By Duy Thai: www.geocities.com/d.

thai Pharmacology Semester 1 page 1 of 5

ENZYMES AS SITES OF DRUG ACTION II

Aspirin
• Aspirin is an example of a drug which acts as an irreversible enzyme inhibitor.
• It inhibits the cyclo-oxygenase enzyme in the production of prostinoids.
• Production of arachidonic acid metabolites:
Aspirin Phospholipid
irreversibly
inhibits cyclo-
oxygenase Phospholipase A2

Arachidonic acid
Cyclo-oxygenase Lipoxygenases

PGG2 Leukotrienes
Lipoxins

PGH2

Prostacyclins Prostaglandins Thromboxane

• Phospholipase A2 is not a good target because arachidonic acid is widely used for many important processes in all
cells of the body.
• Arachidonic acid can act as a second messenger to regulate the function of protein kinases.
• The prostinoids (prostacyclin, prostaglandin, thromboxane) have many actions:
• Control vascular homeostasis
• Cellular proliferation
• Immune and inflammatory mediators
• Reproduction
• Aspirin (acetyl salicylic acid) irreversibly inhibits cyclo-oxygenase by the following mechanism:

Acetyl salicylic acid Salicylic acid

Cyclo-oxygenase Acetylated cyclo-oxygenase

• The acetylated cyclo-oxygenase is non-functional, and remains that way.

• A byproduct, salicylic acid, is a reversible inhibitor of cyclo-oxygenase, although much weaker than aspirin.

Clinical uses of aspirin

• Mild analgesic
• Anti pyretic
• Anti inflammatory agent
• Used in the prevention of acute myocardial infarctions

Acute myocardial infarctions

• AMI’s are often caused by a thrombus occluding one of the coronary vessels.
By Duy Thai: www.geocities.com/d.thai Pharmacology Semester 1 page 2 of 5

• Blood flow to the myocardium is thus reduced and may be insufficient for the needs of the muscle. An infarct
follows.
• Arterial thrombi are composed largely of aggregated platelets and fibrin.
• Low doses (1 per day) of aspirin can effectively block platelet cyclo-oxygenase.
• This blocks the production of TxA2, a major platelet aggregation factor.
• Therefore, aspirin can prevent the formation of arterial thrombi.
• An advantage of a low aspirin dose is that it will not inhibit the vascular endothelial cyclo-oxygenase to a great
extent. Hence, the endothelial cells can still produce prostacyclin, which is one of the anti-coagulant mechanisms
of healthy endothelium.
• Prostacyclin inhibits platelet aggregation.
• How does low doses of aspirin achieve this?
• Irreversible enzyme inhibitors work better the longer they are in contact with the enzyme. This means
that eventually, you do get irreversible inhibition of cyclo-oxygenase in the endothelial cells. The reason
why this is not a problem can be attributed to pharmacokinetics.
• Aspirin is rapidly metabolised by the liver by the first pass effect.
• Aspirin is taken orally and absorbed in the gut.
• When absorbed in the gut, the aspirin travels in the portal vein.
• While in the portal vein, relatively high concentrations of aspirin are exposed to the circulating platelets
in the portal blood.
• The cyclo-oxygenase in the platelets is irreversibly inhibited.
• Because platelets are anucleate, they are unable to resynthesise cyclo-oxygenase. The only way to get
competent platelets is to wait for new ones to be produced in the bone marrow, which takes 5 - 7 days.
Hence, the inhibition of platelets lasts for a few days.
• After the liver has cleared a significant proportion of aspirin from the portal blood, a lower concentration
of aspirin appears in the systemic circulation.
• Low aspirin concentrations will inhibit endothelial cell cyclo-oxygenase very slightly. But since
endothelial cells are able to resynthesise cyclo-oxygenase, this inhibition is of no concern.
Resynthesis is much faster than having to wait for new platelets to be made.
• Hence, endothelial cells recover from inhibition quicker while inhibition of platelets is prolonged.

• There are other irreversible enzyme inhibitors but they are mainly for toxic use (e.g. inhibitors of cholinesterases
are found in insecticides). Very few are used therapeutically.

Drugs affecting blood clotting enzymes

• e.g. Warfarin
• It has a long lasting effect, mainly due to a long half life.
• Warfarin is an analogue of Vitamin K.
• It acts to inhibit the function of vitamin K as a co-factor in the modification of clotting factors.
• Clotting factors made by the liver need to undergo a post-translational modification. This involves the gamma
carboxylation of a glutamate functional group on the clotting factor to become gamma carboxylated glutamic acid.

Clotting factor Glu Clotting factorGla

Vitamin K Vitamin K
(oxidised) (reduced)
(epoxide) (hydroquinone)

Warfarin Warfarin
By Duy Thai: www.geocities.com/d.thai Pharmacology Semester 1 page 3 of 5

Vitamin K
(partially reduced)
(quinone)

• Warfarin prevents the recycling of vitamin K by competing with it for an intracellular enzyme in the liver.
• The clotting factors therefore do not undergo gamma carboxylation.
• Gamma carboxylation gives the clotting factors negatively charges functional groups which can interact
with Ca2+. This enables the clotting factors to work together.

Example of a drug working on an extracellular enzyme

• The renin angiostensin system:
↓ GFR

↓ [Na] in distal tubule

Renin

Angiotensinogen Angiotensin I
Angiotensin converting
enzyme

Angiotensin II

Peripheral Aldosterone
vasoconstriction secretion

↑ Na resorption

↑ BP

• In hypertensive therapy, drugs are used to prevent the formation of angiotensin II by competing for angiotensin
converting enzyme (ACE), which is located on the vascular surface of endothelial cells.
• An example of this type of drug is Captopril
Structurally
Angiotensin I Phe – His – Leu similar to
captopril

ACE cleaves this bond here

Angiotensin II Phe

• Captopril is structurally similar to the peptide fragment produced when angiostensin I is cleaved to angiotensin II.
• It can thus bind competitively to ACE and prevent angiotensin I from binding.
• ACE can also cleave bradykinin, which inactivates it.
By Duy Thai: www.geocities.com/d.thai 1997 Pharmacology Semester 1 page 4 of 6

• Thus, if you inhibit the formation of angiotensin II by inhibiting ACE, you will also increase bradykinin levels.

Drugs targeting signal transduction enzymes

β adrenorecptor

G protein

Phosphodiesterase
Adenylate cyclase

ATP cAMP AMP

Protein kinase A
(cAMP dependent
protein kinase

Phosphorylates intracellular
proteins

Cellular effects

• Protein kinase A has many different effects depending on the tissue:

• In the heart PKA activates L type Ca2+ channels to increase Ca2+
• In adipose tissue, PKA activates lipolysis
• In liver, PKA can activate glycolysis.
• Phosphodiesterase breaks down, cAMP into AMP, thus terminating its action.
• Inhibitors of PDE will thus cause prolongation of the effects mediated by cAMP. PDE also breaks down cGMP to
GMP.

Isoforms of phosphodiesterase
• There are 5 main families differing in their:
1. Affinity for cyclic nucleotides
2. Selectivity for cAMP or cGMP
3. Tissue and cellular distribution
• Different roles in different tissues
• Therefore, we develop drugs which can target a specific isoform, thus limiting the effect to one
tissue.
4. Selectivity for Ca2+/calmodulin
5. Inhibition by selective inhibitors
By Duy Thai: www.geocities.com/d.thai 1997 Pharmacology Semester 1 page 5 of 6

• Note the similarity to receptor molecules. You can get the same function but different subtypes of the receptor,
differing in amino acid sequence.

Inhibitors of phosphodiesterase
• Methylxanthines
• e.g. Theophylline, caffeine
• These drugs are unselective for PDE isoforms, hence they can have wide ranging effects.
• They enhance the effects of both cAMP and cGMP.
• Theophylline is clinically used for bronchodilation in asthmatics
• This is a local effect, but if some of it is swallowed while being inhaled, it can have systemic
effects which lead to a large number of unwanted side effects.
• Some methylxanthines are also adenosine receptor antagonists.
• New generation inhibitors
• e.g. Milrinone, amrinone
• Inhibit the type II isoform of PDE.
• This isoform selectively hydrolyses cAMP and is well expressed in cardiac muscle (although it
can be expressed in other tissues as well).
• They are clinically useful as having a positive inotrophic effect in acute heart failure. (Cardiac
glycosides also have this effect but they are very toxic because they can inhibit the Na+K+
ATPase in other tissues.)

Example of a drug which acts as a functional substrate of an enzyme

• Glycerol trinitrate
• A drug used in angina
• It is a pro drug which is activated in vivo. Administered sublingually. Its activated form is NO (nitric
oxide)
• NO is a substrate activator for the enzyme guanylate cyclase.

Endothelial cell derived

Glycerol trinitrate relaxing factor

NO2- Nitric oxide (NO)

Guanylate cyclase

GTP cGMP

Vascular relaxation

• NO has many other effects:

• It acts as a neurotransmitter
• It is used as a cytotoxic chemical by macrophages
• It can be inhaled directly to cause vasorelaxation of pulmonary capillaries.
• In angina:
• Pain occurs when oxygen supplying the myocardium is insufficient for its needs. (Due to atheroma,
vasoconstriction)
• Organic nitrates cause vasodilation of large veins, hence reducing venous pressure and also reducing
cardiac output (so the heart does not have to pump as hard)
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• They can also cause vasodilation of large coronary collateral vessels, hence improving perfusion to
ischaemic myocardium.