Pharmacology and Therapeutics Pain
Pharmacology and therapeutics 1: Arachidonic acid metabolism and NSAIDs
NSAIDs
- The single most important group of self-prescribed pharmaceuticals
- The most widely used drug class
- A large number of chemically distinct drugs (all of them are chemically diverse but act in the same
manner)
- Some examples include ibuprofen, naproxen, diclofenac, aspirin etc
Activity of NSAIDs
Their main actions:
- Anti-inflammatory drugs – as they inhibit enzymes that are involved in the synthesis of cell
inflammatory mediators (local messenger molecules that immune cells send out to coordinate
inflammatory response). The NSAIDs interfere with the synthesis of these messenger molecules
- Analgesic – the receptor for prostaglandin E2 (a mediator inhibited by NSAIDs) is present on the
nociceptor and facilitates nociceptor activity. Inhibiting the production of prostaglandin E2 results in a
reduction in the facilitation of the nociceptor and therefore a reduction in pain perception hence, why
NSAIDs are analgesics
- Anti-pyretic – reduces fever
Side effects:
- Prolonged use or use at high concs can cause irritation and damage of GI tract leading to ulceration,
damage to the GIT
- Renal complications
- Bleeding
- All these complications occur because the prostaglandins play an important role in the up-keeping of
these organs – the GIT, kidneys, the blood system and inhibition of the prostaglandins by NSAIDs causes
these side effects
NSAIDs: The molecular target
- NSAIDs inhibit the enzyme cyclo-oxygenase (COX) also called prostaglandin H2 synthase
o Called COX because it has cyclooxygenase activity and it also has peroxidase activity so it has two
enzyme catalytic activities
- COX is responsible for one step in the conversion of membrane phospholipids into prostanoids
- Prostanoids will then bind to their receptors and elicit many cellular responses
- (Prostanoids = Prostaglandins and Thromboxane’s)
Prostaglandin synthesis pathway step 1:
- The prostaglandin synthesis starts off with membrane phospholipid
- The membrane phospholipid is cleaved
by the enzyme cytosolic
phospholipase A2 (cPLA2) to release
arachidonic acid
- The phospholipid contains a
phosphate and a glycerol
backbone. Two membrane fatty acids
are at position R1 and R2 of the
phospholipid and these stick into the bilayer. At position three there is a phospholipid head group
which can represented by different chemical structures including serine, ethanolamine, choline, inositol
to give rise to phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl
inositol. These can be the sources of arachidonic acid which is a precursor
,Pharmacology and Therapeutics Pain
- PLA2 cleaves to yield a lyso-phospholipid and a free fatty acid. The lyso-phospholipid has everything
that the membrane phospholipid has except R2, as the R2 is cleaved off (R2 is usually arachidonic acid)
- Arachidonic acid is the precursor for inflammatory mediators called eicosanoids (which include
prostaglandins, thromboxanes, leukotrienes)
- There are many different PLA2 enzymes but the most important one for AA release is cPLA 2
cPLA2 – structural features
- Has a catalytic domain with the catalytic site
consisting of serine and aspartate
- There’s also a c2 domain which binds calcium and
allows membrane binding
- C2 binds to the membrane through a calcium bridge
and the catalytic domain which is quite close to
membrane, processes the phospholipid
- The catalytic diad b/w ser and asp flips b/w two
states
o One in which the Ser has a hydroxy group
o One in which the H is abstracted and is present on the Asp
Phospholipase A2: catalysis
- Hydrolysis reaction happening at the catalytic site
- Initially we have the catalytic diad with serine and aspartate. When the aspartate has an O- , it will
abstract H from serine resulting in a negative
oxygen that can attack the carbon on the
phospholipid
- The phospholipid has two fatty acids (attached
to R1 and R2 in red) and then the head group
at R3 e.g., inositol
- The attack on the carbon results in the
formation of a tetrahedral intermediate (an
acyl intermediate) – the carbon is tetrahedral
(circled in second diagram)
- The phospholipid is now linked to the serine
(unstable intermediate)
- The H gets abstracted from the aspartate
resulting in the third form, and then there is
rearrangement of electrons to break the bond
(shown in red)
- This results in the formation of serine where the R2 phospholipid is still linked and the lyso
phospholipid is released whilst the aspartate is back to its original structure
- The arachidonic acid is then released from
the serine
- The arachidonic acid is still attached to the
serine
- The aspartate abstracts H from water and
this creates a negative charge on the
oxygen which can make a bond to create a
tetrahedral intermediate which is unstable
- Abstraction of the H from aspartate and rearrangement of the electrons result in the breaking of the
bond and therefore serine is released back into its original form as well as aspartate. Arachidonic acid is
also released and this is a precursor to the rest of the pathway
,Pharmacology and Therapeutics Pain
cPLA2 regulation
cPLA2 is regulated in two different ways:
Acute regulation
- In response to calcium which is elevated by cell receptor activation
resulting in membrane binding, the translocation of cPLA2 to
intracellular membranes where it is close to phospholipids
- Ca2+ binding to the C2 domain facilitates membrane binding and
juxtaposes to phospholipid
- Phosphorylation by ERK is required for activity
Regulation of the mRNA expression
- Inflammatory mediators can upregulate cPLA2 expression
- Glucocorticoids (strong anti-inflammatories) can down regulate
cPLA2 expression
- Glucocorticoids can induce a repressor protein (S100 protein) that repressed cPLA2
Prostaglandin synthesis pathway step 2:
- Step 2 in the pathway is the conversion of arachidonic acid into prostaglandin
H2 by an enzyme called prostaglandin H2 synthase via a short-lived
intermediate called Prostaglandin G2
- Prostaglandin H2 synthase has 2 activities: cyclooxygenase activity, which is
the first to take place (generates PGG2) & peroxidase (generates PGH2).
- Prostaglandin H2 synthase is often simply called COX
- It is bound to ER and nuclear envelope membranes
COX: Structural features
- The COX has an EGF domain, a membrane binding domain and a catalytic
domain with a catalytic site comprising of a tyr as well as a haem group
o Haem group is a complex organic ring structure, harbouring an iron
atom
o E.g., in haemoglobin
- The tyr at position 385 sits very close to the substrate entry channel (or the arachidonic acid (AA)
channel)
- The AA is inserted into the enzyme through this channel so the tyr can act as a catalyst for the
conversion of the AA
- There are two isoforms:
- COX-1 and COX-2
- Both exist as homodimers –
dimerization is facilitated by
EGF domain
COX Catalytic reaction
- Haem is needed to create an
initial tyr radical at position 385
- The AA channel accommodates AA
in such a way that is positioned
, Pharmacology and Therapeutics Pain
closely to that tyr and there is abstraction of one of the hydrogens to create a hydroxyl on the tyr and
an AA radical
- There are further reactions where the AA is processed to yield the circled form and this abstracts the H
again from the tyr to recreate tyr and create the prostaglandin G2 which is the precursor for the
peroxide activity of the enzyme
COX-1 and COX-2 localise to nuclear envelope and ER
- They are both localised to the nuclear envelope and the ER
- COX-1 expressed constitutively (all the time) in most tissues
- COX-2 is regulated at the mRNA level
o Its expression is induced by growth factors and inflammatory
mediators such as IL-1, TNF-a, LPS in many cells, including
immune cells, endothelial cells and fibroblasts
o Anti-inflammatory glucocorticoids suppress COX-2 expression
(but not COX-1)
- The intracellular location of these two enzymes after activation of
PLA2 is quite close together
Prostaglandin synthesis pathway step 3: Prostanoid
synthesis by synthases
- There are five different enzymes that that process
the PGH2 into five different types of prostanoid
synthases PGD2, PGF2a, TXA2 (thromboxane), PGI2,
PGE2
- These each have different functions
- They are not always present in all the tissues (there
is some level of tissue specificity)
- There is a different mix of prostanoids being
synthesised in different tissues
- E.g., in mast cells, the PGD2 is present, thromboxane
in platelets
Prostaglandin (PGs) synthesis pathway step 4: transport
- PGs are synthesized inside the cell and they need to be
transported out of the cell
- Recent studies show that they are then transported via
ABC transporters (potential that these transporters
have specificity for PGs
- Area of fundamental research
Pharmacology and therapeutics 1: Arachidonic acid metabolism and NSAIDs
NSAIDs
- The single most important group of self-prescribed pharmaceuticals
- The most widely used drug class
- A large number of chemically distinct drugs (all of them are chemically diverse but act in the same
manner)
- Some examples include ibuprofen, naproxen, diclofenac, aspirin etc
Activity of NSAIDs
Their main actions:
- Anti-inflammatory drugs – as they inhibit enzymes that are involved in the synthesis of cell
inflammatory mediators (local messenger molecules that immune cells send out to coordinate
inflammatory response). The NSAIDs interfere with the synthesis of these messenger molecules
- Analgesic – the receptor for prostaglandin E2 (a mediator inhibited by NSAIDs) is present on the
nociceptor and facilitates nociceptor activity. Inhibiting the production of prostaglandin E2 results in a
reduction in the facilitation of the nociceptor and therefore a reduction in pain perception hence, why
NSAIDs are analgesics
- Anti-pyretic – reduces fever
Side effects:
- Prolonged use or use at high concs can cause irritation and damage of GI tract leading to ulceration,
damage to the GIT
- Renal complications
- Bleeding
- All these complications occur because the prostaglandins play an important role in the up-keeping of
these organs – the GIT, kidneys, the blood system and inhibition of the prostaglandins by NSAIDs causes
these side effects
NSAIDs: The molecular target
- NSAIDs inhibit the enzyme cyclo-oxygenase (COX) also called prostaglandin H2 synthase
o Called COX because it has cyclooxygenase activity and it also has peroxidase activity so it has two
enzyme catalytic activities
- COX is responsible for one step in the conversion of membrane phospholipids into prostanoids
- Prostanoids will then bind to their receptors and elicit many cellular responses
- (Prostanoids = Prostaglandins and Thromboxane’s)
Prostaglandin synthesis pathway step 1:
- The prostaglandin synthesis starts off with membrane phospholipid
- The membrane phospholipid is cleaved
by the enzyme cytosolic
phospholipase A2 (cPLA2) to release
arachidonic acid
- The phospholipid contains a
phosphate and a glycerol
backbone. Two membrane fatty acids
are at position R1 and R2 of the
phospholipid and these stick into the bilayer. At position three there is a phospholipid head group
which can represented by different chemical structures including serine, ethanolamine, choline, inositol
to give rise to phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl
inositol. These can be the sources of arachidonic acid which is a precursor
,Pharmacology and Therapeutics Pain
- PLA2 cleaves to yield a lyso-phospholipid and a free fatty acid. The lyso-phospholipid has everything
that the membrane phospholipid has except R2, as the R2 is cleaved off (R2 is usually arachidonic acid)
- Arachidonic acid is the precursor for inflammatory mediators called eicosanoids (which include
prostaglandins, thromboxanes, leukotrienes)
- There are many different PLA2 enzymes but the most important one for AA release is cPLA 2
cPLA2 – structural features
- Has a catalytic domain with the catalytic site
consisting of serine and aspartate
- There’s also a c2 domain which binds calcium and
allows membrane binding
- C2 binds to the membrane through a calcium bridge
and the catalytic domain which is quite close to
membrane, processes the phospholipid
- The catalytic diad b/w ser and asp flips b/w two
states
o One in which the Ser has a hydroxy group
o One in which the H is abstracted and is present on the Asp
Phospholipase A2: catalysis
- Hydrolysis reaction happening at the catalytic site
- Initially we have the catalytic diad with serine and aspartate. When the aspartate has an O- , it will
abstract H from serine resulting in a negative
oxygen that can attack the carbon on the
phospholipid
- The phospholipid has two fatty acids (attached
to R1 and R2 in red) and then the head group
at R3 e.g., inositol
- The attack on the carbon results in the
formation of a tetrahedral intermediate (an
acyl intermediate) – the carbon is tetrahedral
(circled in second diagram)
- The phospholipid is now linked to the serine
(unstable intermediate)
- The H gets abstracted from the aspartate
resulting in the third form, and then there is
rearrangement of electrons to break the bond
(shown in red)
- This results in the formation of serine where the R2 phospholipid is still linked and the lyso
phospholipid is released whilst the aspartate is back to its original structure
- The arachidonic acid is then released from
the serine
- The arachidonic acid is still attached to the
serine
- The aspartate abstracts H from water and
this creates a negative charge on the
oxygen which can make a bond to create a
tetrahedral intermediate which is unstable
- Abstraction of the H from aspartate and rearrangement of the electrons result in the breaking of the
bond and therefore serine is released back into its original form as well as aspartate. Arachidonic acid is
also released and this is a precursor to the rest of the pathway
,Pharmacology and Therapeutics Pain
cPLA2 regulation
cPLA2 is regulated in two different ways:
Acute regulation
- In response to calcium which is elevated by cell receptor activation
resulting in membrane binding, the translocation of cPLA2 to
intracellular membranes where it is close to phospholipids
- Ca2+ binding to the C2 domain facilitates membrane binding and
juxtaposes to phospholipid
- Phosphorylation by ERK is required for activity
Regulation of the mRNA expression
- Inflammatory mediators can upregulate cPLA2 expression
- Glucocorticoids (strong anti-inflammatories) can down regulate
cPLA2 expression
- Glucocorticoids can induce a repressor protein (S100 protein) that repressed cPLA2
Prostaglandin synthesis pathway step 2:
- Step 2 in the pathway is the conversion of arachidonic acid into prostaglandin
H2 by an enzyme called prostaglandin H2 synthase via a short-lived
intermediate called Prostaglandin G2
- Prostaglandin H2 synthase has 2 activities: cyclooxygenase activity, which is
the first to take place (generates PGG2) & peroxidase (generates PGH2).
- Prostaglandin H2 synthase is often simply called COX
- It is bound to ER and nuclear envelope membranes
COX: Structural features
- The COX has an EGF domain, a membrane binding domain and a catalytic
domain with a catalytic site comprising of a tyr as well as a haem group
o Haem group is a complex organic ring structure, harbouring an iron
atom
o E.g., in haemoglobin
- The tyr at position 385 sits very close to the substrate entry channel (or the arachidonic acid (AA)
channel)
- The AA is inserted into the enzyme through this channel so the tyr can act as a catalyst for the
conversion of the AA
- There are two isoforms:
- COX-1 and COX-2
- Both exist as homodimers –
dimerization is facilitated by
EGF domain
COX Catalytic reaction
- Haem is needed to create an
initial tyr radical at position 385
- The AA channel accommodates AA
in such a way that is positioned
, Pharmacology and Therapeutics Pain
closely to that tyr and there is abstraction of one of the hydrogens to create a hydroxyl on the tyr and
an AA radical
- There are further reactions where the AA is processed to yield the circled form and this abstracts the H
again from the tyr to recreate tyr and create the prostaglandin G2 which is the precursor for the
peroxide activity of the enzyme
COX-1 and COX-2 localise to nuclear envelope and ER
- They are both localised to the nuclear envelope and the ER
- COX-1 expressed constitutively (all the time) in most tissues
- COX-2 is regulated at the mRNA level
o Its expression is induced by growth factors and inflammatory
mediators such as IL-1, TNF-a, LPS in many cells, including
immune cells, endothelial cells and fibroblasts
o Anti-inflammatory glucocorticoids suppress COX-2 expression
(but not COX-1)
- The intracellular location of these two enzymes after activation of
PLA2 is quite close together
Prostaglandin synthesis pathway step 3: Prostanoid
synthesis by synthases
- There are five different enzymes that that process
the PGH2 into five different types of prostanoid
synthases PGD2, PGF2a, TXA2 (thromboxane), PGI2,
PGE2
- These each have different functions
- They are not always present in all the tissues (there
is some level of tissue specificity)
- There is a different mix of prostanoids being
synthesised in different tissues
- E.g., in mast cells, the PGD2 is present, thromboxane
in platelets
Prostaglandin (PGs) synthesis pathway step 4: transport
- PGs are synthesized inside the cell and they need to be
transported out of the cell
- Recent studies show that they are then transported via
ABC transporters (potential that these transporters
have specificity for PGs
- Area of fundamental research
