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.
Definition of antibiotics
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.
Bases of antibiotic selectivity
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 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.
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)
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After the golden era of the 1940s-1950s, the progress in antibiotic discovery has significantly slowed down until the year 2000
Golden era in antibiotic discovery No principally new antibiotics
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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 bacteriaSensitive bacteria
Resistant bacteriaResistant bacteria Resistant bacteriaResistant bacteria
spontaneous mutationin the bacterial gene
acquisition of a resistance gene(often brought by a genetic vector)
vertical transfer(to the descendants)
horizontal transfer (gene exchange)
vertical transfer(to the descendants)
The appearance and spread of antibiotic resistance calls for new antibiotics. Resistance to the available
antibiotics is a result of Darwinian selection
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)
There are three major types of resistance mechanisms:
The number of targets of clinically-useful antibiotics is very limited.
Cell Wall
Biosynthesis
Beta-lactamsGlycopeptides
Bacitracin
Protein synthesis
AminoglycosidesOxazolidinonesTetracyclinesMacrolides
ChloramphenicolLincosamides
Streptogramins
DNA Gyrase Fluoroquinolones
RNA Polymerase Rifampicin
Folate Biosynthesis
Sulfonamides
Membrane
Integrity
Cationic peptidesLipopeptides
Many resistant strains are cross-resistant to different drugs acting upon the same target.
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
New antibiotics: genome-based approaches
Genomics holds a good potential for identifying new drug targets.
New antibiotics: Functional genomics approach
gene
target
lead
lead optimization
drug candidate
preclinical
clinical
HTS enzyme inhibitors
On average: only one lead candidate is developed from 14 high throughput screening experiments!
“easy”
hard
In the functional genomics approach, the enzymatic target is first defined and then inhibitors are searched for in high-throughput screening assays
(HTS).
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.
lead
lead optimization
drug candidate
preclinical
clinical
target mode of action
HTS growth inhibitors
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 Gram-
negative according to a staining procedure developed by Christian Gram in 1884.
Cell wall as antibiotic target
Outside of the cytoplasmic membrane of Gram-positive bacteria lies a thick layer of peptidoglycan which determines the rigidity of the cell wall. In Gram-positive 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-positive bacteria is thick and unstructured
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.
Periplasmic space
Cell wall of Gram-negative bacteria is thin and neatly organized
glycan
tetrapeptide
NAG
NAM
pentaglycine bridge
Peptidoglycan consists of polysaccharide chains cross-linked by
peptide ‘bridges’. 35% to 50% of peptides attached to
polysaccharide chains are crosslinked.
Peptidoglycan composition
Peptide tails protrude from the polysaccharide
helically and thus crosslink to different polysaccharide chains. This accounts for the rigidity of the cell wall.
D-AlaL-Ala
racemaseD-Ala-D-Ala synthetase
UDPMurNAc
UDPL-Ala
UDPD-GluL-Lys
UDP
(1)(2)(3)
UDP
pp
UDPGlcNAc
pp
cytoplasm
membrane
periplasmic space
p p
p
(4)
(8)
(9)
(10)transpeptidase
(5)
(6)
phospholipid
pp
(7)
pp
N-acetyl muramic acidN-acetyl glucosamin
pentaglycin
pentapeptide
The precursor monomers of the
peptidoglycan polymer, disaccharide-
pentapeptides, are synthesized in the cell cytoplasm, transported
across cytoplasmic membrane, and then
attached to the growing peptidoglycan polymer.
Peptidoglycan biosynthesis
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 -lactam antibiotics.
Transpeptidase
D-Ala D-AlaD-Ala
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.
Penicillin was discovered by A. Fleming in 1929
Alexander Fleming
Howard Florey
Ernst ChainPhoto: L. Segovia
-lactam antibiotics
N
SRCONH
CH3
CH3
O CO2H
penicillins
N
SNHCO
OCO
2H
CH3
R
cephalosporins
NO CO
2H
SNH
2
H
CH3
H HOH
carbapenems
NO
N
SO3H
R
O
monobactams
The most important class of antibiotics affecting cell wall biosynthesis are -lactams. -lactam group (a four-atom cyclic amide) is the pharmacophore of all
-lactam antibiotics. -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 -lactam antibiotics are penicillins, cephalosporins, carbapenems and monobactams.
The mechanism of action of -lactam antibiotics is based on the similarity of structures of the C - N bond in the -lactam ring and the
peptide bond connecting two D-alanine residues of the peptidoglycan precursor. TPase recognizes the -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.
NH
C
CH3
C COOH
CH3
NH
C
O
NH C C
H S
R C
O
H
CO N
C
CH
COOH
CH
3
O
Ser
TPase
CH3
D-Ala-D-Ala penicillin
Covalent complex of penicillin with TPase
Mechanism of -lactam action
N
SRCONH
CH3
CH3
O CO2H
Penicillin binding proteins (PBPs)
Several other enzymes of cell wall biosynthesis with a mechanism of action comparable to TPase are also targeted by -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 -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
N
S
H
N
C
H
3
C
H
3
O
C
O
2
H
O
Good activity, but only against Gram-positive bacteria
Acid- and alkali-labile
Sensitive to the action of inactivating penicillinases
N
SH2N
O
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
N
SRCONHCH3
OCO2H
New penicillins
Various penicillins differ mainly by the nature of the N6 side chain R
6-APA
N
S
H
N
C
H
3
C
H
3
O
C
O
2
H
O
Benzyl-penicillin
CH3CH3
CH3
CO2H
Penicillin improvements: better acid stability
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.
amide bond
N
SC O NH
C H3
C H3
O C O2H
CH
N H2
OH
Amoxicillin
N
SCONH
CH3
CH3
O CO2H
CH3OH
N
Cl
Cloxacillin
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
Gram-negative bacteria.
N
S
CH3
CH3
O CO2H
CH
NH2
ampicillinCONH
N
SCONH
CH3
CH3
O CO2H
CH
NH2
OH
amoxicillin
N
S
CH3
CH3
O CO2H
CH
COOH
carbenicillinCONH
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.
N
S
CH3
CH3
O CO2H
CH
NH2
ampicillin
N
SCONHCH3
CH3
O CO2CH2OCOC(CH3)3
CH
NH2
pivampicillin
The pro-drug approach can be used to increase bioavailability of some penicillins
CONH
Penicillin improvements: resistance to -lactamases
The main mechanism of resistance to penicillin is based on the secretion by bacteria of enzymes -lactamases that can hydrolyze amide bond in -lactam ring. The presence of a bulky side chain in the drug may hinder
access of a -lactamase to the amide bond. Therefore, penicillin derivatives containing bulky side chains are fairly resistant to the -lactamase action.
N
SCONH
CH3
CH3
O CO2H
OCH3
OCH3
methicillin
N
SCONH
CH3
CH3
O CO2H
CH3OH
N
Cl
cloxacillin
-lactamase inhibitors
Clavulanic acid
N
O
OCOOH
CH2OH
Sulbactam
N
S
OCOOH
O O
A useful way to overcome -lactamase-based resistance is to administer a -lactam drug in combination with -lactamase inhibitors. Such inhibitors (clavulanic acid,
sulbactam, tazobactam) possess a -lactam ring and generally resemble -lactam antibiotics. They function by binding to the -lactamase enzymes and inactivating the
enzyme without being degraded. Such inhibitors, which look like -lactam antibiotics, have only weak antimicrobial
activity.
Popular combinations are amoxicillin with clavulanate (augmentin) or ampicillin with sulbactam (unasyn).
Other mechanisms of resistance to -lactams
An important mechanism of resistance to -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 -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 have been first obtained from a fungus Cephalosporium acremonium. Similar to penicillins, many cephalosporins are produced semi-synthetically either starting from 7-aminocephalosporanic acid (7-
ACA) or by converting relevant penicillins into cephalosporins.
N
SH2N
OCO2H
O CH3
O
7-aminocephalosporanic acid (7-ACA)
2
3
7
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
Active against Gram-positive cocci and streptococci.
cephalothin cafazolin cephalexin
Cephalosporins are classified by generations
N
SNHC O
O
CO2H
O C H3
O
S S
N N
N
SNHCO
OCO
2H
S CH3
NN
NN
N
SNHCO
OCO
2H
CH3
NH2
I
II
cephaclorcefamandole nofate
N
S
O
CO2H
Cl
NHCO
NH2
N
SNHCO
OCO
2H
CH2S
OCHO
N N
NN
CH3
cefotaxime cefixime
N
SNHC O
O
C O2H
CH2OC OC H
3
N OC H3
N
SNH
2N
SNHCO
O
CO2H
CH2
NOCH2CO
2H
N
SNH
2
Improved activity against some Gram-negatives, for example, H. influenzae.
Better activity for Gram-negatives though, somewhat reduced activity against Gram-positive pathogens.
III
COOH
OH
N
N N
NN
SNHCO
OCO2H
OCH3
S
CH37
Similar to penicillins, cephalosporins can be inactivated by -lactamases.Resistance to -lactamases increases in drugs such as moxalactam which have bulky side chains. The presence of 7--methoxy group
increases moxalactam resistance to -lactamase hydrolysis even further.
Moxalactam
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
Thienamycin penetrates very easily through the outer membrane of Gram-negative bacteria (through porin "holes") . It is resistant to the action of extended spectrum -lactamases (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.
Carbapenems
NO CO2H
SNH2
H
H3C
H HOH
In concentrated solutions, the side amino group of thienamycin can react with the amide bond in the -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.
Imipenem
thienamycin
NO CO2H
SNH2
H
H3C
H HOHimipenem
NH
NHN
O CO2H
SH
H3C
H HOH
doripenemertapenem meropenem
The newer drugs of this class are meropenem, ertapenem and doripenemThe drawback of carbapenems is that they are acid-labile and therefore used only intravenously. In
addition, they are very expensive.
Monobactams
Aztreonam (Azactam)
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.
S
O
3
H
N
O
C
H
3
C
H
3
C
O
O
H
N
H
2
N
O
C
H
3
N
O
S
N
Cell wall inhibitors of non--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.
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.
Cell wall inhibitors of non--lactam type: 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.
Vancomycin
D-Ala-D-Ala vancomycin
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 D-Ala - 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