antibiotics

what gram satin will staphylococcus aureus be

positive

what gram stain will streptococcus be

positive

what gram stain will escherichia coli be

negative

what gram stain will proteus species be 

negative 

what gram stain will salmonella species be

negative

what gram stain will shigella species be

negative

how should antibiotics be designed

• designed to interrupt biological processes which are not found in humans but are essential for survival of bacteria

• this way drug toxicity is minimised 

why is it possible to harm the host organism when trying to kill bacteria 

most processes within mammals and bacteria are very similar 

what are you looking for when making antibiotics 

a degree of selective toxicity 

what is the process of selective toxicity

processes within bacteria which are more susceptible to antibiotics than the equivalent process in the host 

what are some highly selective processes to target as antibiotics 

• unique biological targets

• inhibitors of cell wall metabolism (only bacteria have cell walls)

• inhibitors of dihydropteroate synthesise (an enzyme only present in bacteria 

what are some less selective processes that antibiotics shouldn’t target

• inhibitors of protein synthesis 

• inhibitors of dihydrofolate reductase

• inhibitors of transcription and DNA replication

how have many antibiotics been designed 

designed to chemically perturb some of these unique biological processes which are essential for bacterial replication and/or survival 

what does the cell wall do for bacteria

protects the bacteria from mechanical damage and the ravages of osmotic pressure

what does inhibition of cell wall synthesis lead to

bacterial lysis and death

why is inhibition of cell wall biosynthesis a good antibiotic drug target

mammalian cells dont have/need cell walls

how quickly can prokaryotes replicate by binary fission

as fast as once per 20 mins

why is there less chance of mutations in human cells than in bacteria

human life cycle is much longer (time between both and then giving birth yourself)

under the conditions of replicating quickly what does this mean for prokaryotes adaptation/evolvement to their environment

prokaryotes can rapidly evolve/adapt to their environment

what is a peptidoglycan structure made up of in bacteria cell walls

peptide and sugar units

what are peptide chains bound to in the bacteria cell wall 

NAM sugars 

how is the bacteria cell wall framework meshed together 

by cross-linking between the peptide chains 

why are D enantiomer amino acids incorporated into the bacteria cell wall 

proteases cant recognise them and therefore prevents the hydrolysis of bonds so they cant be broken down 

what is the pentaglycine link inhibited by

penicillin

what is the Gly5 peptide joined to in peptidoglycan cross-linking

joined to the amino group on the lysine side-chain

what is the peptidoglycan cross-linking catalysed by

transpeptidase enzymes

where are transpeptidase enzymes located

on the outer surface of the cytoplasmic membrane

what is penicillin

• a secondary metabolite of certain species of penicillium molds

• produced when they are grown under stress

what are the key structure points in penicillin 

• variable side-chain

• reactive peptide bond of the beta-lactam ring 

• thiazolidine ring 

what is the configuration bond of the amide group in penicillin

planar 

why is the amide group required to be planar in penicillin 

• so that the nitrogen lone pair can conjugate with the carbonyl 

• the nitrogen, carbon and oxygen p-orbitals all have to lie in the same configuration so conjugation can take place 

why is the beta-lactam amide more reactive than a typical amide in penicillin

• because the strained ring system disfavours resonance of the nitrogen lone pair

• bond angle is 90 degrees so there is a huge amount of strain compared to the 109 bond angle it should be 

how does penicillin inhibit bacterial cell wall cross-linking

• the penicillin scaffold has the same scaffold as the peptidoglycan D-Ala-D-Ala

• this means penicillin can fit into the same position in the active site as the natural substrate

• penicillin covalently inhibits bacteria and the bond cant be hydrolysed

what are the certain features of penicillin structure that are essential for its antibiotic activity

• external amide essential

• cis stereochemistry essential- wedged lines both sides of the ring at the top

• lactic essential

• bicyclic system essential

• free acid essential

why cant penicillin G be given orally

• because it degrades in the acidic environment of the stomach

• therefore it must be injected 

which penicillin analogues are more desirable 

• active

• acid-resistant

• and therefore can be delivered orally 

what are the 3 factors involved in explaining why penicillin G is acid sensitive 

1. carbonyl in the beta-lactam ring is highly susceptible to nucleophilic attack 

2. acid-catalysed ring opening of the highly strained 4-membered lactam relics the torsional strains of the fused ring system

3. the neighbouring acyl group can actively participate in an intramolecular mechanism to open the lactam ring

what does ‘acid-catalysed ring opening of the highly strained 4-membered lactam relics the torsional strains of the fused ring system’ mean 

• the Beta-lactam ring has a lot of strain because the small ring forces the bonds into unnatural angles 

• when you add acid this helps open the ring 

• this reduces the strain in not only the ring but also in the whole system 

how can the acid sensitivity problem be tackled 

• attaching an electron withdrawing group to the carbonyl group pulls electron density away from the carbonyl oxygen 

what does attaching an electron withdrawing group to the carbonyl group in penicillin do

reduces its tendency to act as nucleophile thereby reducing/preventing neighbouring group participation 

what does penicillin V have attached 

• an electronegtaive oxygen on the acyl side chain giving it better acid stability than penicillin G

• BUT it is still sensitive to beta-lactamases and is slightly less active than penicillin G 

what do penicillin resistant bacteria produce

beta-lactamase enzymes

what do beta-lactamase enzymes do

catalyse the same ring opening reaction of penicillins that occurs during acid hydrolysis

unlike in peptides transferase what is water able to access and hydrolyse when there is a beta-lactamase enzyme

water is able to access and hydrolyse the ester linkage in the beta-lactamase-penicillin covalent adduct

what is the penicillin adduct 

• the opened beta-lactam ring which is covalently attached to the enzyme 

what are the 2 approaches that can be used to tackle the problem of beta-lactamase sensitivity

1. penicillin analogues which are not recognised by beta-lactamases

2. co-admiistration of beta-lactamase inhibitors

what can be used to provide a ‘steric shield’ towards beta-lactamase binding 

bulky side chains 

why do you need a large bulky group 

a bulky group large enough to ward off the lactamase enzyme but small enough to allow the penicillin to still bind to transpeptidase 

can methicillin be degraded by beta-lactamase

• no

• but its side chain is not electronegative so its sensitive to acid hydrolysis and therefore needs to be injected 

what does oxacillin contain

• a bulky 5-membered electron withdrawing heterocyclic that is resistant to beta-lactamases and acid hydrolysis 

whilst a bulky side chain improves beta-lactamase resistance what can it also lead to 

reduction in transpeptidase binding affinity 

what can be given in combination with penicillin to improve antibiotic efficiency of penicillin

clavulanic acid

what are the certain features of clavulanic acid structure that are essential for its beta-lactamase inhibition

• beta-lactam ring

• R-steriochemistry at positions neighbouring the amide nitrogen

• double bond with Z-configuration branding from the heterocycle

• carboxylic acid gorup

• no substitution at the atom neighbouring the double bonded oxygen

what do some of the more modern penicillins incorporate

both the bulk and electron withdrawing properties into their side chain

what does incorporating the bulk and electron withdrawing properties into penicillins side chain do 

helps to overcome sensitivity to acid hydrolysis and beta-lactamase inactivation simultaneously

what did the first penicillins show against gram positive and gram negative bacteria 

• good activity against gram positive bacteria (despite the thick cell wall)

• poor activity against gram negative bacteria 

what must penicillin reach to exert their mechanism of action 

must reach the cell wall 

what do grape negative bacteria have to stop water and hydrophilic molecules such as penicillins through 

• an outer cell membrane

• it is impervious to water and hydrophilic molecules such as penicillins 

• this means it has become highly resistant to allowing entry for these molecules 

how can penicillins only be transported into the periplasmic space of gram negative bacteria 

through porin structures

what are porins 

water-filled membrane spanning proteins 

what does transport of penicillins depend on

depends on exact structure and charge and has a crucial influence on whether a penicillin is active against gram negative bacteria 

what does the spectrum of activity of any penicillin depend on 

• its structure

• its ability to cross the cell membrane of gram negative bacteria

• its susceptibility to beta lactamases 

• its affinity for the transpeptidase enzyme 

• its rate at which it can be pumped back out of cells by gram negative bacteria 

why are there no clear cut tactics that can be used to improve the spectrum of activity 

the factors vary in importance depending on different bacteria 

when was cephalosporins first activity noted 

in 1940s when a fungus found near sewage outlets in Sardinia stopped the growth of bacteria 

unlike penicillins what do cephalosporins feature

a six-membered ring fused to the beta-lactam and so has less ring strain

why are cephalosporins more stable to acid hydrolysis than the penicillins

because of the six membered ring which has less strain

compare the activity of cephalosporins with penicillins

• lower activity than penicillins

• but broader spectrum of activity 

what bacteria is more specific for cephlasporins 

gram negative beta-lactamases 

what are cephalosporins resistant to

beta-lactamases

what does metabolic hydrolysis of the acetate in cephalosporins reduce 

reduces compound activity 

what would blocking metabolic hydrolysis do to drug activity of cephalosporins

would prolong drug activity

why is cephalothin not that great

its leaving group is readily hydrolysed in vivo

why is cephaloridine better than cephalothin

• has an excellent leaving group (pyridinium) which is not hydrolysed in vivo

• but switterion precludes gut absorption

compare cefalexin to cephaloriidine and cephalothin

• well absorbed

• but reduced activity as methyl group is not a good leaving group

to improve antibiotic activity what must be made 

variants 

what were early penicillins/cephalosporins 

natural products 

how can naturally occurring penicillins be made

can be made biosynthetically by fermentation methods 

how can the acyl side chain in antibiotics be varied 

varied depending on the make-up of the fermentation media 

what are the restrictions to adding acids into the media and why is this a restriction

• only acids of general formula RCH2CO2H can be added 

• this restricts the variety of analogues that can be obtained by this method 

how are penicillin analogues made

semi-synthetically 

what does it mean for something to be made semi-synthetically 

• relying on the microbe as much as possible before doing anything chemically

• the point is you want to die as little as possible chemically 

why is it more efficient to first make and isolate penicillin G by fermentation of penicillium fungi cultures and then selectively hydrolysing the side chain using a penicillin acylase enzyme 

because the fused bicyclic ring structure is immensely difficult to chemically synthesise from scratch 

what can the cephalosporin skeleton be derived from 

the same biosynthetic precursor as penicillin

what has the use of the 6-APA biosynthetic intermediate enabled 

the preparation of a large number of semi-synthetic intermediates 

what can be chemically acylated to give penicillin analogues

6-APA

what does chemically acylating 6-APA overcome

overcomes the limitations of total biosynthesis of penicillin analogues via fermentation

how is 6-APA now produced

produced by enzymatic hydrolysis of the penicillin G fermentation product 

what does cycloserine block 

blocks 2 sequential steps in peptidoglycan biosynthesis 

what is D-Cycloserine mainly used as

an anti-tubercular agent

what is D-Cycloserine a good mimic of

• D-Ala 

• it binds more tightly than D-Ala to both enzymes 

why does D-Cycloserine bind more tightly than D-Ala 

the isoxazole ring holds it in a favoured binding conformation whereas the linear alanine can adopt multiple conformations which may not favour binding to the enzyme 

what is vancomycin

a glycopeptide antibiotic

what dies vancomycin do

inhibits peptidoglycan crosslinking by binding to the D-Ala-D-Ala terminus of the cross linking peptide 

why is vancomycin an unusual inhibitor 

because it inhibits enzymatic cross-linking by binding to the substrate rather than the enzyme 

what is altered in a vancomycin resistant bacteria

• altered peptidoglycan cross-linking processes

• the D-alanine is removed in the cross-linking reaction and is replaced by lactic acid 

what is the ester linkage in vancomycin resistant bacteria tolerated by and what does this mean for vancomycin binding affinity 

• transpeptidase 

• greatly reduces affinity as a key H-bonding interaction has been compromised 

what do different antibiotics target 

different stages of cell wall biosynthesis 

what are antimetabolites

target particular cells at certain stages of metabolism 

why is prontosil active in vivo but not in vitro

because its a prodrug, metabolised by gut bacteria to give biologically active sulfonamide