Targeting bacterial virulence mechanisms for antibiotic development: challenges and opportunities

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Targeting bacterial virulence mechanisms for
antibiotic development challenges and
opportunities
Stephen Lory Harvard Medical School, Boston
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History of antibiotic discovery
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Sales in Billion
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Antibiotic resistance follows their introduction
into therapy
Anti-infective agent Discovery (introduction) Resistance 1st reported Mechanisms of resistance Organisms
Penicillin G 1940 (1943) 1940 Penicillinase S. aureus
Streptomycin 1944 (1947) 1947 S12 ribosomal mutations M. tuberculosis
Tetracycline 1948 (1952) 1952 Eflfux Shigella dysenterie
Erythromycin 1952 (1955) 1956 23S rRNA methylation S. aureus
Vancomycin 1956 (1972) 1988, 2004 D-Ala-D-Ala replacement E. faecalis, S. aureus
Methicillin 1959( 1961) 1961 MecA (PPP2a) S. aureus
Gentamicin 1963 (1967) 1969 Modifying enzymes S. aureus
Nalidixic ac. 1962 (1964) 1966 Topoisomerase mutations E. coli
Cefotaxime 1975 (1981) 1981, 1983 AmpC ß-lactamases, ESBL Enterobacteriaceae
Imipenem 1976 (1987) 1986 Adquired carbapenemases P. aeruginosa, S. marcescens
Linezolid 1979 (2000) 1999 23S RNA mutations S. aureus, E. faecalis
Daptomycin 1980 (2004) 2005 ? S. aureus, E. faecalis
Rafael Cantón, 2006
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Genome derived, target based strategy for
discovery of novel drugs
Essential targets Required under
all conditions Conditional essential Virulence
factors
Mutability and depleteability Fitness in an
infection model
Genome
Broad vs. narrow spectrum
Bioinformatics Interspecies complementation
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Genome derived, target based strategy for
discovery of novel drugs
Essential targets Required under
all conditions Conditional essential Virulence
factors
Mutability and depleteability Fitness in an
infection model
Genome
Broad vs. narrow spectrum
Bioinformatics Interspecies complementation
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Molecular Kochs postulates (Proof that a gene
product is an essential virulence factor) The
pathogenic trait should be associated with the
pathogenic members of the genus, species or
strains. Inactivation of the gene associated
with the pathogenic trait should result in a
measurable loss of pathogenicity or
virulence. Reversion, allelic replacement
(complementation) of the mutated gene should
restore pathogenicity.
Kochs postulates (Proof that a specific
organism caused a disease) The organism occurs
in every case of the disease and under
circumstances and conditions which can account
for the pathological changes and clinical course
of the disease. The organism occurs in no other
disease as a fortuitous and non-pathogenic
colonizer. The organism is isolated, grown in
pure culture and it can induce disease anew.
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New approaches towards drug discovery Model
organisms Pseudomonas aeruginosa -
extracellular pathogen can be genetically
manipulated Chlamydia pneumoniae - obligate
intracellular parasite - no genetic system
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Chlamydia pneumoniae
Causative agent of acute and chronic respiratory
tract infections Bronchitis Pneumonia
Association of with diseases of unknown etiology
Atherosclerosis Multiple sclerosis Reactive
arthritis Lung cancer Macular
degeneration Alzheimers disease Asthma Schizop
hrenia Stroke
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Drug discovery based on expression of virulence
factors in yeast
Target selection
Normal growth
Dividing yeast cells
Effect on viability and infectivity
Growth inhibition Identification of targets
ORFs encoding potential targets
Bacterial target
Effect on target activity
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Chlamydia developmental cycle
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The type III secretion system
Chlamydia in inclusions
P. aeruginosa attached to an epithelial cell
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Lethality of P.aeruginosa protein expressed in
yeast
505 essential protein and virulence factors
screened in yeast- 9 were lethal
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The two activities of ExoS
GTPase activation
ADP-ribosyl transferase
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The ADP-ribosyl transferase activity of ExoS is
responsible for yeast lethality
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Over-expression of the target leads to resistance
to killing
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Ras is the target of ExoS in yeast
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Compounds capable of rescuing yeast cells from
killing by ExoS
Compound library screened 56,000 molecules
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Exosin is a competitive inhibitor of the
ExoS-catalyzed ADP-ribosylation reaction
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Analogues of Exosin protect cells from ExoS
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Exosin analogues
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Inhibitors of ExoS protect CHO cells from killing
by P. aeruginosa
Dead
Live
Staining with 7-Amino- actinomycin D
ExoU
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Summary (I) Expression of a number of P.
aeruginosa essential genes leads to a lethal
phenotype in yeast Over-expression of candidate
yeast genes can be used to identify the target of
cytotoxic proteins A screen of compound libraries
can be used to reverse the lethal phenotype in
yeast Active compounds protect mammalian cells
from the cytotoxic activity of a P.aeruginosa
toxin Yeast Model for human infection?
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Chlamydia developmental cycle
? Genome sequences of most strains available
? No means of genetic manipulation ? No
virulence factors identified to date
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The genetic organization of the type III
secretion system in selected bacteria
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Amino-acid Sequence Alignment and Comparison of
SctW Homologs
Identity 100 67 64 64 45 45
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Surface localized proteins of Chlamydia pneumoniae
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Expression of C. pneumoniae CopN protein blocks
yeast cell division
GFP-CopN
GFP
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Expression of CopN in yeast results in cells
with large buds and abnormally positioned nuclei

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High throughput screen of a compound library for
the rescue of the CopN-induced growth defect in
yeast
Screen Monitor growth (OD at 600 nm) of S.
cerevisiae (PDR1-, PDR3-) in 384 well microtiter
dishes following induction of CopN expression.
Library 50,000 compounds Primary hits 28
compounds Validated hits 12 compounds Available
compounds 8 compounds Confirmed 2 compounds
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Inhibitors of CopN block the formation of large
inclusions in infected cells
CP0433YC1
DMSO
Cm
CP0433YC2
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Role of Type III secretion system in Chlamydia
infection
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Conclusions (II)
Expression of certain Chlamydia proteins leads
to lethality in east CopN function by disrupting
microtubules and causing a cell division arrest
inG2/m phase Compounds that rescue yeast from
CopN lethality prevent infection of mammalian
cells by Chlamydia Chemical knockouts vs.
genetic knockouts Yeast as a tool for drug
discovery
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Acknowledgments
Chlamydia Jin Huang Cammie Lesser ExoS
Anthony Arnaldo Igor Stagljar