Anti-microbial drugs that act via the inhibition of DNA replication

Question 1

Nucleic acid replication as an anti-microbial drug target

Answer 1

Bacterial DNA replication inhibitors - act against DNA gyrase.
Viral DNA replication inhibitors - act against viral DNA polymerase or reverse transcriptase (converts RNA genomes of retroviruses to DNA)
Some eukaryotic parasite are susceptible to inhibitors of bacterial DNA replication as they contain essential endosymbionts ie the apicomplexan plasmid.

Question 2

DNA synthesis

Answer 2

Helicase separates 2 strands.
DNA polymerase replicates the leading strand.
Lagging strand is stabilised by ssDNA binding protein (ssBP).
Lagging strand needs RNA primers and DNA primase for DNA polymerase to generate okazaki fragments.
DNA polymerase replaces RNA primers with DNA.
DNA ligase fills in the gaps.

Question 3

Topo-isomerases

Answer 3

Unwinding the double helix gives tension on the strands.
Topoisomerases facilitate unwinding by making a cut in one strand and ligating it back once the strands relax.
Process is ATP dependant.
DNA gyrase is a topoisomerase II that relieves strain during the unwiding of the helix - It is the target of several antibiotics.

Question 4

Anti-viral Chemotherapy

Answer 4

Viruses are obligate intracellular entities.
They subvert many cellular processes to their own ends, making virus specific targets very rare/
DNA replication, however, is a good target due to virus encoded specific enzymes.
Prevention of the conversion of RNA to DNA can also be a good target in retroviruses.

Question 5

Inhibitors of viral DNA polymerase.

Answer 5

1. Acyclovir
2. Valacyclovir
3. Ganciclovir
4. Foscarnet
   All these drugs act on DNA polymerase, ie DNA viruses.

Question 6

Acyclovir

Answer 6

An acyclic guanosine analogue, useful against herpes virus group (herpes simplex, cytomegalovirus, epstein-barr virus)
A prodrug - not active until triphosphorylated.
Phosphorylation requires viral thymidine kinase to produce acyclovir-monophosphate, and cellular kinases to go on to produce acyclovir di- (and ultimately) tri-phosphate.
Only cells infected with the virus possess the thymidine kinase required for the initial phosphorylation of the drug.
Acyclovir triphosphate inhibits herpes group viral DNA polymerase.
It can also be incorporated into growing DNA chains thus inhibiting DNA chain elongation.
Lacking in oral availability due to low solubility.

Question 7

Valacyclovir

Answer 7

Orally available variant of Acyclovir - adding the valyl-ester increases uptake.
Once in bloodstream it is quickly converted to acyclovir by esterases.
Cnsidered a double pro-drug

Question 8

Ganciclovir

Answer 8

Another acyclic guanosine analogue.
Action similar to Acyclovir - inhibition of viral DNA polymerase
Activation uses thymidine kinase in herpes simplex virus but a specific protein (phosphotranferase - UL97) is responsible for activation in CMV (cytomegalovirus).
I.V administration.

Question 9

Resistance to Acyclovir and Ganciclovir

Answer 9

Resistance can occur to these drugs when mutations occur, either in -

1. The viral thymidine kinase (or phosphotransferase in CMV) preventing conversion of the prodrug.
2. The viral DNA polymerase, preventing binding of the triphosphate.

Question 10

Foscarnet

Answer 10

A phosphoformic acid (pyrophosphate analogue).
Inhibitor of Viral DNA polymerase, RNA polymerase and reverse transcriptase - binds to pyrophosphate site.
No requirement for activation.
Active against HSV, VZV(varicella zoster virus), CMV, EBV, HIV.
Relatively high host toxicity.

Question 11

Nucleoside inhibitors of Reverse Transcriptase

Answer 11

RT is critical to the replication of all retroviruses but not mammalian cells.
It is an attractive drug target.
Both nucleoside and non-nucleoside inhibitors of RT are effective.
AZT is a prototype RT inhibitor.

Question 12

Zidovudine (AZT - azidothymidine)

Answer 12

Deoxythymidine analogue (pro-drug)
Good oral availability and distribution, can cross BBB due to hydrophobic azide group.
Converted to triphosphate by cellular enzymes, inhibits RT and chain elongation.
Chain termination is main effect - lack of 3’OH is key.
5’OH allows incorporation into growing DNA chain.

Question 13

Tenofovir and Emtricitabine

Answer 13

Among the main anti-retrovirals given against HIV infection.
Tenofovir is phosphonate inhibitor of RT.
Emtricitabine is a nucleoside inhibitor of RT requiring phosphorylation.
Termintaion of growing DNA chain.
Together with non-nucleoside RT inhibitor Efavirenz
Provides HAART in a single tablet given once a day.

Question 14

Cidofovir

Answer 14

A cytosine analogue
It is a phosphonate (does not require phosphorylation, already active)
Inhibits viral DNA polymerase and chain elongation.
Active against - CMV, HSV, VZV, EBV, adenovirus, poxvirus, polyoma virus, papilloma virus.

Question 15

Didanosine (ddI)

Answer 15

Synthetic analogue of deoxyadenoside.
Triphosphorylated in cell.
Inhibits RT and chain elongation.
Activity improved if cellular ATP is depleted using hydroxyurea.

Question 16

Nucleoside inhibitors of RT include

Answer 16

Stavudine (d4T)
Zalcitabine (ddC)
Lamivudine (3TC)
Similar mode of action.

Question 17

Resistance to nucleoside reverse transcriptase inhibitors (NRTI)

Answer 17

Resistance comes about due to mutations to the viral RT that decrease binding of the inhibitor.
Different mutations are required for resistance to different drugs.
Example - L74V in didanosine resistance and M184V gives enhanced susceptibility to AZT.
Combinations are therefore preferred in order to decrease the risk of resistance.

Question 18

Non-nucleoside RT inhibitors (NNRTI)

Answer 18

Bind to sites close to but not within the nucleoside binding pocket.
Do not require phosphorylation.
Can be designed with high lipophilicity enabling oral uptake and brain penetration.
Compliments affects of NRTIs - as it binds at different sites, moreover different mutations are required for resistance.
Efavirenz makes HAART together with telofovir and emtricitabine, reduces onset of resistance.
Other non-nucleoside inhibitors of HIV-1 RT include Nevirapine and delavirdine.

Question 19

Inhibitors of bacterial DNA gyrase

Answer 19

DNA gyrase is a DNA topoisomerase II, involved in removing supercoils from double stranded DNA.
DNA is frequently overwound, topoisomerases relieve this.
Gyrase also separates replicated DNA.

Question 20

Quinolones and fluoroquinolones

Answer 20

Synthetic reagents that interfere with DNA gyrase.
Active against gram-negative and gram-positive organisms.
The first quinolone in common use was nalidixic acid.
These quinolones were fine for topical and GI treatments.
Did not reach high systemic levels.
Fluoroderivatives were much better systemically.

Question 21

Fluoroquinolones

Answer 21

Many variations on a theme are sucessfully used.
Resistance relates to changes in the DNA gyrase binding site.
Inhibits DNA gyrase.
Active against gram-negative and gram-positive bacteria.
Antibiotic of choice against anthrax.
Malaria parasites and other apicomplexans are also susceptible to fluoroquinolones (due to apicoplast which is replicated by prokaryotic enzymes including gyrase).

Question 22

Anthrax

Answer 22

Caused by baccillus anthraxis.
Gram positive
The scale of ciprofloxacin increased dramatically following the anthrax scare in 2001.
Anthrax is normally treated with doxyclycline.
Choice of medicine is increasingly politicised.

Question 23

Innovative mettalo-derivatives of ciproflaxacin

Answer 23

Displays upto 100 fold higher activity against plasmodium.
New development.
Selectivity Unclear.