Beta-Lactam Antibiotics

Clinically Important -Lactam Antibiotics

Introduction

-Lactam antibiotics are the most widely produced and used antibacterial drugs in the world, and have been ever since their initial clinical trials in 1941.

-Lactams are divided into several classes based on their structure and function; and are often named by their origin, but all classes have a common -Lactam ring structure.

Beta-Lactam Structure

Classification

Penicillins Natural penicillins

PenG, PenVK, Benzathine Pen, Procaine Pen

Aminopenicillins

Ampicillin, Amoxicillin

Anti-Staph penicillins

Oxacillin, Dicloxacillin

Anti-Pseudomonal

[Carboxy] Ticarcillin

[Ureido] Piperacillin

History

1928- Alexander Fleming discovers a mold which inhibits the growth of staphylococcus bacteria

1940- penicillin is isolated and tested on mice by researchers at Oxford

1941- penicillin mass produced by fermentation for use by US soldiers in WWII

1950s- 6-APA is discovered and semi-synthetic penicillins are developed.

1960s to today- novel -lactams/ -lactamase inhibitors are discovered and modified from the natural products of bacteria

Classification

Cephalosporins

1st Generation

Cephalexin, Cefazolin

2nd Generation

Cefoxitin, Cefuroxime, Cefotetan

3rd Generation

Cefotaxime, Ceftriaxone, Ceftazidime

4th Generation

Cefepime

The Cephalosporins (generalized)

1st Generation Gram (+)

2nd GenerationDecreasing Gram (+)

and Increasing Gram (-)

3rd GenerationGram (-), but also some

GPC

4th Generation Gram (+) and Gram (-)

*Not effective vs. Enterococcus or Listeria

Target- Cell Wall Synthesis

The bacterial cell wall is a cross linked polymer called peptidoglycan which allows a bacteria to maintain its shape despite the internal turgor pressure caused by osmotic pressure differences.

If the peptidoglycan fails to crosslink the cell wall will lose its strength which results in cell lysis.

All -lactams disrupt the synthesis of the bacterial cell wall by interfering with the transpeptidase which catalyzes the cross linking process.

Penicillin binding

protein

Peptidoglycan Synthesis

Peptidoglycan

Peptidoglycan is a carbohydrate composed of alternating units of NAMA and NAGA.

The NAMA units have a peptide side chain which can be cross linked from the L-Lys residue to the terminal D-Ala-D-Ala link on a neighboring NAMA unit.

This is done directly in Gram (-) bacteria and via a pentaglycine bridge on the L-lysine residue in Gram (+) bacteria.

Mechanism

Transpeptidase- PBP

The cross linking reaction is catalyzed by a class of transpeptidases known as penicillin binding proteins

A critical part of the process is the recognition of the D-Ala-D-Ala sequence of the NAMA peptide side chain by the PBP. Interfering with this recognition disrupts the cell wall synthesis.

-lactams mimic the structure of the D-Ala-D-Ala link and bind to the active site of PBPs, disrupting the cross-linking process.

Mechanism of -Lactam Drugs

The amide of the -lactam ring is unusually reactive due to ring strain and a conformational arrangement which does not allow the lone pair of the nitrogen to interact with the double bond of the carbonyl.

-Lactams acylate the hydroxyl group on the serine residue of PBP active site in an irreversible manner.

This reaction is further aided by the oxyanion hole, which stabilizes the tetrahedral intermediate and thereby reduces the transition state energy.

Mechanism of -Lactam Drugs

The hydroxyl attacks the amide and forms a tetrahedral intermediate.

Mechanism of -Lactam Drugs

The tetrahedral intermediate collapses, the amide bond is broken, and the nitrogen is reduced.

Mechanism of -Lactam Drugs

The PBP is now covalently bound by the drug and cannot perform the cross linking action.

Bacterial Resistance

Bacteria have many methods with which to combat the effects of -lactam type drugs.

Intrinsic defenses such as efflux pumps can remove the -lactams from the cell. -Lactamases are enzymes which hydrolyze the amide bond of the -lactam ring, rendering the drug useless.

Bacteria may acquire resistance through mutation at the genes which control production of PBPs, altering the active site and binding affinity for the -lactam .

Range of Activity

-Lactams can easily penetrate Gram (+) bacteria, but the outer cell membrane of Gram (-) bacteria prevents diffusion of the drug. -Lactams can be modified to make use of import porins in the cell membrane.

-Lactams also have difficulty penetrating human cell membranes, making them ineffective against atypical bacteria which inhabit human cells.

Any bacteria which lack peptidoglycan in their cell wall will not be affected by -lactams.

Toxicity

-Lactams target PBPs exclusively, and because human cell membranes do not have this type of protein -lactams are relatively non toxic compared to other drugs which target common structures such as ribosomes.

About 10% of the population is allergic (sometimes severely) to some penicillin type -lactams.

Classes of -Lactams

The classes of -lactams are distinguished by the variation in the ring adjoining the -lactam ring and the side chain at the position.

Penicillin

Modification of -Lactams

-Lactam type antibiotics can be modified at various positions to improve their ability to:

-be administered orally (survive acidic conditions)

-be tolerated by the patient (allergies)

-penetrate the outer membrane of Gram (-) bacteria

-prevent hydrolysis by -lactamases

-acylate the PBPs of resistant species (there are many different PBPs)

Penicillins- Natural

Natural penicillins are those which can be obtained directly from the penicillium mold and do not require further modification. Many species of bacteria are now resistant to these penicillins.

Penicillin G

not orally active

Penicillin G in Acidic Conditions

Penicillin G could not be administered orally due to the acidic conditions of the stomach.

Penicillin V

Penicillin V is produced when phenoxyacetic acid rather than phenylacetic acid is introduced to the penicillium culture. Adding the oxygen decreases the nucleophilicity of the carbonyl group, making penicillin V acid stable and orally viable.

Production

All commercially available -lactams are initially produced through the fermentation of bacteria.

Bacteria assemble the penicillin molecule from L-AAA, L-valine, and L-cysteine in three steps using ACV synthase, IPN synthase, and acyltransferase.

Modern recombinant genetic techniques have allowed the over expression of the genes which code for these three enzymes, allowing much greater yields of penicillin than in the past.

Penicillin Biosynthetic Pathway

o

Penicillins- Extended Spectrum

Extended spectrum penicillins are similar to the aminopenicillins in structure but have either a carboxyl group or urea group instead of the amine

Penicillins- Extended Spectrum

Like the aminopenicillins the extended spectrum drugs have an increased activity against Gram (-) bacteria by way of the import porins.

These drugs also have difficulty penetrating the gut wall and must be administered intravenously if not available as a prodrug.

These are more effective than the aminopenicillins and not as susceptible to -lactamases

Cephalosporins

Cephalosporins were discovered shortly after penicillin entered into widespread product, but not developed till the 1960s.

Cephalosporins are similar to penicillins but have a 6 member dihydrothiazine ring instead of a 5 member thiazolidine ring.

7-aminocephalosporanic acid (7-ACA) can be obtained from bacteria, but it is easier to expand the ring system of 7-APA because it is so widely produced.

Cephalosporins

Unlike penicillin, cephalosporins have two side chains which can be easily modified. Cephalosporins are also more difficult for -lactamases to hydrolyze.

Mechanism of Cephalosporins

The acetoxy group (or other R group) will leave when the drug acylates the PBP.

Cephalosporins- Classification

Cephalosporins are classified into four generations based on their activity.

Later generations generally become more effective against Gram (-) bacteria due to an increasing number of polar groups (also become zwitterions.)

Ceftazidime (3rd gen) in particular can cross blood brain barrier and is used to treat meningitis.

Later generations are often the broadest spectrum and are reserved against penicillin resistant infections to prevent the spread of cephalosporin resistant bacteria.

Carbapenems

Carbapenems are a potent class of -lactams which attack a wide range of PBPs, have low toxicity, and are much more resistant to -lactamases than the penicillins or cephalosporins.

Carbapenems

Thienamycin, discovered by Merck in the late 1970s, is one of the most broad spectrum antibiotics ever discovered.

It uses import porins unavailable to other -lactams to enter Gram (-) bacteria.

Due to its highly unstable nature this drug and its derivatives are created through synthesis, not bacterial fermentation.

Carbapenems

Thienamycin was slightly modified and marked as Imipenem. Due to its rapid degradation by renal peptidase it is administered with an inhibitor called cilastatin under the name Primaxin. Imipenem may cause seizures or sever allergic reactions.

Other modifications of Thienamycin have produced superior carbapenems called Meropenem and Ertapenem, which are not as easily degraded by renal peptidase and do not have the side effects of Imipenem.

-Lactamases

-Lactamases were first discovered in 1940 and originally named penicillinases.

These enzymes hydrolyze the -lactam ring, deactivating the drug, but are not covalently bound to the drug as PBPs are.

Especially prevalent in Gram (-) bacteria.

Three classes (A,C,D) catalyze the reaction using a serine residue, the B class of metallo- -lactamases catalyze the reaction using zinc.

-Lactamase Inhibitors

There are currently three clinically available -lactamase inhibitors which are combined with -lactams; all are produced through fermentation.

These molecules bind irreversibly to -lactamases but do not have good activity against PBPs. The rings are modified to break open after acylating the enzyme.

-Lactam/Inhibitor combinations

Aminopenicillins:

ampicillin-sulbactam = Unasyn

amoxicillin-clavulante = Augmentin

Extended-Spectrum Penicillins

piperacillin-tazobactam = Zosyn

ticarcillin-clavulanate = Timentin

Summary

-Lactam antibiotics have dominated the clinical market since their introduction in the 1940s and today consist of nearly of the market.

Development of natural products such as penicillin G into more potent forms through rational modification has increased the range of activity of these drugs, although this has led to some toxicity problems.

Widespread use of -lactams has led to the development of resistant strains, new modifications are necessary in order for -lactams to remain viable.

Mechanisms of antimicrobial resistance

Drug-modifying

enzymes(e.g., - lactamases,

aminoglycoside-

modifying enzymes)

Altered

drug

targets (e.g., PBPs

ribosomes,

DNA gyrase)

Altered

uptake or

accumulation of

drug(e.g., altered porins,

membrane

efflux pumps)

Cell Wall Assembly

Layer of cell wall

with cross links

of 5 glycines

(gray)

Second layer of cell

wall cross-linked to

the lower layer

Transpeptidase (PBP) forms a 5-glycine bridge between peptides

A subunit is added

to the growing chain

5-glycine crosslinking bridges cannot form in the presence of a beta-lactam, and the cell wall is deformed and weakened