Chemotherpaeutic Antibiotics: Nucleic Acid Inhibition and Membrane Damage (DONE)

Question 1

The targets of nucleic acid inhibition

Answer 1

DNA replication/transcription
DNA gyrase (type 2 topoisomerase): quinolones
RNA polymerase inhibition: rifampicin
Folic acid biosynthesis: sulphonamides, dapsone, trimethoprim
Others: nitrofurantoin, metronidazole

Question 2

DNA gyrase

Answer 2

The replication of the bacterial chromosome requires separation of the 2 highly intertwined parental strands from one another
Separation of strands folded in a helix generates loops, termed positive supercoiled twists, in the single strands
DNA gyrase relaxes positively supercoiled DNA

Question 3

How does DNA gyrase relax DNA?

Answer 3

By periodically breaking phosphodiester bonds in one of the strands of the double helix
Introducing negative supercoils
Resealing the nick

Question 4

DNA gyrase structure

Answer 4

DNA gyrase is composed of 4 subunits: 2 gyrase A subunits, 2 gyrase B subunits, encoded respectively by gyrA and gyrB
Topoisomerase IV is composed of 4 subunits: 2 ParC subunits, 2 ParE subunits

Question 5

DNA gyrase subunits

Answer 5

A subunit: The A subunits are involved in the DNA breakage and resealing events associated by supercoiling
B subunit: The B subunits are responsible for ATP hydrolysis, and stabilizing the interaction with quinolones

Question 6

Topoisomerase IV

Answer 6

In gram positive, topoisomerase IV is responsible for unlinking newly replicated daughter chromosomes and resolving knots that result from recombination events

Question 7

DNA gyrase-selectivity

Answer 7

Eukaryotic topoisomerase II displays activities that are homologous to those mediated by bacterial gyrase
However, the eukaryotic structure is a dimer with two identical subunits
The structural differences between the bacterial and mammalian topoisomerase II enzymes account for the selective action of the quinolones against bacterial gyrase

Question 8

DNA gyrase- inhibition: quinolones

Answer 8

Do not bind directly to either the gyrase A or B subunits, or to the intact enzyme
Gyrase stimulates the binding of quinolones to dsDNA
Gyrase cleaves dsDNA to produce exposed single-stranded regions that constitute binding sites for the antibiotics

Question 9

Quinolone action

Answer 9

Key step in quinolone action is the trapping gyrase or topoisomerase IV on DNA as tertiary drug enzyme DNA complexes
Quinolones stabilized the stranded break in DNA created by gyrase so that re-ligation becomes unfavourable
The tertiary complex blocks transcription (DNA polymerase) and more importantly DNA replication

Question 10

Examples of quinolones

Answer 10

Nalidix acid- uncomplicated UTI
Norfloxacin- uncomplicated UTI
Ofloxacin- UTI, lower RTI
Levofloxacin- CA pneumonia
Ciprofloxacin- broad spectrum gram negative

Question 11

RNA polymerase role and inhibition

Answer 11

Bacterial DNA-dependent RNA polymerase mediates the transcription cycle and represents a major point of regulation for prokaryotic gene expression

Question 12

Rifampicin

Answer 12

Semi-synthetic rifampicin binds close to the active site of the catalytic B-subunit and inhibits initiation of RNA synthesis by physically preventing extension of RNA products beyond a length of 2-3 nucleotides

Question 13

Indications of rifampicin

Answer 13

Brucellosis, legionella, prophylaxis of meningococcal meningitis, TB, endocarditis

Question 14

Folic acid synthesis

Answer 14

Folic acid is required for growth by both bacterial and mammalian cells
Conversion of dihydrofolic acid to tetrahydrofolic acid is necessary for the synthesis of certain amino acids, purines, thymidine and DNA
Animals and man acquire folate through the diet
Folate is accumulated by mammalian cells, but not by bacteria, which must synthesize the compound intracellularly
This is the basis of the selective toxicity of the sulphonamides

Question 15

Folic acid synthesis inhibition

Answer 15

Sulphonamides: mainly bacteriostatic
Structural analogues of para-aminobenzoic acid
Act as alternative substrates for dihydropteroate synthase
DHPS catalyses the formation of the folate intermediate dihydropteroic acid
Inhibition of bacterial growth results from the formation of inactive folate-like analogues

Question 16

Trimethoprim

Answer 16

Bacteriostatic or bactericidal depending on the growth conditions
Selectively inhibits dihydrofolate reductase
DHFR reduces dihydrofolic acid to tetrahydrofolic acid

Question 17

Nitrofurantoin

Answer 17

The reduced form of nitrofurantoin is highly reactive and attack ribosomal proteins, DNA, respiration and pyruvate metabolism
This is made possible by the rapid reduction of nitrofurantoin inside the bacterial cell, as compared to the mammalian cell

Question 18

Metronidazole

Answer 18

Taken up by diffusion and selectively absorbed by anaerobic bacteria, once taken up by anaerobes it is non-enzymatically reduced generating reactive intermediates reacting with cysteine-bearing enzymes
Metronidazole metabolites are taken up into bacterial DNA, and form unstable molecules
Used for anaerobic infections including dental

Question 19

Membrane damage

Answer 19

Colistin/polymyxin B
Binds to lipopolysaccharide (LPS) in the outer membrane of gram negative bacteria, disrupts both the outer and inner membrane
The hydrophobic tail is important in causing membrane damage, suggesting a detergent-like mode of action

Question 20

Daptomycin-lipopeptide

Answer 20

Disrupt multiple aspects of bacterial cell membrane function
Binds to the membrane and cause rapid depolarization, resulting in a loss of membrane potential leading to inhibition of protein, DNA and RNA synthesis