1. An Introduction to Medicinal Chemistry 3/e PROTEINS AS DRUG TARGETS:
Patrick
An Introduction to Medicinal Chemistry 3/e
Chapter 6
PROTEINS AS DRUG TARGETS:
RECEPTOR STRUCTURE & SIGNAL TRANSDUCTION
Part 2: Sections

2. Contents
Part 2: Sections
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]

3. 3. G-protein-coupled receptors (7-TM receptors)
3.1 Structure - Single protein with 7 transmembrane regions
Extracellular
loops
HO2C
NH2
VII VI V IV
III II I
Membrane N
-Terminal chain
Transmembrane
helix
G-Protein
binding region
Variable
intracellular loop
Intracellular loops C
-Terminal chain

4. 3. G-protein-coupled receptors (7-TM receptors)
3.2 Ligands
Monoamines e.g. dopamine, histamine, noradrenaline, acetylcholine (muscarinic)
Nucleotides
Lipids
Hormones
Glutamate
Ca++

5. 3. G-protein-coupled receptors (7-TM receptors)
3.3 Ligand binding site - varies depending on receptor type
Ligand B D C A
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

6. 3. G-protein-coupled receptors (7-TM receptors)
3.4 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 - better template

7. 3. G-protein-coupled receptors (7-TM receptors)
3.4 Bacteriorhodopsin & rhodopsin family

8. 3. G-protein-coupled receptors (7-TM receptors)
3.5 Receptor types and subtypes
Reflects differences in receptors which recognise the same ligand
Receptor
Types
Subtypes
Alpha (a)
Beta (b)
Adrenergic
Cholinergic
a1, a2A, a2B, a2C
b1, b2, b3
Nicotinic
Muscarinic
M1-M5

9. 3. G-protein-coupled receptors (7-TM receptors)
3.5 Receptor types and subtypes
Receptor types and subtypes not equally distributed amongst tissues.
Target selectivity leads to tissue selectivity
Heart muscle - b1 adrenergic receptors
Fat cells - b3 adrenergic receptors
Bronchial muscle - a1& b2 adrenergic receptors
GI-tract - a1 a2 & b2 adrenergic receptors

10. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
GS-Protein
- membrane bound protein of 3 subunits (a, b, g)
- aS subunit has binding site for GDP
-GDP bound non covalently b g a
GDP

11. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein
Ligand
Ligand
binding
Induced
fit ß a g
G-protein
binds
Induced
fit for ß a g
GDP
GTP
Receptor G
Protein
Cell membrane ß a g
Binding site for G-protein opens
G-Protein alters shape
GDP binding site distorted
GDP binding weakened
GDP departs
= GDP

12. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
a) Interaction of receptor with Gs-protein ß a g ß a g ß a g
GTP binds
Fragmentation
and release
Induced fit
G-protein alters shape
Complex destabilised
Binding site recognises GTP
Process repeated for as long as ligand bound to receptor
Signal amplification - several G-proteins activated by one ligand
as Subunit carries message to next stage

13. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
as-subunit
Adenylate cyclase
GTP
GDP
b) Interaction of as with adenylate cyclase
Binding site
for as subunit
GTP hydrolysed
to GDP catalysed
by as subunit
Active site
(open) P
cyclic AMP
ATP
Active site
(closed)
Binding
Induced
fit
Active site
(closed)
cyclic AMP
ATP
Signal
transduction
(con)
as Subunit changes shape
Weaker binding to enzyme
Departure of subunit
Enzyme reverts to inactive
state
as Subunit recombines with b,g dimer
to reform Gs protein

14. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
b) Interaction of as with adenylate cyclase
Several 100 ATP molecules converted before as-GTP deactivated
Represents another signal amplification
Cyclic AMP becomes next messenger (secondary messenger)
Cyclic AMP enters cell cytoplasm with message

15. 3. G-protein-coupled receptors (7-TM receptors)
3.6 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

16. Kinase: Any of various enzymes that catalyze the transfer of a phosphate group from a donor, such as ADP or ATP, to an acceptor.
Phosphatase: Any of numerous enzymes that catalyze the hydrolysis of esters of phosphoric acid and are important in the absorption and metabolism of carbohydrates, nucleotides, and phospholipids and in the calcification of bone.
Phosphorylase: An enzyme that catalyzes the production of glucose phosphate from glycogen and inorganic phosphate.

17. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA)
cyclic AMP
ATP
Adenylate
cyclase
Protein
kinase
Activation
Enzyme
(inactive)
Enzyme
(active) P
Chemical
reaction

18. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA)
Protein kinase A protein subunits
- 2 regulatory subunits (R) and 2 catalytic subunits (C)
cAMP
catalytic subunit R C C R
cAMP
binding
sites
Note
Cyclic AMP binds to PKA
Induced fit destabilises complex
Catalytic units released and activated

19. 3. G-protein-coupled receptors (7-TM receptors)
3.6 Signal transduction pathway
c) Interaction of cyclic AMP with protein kinase A (PKA) C
Protein
+ ATP
+ ADP P
Phosphorylation of other proteins and enzymes
Signal continued by phosphorylated proteins
Further signal amplification

20. 3. G-protein-coupled receptors (7-TM receptors)
3.7 Glycogen metabolism - triggered by adrenaline in liver cells

21. 3. G-protein-coupled receptors (7-TM receptors)
3.7 Glycogen metabolism - triggered by adrenaline in liver cells
Coordinated effect - activation of glycogen metabolism
- inhibition of glycogen synthesis
Adrenaline has different effects on different cells activates fat metabolism in fat cells

22. 3. G-protein-coupled receptors (7-TM receptors)
3.8 GI proteins
Binds to different receptors from those used by Gs protein
Mechanism of activation by splitting is identical
aI subunit binds adenylate cyclase to inhibit it
Adenylate cyclase under dual control (brake/accelerator)
Background activity due to constant levels of as and ai
Overall effect depends on dominant G-Protein
Dominant G-protein depends on receptors activated

23. 3. G-protein-coupled receptors (7-TM receptors)
3.9 Phosphorylation
Prevalent in activation and deactivation of enzymes
Phosphorylation radically alters intramolecular binding
Results in altered conformations O N H 3
Active site
closed O N H 3 P
Active site
open N H 3 O P

24. 3. G-protein-coupled receptors (7-TM receptors)
3.10 Drugs interacting with cyclic AMP signal transduction
Cholera toxin - constant activation of c.AMP - diahorrea
Theophylline and caffeine inhibit phosphodiesterases
- phosphodiesterases responsible for metabolising cyclic AMP
- cyclic AMP activity prolonged

25. 3. G-protein-coupled receptors (7-TM receptors)
3.11 Signal transduction involving phospholipase C (PLC)
Gq proteins - interact with different receptors from GS and GI
Split by same mechanism to give aq subunit
aq Subunit activates or deactivates PLC (membrane bound enzyme)
Reaction catalysed for as long as aq bound - signal amplification
Brake and accelerator
Active site
(open) a
PLC a
Active site
(closed)
PLC a
PLC DG
PIP2
IP3
Active site
(closed)
enzyme
deactivated a
PLC
aq departs
GTP hydrolysis a
PLC
PIP2 DG
IP3
Phosphate
Binding weakened

26. 3. G-protein-coupled receptors (7-TM receptors)
3.11 Signal transduction involving phospholipase C (PLC)
Phosphatidylinositol diphosphate
(integral part of cell membrane)
Inositol triphosphate
(polar and moves
into cell cytoplasm)
Diacylglycerol
(remains in membrane)
R= long chain hydrocarbons

27. 3. G-protein-coupled receptors (7-TM receptors)
3.12 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
PKC DG
Binding
site for DG
Cell membrane
Cytoplasm
PKC moves
to membrane
PKC DG
Cytoplasm
DG binds to
DG binding site
Active site
closed
PKC DG
Cytoplasm
Induced fit
opens active site
Enzyme
(inactive)
(active)
Chemical
reaction

28. 3. G-protein-coupled receptors (7-TM receptors)
3.12 Action of diacylglycerol
Drugs inhibiting PKC - potential anti cancer agents
Bryostatin (from sea moss)

29. 3. G-protein-coupled receptors (7-TM receptors)
3.13 Action of inositol triphosphate
IP3 - hydrophilic and enters cell cytoplasm
Mobilises Ca2+ release in cells by opening Ca2+ ion channels
Ca2+ activates protein kinases
Protein kinases activate intracellular enzymes
Cell chemistry altered leading to biological effect

30. 3. G-protein-coupled receptors (7-TM receptors)
3.13 Action of inositol triphosphate
Cytoplasm
Cell membrane
IP3
Calcium
stores
Calmodulin
Ca++
Calmodulin
Ca++
Activation
Protein
kinase
Activation
Protein
kinase
Enzyme
(active)
(inactive) P
Enzyme
(inactive)
(active) P
Chemical
reaction
Chemical
reaction

31. 3. G-protein-coupled receptors (7-TM receptors)
3.14 Resynthesis of PIP2
IP3 + DG
PIP2
several
steps
Li+ salts
Inhibition
Lithium salts used vs manic depression