Eicosanoid Synthesis

Prostaglandins & related compounds: "local hormones"

synthesized from the polyunsaturated fatty acid
arachidonate.
 They have specific effects on target cells close to their
site of formation.
They are rapidly degraded, so they are not transported
to distal sites within the body.
 Examples: prostaglandins, prostacyclins, thromboxanes,
leukotrienes, epoxyeicosatrienoic acids (EETs).
 Roles in inflammation, fever, regulation of blood
pressure, blood clotting, immune system modulation,
control of reproductive processes & tissue growth,
sleep/wake cycle regulation.
 COOH

A r a c h id o n ic a c id

Prostaglandins & related compounds are collectively

known as eicosanoids.
They are produced from arachidonic acid, a 20-C poly-
unsaturated fatty acid (5,8,11,14-eicosatetraenoic acid).

PGE2 (prostaglandin COOH

E2) is an example of
a prostaglandin. HO
OH
PGE2
 O

COOH

PGE2
(prostaglandin E2). HO
OH
PGE2

Prostaglandins all have a cyclopentane ring.

 A letter code is based on ring modifications (e.g.,
hydroxyl or keto groups).
 A subscript refers to the number of double bonds in
the two side-chains.
Thromboxanes are similar but have instead a 6-member
ring.
Prostaglandin receptors:
 Prostaglandins & related compounds are transported
out of the cells that synthesize them.
 Most affect other cells by interacting with plasma
membrane G-protein coupled receptors.
Depending on the cell type, the activated G-protein
may stimulate or inhibit formation of cAMP, or may
activate a phosphatidylinositol signal pathway
leading to intracellular Ca++ release.
 Another prostaglandin receptor, designated PPAR,
is related to a family of nuclear receptors with
transcription factor activity.
 Prostaglandin receptors are specified by the same
letter code. E.g.:
• Receptors for E-class prostaglandins are EP.
• Thromboxane receptors are designated TP.
 Multiple receptors for a prostaglandin are specified
by subscripts (E.g., EP1, EP2, EP3, etc.).
• Different receptors for a particular prostaglandin
may activate different signal cascades.
• Effects may vary in different tissues, depending on
which receptors are expressed.
 S ite o f cleavage b y
P h o sp h olipase A 2 O
The fatty acid
arachidonate O H 2C O C R1
is often esterified R2 C O C H O
to OH on C2 of H OH
CH 2 O P O
glycerophospho- H
H
OH
lipids, especially S ite o f cleavage b y O- OH OH
phosphatidyl P h o sp h olipase C H OH
inositol. H H
P h o sp h a tid y l in o sito l
Arachidonate is released from phospholipids by hydrolysis
catalyzed by Phospholipase A2.
This enzyme hydrolyzes the ester linkage between a fatty
acid and the OH at C2 of the glycerol backbone, releasing
the fatty acid & a lysophospholipid as products.
Corticosteroids are anti-inflammatory because they
prevent inducible Phospholipase A2 expression, reducing
arachidonate release.
There are multiple Phospholipase A2 enzymes, subject to
activation via different signal cascades.
 The inflammatory signal platelet activating factor is
involved in activating some Phospholipase A 2 variants.
 Attempts have been made to develop drugs that inhibit
particular isoforms of Phospholipase A2, for treating
inflammatory diseases.
Success has been limited by the diversity of
Phospholipase A2 enzymes, and the fact that
arachidonate may give rise to inflammatory or
anti-inflammatory eicosanoids in different tissues.
 O

O H2C O C R2

R1 C O CH O

H2C O P O

Phosphatidyl cleavage by O H
inositol signal Phospholipase C OH OPO 3 2
H OH
cascades may OH H
lead to release H H
PIP 2
of arachidonate. phosphatidylinositol- H OPO 3 2
4,5-bisphosphate

After PI is phosphorylated to PIP2, cleavage via

Phospholipase C yields diacylglycerol (and IP3).
Arachidonate release from diacylglycerol is then catalyzed
by Diacylglycerol Lipase.
 leukotrienes

Linear pathway Lipoxyganase

phospholipids arachidonate diacylglycerol
Two major Cyclic pathway PGH2 Synthase
pathways of
eicosanoid Prostacyclin prostaglandin H2 Thromboxane
metabolism. Synthase Synthase
Cyclic prostacyclins thromboxanes
pathway: other prostaglandins

Prostaglandin H2 Synthase catalyzes the committed step

in the “cyclic pathway” that leads to production of
prostaglandins, prostacyclins, & thromboxanes.
Different cell types convert PGH2 to different compounds.
 COOH

PGH2 Synthase is a arachidonic acid

heme-containing 2 O2 Cyclooxygenase
dioxygenase, bound
to ER membranes. O
COOH

(A dioxygenase O
incorporates O2 into PGG2 OOH
a substrate).
2 e Peroxidase
PGH2 Synthase
O
exhibits 2 activities: COOH

cyclooxygenase & O
peroxidase. PGH2
OH
 COOH

PGH2 Synthase
arachidonic acid
(expressing both
cyclooxygenase & 2 O2 Cyclooxygenase
peroxidase activities) O
COOH
is sometimes referred
to as Cyclooxygenase, O
abbreviated COX. PGG2 OOH
The interacting 2 e Peroxidase
cyclooxygenase and
O
peroxidase reaction COOH
pathways are complex.
O
PGH2
OH
 COOH
A peroxide (such as that
generated later in the
reaction sequence) arachidonic acid
oxidizes the heme iron. 2 O2 Cyclooxygenase
The oxidized heme
O
accepts an electron from COOH
a nearby tyrosine
O
residue (Tyr385).
PGG2 OOH
The resulting tyrosine 2 e Peroxidase
radical is proposed to
extract a H atom from O
COOH
arachidonate, generating
a radical species that O

reacts with O2. PGH2

OH
The signal molecule ·NO (nitric oxide) may
initiate prostaglandin synthesis by reacting with
superoxide anion (O2·) to produce peroxynitrite,
which oxidizes the heme iron enabling electron
transfer from the active site tyrosine.
Prostaglandin synthesis in response to some
inflammatory stimuli is diminished by inhibitors
of Nitric Oxide Synthase.
 PDB 1PGE
Membrane-
binding
domain:
4 short heme
amphipathic
ibuprofen
-helices that analog
insert into one
leaflet of the membrane
bilayer, facing binding
domain Prostaglandin H2 Synthase
the ER lumen.
Arachidonate, derived from membrane lipids, approaches
the heme via a hydrophobic channel extending from the
membrane-binding domain.
In the image above, the channel is occupied by an
inhibitor, an ibuprofen analog.
Non-steroidal anti-inflammatory drugs H3C CH3
(NSAIDs), such as aspirin and derivatives CH

of ibuprofen, inhibit cyclooxygenase CH2

activity of PGH2 Synthase.
 They inhibit formation of prostaglandins
involved in fever, pain, & inflammation.
 They inhibit blood clotting by CH
H3C COOH
blocking thromboxane formation
in blood platelets. Ibuprofen

Ibuprofen and related compounds block the

hydrophobic channel by which arachidonate
enters the cyclooxygenase active site.
 COOH O

O C CH3
+ Enz-Ser CH2 OH

Aspirin PGH2 Synthase (active)

COOH
O
OH
+ Enz-Ser CH2 O C CH3

Salicylic acid Acetylated PGH2 Synthase (inactive)

Aspirin acetylates a serine hydroxyl group near the active

site, preventing arachidonate binding.
The inhibition by aspirin is irreversible.
However, in most body cells re-synthesis of PGH2 Synthase
would restore cyclooxygenase activity.
Thromboxane A2 stimulates blood platelet aggregation,
essential to the role of platelets in blood clotting.
 Many people take a daily aspirin for its anti-clotting
effect, attributed to inhibition of thromboxane
formation in blood platelets.
 This effect of aspirin is long-lived because platelets
lack a nucleus and do not make new enzyme.
Two isoforms of PGH2 Synthase are COX-1 & COX-2
(Cyclooxygenase 1 & 2):
 COX-1 is constitutively expressed at low levels in
many cell types.
 COX-2 expression is stimulated by growth factors,
cytokines, and endotoxins.
Different localization of these isoforms within a cell,
coupled to localization of enzymes that convert the
product PGH2 into particular prostaglandins or
thromboxanes, may result in COX-1 & COX-2 yielding
different ultimate products.
 COX-1 is essential for thromboxane formation in
blood platelets, and for maintaining integrity of the
gastrointestinal epithelium.
 COX-2 levels increase in inflammatory diseases such
as arthritis.
Inflammation is associated with up-regulation of
COX-2 & increased amounts of particular prostaglandins.
 COX-2 expression is increased in some cancer cells.
Angiogenesis (blood vessel development), which is
essential to tumor growth, requires COX-2.
Overexpression of COX-2 leads to increased expression
of VEGF (vascular endothelial growth factor).
Regular use of NSAIDs has been shown to decrease the
risk of developing colorectal cancer.
Most NSAIDs inhibit both COX I & COX II.
Selective COX-2 inhibitors have been developed, e.g.,
Celebrex and Vioxx.
 COX-2 inhibitors are anti-inflammatory & block pain,
but are less likely to cause gastric toxicity associated
with chronic use of NSAIDs that block COX-1.
 A tendency to develop blood clots when taking some
of these drugs has been attributed to:
• decreased production of an anti-thrombotic (clot
blocking) prostaglandin (PGI2) by endothelial cells
lining small blood vessels
• lack of inhibition of COX-1-mediated formation
of pro-thrombotic thromboxanes in platelets.
Some evidence suggests the existence of a third
isoform of PGH2 Synthase, designated COX-3, with
roles in mediating pain and fever, and subject to
inhibition by acetaminophen (Tylenol).
Acetaminophen has little effect on COX-1 or COX-2,
and thus lacks anti-inflammatory activity.

Explore the structure of PGH2 Synthase-1 (COX-1)

crystallized with bound iodosuprofen, a derivative of
ibuprofen.
 leukotrienes

Linear pathway Lipoxyganase

phospholipids arachidonate diacylglycerol
Cyclic pathway PGH2 Synthase

Prostacyclin prostaglandin H2 Thromboxane

Synthase Synthase
prostacyclins thromboxanes
other prostaglandins

The 1st step of the Linear Pathway for synthesis of

leukotrienes is catalyzed by Lipoxygenase.
Mammals have a family of Lipoxygenase enzymes that
catalyze oxygenation of various polyunsaturated fatty acid
at different sites. Many of the products have signal roles.
 Catalyzed by 5-Lipoxygenase:
COOH
E.g., 5-Lipoxygenase,
found in leukocytes,
catalyzes conversion O2 arachidonate
of arachidonate to
5-HPETE OOH
(5-hydroperoxy- COOH
eicosatetraenoic acid).
5-HPETE is converted 5-HPETE
to leukotriene-A4, H2O
which in turn may be O
COOH
converted to various
other leukotrienes.
leukotriene-A 4
A non-heme iron is the
prosthetic group of membrane-
binding
Lipoxygenase enzymes. domain
Ligands to the Fe include
3 His N atoms & the
C-terminal carboxylate O.
substrate
The arachidonate substrate analog
binds in a hydrophobic adjacent to
Fe (green)
pocket, adjacent to the
catalytic iron atom.
O2 is thought to approach
from the opposite side of 15-Lipoxygenase PDB 1LOX
the substrate than the iron,
for a stereospecific reaction.
 COOH
Lipoxygenase reaction
starts with extraction of
H from arachidonate, O2 arachidonate
with transfer of the e to
OOH
the iron, reducing it from
Fe3+  Fe2+. COOH

The resulting fatty acid

radical reacts with O2 to 5-HPETE
form a hydroperoxy group.
Which H is extracted, & the position of the hydroperoxy
group, varies with different lipoxygenases (e.g.,
5-Lipoxgenase shown here, 15-Lipoxygenase, etc.)
Additional reactions then yield the various leukotrienes.
Leukotrienes have roles in inflammation and induce
asthmatic constriction of the bronchioles.
Some leukotrienes act via specific G-protein coupled
receptors (GPCRs) in the plasma membrane.
Anti-asthma medications include:
 inhibitors of 5-Lipoxygenase, e.g., Zyflo (zileuton)
 drugs that block leukotriene-receptor interactions.
E.g., Singulair (montelukast) & Accolate (zafirlukast)
block binding of leukotrienes to their receptors on the
plasma membranes of airway smooth muscle cells.
5-Lipoxygenase requires the membrane protein FLAP
(5-lipoxygenase-activating protein).
FLAP binds arachidonate, facilitating its interaction with
the enzyme.
A complex including membrane-
binding
5-Lipoxygenase, domain
FLAP, & Phospholipase
A2 forms in association
with the nuclear envelope
during leukotriene substrate
synthesis in leukocytes. analog
adjacent to
A -barrel domain at Fe (green)
the N-terminus of
Lipoxygenase enzymes
has a role in membrane
binding. 15-Lipoxygenase PDB 1LOX
 COOH
COOH

O
A r a c h id o n ic a c id 14,15-EET

Cytochrome P450 epoxygenase pathways:

Epoxyeicosatrienoic acids (EETs) and
hydroxyeicosatrienoic acids are formed from
arachidonate by enzymes of the cytochrome P450 family.
Other members of the cytochrome P450 family participate
in a variety of oxygenation reactions, including
hydroxylation of sterols.
Shown is an EET COOH
produced from
arachidonate by activity
of a cytochrome O
P450 epoxygenase. 14,15-EET

(14,15-epoxyeicosatrienoic acid)

EETs are modified by additional enzyme-catalyzed

reactions to produce many distinct compounds.
They may be incorporated into phospholipids, and
released by action of phospholipases.
EETs have roles in regulating cellular proliferation,
inflammation, peptide hormone secretion, & various signal
pathways relevant to cardiovascular and renal functions.
 Synthesis of
CH2O2C-R
Eicosanoids
CHO2C In cell membrane
O +
CH2O-P-O-CH2CH2NR'3 R’= H or CH3
O-
Hydrolysis of sn-2 ester bond
by phospholipase A2 (PLA2)

-
O2C

Arachidonate
 Synthesis of
Eicosanoids:
PLA2 Activation
Various stimuli: Activation of
Hormones, autacoids, etc. Membrane-bound PLA2
Receptors Activity

Ca+2

Arachidonate release and eicosanoid synthesis

are important mediators of tissue injury and inflammation
 Synthesis of
Eicosanoids:
Prostaglandin Synthesis
Prostaglandin
endoperoxide CO2-
O
O CO2- synthetase
O
O
(Cyclooxygenase) H
O=O

Cyclic Cyclooxygenase
endoperoxide

CO2- O CO2-
O Hydroperoxidase
O O

OH O-O-H PGG2
PGH2 Hydroperoxide
Prostaglandin endoperoxide synthetase (also called
cyclooxygenase) possesses both cyclooxygenase and
hydroperoxidase activity
Two forms of cyclooxygenase: COX -1 - constitutively expressed
COX -2 - inducible
Cyclooxygenase (COX) Inhibitors
Nonsteroidal antiinflammatory drugs:
CO2H
CO2H
COX COX
HOH2C
OCOCH3
Ser-530 CH2OCOCH3
O - CCH3
Acetylsalicylic acid O
(aspirin)
Irreversible inhibition of COX by acetylation
of the active site

Actions of Aspirin:
Antiinflammatory (COX-2 inhibition)
GI injury (COX-1 inhibition)
 COX-2 Selective
Inhibitors
F3C
O

O N
N
CH3

SO2CH3
SO2NH2
Rofecoxib (Vioxx)
Celecoxib (Celebrex)

Glucocorticoids block COX-2 expression

 Prostaglandins
O
CO2H

HO PGE2
OH
O CO2-
HO
O
CO2H
OH
PGH2
O
OH
PGD2
HO

Prostaglandins exhibit a variety CO2H

of actions on different tissues

HO
OH PGF2
 Prostacyclin and
Thromboxanes
OH

O CO2- Prostacyclin
HO2C OH
synthase
O O

PGH2 OH Prostacyclin (PGI2):

Blocks platelet
Thromboxane aggregation
synthase
OH

CO2- Non-Enzymatic CO2-

O

O HO O
OH OH

Thromboxane A2 (TxA2): Thromboxane B2 (TxB2):

Promotes platelet inactive
aggregation (t1/2 = 30 sec.)
 Leukotriene
Biosynthesis
CO2H
OOH
CO2H
Leukotrienes are
5-Lipoxygenase important mediators
of inflammation

Arachidonic acid e 5-Hydroperoxyeicosa-

A s 6,8,11,14-tetraenoic acid
LT ola
r (5-HPETE)
yd
H
5-Lipoxygenase
CO2H O
OH CO2H

Leukotriene B4 (LTB4) Leukotriene A4 (LTA 4)

Gly
Glutathione
Glu
Cys
LTC4 synthase
Cys
S - Glu S
CO2H - Gly CO2H
OH OH
Leukotriene C4 (LTC4)
Leukotriene E4 (LTE4) Cysteinyl leukotrienes
 Leukotriene
Biosynthesis (Cont’d)
CO2H
CO2H
12-Lipoxygenase

HOO

Arachidonic acid 12-Hydroperoxyeicosa-

5,8,10,14-tetraenoic acid
(12-HPETE)

CO2H

HO 12-Hydroxyeicosa-
5,8,10,14-tetraenoic acid
(12-HETE)
 Leukotriene
Biosynthesis Inhibition
S OH
CH-N-CONH
2

CH3
Zileuton (Zyflo)

An inhibitor of 5-lipoxygenase
Used in the treatment of asthma
Eicosanoids

 Localized, short-lived hormone-like

activities (a few seconds)
 Very low cellular concentrations
 Action initiated by signal transduction
 Like hormones
Eicosanoid study

 Pioneered and continued by Swedish

biochemists
 Ulf von Euler, Sune Bergstrom & Bengt
Samuelsson

 Virtually in all mammalian tissue/organs

Subclasses

 Prostaglandin, thromboxanes and

leukotriene
 Derived from 20-carbon, polyunsaturated
arachidonic acid (20:4Δ5,8,11,14)
COX: cyclooxygenase etc.
 Prostaglandin synthase = COX
 Oxygenase and peroxidase activity
 Forms five-membered cyclic structure
 PGE2 and PGD2: in sleep center in brain
 PGD2: promotes sleep
 PGE2: induces wakefulness
COX enzymes
 Isoenzymes in humans and other animals
 COX-1: constitutive in most tissue at all
times
 COX-2: induced by infammatory stimuli
 Elevated levels of prostaglandin
 Pain and swelling: inflammatory response
Painkillers
 Nonsteroidal anti-inflammatory drug =
NSAID
 Inhibition of COX-1 and COX-2
 Acetaminophen: inhibits COX-3

 Side effects: gastric problems

COX inhibitors
 More selective for COX-2, not on COX-1
 Rheumatoid arthritis, osteoarthritis, and
acute pain
 May increase risk of heart attack and
stroke
COX inhibitors
 May prevent some cancers and
Alzheimer’s disease
 COX-2 overexpressed in some cancer cells
 Overproduction of prostaglandins
 Promote tumor and cell proliferation

 Treatment of colorectal cancer

Leukotrienes
 Cause contraction of smooth muscle
 Especially in lungs

 Asthma attack and allergic reaction

 May be caused by overproduction of
leukotriene