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

thai 1997 Pharmacology Semester 1 page 1 of 6

ENZYMES AS SITES OF DRUG ACTION I

Targets of drug action

• Drugs which are developed can act on 4 types of proteins:
• Surface receptors
• Ion channels
• Transporters/carriers
• Enzymes
• The reasons why proteins (and I will refer specifically to enzymes here) make good targets is because:
• They have high structural specificity
• Tissue specific expression
• Species differences in enzyme properties.
• Although the enzyme may have the same name and same function, there are slight chemical
differences between species which is exploited by species specific drugs.
• Enzymes act as catalysts for reactions
• Some enzymes work in cascade reactions.
• We must target the rate limiting enzyme because this enzyme limits the rate of all the other
enzymes in the reaction cascade.
• Isoforms
• Most enzymes have more than one isoform.
• The development of drugs which target specific isoforms can help in:
• Improving tissue selectivity
• Reducing side effects.

Mechanisms by which drugs interact with an enzyme

• Competitive inhibitors
• Bind to the same binding site as the substrate.
• Binding is surmountable
• Non-competitive inhibitors
• Does not bind to the same binding site as the substrate.
• Pseudo-irreversible inhibitors
• Have high affinity to an enzyme and a slow off rate. The binding is non-covalent but because the affinity is
so strong, they can be considered irreversible.
• Irreversible inhibitors
• Bind to the enzyme with strong covalent bonds
• Allosteric activation
• Does not participate in a reaction.
• Binds to the enzyme and increases its catalytic ability.
• Binds to a site different from the substrate binding site.
• Substrates and false substrates
• Drugs can act as substrates, substituting for endogenous substrates with biological activity.

Types of enzymes targeted by drugs

• Enzymes regulating cellular metabolism
• Enzymes that pump ions (ion channel ATPases)
• Enzymes involved in homeostatic regulation
• Neurotransmitter/hormone synthesis
• Degradation or action of regulatory factors
• Blood clotting enzymes
By Duy Thai: www.geocities.com/d.thai 1997 Pharmacology Semester 1 page 2 of 6

Cholesterol synthesis and hypercholesterolaemia

• Cholesterol synthesis occurs in all cells in the body, although the liver has a greater capacity to do this than other
cells.

Acetyl CoA

HMG CoA
HMG CoA Reductase
Mevalonate

Farnesyl-P-P

Squalene

Cholesterol
• The cholesterol which is formed is used for a variety of things:
• Steroid hormones
• Bile acids
• Constituent of cell membranes, making them fluid
• HMG CoA reductase is the rate limiting enzyme of cholesterol synthesis.
• In people with hypercholesterolaemia, we inhibit this enzyme so that production of cholesterol ceases.
• Inhibitors of HMG CoA reductase are:
• Mevastatin
• Lovastatin
• These 2 drugs act globally (there is no need to be tissue specific) because we want to reduce cholesterol
synthesis is all cells of the body.
• The drugs have close structural resemblance to the substrate of this enzyme, namely HMG CoA.
• By binding to the same binding site on the enzyme, HMG CoA cannot move to the next step of
the synthesis pathway.
• Treatment of a cholesterol synthesis inhibitor drug is not enough to reduce the hypercholesterolaemia. The drugs are
usually used in conjunction with drugs which chelate bile salts.
• The bile salts are prevented from being absorbed and going back to the liver (enterohepatic circulation).
They thus remain in the gut to be excreted.
• Remember that bile salts are a major source of cholesterol in the body.

Antiviral drugs
• The 2 antiviral drugs mentioned today are:
• Acyclovir
• Used against herpes virus
• Zidovudine (AZT)
• Used against HIV
• Both drugs show species selectivity. You cannot use one Acyclovir against HIV
• Both drugs are prodrugs.
• They are administered in an inactive form and are activated in the body, either by human enzymes or viral
enzymes.
• Thus, the drugs act as substrates for one set of enzymes and when activated, act as inhibitors to another set
of enzymes.
By Duy Thai: www.geocities.com/d.thai Pharmacology Semester 1 page 3 of 6

• Acyclovir is an analogue of a guanine base

• Zidovudine is an analogue of a thymidine base
• Thus, you can predict that the drugs are involved in inhibiting nucleic acid synthesis by competing with the guanine
base or thymidine base.

Acyclovir
• Targets the herpes virus
• Administered as a pro drug
• The active form is acyclovir triposphate (-P-P-P).
• Mechanism of action:

Acyclovir
Herpes virus kinases

Acyclovir - P
Human kinases

Acyclovir -P-P-P

DNA polymerase

Viral DNA Copy

• The inactive Acyclovir is selectively activated by the viral kinase.
• Thus, if there were no herpes viral infection, acyclovir would not be activated because it needs specifically
the herpes virus kinase.
• Acyclovir -P-P-P inhibits the viral DNA polymerase by being a competitive inhibitor to the other DNA bases,
namely guanine.
• Here, we have species selectivity between the human and the virus.
• Both humans and virus have DNA polymerase.
• If acyclovir were to act on human DNA polymerase, this would be bad.
• Luckily, acyclovir is designed to be specific for the virus DNA polymerase, and so the human
DNA polymerase is safe

Zidovudine (AZT)
• Targets HIV and other retroviruses.
• A retrovirus is a virus which contains a strand of RNA
• The virus has a special enzyme, reverse transcriptase (humans do not have this enzyme) which allows the
RNA to be “reverse transcribed” into DNA.
• The DNA is incorporated into host cell DNA.
• Whenever the host cell transcribes a section of DNA, the viral DNA is also transcribed along with it.
Transcription of the viral DNA produces viral RNA. Translation of the RNA produces viral proteins
which are necessary for the virus’s viability.
By Duy Thai: www.geocities.com/d.thai 1997 Pharmacology Semester 1 page 4 of 6

• Mechanism of action

AZT

AZT -P-P-P

Viral reverse transciptase

Viral RNA Viral DNA

Incorporated in to
host DNA

Replication of viral
RNA and protein
• AZT is activated by human kinases. Hence it is activated everywhere in the body, even when there is no viral
infection.
• However, if there is no viral infection, the active form of AZT has got nothing to do because it is species selective for
the enzyme reverse transcriptase.
• Humans do not have this enzyme, so AZT has nothing to bind to unless there is a viral infection by a
retrovirus. Only retroviruses have reverse transcriptase.
• Notice how the drug is species selective between humans and other viruses.
• AZT inhibits reverse transcriptase by preventing the addition of nucleotide bases, particularly thymidine.

• What we have just seen in the 2 examples given are:

• Competition for a natural substrates (the nucleotide bases)
• Species selectivity between humans and virus, and between virus and virus
• Cellular selectivity
• e.g. Acyclovir does not work in all cells of the body. It only works in virally infected cells because
it required the viral kinase to activate it.

A recent breakthrough in the fight against HIV - Protease inhibitors

• After the HIV has reverse transcribed its RNA into DNA, the DNA strand is incorporated into the host cell DNA.
• The viral DNA codes for polyproteins.
• 1 gene codes for several proteins strung together.
• Proteases are used to chop up this string of proteins so that individual proteins are produced.
• Human proteases chop up the polyproteins to produce coat proteins.
• Coat proteins are the proteins found on the surface of the virus
• Viral proteases chop up the polyproteins to produce core proteins.
By Duy Thai: www.geocities.com/d.thai Pharmacology Semester 1 page 5 of 6

• Core proteins are found inside the virus particle.

• Protease inhibitors are drugs which inhibit the protease enzyme.
• If we inhibit the human protease, the virus will lack surface proteins. However, the proteases in other,
healthy cells are also inhibited, which is not a good thing.
• Drugs have been developed which specifically target the viral proteases. This prevents formation of the
core proteins.
• The virus particle which is formed, thus has an intact surface coating but lacks core proteins,
rendering it inactive.
• An example of a protease inhibitor is Saquinavir
• Saquinavir competitively inhibits the active site of the protease.
• It mimics the cleavage site of the viral polyprotein.
• Protease inhibitors are often used in conjunction with AZT because resistance can develop against the protease
inhibitor (via mutations in the polyprotein).

Antibacterial drugs - Folic acid metabolism inhibitors

• Folic acid is used in the synthesis of bases (e.g. guanine, thymidine, uracil, cytosine, adenine) of nucleotides which
are used in DNA and RNA synthesis.
• The structure of folic acid is like so:

Pteridine ring pABA Glutamic acid

• Mammalian cells cannot make folic acid. They obtain it from their diet.
• Hence, mammalian cells do have a folic acid transporter molecule.
• Bacteria do not have a folic acid transporter because they don’t need it.
• Bacteria have enzymes which are capable of making folic acid de novo.
• These enzymes are good targets for antibacterial drugs because the enzymes are only found in bacteria.
• An example of this type of drug is Sulphanilamide
• It is an analogue of the pABA portion of folic acid.
• It competitively inhibits the enzyme by binding to the substrate binding site, thus preventing the actual
pABA molecule to bind and form folic acid.
• Other antibacterial drugs;
• Trimethoprim
• Methotrexate
• Both these drugs do not prevent the synthesis of folic acid. Instead, they prevent the recycling of it.
Methotrexate
Trimethoprim

O DHFR = Dihydrofolate reductase

DHFR DHFR

Folic acid FH2 FH4

FH4-C

dTMP dUMP

• Both bacteria and humans have DFHR but the enzymes are slightly different structurally, therefore selective drugs
can be developed to target either the human form or the bacterial form.
• Methotrexate and Trimethoprim are slightly different.
• They do not differ in mechanism of action. Instead, they differ in context of use.
By Duy Thai: www.geocities.com/d.thai 1997 Pharmacology Semester 1 page 6 of 6

• Trimethoprim is selective for the bacterial form of DHFR.

• Hence, it is used as an antibacterial drug
• Methotrexate is mainly selective for the human form of DHFR
• Methotrexate is used in chemotherapy to prevent the synthesis of nucleotides required for DNA
synthesis in rapidly dividing mammalian cells (cells which are out of control).

Enzymes that pump ions

• e.g. Digitoxin
• Digitoxin belongs to a family known as cardiac glycosides.
• It is used in cardiac failure to improve the cardiac output of the heart by increasing the force of contraction.
• It acts by inhibiting the Na+K+ATPase, particularly in cardiac muscle cells.
• Mechanism of action:
• In a cardiac myocyte, a low intracellular Na+ concentration allows Na+ to flow down its concentration
gradient into the cell. This helps to remove a Ca2+ load in the cell.
• The Na+ enters the cell via a Na+ Ca2+ counter exchange.
• Na+ flows in, Ca2+ flows out
• When the Na+ flows into the cell, its concentration is prevented from rising by its removal via a Na+ K+
ATPase which pumps Na+ out of the cell (against its concentration gradient) and K+ into the cell (also
against its concentration gradient).
• Digitoxin binds to the Na+ K+ ATPase pump and shuts it.
• Digitoxin is acting as an allosteric enzyme inhibitor. It does not occupy the ATP binding site.
• If the pump is inactivated, the Na+ concentration in the cell will rise. An increase in intracellular
Na+ concentration reduces the concentration gradient, and so less Na+ ions flow in.
• Less Na+ flowing in means less Ca2+ flowing out and so Ca2+ levels rise.
• The Ca2+ is sequestered by the sarcoplasmic reticulum
• The next time there is a depolarisation, more Ca2+ can be released, resulting in a stronger force of
contraction.

Cardiac glycosides and heart failure

• In heart failure, the heart is unable to deliver enough blood to match the body’s requirement.
• Heart failure can be caused by:
• Heart valve disease
• Damage, ischaemia
• Increased peripheral resistance due to peripheral vascular disease
• Cardiac glycosides help by:
• Increasing the force of contraction
• This increases the cardiac output, resulting in improved perfusion of peripheral tissues
• It reduces the venous pressure (which is high in heart failure) by allowing more venous blood to go to the
heart. This relieves the oedema.