Download The Need for Constant Renewal of the Antibacterial

What future for antibiotics?
Anthony W. Smith, BPharm, PhD, MRPharmS
Dean
The School of Pharmacy
University of London
World Health
Antibiotic targets
• Peptidoglycan synthesis
• Beta-lactams, glycopeptides
• RNA polymerase
• Protein synthesis
• Aminoglycosides, tetracyclines,
macrolides, oxazolidinones,
• Nucleic acid synthesis
• Fluoroquinolones
Beta-lactams and glycopeptides
inhibit peptidoglycan synthesis
NAG
NAM
Glycopeptides
Bind terminal D-Ala-D-Ala Prevent
subunit incorporation
NAG
NAM
NAG
NAM
Beta-Lactams
Bind ‘PBPs’
Prevent trans and carboxypeptidation
NAG
NAM
b-Lactams - mode of action
O
R
H
N
H
H
N
O
O
H
HN
R
H
O
NH
O
O
COOH
D-Ala-D-Ala
HO
O
H
N
NH2
Amoxicillin
S
N
O
CH3
CH3
COOH
•
Is the emergence of antibiotic
resistance inevitable?
“Natural selection makes antibiotic
resistance inevitable, rendering any
antibiotic less profitable over time; the
situation is exacerbated by the overuse
and misuse of currently available
antibiotics.” Stephen J. Projan, 43rd
ICAAC, Chicago 2003
Emergence of antibiotic resistance by
selective pressure
• EDG Murray (1917 – 1954) strain collection
– ‘pre-antibiotic era’ isolates of enteric bacteria
practically fully sensitive to a range of antibiotics
– 2/433 penicillin resistant
– 9/433 tetracycline resistant
• Rapid emergence of resistance of the past 60 years
–
–
–
–
Mutation
Genetic re-arrangement
Genetic resistance determinants
Resistance genes can be spread by horizontal transfer
Hospital-acquired infections to drugresistant bacteria (USA 2002)
Pathogen
Antibiotic
Estimated cases
Staphylococcus aureus
methicillin
102,000
Coag –ve staphylococci
methicillin
130,000
Enterococci
vancomycin
26,000
Pseudomonas aeruginosa
ceftazidime
12,000
Escherichia coli
ampicillin
65,000
Pseudomonas aeruginosa
imipenem
16,000
Klebsiella pneumoniae
ceftazidime
11,000
Evolution of antibiotic resistance in
Staphylococcus aureus
oxacillin, flucloxacillin
penicillin
methicillin
Penicillin resistance
1940
vancomycin
teicoplanin
1950
methicillin resistance
1960
MRSA
1970
gentamicinresistant
MRSA
Epidemic
1980 strains 1990
1996 2000 2002
GISA
VRSA
MRSA in Europe (2002 data)
•
•
•
•
•
•
•
•
Greece
UK
Germany
Spain
Belgium
Czech Republic
Netherlands
Sweden
48.6%
44.5%
27.2%
23.5%
19.2%
6.2%
1.0%
0.7%
Source: European Antimicrobial Resistance Surveillance System
Antibiotics – mechanisms of
resistance
• Alteration in target site
• Alteration in access to the target site
• Production of inactivating enzymes
Resistance to antibacterial agents
Mechanism of resistance
Altered
target
b-lactams
Glycopeptides
Aminoglycosides
Tetracyclines
Chloramphenicol
Macrolides
Fusidic acid
Oxazolidinones
Quinolones
Rifampicin







Altered Inactivation
uptake







b-lactam antibiotics and
b-lactamase
• Chromosomal b-lactamase
– inducible
– Staphylococcus aureus, some Gramnegatives including Pseudomonas sp. and
Enterobacteriacae
• Plasmid-mediated b-lactamase
– constitutive
– TEM type most common, also extended
spectrum
R H
H
S
N
O
O
Serine
H
OH
-
O
Arginine
Lysine
X
Serine/Histidine
Charge stabilisation by
positively-charged lysine and
arginine residues
Lactam attack by
serine residue
R H
Serine
O
Serine/Histidine
H
S
N
O
X
O
-
O
Arginine
Lysine
R H H
HO
S
+
HN
O
COOH
An inactive penicillanoic acid
b-lactamase
b-lactamase inhibitors
O
OH
Clavulanic acid
N
O
O
O
O
O
S
N
COOH
S
CH3
CH3
COOH
Sulbactam
N
O
N
O
N
CH3
COOH
Tazobactam
N
Altered penicillin-binding
proteins (PBPs)
– transpeptidases and carboxypeptidases
required for cross-linking and ‘pruning’
of peptidoglycan (PG).
– PG synthesis has to be carefully regulated
to avoid over extensive cross-linking
– PBP2a – methicillin resistance in S.
aureus
Glycopeptides block
carboxypeptidase/transpeptidase
NAG
Glycopeptides
Bind terminal D-Ala-D-Ala Prevent
subunit incorporation
NAM
Glycopeptide resistance
Unstable complex with 4
hydrogen bonds
Stable complex with 5
hydrogen bonds – inhibits
transpeptidation
O
O
O
O
HN
O
O
O
NH
NH
O
O
Vancomycin
NAG
L-lys
L-lys
Vancomycin
D-glu
D-glu
L-ala
L-ala
NAM
NAG
NAM
Resistance by efflux pumps
• Antibiotics pumped out of cell
– can explain resistance to structurally unrelated agents
eg tetracyclines and quinolones
Tetracycline
transported
into cell
Tetracycline pumped
out of cell
Drug does not reach optimum concentration
Are there more targets for
antibiotics?
• Metabolic enzymes are attractive targets
– Central role in microbial physiology
– High conservation among various pathogens
– Enzyme assays suitable for high through-put
screening
– Numerous targets must exist?
The promise of ‘…omics’?
• Genomic approach
– Gene-by gene strategies to identify those
essential for in vivo growth
Search in Salmonella
• Phenotypes of metabolic mutants in vivo
– Essential, contributing and dispensable
• In vivo proteomics
– Recover Salmonella from caecum or spleen
– Identify expressed proteins by mass spec
Nature (2006) 440,303-307
Shortage of new targets
• 155 ‘promising’ target candidates
– 64 conserved in diverse set of major human
pathogens S. aureus, E. faecalis, S. pneumoniae
and H. influenzae
– Almost all belong to pathways already
exploited by antibiotics
• 8 new candidates have very high sequence
identities with human enzymes
– Toxicity
Nature (2006) 440,303-307
RNA interference
• ‘Knock-down’ technology
– Shot-gun clone into inducible anti-sense
expression vector
– Replica plate and examine for growth or no
growth under inducing conditions
– Fatty acid biosynthesis inhibition (Merck)
– Lipid A biosynthesis (Chiron/Novartis)
– Novel ribosomal sites (Pleuromutilins/GSK)
Platensimycin and fatty acid
biosynthesis
• Produced by Streptomyces platensis
• Identified from a screen of 250,000 extracts
from drug-producing micro-organisms
• Only third new class of molecule (linezolid
and daptomycin) in 40 years
Nature (2006) 441,293-294
Antibiotics in 2006
• A role for screening against compound
libraries, particularly natural products
• Maintain the activity of existing agents
– Rational prescribing
– Completing courses of treatment