Definition of antibiotics
Antibiotics are compounds of natural, semi-synthetic, or synthetic origin
which inhibit growth of microorganisms without significant toxicity to the
human or animal host.
The key concept of antibiotic therapy is selectivity.
The independent evolutionary history of bacterial (prokaryotic) and host
(eukaryotic) cells led to a significant difference in cell organization, biochemical
pathways and structures of proteins and RNA.
These differences form the basis for drug selectivity.
Bases of antibiotic selectivity
1. The target of an antibiotic can be present only in bacteria
but not in the eukaryotic host.
2. The target in bacteria is different from the homologous
target in the eukaryotic host.
Modern genomics provide a great tool for identifying targets of new
selective antibiotics
Selectivity of antibiotics is not ‘natural’
Natural antibiotics are weapons that bacteria or fungi use to compete
with other microorganisms.
Selectivity is not a ‘natural’ feature of antibiotics.
Most of clinically-useful antibiotics are fortuitously selective
antibacterials.
Many antibiotics are omni-potent and inhibit growth of a wide variety
of organisms. Such antibiotics can be developed into selective drugs
through modification of their chemical structures.
Antibiotics are classified as bacteriostatic or bactericidal.
Bacteriostatic drugs make bacteria
dormant, but do not kill them.
Most bacterial cells resume growth
after removal of the antibiotic
(e.g. chloramphenicol)
Bactericidal drugs kill bacteria
(e.g. ciprofloxacin)
Antibiotics with a bactericidal mode of action are preferred, especially for
treatment of immunocompromised patients. The mode (static vs. cidal) of
antibiotic action may differ for different pathogens and may depend on the drug
concentration.
The basis of bactericidal versus bacteriostatic effects is poorly understood but
maybe related to the accumulation of reactive oxygen radicals in the bacterial
cells upon treatment with bactericidal drugs.
After the golden era of the 1940s-1950s, the progress in antibiotic
discovery has significantly slowed down until the year 2000
2000
2003
linezolid
daptomycin
1962
1958
1947
aminoglycosides
tetracycline
1952
1942
β-lactams
No principally new antibiotics
macrolides
glycopeptides
streptogramins
lincosamides
1920
sulfonamides
1949
Golden era in antibiotic discovery
Growing resistance
The appearance and spread of antibiotic resistance
calls for new antibiotics. Resistance to the available
antibiotics is a result of Darwinian selection
There are two major mechanisms by which bacteria can become resistant to
antibiotics:
1. Mutation of a “normal” bacterial gene resulting in antibiotic resistance.
2. Acquisition of a resistance gene from the environment.
Sensitive bacteria
spontaneous mutation
in the bacterial gene
acquisition of a resistance gene
(often brought by a genetic vector)
Resistant bacteria
vertical transfer
(to the descendants)
Resistant bacteria
horizontal transfer
(gene exchange)
vertical transfer
(to the descendants)
There are three major types of resistance mechanisms:
  Modification of the drug (aminoglycoside modifying enzymes)
  Modification of the drug target (Erm methylases, target site
mutations)
  Reducing the drug’s intracellular concentration: drug-specific
transporters (mefA), multi-drug transporters (bmr), reduced drug
uptake (mutations in porins)
The number of targets of clinically-useful antibiotics is very limited.
Cell Wall
Biosynthesis
Beta-lactams
Glycopeptides
Bacitracin
Protein synthesis
Aminoglycosides
Oxazolidinones
Tetracyclines
Macrolides
Chloramphenicol
Lincosamides
Streptogramins
DNA Gyrase
RNA Polymerase
Fluoroquinolones
Rifampicin
Folate
Biosynthesis
Sulfonamides
Membrane
Integrity
Cationic peptides
Lipopeptides
Many resistant strains are cross-resistant to different drugs acting upon
the same target.
New antibiotics
Originally, screening for new antibiotics was based on testing the
bacterial or fungal extracts.
A number of drugs developed in the middle of the 20th century still
remain among the best.
These include aminoglycosides, cephalosporins, tetracyclines,
macrolides, etc.
There is now a renewed interest in screening natural sources
(especially, non-traditional, e.g. marine microorganisms)
New antibiotics: genome-based approaches
Genomics holds a good potential for identifying new drug targets.
New antibiotics: Functional genomics approach
In the functional genomics approach, the enzymatic target is first defined and
then inhibitors are searched for in high-throughput screening assays (HTS).
gene
target
“easy”
HTS
enzyme inhibitors
lead
lead optimization
drug candidate
hard
preclinical
clinical
On average: only one lead candidate is developed from 14 high throughput
screening experiments!
New antibiotics: Reverse genomics approach
In the reverse genomics approach the lead is first identified in the
whole cell assay and then the target is searched for.
HTS
growth inhibitors
lead
lead optimization
drug candidate
preclinical
clinical
target
mode of action
Cell wall as antibiotic target
Most of the bacteria have a rigid cell wall which
protects the cell from changes in osmotic pressure.
Presence of the cell wall is critical for the survival of
bacterial cell.
The structure and composition of bacterial cell wall
is dramatically different from the cell envelope of the
eukaryotic cell. Therefore, enzymes of cell wall
biosynthesis are unique to bacteria and presents an
excellent target for antibiotics.
According to the structure of their cell wall, all
bacteria are divided into Gram-positive and Gramnegative according to a staining procedure
developed by Christian Gram in 1884.
Cell wall of Gram-positive bacteria is thick and unstructured
Outside of the cytoplasmic membrane of Gram-positive bacteria lies a thick
layer of peptidoglycan which determines the rigidity of the cell wall. In Grampositive bacteria, peptidoglycan accounts for 50% of the cell weight and up to
90% of the weight of the cell wall. Peptidoglycan layer is 20-80 nm thick and
rather unstructured.
Cell wall of Gram-negative bacteria is thin and neatly organized
Periplasmic
space
The cell wall of Gram-negative bacteria consists of the cytoplasmic membrane,
a thin layer of peptidoglycan, and an outer membrane. The area between the
cytoplasmic membrane and peptidoglycan layer is called the periplasmic space.
Peptidoglycan composition
NAG
Peptidoglycan consists of
polysaccharide chains cross-linked by
tetrapeptide
peptide ‘bridges’.
35% to 50% of peptides attached to
polysaccharide chains are crosslinked.
glycan
NAM
pentaglycine bridge
Peptide tails protrude from
the polysaccharide
helically and thus crosslink
to different polysaccharide
chains. This accounts for
the rigidity of the cell wall.
Peptidoglycan biosynthesis
The precursor
monomers of the
peptidoglycan polymer,
disaccharidepentapeptides, are
synthesized in the cell
cytoplasm, transported
across cytoplasmic
membrane, and then
attached to the growing
peptidoglycan polymer.
Transpeptidase
D-Ala
D-Ala
D-Ala
TPase
TPase
TPase
Crosslinking between glycan strands is catalyzed by the enzyme called transpeptidase
(TPase). In the course of the reaction, TPase hydrolyzes peptide bond between two
terminal D-Ala residues of the precursor and forms a transient covalent link with the
precursor peptide. The intermediate is then resolved with the formation of a new peptide
bond.
The reaction of formation of the covalent intermediate is targeted by β-lactam antibiotics.
Penicillin was discovered by A. Fleming in 1929
Howard Florey
Alexander Fleming
Photo: L. Segovia
Ernst Chain
Development of methods for growing Penicillium notatum and purifying
penicillin by Florey and Chain made it into a drug. The deep
fermentation method, the use of corn steep liquor and the discovery of
P. chrysogenum by Mary Hunt made the commercial production of
penicillin possible.
β-lactam antibiotics
carbapenems
penicillins
S
RCONH
CH3
CH3
N
O
CO2 H
H
H3C
OH H H
NH2
N
O
cephalosporins
CO2 H
monobactams
O
S
RNHCO
S
N
N
R
O
CO2H
CH3
N
O
SO 3 H
The most important class of antibiotics affecting cell wall biosynthesis are βlactams. β-lactam group (a four-atom cyclic amide) is the pharmacophore of all
β-lactam antibiotics. β-lactam rings were unknown before the discovery of
penicillin and it took big effort to determine the structure of the drug.
The most important classes of β-lactam antibiotics are penicillins,
cephalosporins, carbapenems and monobactams.
Mechanism of β-lactam action
The mechanism of action of β-lactam antibiotics is based on the
similarity of structures of the C - N bond in the β-lactam ring and the
peptide bond connecting two D-alanine residues of the peptidoglycan
precursor. TPase recognizes the β-lactam as its substrate and forms a
covalent bond with the antibiotic molecule. This adduct is very stable and
because of that TPase is irreversibly inactivated.
penicillin
D-Ala-D-Ala
CH3
N
H
C
CH 3
C
N
H
C
S
RCONH
N
COOH
O
O
CH3
CH3
CO2 H
Covalent complex of penicillin with TPase
R
C
NH
H
C
H S
C
C
O
O
C
N
O
Ser
TPase
CH3
CH3
CH
COOH
Penicillin binding proteins (PBPs)
Several other enzymes of cell wall biosynthesis with a mechanism of
action comparable to TPase are also targeted by β-lactams.
These proteins can be detected on a gel by their ability to bind penicillin.
These proteins are called ‘penicillin binding proteins’ or PBPs.
Penicillins : penicillin G
In penicillins, the β-lactam ring is fused to thiazolidine ring.
Originally, penicillin was produced in the form of Penicillin G
(benzylpenicillin) by fermenting Penicillium mold in the presence of phenyl
acetic acid
O
S
HN
N
O
CH3
CH3
CO2 H
Good activity, but only against Gram-positive bacteria
Acid- and alkali-labile
Sensitive to the action of inactivating penicillinases
6-APA
Presently, many penicillins are produced semisynthetically starting from
6-aminopenicillanic acid (6-APA) as a precursor.
6-APA can be generated from penicillin G by cleaving off the benzyl
moiety of penicillin G.
Various new side chains can be then attached to the penicillin molecule
through the amino group of 6-APA
Benzyl-penicillin
New penicillins
6-APA
O
S
HN
N
O
CH3
CH3
CO2 H
S
H 2N
CH3
CH3
O
N
CO2H
S
R C O NH
O
N
CH3
CH3
CO2H
Various penicillins differ mainly by the nature of the N6 side chain R
Penicillin improvements:
better acid stability
Amoxicillin
Cloxacillin
HO
HO
C O NH
CH
NH
CH
S
CH
N
2
O
3
3
CO H
2
CH3
N
CONH
Cl
O
S
N
CH3
CH3
CO2 H
amide bond
The amide bond in the β-lactam ring is highly strained and relatively
unstable in acidic solutions. The rate of acid hydrolysis depends on the
chemical nature of the side chain. Electron-withdrawing side chains
decrease the rate of acid hydrolysis. Because of that, amoxicillin or
cloxacillin are more acid-stable: they can withstand the acidic pH of the
stomach and can be used orally.
Penicillin improvements:
broader spectrum
Penicillins enter the periplasmic space of Gram-negative bacteria through the
‘holes’ in the outer membrane (porins). Hydrophobic side chains (e.g. benzyl
group in penicillin G) interfere with passage through porins.
More polar groups, such as -NH2 or -COOH facilitate crossing the outer
membrane and increase access of β-lactmas to the periplasmic space of Gramnegative bacteria.
ampicillin
CH
S
CONH
O
CO2 H
amoxicillin
HO
S
C ONH
CH
O
C O2 H
carbenicillin
S
CONH
COOH
CH3
C H3
N
N H2
CH
CH3
N
NH2
CH3
N
O
CH3
CH3
CO2 H
The pro-drug approach can be used to increase bioavailability
of some penicillins
Ampicillin has a very broad spectrum of activity. It can be used orally or
parenterally. But it has low bioavailability.
A more lipophilic pro-drug, pivampicillin, has a better oral bioavailability.
Pivampicillin is an ester of ampicillin; the ester bond is slowly
hydrolyzed in the blood resulting in the release of the active ampicillin.
ampicillin
CH
S
CONH
N
NH2
O
CH3
CH3
CO2 H
pivampicillin
CH
NH2
S
CONH
N
O
CH3
CH3
CO2CH2OCOC(CH3)3
Penicillin improvements:
resistance to β-lactamases
The main mechanism of resistance to penicillin is based on the secretion by
bacteria of enzymes β-lactamases that can hydrolyze amide bond in β-lactam
ring. The presence of a bulky side chain in the drug may hinder access of a
β-lactamase to the amide bond. Therefore, penicillin derivatives containing
bulky side chains are fairly resistant to the β-lactamase action.
methicillin
HO
OCH3
S
CONH
N
OCH3
O
CH3
CH3
CO2H
CH3
cloxacillin
N
CONH
Cl
O
S
N
CH3
CH3
CO2H
β-lactamase inhibitors
A useful way to overcome β-lactamase-based resistance is to administer a β-lactam
drug in combination with β-lactamase inhibitors. Such inhibitors (clavulanic acid,
sulbactam, tazobactam) possess a β-lactam ring and generally resemble β-lactam
antibiotics. They function by binding to the β-lactamase enzymes and inactivating the
enzyme without being degraded.
Such inhibitors, which look like β-lactam antibiotics, have only weak antimicrobial
activity.
Popular combinations are amoxicillin with clavulanate (augmentin) or ampicillin with
sulbactam (unasyn).
Clavulanic acid
Sulbactam
CH2OH
O
O
O
O
S
N
COOH
O
N
COOH
Other mechanisms of resistance to β -lactams
An important mechanism of resistance to β-lactams involves mutations in
transpeptidases and other penicillin-binding proteins (PBPs) involved in
bacterial cell wall biosynthesis.
Resistance mechanism found in methicillin-resistant Staphylococcus
aureus (MRSA) is based on acquisition of a mecA gene which encodes a
resistant mutant protein, PBP2’. PBP2’ has a very low affinity for β-lactam
antibiotics and can support cell wall biosynthesis even when all other PBPs
are covalently inactivated by the drug.
Genetic analysis show that mecA gene has been independently transferred
to S. aureus at least 5 times resulting in 5 independent lineages of MRSA
Cephalosporins
Cephalosporins have been first obtained from a fungus Cephalosporium
acremonium. Similar to penicillins, many cephalosporins are produced
semi-synthetically either starting from 7-aminocephalosporanic acid (7ACA) or by converting relevant penicillins into cephalosporins.
7-aminocephalosporanic acid (7-ACA)
S
7
H 2N
3
N
O
O
CH3
2
CO2H
O
The activity of cephalosporins is modulated not only by the nature of
substitutions R2 at C7 (as in penicillins) but also by the side chain R1 at C3.
Cephalosporins are classified by generations
cephalothin
I
cafazolin
NH
S
N HC O
S
cephalexin
N
N
O
CH
O
CO H
2
N
N
3
S
N HC O
N
N
S
S
CH
O
CO H
2
O
2
3
N
O
N N
CO H
2
Active against Gram-positive cocci and streptococci.
cefamandole nofate
OCHO
S
NHCO
II
N
CO H
2
CH
3
cephaclor
2
S
N HC O
N N
CH S
2
O
NH
S
N HCO
N
N N
Cl
O
HC
3
CO H
2
Improved activity against some Gram-negatives, for example, H. influenzae.
N OC H
III
N
H N
2
S
cefotaxime
3
N OC H C O H
2 2
S
N HC O
N
N
C H OC O C H
2
3
O
CO H
2
H N
2
S
cefixime
S
N HC O
N
CH
O
CO H
2
2
Better activity for Gram-negatives though, somewhat reduced activity against Grampositive pathogens.
Moxalactam
COOH
OCH3
NHCO
S
7
N
HO
CH3
S
O
CO2H
N
N
N N
Similar to penicillins, cephalosporins can be inactivated by β-lactamases.
Resistance to β-lactamases increases in drugs such as moxalactam
which have bulky side chains. The presence of 7-α-methoxy group
increases moxalactam resistance to β-lactamase hydrolysis even further.
Carbapenems
Carbapenems combine chemical features of penicillins and cephalosporins.
Prototype carbapenem thienamycin, has been isolated from Streptomyces
cattleva. It exhibits excellent activity against a broad spectrum of Gram-positive
and Gram-negative organisms.
thienamycin
H
H 3C
OH H H
S
O
NH2
N
CO2H
Thienamycin penetrates very easily through the outer membrane of Gram-negative
bacteria (through porin "holes") . It is resistant to the action of extended spectrum βlactamases (ESBL) which can inactivate penicillins and cephalosporins. In contrast
to penicillins and cephalosporins which target only PBPs, carbapenems can target
another enzyme of the cell wall biosynthesis, Ld transferase (LdT) which sometimes
can help cell to bypass the need for TPase. Therefore, carbapenems shows
excellent activity against some Gram-positive strains which developed resistance to
penicillins and cephalosporins.
Imipenem
H
H 3C
OH H H
thienamycin
S
O
N
H
NH2
H 3C
CO2H
OH H H
imipenem
S
O
N
N
H
NH
CO2H
In concentrated solutions, the side amino group of thienamycin can react with
the amide bond in the β-lactam ring of another thienamycin molecule making
the drug unstable in concentrated solutions. This problem has been solved in
the thienamycin derivative, imipenem by modifying the side chain.
Imipenem was the first parenteral carbapenem.
meropenem
ertapenem
doripenem
The newer drugs of this class are meropenem, ertapenem and doripenem
The drawback of carbapenems is that they are acid-labile and therefore used only intravenously. In
addition, they are very expensive.
Monobactams
Aztreonam (Azactam)
O
S
H2N
N
N
CH3
N
O
H3C
H3 C
N
O
SO3H
COOH
Monobactams were developed as narrow-spectrum antibiotics specifically
targeting aerobic Gram-negative bacteria. Monobactams are particularly
useful for the treatment of individuals allergic to penicillin. Such patients can
still be treated with the monobactams, which are sufficiently structurally
different to not induce allergic reaction.
Cell wall inhibitors of non-β-lactam type: Bacitracin
A number of peptide antibiotics affect cell wall biosynthesis.
Bacitracin, a polypeptide antibiotic produced by licheniformis group of
Bacillus subtilis, inhibits cell wall synthesis by interfering with
dephosphorylation of the lipid carrier that moves the peptidoglycan
precursors across the cytoplasmic membrane. Blocking regeneration of the
lipid carrier aborts cell wall synthesis.
Side effect of bacitracin is based on its interference with sterol biosynthesis
in mammalian cells which accounts for its certain toxicity in humans.
Therefore, it is used exclusively in topical formulations.
Cell wall inhibitors of non-β-lactam type: Vancomycin
An important class of antibiotics that interfere with synthesis of cell wall are
glycopeptides. The most important of them is vancomycin.
Vancomycin is isolated from a bacterium Nocardia orientalis.
Vancomycin is a tricyclic glycopeptide with a large molecular weight of 1449 D.
It is active against most Gram-positive bacteria.
It is especially important in the treatment of infections due to methicillin- and
cephalosporin-resistant organisms.
Vancomycin is bactericidal against most of the susceptible bacteria.
Vancomycin
D-Ala-D-Ala
vancomycin
Vancomycin binds very tightly to the D-Ala - D-Ala residues at the ‘end’ of the
peptidoglycan precursor peptide. Because of that, the peptide bond between
two D-Ala residues becomes inaccessible to TPase so that peptidoglycan
strands cannot be crosslinked.
Resistance to vancomycin
Vancomycin was kept as a ‘reserve’ antibiotic and was usually prescribed only
when other drugs proved to be inactive. However, even in spite of its relatively
infrequent usage, resistant strains eventually appeared.
Vancomycin resistant enterococci (VRE) account now for up to 25% hospital
strains of enterococci.
First vancomycin resistance appeared in staphylococci in ‘vancomycin
intermediate susceptible S. aureus’ - VISA.
VISA cells have abnormal peptidoglycan: the cell wall is thicker and less
crosslinked. Therefore more D-Ala - D-Ala remain in the cell wall and are
exposed. They work as vancomycin trap (sponge). However, because of the
abnormal cell wall, VISA strains are sick (high fitness cost) and do not spread
very rapidly.
In 2002 the first Vancomycin-resistant S. aureus (VRSA) strain was reported.
The Van gene they have acquired replaces D-Ala - D-Ala in the precursor for DAla - D-lactate. Since Van genes are active only when cells are exposed to
vancomycin, VRSA strains are more fit than VISA.
New glycopeptides
Newer versions of glycopeptide antibiotics include teicoplanin and
dalbavancin (ZevenTM). These drugs are similar to vancomycin but have
additional hydrophobic side chains.
Teicoplanin and dalbavancin are active against all vancomycin-sensitive
strains, plus against a number of resistant strains and do not induce
expression of VanB resistance gene.
Both teicoplanin and dalbavancin are rapidly cidal.
Dalbavancin is very tightly bound to serum proteins and is therefore very
stable. Because of that, it can be administered once a week.
dalbavancin