02/11/15
Introduc/on to an/microbial
chemotherapy MD2001
Professor Stephen H. Gillespie
Learning objec/ves
Chemotherapy
• Define the principles of an/-bio/c
chemotherapy
• List the main classes of an/bacterial agents
• Describe the principle of selec/ve toxicity
giving examples
• Outline the main mechanisms of ac/on of the
main an/bio/c classes
• The term chemotherapy is now applied to the
use of chemicals (either natural of synthe/c)
to inhibit the growth/replica/on of 'invading
organisms' or cancerous cells within the body.
• An/bio/cs and an/-bacterials can be used
interchangeably
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The colonial challenge
Discovering the concept of
an/bio/cs
Erhlich’s discoveries
• Discovered and established the concept of
selec/ve toxicity
• Trypanosomes were killed by Salvarsan
• Trypanosomes could become resistant
• Trypanosomes resistant to one agent
remained suscep/ble to others
Domagk and ra/onal development of
an/bio/cs
• Pursued a series of dyes as
poten/al an/bacterials
• Discovered prontosil
(Sulfonamidochrysoidine
(KI-730) : a red dye that
inhibited bacteria
• Jacques and Thérèse
Trefouel (Ins/tut Pasteur)
found that prontosil was
metabolised to
sulphanilamide which was
not a dye but s/ll ac/ve
against bacteria
Domagk’s achievement
Penicillin
• Developed the concept of ra/onal design of
an/-bacterials
• Went on to develop an/-tuberculosis agents
• A chance discovery
• Unable to create
enough material to
make treatment
possible
• Published the discovery
as a method to select
bacteria on agar
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02/11/15
Florey and Chain
• Recognised the value of
penicillin as a poten/al
treatment of bacterial
infec/on
• Used large fermenters to
create enough star/ng
material to allow extrac/on
of penicillin
• They worked in great
secrecy during the war,
later in America
• The urine of treated
pa/ents was collected and
penicillin re-extracted
The penicillin nucleus and the
semi-synthe/c revolu/on
Pre-1946
• Pelargonium roots
• Cod liver oil
• Gold
Penicillin G
Penicillin nucleus
Ampicillin
Waksman and Schatz
• An/bio/c discovery based
on a theory that soil
organisms may have
produced agents to kill
mycobacteria which were
derived from soil organisms
• Thousands of soil samples
were screened for the
presence of factors that
inhibit mycobacteria
• Streptomycin discovered
PRESENTER: KASHA SINGH
The streptomycin trial
– the long term results
The streptomycin trial
Treatment
group
Bed rest
Streptomycin
Total
52
55
Deaths
assessed at 6m
14
4
Treatment Total
group
Deaths
assessed
6m
5 yrs
Bed rest
52
27% 62%
Streptomycin 55
7%
58%
35 of 41 streptomycin pa/ents tested had developed
resistance
27%
7%
P = 0.01
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Summary to date
SELECTIVE TOXICITY
• An/bio/cs can be developed based on a
theory of ac/vity – even though it is
subsequently proved to be false
• An/bio/cs can be developed as a result of
chance occurances
• An/bio/cs can be discovered on the basis of
systema/c screening of natural products
• 1) Central to the use of chemotherapeu/c agents is
the concept of SELECTIVE TOXICITY. These drugs are
intended to be toxic to the invading organism or
cancerous cell but be rela/vely harmless to the host
or normal cells.
• 2) This approach depends upon the existence of
biochemical differences between the target group of
cells and the host.
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Examples of different selec/ve
toxicity
• Penicillins: in the absence of allergy have very
low toxicity and high doses can be used
• Aminoglycosides have a narrow THERAPEUTIC
INDEX thus the dose that causes toxicity is
very close to the therapeu/c dose
• For an/-tuberculosis drugs such as isoniazid
and pyrazinamide a number of pa/ents will
develop hepatotoxicity that is not dose related
and may require treatment to be stopped
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02/11/15
An/bio/cs ac/ve against the cell
membrane
Figure 44.1 Diagrams of structure and metabolism of a bacterial cell. A Schematic representation of a bacterial cell. B Flow diagram showing the synthesis of the main
types of macromolecule of a bacterial cell. Class I reactions result in the synthesis of the precursor molecules necessary for class II reactions, which result in the synthesis
of the constituent molecules; these are then assembled into macromolecules by class III reactions. (Modified from: Mandelstam J, McQuillen K, Dawes I (eds) 1982
Biochemistry of bacterial growth, Blackwell Scientific, Oxford.)
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PEPTIDOGLYCANS
Drug class
Target
Mechanisms
Examples
Beta-lactam and
cephalosporin
Penicillin binding
proteins
Preven/ng
pep/doglycan crosslinking
Penicillin G
Flucloxacillin
Tazobactam
Glycopep/de
C-terminal D-AlaD-Ala
Prevents
transglycola/on and
transpep/da/on
Vancomycin
Teicoplanin
Cyclic pep/de
C 55-
isoprenyl
pyrophosphatE
Prevents carriage of
building-blocks of
pep/doglycan
bacterial cell wall
outside of the inner
membrane.
Bacitracin
Polymyxin
Phosphonic acids
murA protein
Inhibits first stage of
pep/doglycan
synthesis
Fosphomycin
Lipopep/des
Cell wall stress
simulon
Calcium-dependent
membrane
depolarisa/on
Daptomycin
• 1) They make up the cell wall of bacteria and do not occur in
eukaryotes.
• 2) Cell wall is made up from various numbers of strands of
pep/doglycans
• 3) The strands are made up mul/ples of amino-sugars; NACETYLGLUCOSAMINE & N-ACETLYMURAMIC ACID dimers.
The n-acetylmuramic acid has a short pep/de side chain
(hence pep/doglycan).
• 4) The pep/de side chains are cross-linked to form a
laqcework.
• 5) Cross-linking gives the cell all its strength.
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Penicillins
• 1) Penicillins G & V.
• 2) β-Lactamase-resistant Penicillins
• Methicillin, Oxacillin, Nafcillin, Cloxacillin,
Dicloxacillin.
• 3) Broad-spectrum penicillins
• Ampicillin & Amoxicillin.
• 4) Extended-spectrum penicillins
• Carbenicillin, Ticaracillin, Azlocillin, Piperacillin.
Figure 44.2 Schematic diagram of a single layer of peptidoglycan from a bacterial cell (e.g. Staphylococcus aureus) showing the site of action of the β-lactam
antibiotics (more detail in Fig. 44.3). In Staphylococcus aureus the peptide cross-links consist of five glycine residues. Gram-positive bacteria have several layers of
peptidoglycan. (NAMA, N-acetylmuramic acid; NAG, N-acetylglucosamine.)
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Figure 44.3 Schematic diagram of the biosynthesis of peptidoglycan in a bacterial cell (e.g. Staphylococcus aureus) with the sites of action of various antibiotics. The
hydrophilic disaccharide-pentapeptide is transferred across the lipid cell membrane attached to a large lipid (C55 lipid) by a pyrophosphate bridge (-P-P-). On the outside,
it is enzymically attached to the 'acceptor' (the growing peptidoglycan layer). The final reaction is a transpeptidation, in which the loose end of the (gly)5 chain is attached
to a peptide side-chain of an M in the acceptor and during which the terminal amino acid (alanine) is lost. The lipid is regenerated by loss of a phosphate group (Pi) before
functioning again as a carrier. (M, N-acetylmuramic acid; G, N-acetylglucosamine.)
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CEPHALOSPORINS
Bacterial Folate Antagonists.
• 1) Come from the fungus Cephalosporium
Acremonium.
• 2) Work by the same mechanisms as penicillins
• 3) Classified by generaTons in the order in which
they were developed.
• 1st, 2nd and 3rd generaTon.
• 4) Now can be termed by means of
administraTon; Oral is Cephalexin, Parenteral are
Cefuroxime & Cefotaxime
1) SULPHONAMIDES & TRIMETHOPRIM
• These are an/bio/cs which act through an inhibi/on of the
folate pathway in bacteria.
2) Folate system important in cell metabolism
3) Bacteria must make their own supply but we don't as we get
it in diet.
4) This makes bacteria suscep/ble to drugs which interfere with
folate metabolism: thus we have our 'selec/ve toxicity' target.
5) Sulphonamides mark the beginning of an/microbial
chemotherapy da/ng back to the 1930s and preceding the
penicillins.
Figure 45.1 Structures of two representative sulfonamides and trimethoprim. The structures illustrate the relationship between the sulfonamides and the p-aminobenzoic
acid moiety in folic acid (orange box), and the possible relationship between the antifolate drugs and the pteridine moiety (orange). Co-trimoxazole is a mixture of
sulfamethoxazole and trimethoprim.
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AMINOGLYCOSIDES
Streptomycin, Kanamycin, Neomycin, Gentamycin
• 1) Form Ionic bonds at the cell surface
• 2) Penetrate the cell wall by a a transport
mechanism across the cell membrane.
• 3) Diffuse into the cytoplasm and then binds
to the bacterial ribosomes.
Figure 45.2 The action of sulfonamides and trimethoprim on bacterial folate synthesis. See Figure 21.2 for more detail of tetrahydrofolate synthesis, and Table 44.1 for
comparisons of antifolate drugs. (PABA, p-aminobenzoic acid.)
Figure 44.4 Schematic diagram of bacterial protein synthesis indicating the points at which antibiotics inhibit the process.
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Chloramphenicol,
Erythromycin & Clindamycin
Inhibi/on of protein synthesis
TETRACYCLINES
• 1) Bind to the ribosomes
• (i) At the interface between the assembled
30s and 50s subunits.
• (ii) Directly to the individual subunits.
• 2) Inhibits protein synthesis by
• Misreading of mRNA.
• 1) They prevent arachment of the tRNA to the
acceptor (A) site on the mRNA-ribosomal complex.
• 2) This prevents the addi/on of amino acids to the
pep/de chain.
• 3) Unlike the aminoglycosides, they are only weakly
bound to the ribosomes.
• 4) Differences in the ac/vity of individual
tetracyclines are related to their solubility in the lipid
membrane of the bacteria.
• Prevent the addi/on of new amino acids to the
growing pep/de chain by binding to the ribosomes.
• This prevents associa/on of the pep/dyl-transferase
with the amino acid and no pep/de bond is formed
i.e. no transpep/da/on.
• May also prevent transloca/on of the ribosome
down the mRNA template (Erythromycin).
FLUOROQUINOLONES.
• These are synthe/c an/bio/cs recently introduced into clinical
prac/ce.
• Broad-spectrum agents:
• Ciprofloxacin, ofloxacin, norfloxacin.
• Narrower-spectrum drugs:
• Cinoxacin and nalidixic acid (first introduced and is not
fluorinated).
• 1) Act by inhibi/ng bacterial DNA Topoisomerase II also
known as DNA gyrase.
• 2) This enzyme catalyses the introduc/on of nega/ve
supercoil in DNA permiqng transcrip/on and replica/on.
Figure 44.6 Schematic diagram of the action of DNA gyrase: the site of action for quinolone antibacterials. A Conventional diagram used to depict a bacterial cell and
chromosome (e.g. Escherichia coli). Note that the E. coli chromosome is 1300 mm long and is contained in a cell envelope of 2 μm × 1 μm; this is
approximately equivalent to a 50 m length of cotton folded into a matchbox. B Chromosome folded around RNA core, and then supercoiled by DNA gyrase (topoisomerase
II). Quinolone and antibacterials interfere with the action of this enzyme. (Modified from: Smith J T 1985 In: Greenwood D, O'Grady F (eds) Scientific basis of antimicrobial
therapy. Cambridge University Press, p. 69.)
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Figure 45.4 A simplified diagram of the mechanism of action of the fluoroquinolones. A An example of a quinolone (the quinolone moiety is shown in orange). B Schematic
diagram of (left) the double helix and (right) the double helix in supercoiled form. (See also Fig. 44.6.) In essence, the DNA gyrase unwinds the RNA-induced positive
supercoil (not shown) and introduces a negative supercoil.
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02/11/15
Learning objec/ves
• Define the principles of an/-bio/c
chemotherapy
• List the main classes of an/bacterial agents
• Describe the principle of selec/ve toxicity
giving examples
• Outline the main mechanisms of ac/on of the
main an/bio/c classes
37
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