b
synthase
Inhibits (active) Catalytic Inhibitor (inactive)
C subunit of
glycogen PKA
Glycogen Inhibitor-P Phosphatase
synthesis synthase-P (active) (inhibited)
(inactive)
Phosphorylase Phosphorylase
kinase (inactive) kinase-P (active)

Effect of adrenaline is to
Phosphorylase b Phosphorylase a
activate glycogen (inactive) (active)
Activates
metabolism and inhibit glycogenolysis
Glycogen Glucose-1-phosphate
glycogen synthesis
SHC4031 Dr. Duncan Gill Slide 29
Glycogenolysis: chemistry

ATP

Glucose-1-phosphate

SHC4031 Dr. Duncan Gill Slide 30

Transduction involving Gi proteins

• Bind to different receptors from those used by Gs

proteins:
 a2-adrenoreceptor (clonidine, lofexidine)
– M3 and M4 muscarinic receptor (chlorpromazine, tropicamide)
– Dopamine D2, D3, D4 receptor (ropinirole, apomorphine)
• Mechanism of activation is identical
• ai-subunit binds to adenylate cyclase and inhibits it
• Adenylate cyclase is under dual control
(brake/accelerator)
• Overall effect depends on dominant a-subunit
• Dominant a-subunit depends on receptors activated
SHC4031 Dr. Duncan Gill Slide 31
 Gq proteins

• Receptors binding Gq protein include:

 a1-adrenoceptor (pseudoephedrine)
– Histamine H1 (cetirizine, loratidine, etc.)
– Serotonin 5-HT2 (aripiprazole)
– Muscarinic M1, M3, M5 (ipatropium, tiotropium)
• Gq protein also made up of three subunits a, b, g
 a-subunit activates phospholipase C.

SHC4031 Dr. Duncan Gill Slide 32

Signal transduction: Gq protein
Gq proteins - interact with receptors different to those which interact with GS
Ligand binding triggers dissociation by a similar mechanism to give an  q subunit
 q subunit activates or deactivates phospholipase C (PLC)
• membrane bound enzyme
• hydrolyses phospholipids

Phospholipase 1
Phospholipase 1
Phospholipase D

Phospholipase C

Reaction catalysed for as long as  q subunit bound

• signal amplification
SHC4031 Dr. Duncan Gill Slide 33
Phospholipase C and inositols

Extracellular PIP2 DAG

Intracellular
Protein
Kinase C
IP3

Phosphorylation
IP3 sensitive3 of substrates
Ca2+ channel

Endoplasmic
PIP2 = phosphatidylinositol
4,5-bisphosphate reticulum
IP3 = inositol triphosphate Ca2+
SHC4031 Dr. Duncan Gill Slide 34
Phospholipase C and inositols

Phospholipase C

SHC4031 Dr. Duncan Gill Slide 35

Diacyl glyceride as secondary messenger: protein kinase C

Regulatory
subunit DAG
C
DAG
DAG

DAG
C
Catalytic DAG
subunit

Ras
GRP
Ras GRP – P

SHC4031 Dr. Duncan Gill Slide 36

DAG and protein kinase C
Ras GRP is a protein involved in cancer
Regulated by DAG
• other compounds such as phorbols can mimic DAG and cause cancer

http://www.nature.com/nrc/journal/v7/n4/fig_tab/nrc2110_F4.html
Phorbol myristate

SHC4031 Dr. Duncan Gill Slide 37

Transduction involving Gq proteins

Drugs inhibiting PKC - potential anti-cancer agents

Bryostatin 1 (from sea moss)

SHC4031 Dr. Duncan Gill Slide 38

Action of inositol triphosphate

Cell membrane

IP3
IP3 hydrophilic: enters cytolasm Cytoplasm

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

Enzyme Enzyme Enzyme Enzyme

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

Enzyme-catalysed Enzyme-catalysed
reaction reaction

SHC4031 Dr. Duncan Gill Slide 39

Tyrosine kinase-linked receptors

messenger
messenger
induced
fit

active site
closed closed
open

intracellular reaction

• Protein serves dual role - receptor plus enzyme

• Receptor binds messenger leading to an induced fit
• Protein changes shape and opens intracellular active site
• Reaction catalysed within cell
• Over-expression related to several cancers
SHC4031 Dr. Duncan Gill Slide 40
Tyrosine kinase catalytic reaction

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

ATP ADP
OH O P

Tyrosine Phosphorylated
residue tyrosine
residue

SHC4031 Dr. Duncan Gill Slide 41

Epidermal growth factor (EGF) signaling pathway

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 OH OH OH PO PO PO OP
OH

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

SHC4031 Dr. Duncan Gill Slide 42

Growth factor signalling pathway

• Activation of ras initiates as sequence of

kinase activations (MAPK / ERK pathway)
OP
Ras • Up-regulates gene transcription
PO PO
OP OP
PO PO PO OP Gene transcription

Raf (inactive) Raf (active)

Mek (inactive) Mek (active)

Map kinase (inactive) Map kinase (active)

Transcription Transcription
factor (inactive) factor (active)
SHC4031 Dr. Duncan Gill Slide 43
MAPK / ERK pathway kinases as drug targets
• Role of MAPK / ERK in up-regulating gene transcription suggests
inhibition of enzymes may be useful in treating cancers.
• In many cancers a kinase on the pathway is mutated.
• Several Raf kinase inhibitors: sorafenib, dabrafenib, vemurafenib.
• Mek inhibitors include cobimetinib, trametinib.

SHC4031 Dr. Duncan Gill Slide 44