I9- drugs used in bacterial infections that inhibit cell wall synthesis

Why do antibiotics target cell wall synthesis and what is the structure of the cell wall?

• The cell wall is made from peptidoglycan , gives structural strength and prevents bacteria from bursting

Peptidoglycan consists of:

• Alternating sugar molecules: NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid).

• Peptide chains attached to NAM.

• Contain both d and L amino acids

• Cross-links between peptide chains, which strengthen the bacterial cell wall

The final cross-linking step is called transpeptidation.

It is catalysed by enzymes called transpeptidases, which are also known as penicillin-binding proteins (PBPs).

What is transpeptidation, and why is it important?

Transpeptidase is a bacterial enzyme, also known as a penicillin-binding protein (PBP), responsible for cross-linking peptidoglycan chains to strengthen the bacterial cell wall.

• This enzyme is inhibited by most of the beta-lactam antibiotics ( apart from vancomycin )

• this is where penicillin works /attacks

What is the mechanism of action for transpeptidase?

Mechanism of action (step by step)

1. Substrate recognition: Transpeptidase recognises the terminal D-Ala-D-Ala sequence on a peptidoglycan peptide chain.

2. Nucleophilic attack: A serine residue in the enzyme's active site attacks the carbonyl carbon of the peptide bond between the two D-alanine residues.

3. Release of D-alanine: The terminal D-alanine is released, leaving the remaining peptide chain covalently attached to the enzyme.

4. Second nucleophilic attack: An amino group from a neighbouring peptide chain (e.g. glycine) attacks the carbonyl carbon of the enzyme-bound intermediate.

5. Cross-link formation: A peptide bond forms between the two peptidoglycan chains, releasing the enzyme.

6. Result: The peptidoglycan chains become cross-linked, providing strength and rigidity to the bacterial cell wall.

Why is cell wall synthesis a good drug target?

• Human cells do not have peptidoglycan cell walls.-

• Therefore, antibiotics can inhibit bacterial cell wall synthesis without directly targeting an equivalent structure in human cells.

• This is an example of selective toxicity.

How do beta-lactam antibiotics work?

• They inhibit the transpeptidase mechanism of action

What happens when penicillin is present?

1. Penicillin resembles the D-Ala-D-Ala substrate recognised by transpeptidase.

2. The serine residue attacks the carbonyl carbon of penicillin's β-lactam ring.

3. The β-lactam ring opens.

4. Penicillin becomes covalently attached to the enzyme.

5. The enzyme cannot complete the cross-linking reaction.

6. Peptidoglycan synthesis is disrupted, weakening the bacterial cell wall.

7. Growing bacteria may undergo cell lysis and die.

Key point: β-lactam antibiotics are bactericidal because they inhibit the cross-linking of peptidoglycan.

They are most effective against actively growing bacteria.

Why is the beta - lactam ring so important?

• makes it less susceptible to nucleophilic attack,but the ring can behave differently

  Structural feature	Why is it important?
  Four-membered β-lactam ring	Produces considerable ring strain, increasing reactivity.
  Restricted amide geometry	Reduces resonance stabilisation between nitrogen and the carbonyl group.
  Electrophilic carbonyl carbon	Allows nucleophilic attack by the serine residue of transpeptidase.
  Free carboxylic acid group	Important for recognition and binding to the target enzyme.
  Acylamino side chain	Helps determine antibacterial activity, stability and spectrum.

Why does the ring strain increase activity ?

• The ring is strained due to the angles

• Amide resonance is reduced.

• The carbonyl carbon becomes more electrophilic.

• The serine residue of transpeptidase can attack it more readily.

Remember:

The β-lactam ring must be reactive enough to inhibit transpeptidase, but stable enough to survive until it reaches the bacterial enzyme.

This balance explains many of the structural modifications covered in your lectures.

What is the basic structure of penicillin and its SAR?

All penicillins are derived from 6-aminopenicillanic acid (6-APA).

This contains:

• A four-membered β-lactam ring.

• A five-membered thiazolidine ring containing sulfur.

• A free carboxylic acid group.

• An amino group at position 6, which can be modified to form different penicillins.

SAR

Structural feature	Function	Can it be modified?
β-lactam ring (E)	Essential for inhibition of transpeptidase	Must be retained
Free (E) carboxylic acid	Important for binding to the target enzyme	Must be present in the active drug
Bicyclic ring system	Increases β-lactam ring strain and reactivity	Some structural variation is possible
Acylamino ( E) group at C6	Important for antibacterial activity	The attached R group can be changed
Sulfur atom	Part of the thiazolidine ring	Not essential in all β-lactam structures
C6 stereochemistry	Determines whether the molecule fits the enzyme	Must be appropriate for activity

Why is the R group important?

• It is the part that medicinal chemists modify

Changing the R group can improve :

1. Acid stability — whether the drug survives stomach acid.

2. β-lactamase resistance — whether bacterial enzymes can destroy the antibiotic.

3. Antibacterial spectrum — which bacteria the drug can kill.

4. Pharmacokinetic properties — including absorption and distribution.

What is penicillin G and its limitations?

• prototype penicillin

• active against G +ve and some g -ve

• active against anaerobic bacteria

• bactericidal and active only against rapidly dividing bacteria

• susceptible to all beta-lactamases

• orally inactive due to acid sensitivity

• may cause allergic reactions

It has three major limitations.

Limitation	What happens?	What modification is needed?
Acid sensitivity	The β-lactam ring can open in acidic conditions, inactivating the drug.	Introduce an electron-withdrawing side chain.
β-lactamase sensitivity	Bacterial β-lactamase enzymes hydrolyse the β-lactam ring.	Introduce bulky groups to protect the ring.
Limited Gram-negative activity	The drug has limited ability to cross the Gram-negative outer membrane.	Introduce more hydrophilic side chains.

What causes acid sensitivity?

• It can be unstable because the beta-lactam ring can undergo acid-catalysed ring openings

Three factors that affect it :

1. Ring strain - The four-membered β-lactam ring is highly strained, making it susceptible to nucleophilic attack.

2. Carbonyl reactivity - Reduced amide resonance makes the β-lactam carbonyl carbon electrophilic.

3. Acyl side chain - The side-chain amide oxygen can act as a nucleophile and attack the β-lactam carbonyl, causing ring opening.

How can we overcome acid sensitivity?

Introduce electron-withdrawing groups (EWGs) into the penicillin side chain.

How does this work?

1. Electron-withdrawing groups reduce electron density on the side-chain amide oxygen.

2. This makes the oxygen less nucleophilic.

3. It is less likely to attack the β-lactam carbonyl.

4. Acid-catalysed ring opening is reduced.

5. The penicillin becomes more acid-stable and suitable for oral administration.

What causes beta-lactamase sensitivity?

Some bacteria produce β-lactamase enzymes, which hydrolyse the β-lactam ring and inactivate penicillin.

Mechanism:

1. β-Lactamase recognises and binds penicillin.

2. The active-site serine attacks the β-lactam carbonyl.

3. The β-lactam ring opens.

4. Water hydrolyses the enzyme-bound intermediate.

5. Inactive penicilloic acid is released, and β-lactamase is regenerated.

Result: Penicillin is destroyed before it can inhibit bacterial transpeptidase.

How can we overcome beta-lactamase sensitivity?

Method 1: Introduce bulky side chains (steric hindrance)

Add bulky groups near the β-lactam ring to prevent β-lactamase from accessing it.

How does this work?

1. Bulky groups are introduced into the R side chain.

2. These groups create steric hindrance around the β-lactam ring.

3. Susceptible β-lactamases cannot easily access the β-lactam carbonyl.

4. Penicillin is protected from enzymatic hydrolysis.

5. The antibiotic can still bind to susceptible bacterial PBPs.

Examples:

Drug	Structural modification	Benefit
Methicillin	Bulky aromatic side chain containing methoxy groups	Resistance to certain β-lactamases
Oxacillin	Bulky isoxazolyl side chain	β-Lactamase resistance and acid stability
Cloxacillin	Substituted isoxazolyl side chain	β-Lactamase resistance
Flucloxacillin	Bulky isoxazolyl side chain containing halogens	β-Lactamase resistance and oral activity

Limitation: Introducing bulky groups can reduce antibacterial activity or restrict the spectrum because the antibiotic may bind less effectively to certain PBPs.

What is another way to overcome beta-lactamase activity?

Method 2: Co-administer a β-lactamase inhibitor

Instead of modifying the antibiotic, administer another drug that inhibits β-lactamase.

How does this work?

1. The β-lactamase inhibitor binds to the bacterial β-lactamase enzyme.

2. The inhibitor prevents susceptible β-lactamases from destroying the antibiotic.

3. The antibiotic remains active.

4. It can inhibit bacterial PBPs and prevent cell wall cross-linking.

Examples:

Antibiotic	β-Lactamase inhibitor	Combination
Amoxicillin	Clavulanic acid	Co-amoxiclav
Ampicillin	Sulbactam	Ampicillin/sulbactam
Piperacillin	Tazobactam	Piperacillin/tazobactam

Your lecture also discusses newer β-lactamase inhibitors, including avibactam, relebactam and vaborbactam.

Key point: Bulky side chains protect the antibiotic from β-lactamase attack, whereas β-lactamase inhibitors target the resistance enzyme itself.

Neither approach overcomes every type of β-lactamase resistance.

What causes a limited spectrum of activity?

This is mainly due to differences in bacterial cell envelope structure.

Gram-positive bacteria:

• Have a thick peptidoglycan layer.

• Do not have an outer membrane.

• PBPs are relatively accessible to penicillin.

Gram-negative bacteria:

• Have an additional outer membrane.

• Antibiotics must pass through this membrane to reach PBPs.

• Hydrophilic antibiotics can enter through water-filled porin channels.

• Entry depends on the antibiotic's size, shape, charge and hydrophilicity.

Result: Penicillin G has limited penetration through many Gram-negative outer membranes.

How can we overcome a limited spectrum of activity?

Introduce hydrophilic groups into the R side chain to improve penetration through suitable Gram-negative porins.

Examples

Drug class	Examples	Structural modification	Benefit
Aminopenicillins	Ampicillin, amoxicillin	Addition of an amino group	Increases hydrophilicity and improves Gram-negative penetration
Carboxypenicillins	Carbenicillin, ticarcillin	Addition of a carboxylic acid group	Increases hydrophilicity and Gram-negative activity
Ureidopenicillins	Piperacillin, azlocillin, mezlocillin	Longer ureido-containing side chains	Improved interactions with certain PBPs and broader antibacterial activity

summary problems with penicillin

Problem	What causes it?	How do we overcome it?	Drug examples
1. Acid sensitivity	Acid-catalysed β-lactam ring opening, assisted by the side-chain amide oxygen	Introduce electron-withdrawing groups to reduce side-chain oxygen nucleophilicity	Penicillin V, ampicillin, amoxicillin
2. β-Lactamase sensitivity	Bacterial β-lactamase hydrolyses the β-lactam ring	Introduce bulky side chains for steric protection OR administer β-lactamase inhibitors	Flucloxacillin; co-amoxiclav
3. Limited Gram-negative activity	Poor penetration through the Gram-negative outer membrane	Introduce hydrophilic side chains and optimise PBP binding	Ampicillin, amoxicillin, piperacillin

Why were ampicillin and amoxicillin developed ?

• They belong to the aminopenicillin class

Ampicillin

• An amino group is introduced at the alpha carbon of the side chain

• This is beneficial for the amino group in acidic conditions, as it becomes protonated: NH₂ → NH₃⁺, reducing nucleophilicity and making beta-lactam opening less likely

• The amino group increases hydrophilicity, improving penetration into susceptible Gram-negative bacteria

Amoxicillin

• It contains the same alpha-amino group as ampicillin, with an OH group on its aromatic group

• This is beneficial because the hydroxyl group changes the physicochemical properties of the molecule and contributes to improved oral absorption
  
  

Comparison

Characteristic	Ampicillin	Amoxicillin
α-Amino group	Present	Present
Additional phenolic –OH	Absent	Present
Acid stability	Improved	Improved
Gram-negative activity	Broader than penicillin G	Broader than penicillin G
Oral bioavailability in lecture	33–54%	74–80%
β-lactamase resistance	Susceptible	Susceptible

How does base-catalysed degradation occur?

Penicillin can also undergo degradation in alkaline conditions

Step 1: Hydroxide ions (OH⁻) attack the electrophilic β-lactam carbonyl carbon.

Step 2: A tetrahedral intermediate forms.

Step 3: The β-lactam ring opens.

Step 4: Penicilloic acid is formed.

Step 5: Penicilloic acid can undergo further degradation, producing inactive penilloic acid and subsequently other degradation products.

What ph provides the best stability ?

• pH 6- 7 most favourable range

• penicillin solutions are often buffered ang used promptly after preparation

What is beta-lactamase, and how does it cause antibiotic resistance?

• Some bacteria produce enzymes called beta-lactamases

These enzymes hydrolyse the β-lactam ring, inactivating the antibiotic.

How does β-lactamase work?

1. The β-lactam antibiotic enters the enzyme's active site.

2. A serine residue hydroxyl attacks the β-lactam carbonyl.

3. The β-lactam ring opens. leaving a acyl-enzyme intermediate

4. Water hydrolyses the enzyme-bound intermediate.

5. The inactive antibiotic is released.

6. The β-lactamase enzyme is regenerated and can destroy another antibiotic molecule.

Why is this a problem - Comparison: Transpeptidase versus β-lactamase?

Both transpeptidase and many β-lactamases contain an active-site serine.

However, their reactions have different outcomes.

Comparison: Transpeptidase versus β-lactamase

Feature	Transpeptidase	Serine β-lactamase
Normal function	Cross-links peptidoglycan	Hydrolyses β-lactam antibiotics
Active-site residue	Serine	Serine
Reaction with penicillin	β-Lactam ring opens	β-Lactam ring opens
Covalent intermediate	Forms	Forms
Subsequent hydrolysis	Very slow	Rapid
Overall result	Enzyme inhibited	Antibiotic inactivated

Key note: The challenge for medicinal chemists: Design a penicillin that can still bind to transpeptidase but is less easily attacked by β-lactamase.

What are the classes of beta- lactamases?

Ambler classification by molecular structure
• Classes A, C and D are serine enzymes, mechanistically close to the transpeptidase:
Extended-spectrum beta-lactamase (ESBL – Class A). Klebsiella pneumoniae carbapenemase (KPC, a class A carbapenemase.

• Class B are metalloenzymes and use zinc rather than a serine: NDM (New Delhi metallo-β-lactamase)
• No currently available inhibitor works against class B

How can you reduce the nucleophilic attack to be more beta-lactamase resistant?

• Introduce steric bulk, creating steric hindrance

• by attaching a substituted aromatic R group to the amide, preventing beta-lactamase from accessing and hydrolysing the beta-lactam ring effectively

Note - it could also prevent antibiotic ot binding effectively to its target transpeptidase, not always a better antibitic

Why are isoxazole rings useful?

• They are a five-membered aromatic ring containing oxygen and nitrogen

• It is electron-withdrawing, so it reduces acid sensitivity ( more stable in stomach acid )

• It has steric protection ( shields the beta-lactam ring from the enzymes)

• Halogens introduced affect lipophilicity, absorption, protein binding, and distribution

• Low activity compared with original penicillins

Comparison of isoxazolyl penicillins

Drug	Key structural feature	Main advantage
Oxacillin	Isoxazolyl side chain	Acid stability and penicillinase resistance
Cloxacillin	Chlorinated isoxazolyl side chain	Penicillinase resistance
Flucloxacillin	Chlorine and fluorine substituents	Penicillinase resistance and useful pharmacokinetic properties

How can you deactivate beta-lactamase?

• Introducing steric bulk or using an isoxazolyl group reduces the intrinsic activity of the penicillin

• The best broad-spectrum orally active antibiotics, e.g. ampicillin and amoxicillin, are not β-lactamase resistant

Co-administration of an inhibitor
• clavulanic acid isolated from Streptomyces clavuligerus
• weak antibacterial activity
• irreversible inhibitor of β-lactamase
• structurally related to all penicillins
• important structural feature is the enol ether (in Z configuration)

• cant inhibit all beta lactamases

Describe the mechanism by which clavulanic acid inhibits beta -lactamase ?

1. The active-site serine residue of β-lactamase nucleophilically attacks the carbonyl carbon of clavulanic acid's β-lactam ring.

2. The β-lactam ring opens, forming a covalent enzyme–inhibitor complex.

3. The remaining five-membered ring opens, and the molecule undergoes structural rearrangement to form an imine intermediate.

4. Further proton transfers and rearrangement produce an enamine intermediate.

5. These reactions can irreversibly inactivate β-lactamase, preventing it from breaking down susceptible β-lactam antibiotics.

What are other beta-lactam inhibitors

1.Sulbactam

Sulbactam inhibits certain β-lactamases.

The lecture describes it as having broader activity than clavulanic acid but lower potency.

It can be combined with ampicillin.

2.Tazobactam

Tazobactam is another β-lactamase inhibitor.

It is commonly combined with piperacillin.

Piperacillin + tazobactam provides broad-spectrum antibacterial treatment against susceptible organisms.

3.Enmetazobactam

Enmetazobactam is a structurally modified analogue of tazobactam.

What is special about its structure?

It is zwitterionic.

This means the molecule contains both positive and negative charges.

The lecture highlights that its overall neutral charge can facilitate penetration into bacterial cells.

What are some non-beta-lactam inhibitors?

1. diazabicyclooctane class ( dbo)

• They lack a β-lactam ring.

• They also inhibit β-lactamases through a different type of covalent reaction.

How do diazabicyclooctanes inhibit β-lactamase?

Step 1: The inhibitor binds to the β-lactamase active site.

Step 2: The active-site serine attacks the inhibitor's carbonyl.

Step 3: A covalent carbamylated enzyme intermediate forms.

Step 4: The enzyme is inhibited.

Step 5: The reaction can slowly reverse, releasing intact inhibitor.

Why is this important?

Unlike classical mechanism-based inhibitors such as clavulanic acid, these compounds can be regenerated following reversible enzyme inhibition.

What β-lactamase classes do they inhibit?

The lecture identifies activity against:

• Class A

• Class C

• Some class D enzymes

They do not provide reliable activity against class B metallo-β-lactamases.

Example combination: Ceftazidime + avibactam,Avibactam

• Relebactam

What are non-beta-lactam inhibitors?

2. Boronates

• Vaborbactam contains a boron atom.

• Boron is electron-deficient, making it susceptible to nucleophilic attack.

How does vaborbactam inhibit β-lactamase?

Step 1: Vaborbactam enters the β-lactamase active site.

Step 2: The active-site serine oxygen attacks the electron-deficient boron atom.

Step 3: A reversible covalent tetrahedral adduct forms.

Step 4: The enzyme is inhibited.

Step 5: The interaction can reverse, releasing the inhibitor.

Why is the tetrahedral structure important?

It resembles the tetrahedral intermediate formed during the normal hydrolysis reaction catalysed by serine β-lactamases.

This allows vaborbactam to inhibit the enzyme effectively.

Which antibiotic is vaborbactam combined with?

Meropenem.

Meropenem + vaborbactam is used against certain resistant bacterial infections.

Why are many penicillins more effective against Gram-positive bacteria?

Gram-positive bacteria lack an outer membrane, making their PBPs (penicillin-binding proteins ) more accessible to suitable penicillins.

Q2. Why are Gram-negative bacteria less susceptible to some penicillins?

A: Their outer membrane restricts antibiotic entry, preventing sufficient concentrations from reaching the PBPs.

How do hydrophilic penicillins enter Gram-negative bacteria?

• Many enter through porins,which are water-filled protein channels in the outer membrane

What are bacterial porins?

Water-filled protein channels in the outer membrane of Gram-negative bacteria that allow certain molecules to enter

What properties influence antibiotic passage through porins?

What determines whether a drug can pass through a porin?

Property	Effect on penetration
Molecular size	Smaller molecules generally pass more easily.
Molecular shape	Compact molecules are more likely to fit through the channel.
Hydrophilicity	Hydrophilic molecules are generally more compatible with aqueous pores.
Net charge	Influences interactions with charged residues within the porin.
Steric bulk	Bulky structures may prevent passage.

How can bacteria reduce antibiotic entry without altering PBPs?

1. Reduced porin expression

Bacteria produce fewer porin channels.

This reduces the amount of antibiotic entering the cell.

2. Porin mutations

Structural changes in porins prevent or reduce antibiotic passage.

3. Efflux pumps

Bacteria use membrane transport proteins to remove antibiotics actively.

This reduces the intracellular or periplasmic antibiotic concentration to act on their targets

summaries - comparing penicillins

1. Comparing the different penicillins

Drug	Important structural characteristic	Why is it useful?	Main limitation
Penicillin G	Benzyl side chain	Original penicillin with good activity against susceptible Gram-positive bacteria	Acid-sensitive and β-lactamase-sensitive
Penicillin V	Phenoxymethyl side chain	Electron-withdrawing oxygen improves acid stability	Still susceptible to β-lactamases
Ampicillin	α-Amino group	Improves acid stability and permits activity against some Gram-negative bacteria	Susceptible to β-lactamases
Amoxicillin	α-Amino group and para-hydroxyl group	Improved oral absorption compared with ampicillin	Susceptible to β-lactamases
Methicillin	Bulky substituted aromatic side chain	Resistant to many penicillinases	Acid-sensitive; ineffective against MRSA
Flucloxacillin	Bulky isoxazolyl side chain containing halogens	Acid-stable and resistant to many penicillinases	Poor Gram-negative activity
Oxacillin	Isoxazolyl side chain	Acid-stable and penicillinase-resistant	Relatively narrow spectrum
Cloxacillin	Chlorinated isoxazolyl side chain	Acid-stable and penicillinase-resistant	Relatively narrow spectrum

summary - beta lactamas einhibitors

Drug	Structural class	Mechanism	Example combination
Clavulanic acid	β-Lactam-based inhibitor	Mechanism-based inactivation of susceptible β-lactamases	Amoxicillin
Sulbactam	β-Lactam-based inhibitor	Mechanism-based inhibition	Ampicillin
Tazobactam	β-Lactam-based inhibitor	Mechanism-based inhibition	Piperacillin
Enmetazobactam	β-Lactam-based inhibitor	Inhibits susceptible β-lactamases; zwitterionic structure aids penetration	Cefepime
Avibactam	Diazabicyclooctane	Reversible covalent carbamylation of active-site serine	Ceftazidime
Relebactam	Diazabicyclooctane	Reversible covalent carbamylation of active-site serine	Imipenem/cilastatin
Vaborbactam	Boronate	Reversible tetrahedral adduct with active-site serine	Meropenem

Important for your flashcards: Focus on the relationship between the inhibitor's chemical structure, mechanism of inhibition and reason for combination therapy.