I 8 - Drugs used in the treatment of bacterial infections 1 - inhibitor of metabolism

What are the origins of antibiotics

Natural

• Made by soil bacteria and fungi
• Benzylpenicillin, erythromycin, vancomycin

semi -synthetic

• A natural core, chemically modified
• Amoxicillin from 6-aminopenicillanic acid (6-APA), amikacin from kanamycin

Fully synthetic
• No natural parent at all
• Sulfonamides, trimethoprim, quinolones
• Nitrofurantoin, metronidazole, oxazolidinones
• Strictly, these are antibacterials, not antibiotics

What do we have to consider when using antibiotics?

• Most processes in mammals and bacteria are very similar; if we try to kill bacteria, we may also harm the host organism

• Need to find a degree of selective toxicity - processes within bacteria which are more susceptible to antibiotics than the processes in the host

What are highly selective targets in bacteria?

Target	Why is it highly selective?
Bacterial cell wall synthesis	Human cells lack cell walls.
Dihydropteroate synthase (DHPS)	This enzyme is present in bacteria but absent in humans.

Note - preferred as they allow bacteria to be inhibited with less harm to the host

What are less selective targets in bacteria ?

Less selective targets

These involve processes present in both bacteria and humans:

• Protein synthesis.

• Dihydrofolate reductase (DHFR).

• Transcription.

• DNA replication.

What antibiotic drug classes can be identified from name endings ?

Drug name ending	Drug class	Examples
-cillin	Penicillins	Amoxicillin, flucloxacillin
cef- / ceph-	Cephalosporins	Cefalexin, ceftriaxone
-penem	Carbapenems	Meropenem, ertapenem
-bactam	Beta-lactamase inhibitors	Tazobactam, avibactam
-floxacin	Fluoroquinolones	Ciprofloxacin, levofloxacin
-mycin	Often products derived from Streptomyces	Streptomycin
-micin	Often products derived from Micromonospora	Gentamicin

What are the main targets for antibacterial drugs ?

Main targets

1. Cell wall: Inhibition of cell wall synthesis.

2. Ribosomes: Inhibition of bacterial protein synthesis.

3. DNA/RNA: Inhibition of DNA replication or transcription.

4. Plasma membrane: Disruption of membrane function.

5. Metabolic enzymes: Inhibition of essential metabolic pathways.

What are the structures of Gram-positive and Gram-negative bacteria?

Feature	Gram-positive bacteria	Gram-negative bacteria
Cell membrane	One cytoplasmic membrane	Inner and outer membranes
Peptidoglycan	Thick layer	Thin layer
Outer membrane	Absent	Present
Lipopolysaccharide (LPS)	Absent	Present in outer membrane
Drug entry	Generally fewer barriers	Outer membrane restricts drug entry
Periplasmic space	Less prominent	Contains enzymes such as beta-lactamases

What are the main structural differences between gram positive and gram negative bacteria ?

Gram-positive bacteria

Structure:

• One cytoplasmic membrane.

• Thick peptidoglycan layer.

• No outer membrane.

Gram-negative bacteria

Structure:

• Inner cytoplasmic membrane.

• Thin peptidoglycan layer.

• Outer membrane containing lipopolysaccharides (LPS).

Why are Gram-negative bacteria harder for antibiotics to target ?

• The outer membrane acts as an additional protective barrier.

• Some drugs must enter through porin channels.

• Beta-lactamases in the periplasm can break down susceptible beta-lactam antibiotics.

What is Prontosil, and how was it made active?

• Early antibacterial drug developed from azo dye

• found it was effective in vivo but inactive in vitro

• Found that it was metabolised by azoreductase into an active antibiotic ( it has an azo bond which is cleaved and then releases sulfanilamide)

• Prontosil acts as a prodrug

Why do bacteria need folate ?

• Bacteria require folate to produce tetrahydrofolate (THF).

• THF transfers one-carbon units needed for the synthesis of thymidine and other DNA components.

• Therefore, folate metabolism is essential for bacterial DNA synthesis and growth.

note: needed for DNA synthesis

What is the folate pathway - describe the steps ?

• Pathway that synthesises tetrahydrofolate, which is needed for DNA synthesis

• Sulfanilamide inhibits this process

Step 1 – Formation of dihydropteroate

• Para-aminobenzoic acid (PABA) combines with dihydropterin pyrophosphate.

• The enzyme dihydropteroate synthase (DHPS) catalyses the reaction.

• This produces dihydropteroate.

Step 2 – Formation of dihydrofolate

• Glutamate is added to dihydropteroate.

• This produces dihydrofolate (DHF).

Step 3 – Formation of tetrahydrofolate

• Dihydrofolate is reduced to tetrahydrofolate (THF).

• The reaction is catalysed by dihydrofolate reductase (DHFR).

• NADPH provides the reducing power.

Step 4 – DNA synthesis

• Tetrahydrofolate provides one-carbon units needed for thymidine synthesis.

• Thymidine is required for DNA synthesis.

How does folate metabolism differ in bacteria and humans?

Bacteria	Humans
Must synthesise folate.	Obtain folate from the diet.
Have DHPS.	Do not have DHPS.
Have DHFR.	Have DHFR.
Susceptible to sulfonamides targeting DHPS.	Lack the DHPS target of sulfonamides.

What are sulfonamides, and how do they work?

What are sulfonamides?

Sulfonamides are synthetic, bacteriostatic antibacterial drugs that inhibit bacterial folate synthesis.

How do sulfonamides work?

1. Sulfonamides have a similar chemical structure to PABA.

2. They act as competitive inhibitors of dihydropteroate synthase (DHPS).

3. They compete with PABA for the enzyme's active site.

4. DHPS may incorporate the sulfonamide instead of PABA, producing a compound that cannot undergo further transformation.

5. This interrupts the synthesis of dihydrofolate and tetrahydrofolate.

6. Without sufficient THF, bacteria cannot efficiently synthesise thymidine for DNA.

7. This inhibits bacterial growth.

Why can sulfonamides bind to DHPS?

Sulfonamides mimic PABA through similar interactions, including:

• Hydrogen bonding.

• Ionic interactions.

• π–π interactions.

These interactions help sulfonamides bind to the enzyme.

Are sulfonamides bacteriostatic or bactericidal?

Bacteriostatic.

• They inhibit bacterial growth and multiplication rather than directly killing bacteria.

• The host immune system can then eliminate the bacteria.

What structural features are essential for sulfonamide activity - SAR?

Structural feature	Importance
N4 amino group	Essential for antibacterial activity and must remain unsubstituted in the active drug.
Para relationship	The amino group must be para to the sulfonamide group to mimic PABA.
N1 sulfonamide nitrogen	Must retain a hydrogen; may be primary or secondary.
N1 substituent	Main position for structural modification to improve drug properties.- can be modified to improve activity

What effects does introducing electron-withdrawing groups on the N1 substituent have on activity?

Electron-withdrawing heterocyclic groups :

• Increase the acidity of the sulfur-linked amide ( n-h), lowering the pKa towards PABA ( closest to 4.9 is better for binding )

• The pKa affects how much of the drug is ionised at physiological pH - this is increased

• Compared to the carboxylic acid in PABA, enhances antimicrobial activity

• Enhances water solubility

• Higher potency and better solubility in urine

What is another enzyme target in the folate pathway, and how does trimethoprim work?

What is DHFR?

Dihydrofolate reductase (DHFR) is an enzyme that converts dihydrofolate (DHF) into tetrahydrofolate (THF).

• THF is essential for DNA synthesis.

• Both bacteria and humans have DHFR.

Mechanism of action of trimethoprim

1. Trimethoprim binds to bacterial DHFR.

2. It inhibits the reduction of DHF to THF.

3. This reduces the availability of THF.

4. Consequently, bacteria cannot produce sufficient thymidine for DNA synthesis.

5. Bacterial growth is inhibited.

why is trimethoprim selective for bacteria?

• Bacterial and human DHFR have slightly different structures.

• These differences allow trimethoprim to bind much more strongly to bacterial DHFR.

• The lecture states that bacterial DHFR is approximately 100,000 times more susceptible to trimethoprim.

Other drugs that inhibit DHFR

Drug	Main target	Clinical application
Trimethoprim	Bacterial DHFR	Antibacterial treatment
Methotrexate	Human DHFR	Cancer treatment
Pyrimethamine	Plasmodial DHFR	Antiprotozoal treatment

What is sequential blocking?

Sequential blocking occurs when two drugs inhibit different stages of the same metabolic pathway.

• Use a combination of the drugs. Co-trimoxazole is a combination of two antibacterial drugs:

  • Sulfamethoxazole – a sulfonamide.

  • Trimethoprim – a DHFR inhibitor.

How does co-trimoxazole work ?

Step 1 – Sulfamethoxazole

• Competitively inhibits DHPS.

• Prevents the normal formation of dihydropteroate.

• Reduces the production of dihydrofolate.

Step 2 – Trimethoprim

• Inhibits DHFR.

• Prevents remaining dihydrofolate from being converted into tetrahydrofolate.

Step 3 – Combined effect

• THF production is strongly reduced.

• Thymidine and DNA synthesis are impaired.

• Bacterial growth is inhibited.

drug summary

rug	Drug class	Target enzyme	Mechanism of action	Main effect
Sulfonamides	Antimetabolite antibacterials	DHPS	Mimic PABA and competitively inhibit folate synthesis	Bacteriostatic
Sulfamethoxazole	Sulfonamide	DHPS	Prevents normal dihydropteroate formation	Bacteriostatic
Trimethoprim	DHFR inhibitor	Bacterial DHFR	Prevents DHF conversion to THF	Bacteriostatic
Co-trimoxazole	Combination antibacterial	DHPS + DHFR	Sequential inhibition of bacterial folate synthesis	Synergistic; may be bactericidal
Methotrexate	Antifolate	Human DHFR	Inhibits folate metabolism and DNA synthesis	Anticancer
Pyrimethamine	Antifolate	Plasmodial DHFR	Inhibits folate metabolism in parasites	Antiprotozoal

summary - key concepts

• Selective toxicity – why certain antibiotics target bacteria rather than human cells.

• Gram-positive vs Gram-negative bacteria – structural differences and their effect on drug entry.

• Prontosil – why it is a prodrug and how it forms sulfanilamide.

• The bacterial folate pathway – the steps from PABA to THF and its role in DNA synthesis.

• Sulfonamide mechanism of action – competitive inhibition of DHPS.

• Sulfonamide SAR – the importance of N1, N4, the para relationship and pKa.

• Trimethoprim mechanism of action – inhibition of bacterial DHFR.

• Selective inhibition of DHFR – differences between trimethoprim, methotrexate and pyrimethamine.

• Co-trimoxazole – sequential blocking and synergism.

• Antibacterial resistance – mutations and overproduction of DHFR