REVIEW

Targeting virulence: a new paradigm for

antimicrobial therapy
Anne E Clatworthy, Emily Pierson & Deborah T Hung

Clinically significant antibiotic resistance has evolved against virtually every antibiotic deployed. Yet the development of new
classes of antibiotics has lagged far behind our growing need for such drugs. Rather than focusing on therapeutics that target in
vitro viability, much like conventional antibiotics, an alternative approach is to target functions essential for infection, such as
virulence factors required to cause host damage and disease. This approach has several potential advantages including expanding the
repertoire of bacterial targets, preserving the host endogenous microbiome, and exerting less selective pressure, which may result
in decreased resistance. We review new approaches to targeting virulence, discuss their advantages and disadvantages, and propose
that in addition to targeting virulence, new antimicrobial development strategies should be expanded to include targeting bacterial
gene functions that are essential for in vivo viability. We highlight both new advances in identifying these functions and prospects for
antimicrobial discovery targeting this unexploited area.

Since the introduction of penicillin, the deployment of any novel antibio-
tic has been followed by the evolution of clinically significant resistance
to that antibiotic in as little as a few years (Fig. 1)1. It is clear that we are
in a race to develop new antimicrobials to supplement our dwindling
antibiotic arsenal for combating the growing emergence of antibiotic-
resistant strains. Currently, we are losing this race. The Infectious Disease
Society of America estimates that 70% of hospital-acquired infections
in the United States are resistant to one or more antibiotics. Yet with
the exception of the recent development of the narrow-spectrum drugs
daptomycin and linezolid, there have been no new classes of clinically
relevant antibiotics discovered in over 40 years.
Traditional antibiotics have been identified for their ability to kill
bacteria (bacteriocidal) or inhibit growth (bacteriostatic). They act by
inhibiting bacterial functions (such as cell wall synthesis, DNA replica-
tion, RNA transcription and protein synthesis) that are essential for in
vitro, logarithmic growth (Fig. 2). Although it is clear that antibiotics
aimed at cellular viability have historically been highly effective, these
modes of action impose selective pressure that fosters the growth of
antibiotic-resistant strains. Given the current gap between our ability to
develop novel antibiotics and the real need for such drugs, the threat of
a postantibiotic era is looming large on the horizon. Therefore, in addi-
tion to compounds that act by targeting in vitro cell growth, we must
consider developing antimicrobials that have novel modes of action. New
approaches have been revealed by the tremendous effort over the past few
decades to understand how bacteria cause disease. This effort to under-
Department of Molecular Biology and Center for Computational and Integrative
Biology, Massachusetts General Hospital, 185 Cambridge St., Boston,
Massachusetts 02114, USA; Department of Microbiology and Molecular
Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts
02115, USA; and the Broad Institute of MIT and Harvard, 7 Cambridge Center,
Cambridge, Massachusetts 02142, USA. Correspondence should be addressed to
D.T.H. (hung@molbio.mgh.harvard.edu).
Published online 20 August 2007; doi:10.1038/nchembio.2007.24
stand pathogenesis has begun to elucidate mechanisms that could be
targeted to clear the infection in lieu of targeting simply in vitro bacterial
viability. Instead, targeting bacterial virulence or disrupting the interac-
tion between the host and the pathogen are attractive options that are
increasingly being explored.
The conventional concept of virulence is defined by the ability of a
pathogen to cause disease; virulence determinants are defined as bacte-
rial factors (for example, toxins, cytolysins or proteases) or mechanisms
that actively cause damage to host tissues. Thus, efforts to develop anti-
virulence therapies are geared at ‘disarming’ the pathogen by inhibiting
virulence factors that can cause direct harm to the host. In theory then,
compounds that target virulence create an in vivo scenario that is similar
to vaccination with a live, attenuated strain. The bacteria are eventually
cleared by the host immune response with little to no impact on the
normal human microbiota. A potential (though yet unproven) advantage
of this approach is that new antimicrobials aimed at inhibiting virulence
rather than growth may impose weaker selective pressure for the develop-
ment of antibiotic resistance relative to current antibiotics.
Here we review recent strategies that target various pathways related to
virulence, including inhibiting toxin function, toxin delivery, regulation
of virulence expression, and bacterial adhesion (summarized in Table 1
and Figure 3). Though this review is not entirely comprehensive, we
highlight some of the major approaches being taken to target virulence.
At the end of this review, we explore an even broader framework for new
antimicrobial development that includes not only the strategy of target-
ing virulence but also the strategy of targeting bacterial in vivo essential
gene functions—gene functions that are required for survival within the
host yet that are distinct from those required for in vitro viability. The
targets of both of these therapeutic strategies (that is, virulence factors
and in vivo essentials) are required for infection, yet the utility of these
strategies has only recently begun to be explored. Given the need for
antibiotics with novel modes of action, consideration of these approaches
is warranted.

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007 541

REVIEW

Antibiotic deployment a search for small molecules that would pro-

tect the population from anthrax, Merck has
Tetracycline
identified a hydroxymate (LFI) that inhibits LF
Chloramphenicol Vancomycin
protease activity and promotes cellular survival
Streptomycin Ampicillin
in a macrophage cytotoxicity assay9. LFI (Fig.
Sulfonamides Erythromycin Cephalosporins Daptomycin
3b) binds the active site of Bacillus anthracis
Penicillin Methicillin Linezolid
LF and offers complete protection from spore
infection when administered to mice in combi-
1930 1935 1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005
nation with ciprofloxacin 66 h postinfection in
a model in which ciprofloxacin alone only offers
Sulfonamides Chloramphenicol Ampicillin Vancomycin Linezolid
50% protection from lethality9. Along similar
Penicillin Streptomycin Erythromycin lines, a mixture-based peptide library was used
Daptomycin
Tetracycline Methicillin to determine the optimal peptide substrate
Cephalosporins sequence for LF, which was then used to design
Antibiotic resistance observed peptide analogs that inhibit LF activity in vitro
and protect macrophages from LF-induced
Figure 1 Timeline of antibiotic deployment and the evolution of antibiotic resistance. The year each cytolysis10.
antibiotic was deployed is depicted above the timeline, and the year resistance to each antibiotic was Compounds that inhibit anthrax toxin are
observed is depicted below the timeline (with the caveat that the appearance of antibiotic resistance not limited to targeting LF or EF. PA is also
does not necessarily imply that a given antibiotic has lost all clinical utility). a potential target that could be inhibited in
multiple ways. Strategies include inhibition of
binding of PA to its host receptor, processing of
Inhibition of toxin function PA by host proteases, binding of processed PA to LF or EF, or transloca-
Many pathogenic bacteria cause damage to host tissues through the tion of LF or EF by physically blocking the PA heptamer pore8. Recent
release of toxins, which are proteins that act to perturb host cell functions work indicates that a phenylalanine clamp controls protein translocation
and may ultimately result in host cell death. Thus, an obvious approach through the heptameric PA pore and that this clamp can be targeted with
to inhibiting bacterial virulence is disruption of toxin function, which small molecules that block the pore11. Alternatively, one could interfere
can occur in a direct manner by inhibition of the toxin activity itself, or with toxin translocation by inhibiting formation of the pore itself by
in an indirect manner, by modulating the host response to the toxin. inhibiting endosome acidification12,13 or by inhibiting PA heptamer
In fact, direct inhibition is the basis for the historical use of antitoxins assembly. Cisplatin seems to prevent both LF and EF toxicity by inhibit-
(antibodies) against toxins such as diptheria, botulinus, tetanus and other ing heptamer assembly, and simultaneous administration of cisplatin
toxins. Recently, much effort has been focused on inhibiting the effects of with a lethal dose of anthrax lethal toxin has been shown to be protec-
the three proteins that comprise anthrax toxin: lethal factor (LF), edema tive in rodent models, though a delay in cisplatin administration was
factor (EF) and protective antigen (PA) (Fig. 3a). shown to be ineffective14. This result should be contrasted with the result
Though each toxin component alone is nontoxic, the pairing of either obtained in ciprofloxacin and LFI combination therapy, which works
LF or EF with PA results in characteristic toxin activity that is ultimately even in delayed administration. This difference underscores the concept
responsible for the pathology of the disease2. Much is already understood that the specific mechanism of virulence inhibition may determine the
about toxin entry into mammalian cells. First, individual PA monomers efficacy of a particular small molecule in targeting virulence at different
diffuse to the surface of mammalian cells where they are proteolytically stages of infection (see below).
cleaved by host proteases. Cleaved PA spontaneously oligomerizes into Rather than directly inhibiting toxin function, one could block the
heptamers, which can bind either LF or EF. PA-EF PA-LF complexes are downstream effects of the toxin by targeting host proteins. For example, in
then endocytosed and trafficked to the endosome, where a drop in the pH secretory diarrheas and cholera, Cl– secretion is central to intestinal fluid
triggers a conformational change in PA that converts it to a transmem- secretion (the disease pathology); thus inhibitors of Cl– channels may
brane pore. Ultimately LF and EF are translocated through PA’s pore into have therapeutic value in blocking these diarrheas. A screen for inhibi-
the host cytosol where they exert their respective toxic effects (Fig. 3a). tors of the cystic fibrosis transmembrane conductance regulator (CFTR)
EF is a calmodulin-dependent adenylate cyclase whose action is known protein15, a cAMP-activated Cl– channel that is defective in individuals
to result in prolonged increases in cyclic AMP levels, but it is otherwise with cystic fibrosis, found that compounds of the 2-thioxo-4-thiazolidi-
poorly understood. The exact mechanism by which LF, a Zn2+ protease, none class inhibit the CFTR protein. The most potent thiazolidinone
exerts its cytotoxic effect in vivo is also not clearly understood. Genetic compound was evaluated for its ability to inhibit mouse intestinal fluid
evidence suggests that LF-mediated cell death is dependent on suscep- secretion after oral administration of cholera toxin. A single intraperito-
tible alleles of the Nlrp1b gene, as well as the gene encoding caspase-1 in neal injection of the CFTR inhibitor decreased the level of fluid secretion
murine macrophages3. LF can also cleave the N termini of several mito- by more than 90% after administration of cholera toxin. Although it is
gen-activated protein (MAP) kinase kinases4,5. However, it is unclear how still unclear whether administration of this compound can positively
LF-mediated MAPKK cleavage is related to cytotoxicity, though small influence the resolution of infection with Vibrio cholerae, this finding
molecules that activate MAP kinase cascades have been shown to protect underscores the notion that new therapeutics aimed at targeting virulence
mouse macrophages from LF-induced cell death6. Apart from causing cell need not be absolutely restricted to pathogen proteins.
death, LF can also paralyze actin-based motility in neutrophils—an effect
that has been correlated with decreased levels of Hsp27 and p38 MAP Targeting bacterial toxin delivery
kinase phosphorylation in LT-treated, cultured neutrophils7. In addition to targeting bacterial toxin function, one could also inhibit
Regardless of the precise mechanism of activity of LF, it has certainly virulence by interfering with appropriate delivery of the toxin to its
been the focus of great attention as a target for new antimicrobials8. In site of action. This principle has been applied for several decades in the

542 VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY


treatment of the antibiotic-associated diarrheal disease caused by
Clostridium difficile16. Cholestyramine simply binds the clostridial tox-
ins, preventing their delivery or accessibility to the intestinal epithelium
and thus blunting their toxic effects. Cholestyramine binds toxin B (a
cytotoxin) and likely toxin A (an enterotoxin) as well, though its use has
been limited to relatively mild cases of the disease.
A more recent application of this principle is prevention of toxin (or
effector) delivery by inhibiting bacterial secretion systems. Many proteins
involved in bacterial secretion are specific to prokaryotes and are therefore
rational candidates to target with novel antimicrobials. Recently, there has
been interest in targeting the type III secretion system (T3SS) common
to Yersinia spp., Pseudomonas aeruginosa, pathogenic Escherichia coli,
Shigella spp., Salmonella spp. and Chlamydia spp. The T3SS is a syringe-
like apparatus that facilitates the injection of bacterial effectors from these
species directly into the host cytosol (Fig. 3a)17. Depending on the spe-
cies, T3SS effectors have been implicated in perturbing a variety of host
cellular processes, including cytoskeletal dynamics, gene expression, cell
cycle progression and apoptotic cell death programs.
Chemical screens for inhibitors of the T3SS in Yersinia pseudotuber-
culosis identified acylated hydrazones of different salicylaldehydes18,19.
One compound was found to directly target the Y. pseudotuberculosis
T3SS, thereby preventing effector molecule translocation and attenuat-
ing the pathogen in a cell culture model of infection without interfering
with in vitro growth19. Compounds of this class have also been found to
inhibit intracellular replication and infectivity, and translocation of the
T3SS effectors IncG and IncA during early and middle stages of infec-
tion of Chlamydia trachomatis (Fig. 3c)20. They have also been shown to
interfere with the life cycle of Chlamydia pneumoniae21. Very recently,
acylated hydrazones of different salicyclaldehydes have also been shown
to be effective at inhibiting both the secretion of T3SS effectors and the
invasion of Salmonella enterica serovar Typhimurium into cultured epi-
thelial cells22. However, compounds of this class were only able to sup-
press T3SS-dependent secretory and inflammatory responses in a bovine
intestinal loop model of S. enterica serovar Typhimurium infection if
the bacteria were first preincubated with the compounds. Simultaneous
infection and administration of these compounds was ineffective in
preventing fluid accumulation and neutrophil influx into the infected
intestinal loops22. Thus, while it seems that acylated hydrazones of dif-
ferent salicyaldehydes may effectively target the T3SS of many different
pathogens, more work will need to be done to evaluate whether this class
of compounds has broad-spectrum therapeutic value.
Targeting the regulation of virulence expression
In addition to targeting toxin function or delivery, disrupting the regula-
tory mechanisms that result in virulence expression is attractive because
it would prevent the formation of toxin. To some degree, the use of the
protein synthesis inhibitor clindamycin, in addition to a cell wall anti-
biotic such as penicillin, in the treatment of streptococcal toxic shock
syndrome to prevent the production of toxin (a phenomenon known
as the “Eagle effect”) is a clinical application of this principle23. Recent
advances in our understanding of virulence regulation have identified
many different regulatory steps that could be targeted.
Most efforts to inhibit the regulation of virulence factor expression
have focused on interfering with quorum sensing (Fig. 3a), a mode of
bacterial communication used by multiple bacterial species to regulate
processes such as bioluminescence, antibiotic synthesis, biofilm forma-
tion and virulence factor expression as a function of population den-
sity24. Quorum sensing is a phenomenon whereby bacteria can sense
their population density by releasing diffusible signaling molecules that
accumulate in the population and surrounding environment. These sig-
naling molecules increase in concentration as the bacterial population
NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007
REVIEW
Inhibition of
Inhibition of DNA or RNA synthesis protein synthesis
dTTP
GTP
Bacterial cell
DHP DHF
dTMP
THF dUMP Inhibition of
Inhibition of cell wall synthesis
folate synthesis
Depolarization of
membrane potential
K+
K+ K+
K+
Figure 2 Traditional targets of antibacterial compounds. Traditional
antibiotics function by inhibiting DNA or RNA synthesis (for
example, fluoroquinolones), inhibiting protein synthesis (for example,
aminoglycosides), inhibiting cell wall synthesis (for example, β-lactams),
inhibiting folate synthesis (for example, sulfa drugs), or depolarizing
membrane potential (daptomycin).
expands until a critical threshold concentration is reached. At critical
threshold concentrations (when the bacteria are ‘quorate’), quorum
sensing signaling molecules can influence the expression of a variety of
different genes, including the expression of genes involved in virulence.
Teleologically, the bacteria may begin to express virulence factors only
when they have reached a significantly large population size to cause an
‘effective’ infection. Thus, by interfering with quorum sensing pathways,
one could inhibit a bacterial population’s ability to monitor the number
of cells within that population and thereby interfere with quorum sensing
activation of virulence factor expression.
In many Gram-negative bacteria, quorum sensing is mediated by acyl-
homoserine lactone molecules (AHLs) that are synthesized and recog-
nized by quorum sensing circuits composed of LuxI and LuxR homologs
(Fig. 3a)24. LuxI homologs synthesize AHL molecules from the common
metabolic intermediate S-adenosylmethionine (SAM) and an acyl-acyl
carrier protein. At critical threshold concentrations, AHL molecules
are recognized by their cognate transcriptional activator (LuxR homo-
logs), which in turn regulates the transcription of genes associated with
virulence. Thus, one could inhibit AHL-mediated quorum sensing by
inhibiting the enzymes that synthesize quorum sensing molecules (LuxI
homologs). In vitro, the activity of the P. aeruginosa LuxI homolog RhlI
can be inhibited with analogs of SAM such as S-adenosylhomocysteine,
sinefungin and S-adenosylcysteine25, resulting in the inability to synthe-
size quorum sensing molecules. Though SAM is a common metabolic
intermediate, it is possible that improved analogs of SAM could be gener-
ated that could interfere with LuxI-mediated AHL synthesis but not host
metabolic processes that rely on SAM.
Alternatively, one could inhibit quorum sensing by interfering with
the concentration of the AHL signaling molecules through degradation.
For example, Gram-positive Bacillus species produce acylhomoserine
lactonase, an enzyme that hydrolyzes the lactone ring of AHLs, thereby
rendering them unable to mediate signaling26,27. Tobacco plants engi-
neered to express AHL-lactonase show an enhanced resistance to Erwinia
543
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Table 1 Targets of antivirulence inhibitors, their modes of action and their effects on infection in vivo
Targets
Toxin function
B. anthracis lethal factor (LF)
B. anthracis protective antigen (PA)
Toxin delivery
Type III secretion system (T3SS)
Regulators of virulence expression
V. cholerae ToxT
Quorum sensing: LuxR homologs
Quorum sensing: S. aureus AgrC
Adhesion
Pilin chaperones (for example, PapD
in UPEC)
Inhibitors (examples)
(2R)-2-[(4-fluoro-3-methylphenyl)
sulfonylamino]-N-hydroxy-2-
(tetrahydro-2H-pyran-4-yl)
acetamide (LFI)
Peptide analogs derived from
the optimal peptide substrate
sequence
Cisplatin
Hexa-D-arginine
Acylated hydrazones of different
salicylaldehydes (for example,
INP0400)
Virstatin
Structural analogs of AHLs (for
example, halogenated furanones)
Inhibitory autoinducing peptides
(AIPs)
Pilicides: bicyclic 2-pyridones
and N-substituted amino acid
derivatives
Modes of action in vitro
Binds LF active site and inhibits
LF protease activity
Inhibit LF cleavage of MKKs
in vitro
Inhibits LF translocation into the
host cytosol
Inhibits PA processing
Prevent effector molecule
translocation
Prevents expression of the
toxin-coregulated pilus and
cholera toxin in V. cholerae
Accelerate turnover of LuxR
homologs; inhibit expression of
quorum sensing regulated genes;
inhibit the production of
carbapenem in E. carotovora and
virulence factors in P. aeruginosa
Inhibits agr locus activation
Inhibit pilus assembly
Effect on in vivo (or ex vivo)
infection References
Protects mice from spore infection 9
when used in combination with
ciprofloxacin; provides complete
protection in mice immunized with
LF and PA
Protect macrophages from 10
LF-induced cytolysis ex vivo
Protective when administered 14
simultaneously with anthrax lethal
toxin in murine models
Delays lethal toxin-induced toxemia 62
in rodent models and cultured
macrophages
Attenuate Y. pseudotuberculosis 18–22
infection and inhibit intracellular
replication of C. trachomatis and
C. pneumoniae ex vivo; preincubation
of compound with bacteria before
infection suppresses secretory and
inflammatory responses in a bovine
intestinal loop model of S. enterica
serovar Typhimurium infection
Protects infant mice from intestinal 44
colonization with V. cholerae
Promote clearance of P. aeruginosa 30–32,
from the lungs of mice in a 34–38
pulmonary infection model;
increase the survival time of mice
in a lethal P. aeruginosa lung
infection model
Administration of inhibitory AIP 41–43
to mice during S. aureus infection
inhibits abscess formation
Bicyclic 2-pyridones inhibit 47,48
adhesion of E. coli to bladder
carcinoma cells ex vivo

carotovora infection27, which demonstrates that degradation of the AHL
signal is a viable approach to attenuating infection with E. carotovora.
Mammalian sera also show strong AHL lactonase activity that is similar
to the activity of paraoxonases28, which themselves have lactonase activi-
ty against AHLs in vitro29. Thus, interference with quorum sensing by
increasing degradation of AHL molecules may be a natural phenome-
non used by multiple species. Though it has not been exploited in anti-
microbial discovery, it represents an alternate mechanism for targeting
virulence.
Finally, one could inhibit AHL-mediated quorum sensing by interfer-
ing with the recognition of and subsequent transcriptional response to
the signaling molecules by LuxR homologs. For example, a number of
structural analogs of AHLs have been shown to inhibit the expression of
quorum sensing regulated genes and virulence factor production as well
as biofilm formation in P. aeruginosa30–32. In particular, one class of struc-
tural analogs of AHLs, halogenated furanones, have been shown to have
quorum sensing inhibitory properties33 and seem to function by accele-
rating the turnover of LuxR homologs34. In E. carotovora, halogenated
furanones have been shown to inhibit the production of carbapenem,
which is regulated by the LuxR homolog CarR35. In P. aeruginosa, haloge-
nated furanones inhibit production of exotoxins, repress the expression
of quorum sensing regulated genes, and increase the susceptibility of
P. aeruginosa biofilms to tobramycin in vitro36,37. In vivo, administration
of synthetic halogenated furanones promotes clearance of P. aeruginosa
from the lungs of infected mice37,38 and increases the survival time of
mice in a lethal P. aeruginosa lung infection model38. However, AHL
molecules themselves as well as other structural variant lactones are base-
labile and are substrates for mammalian paraoxonases28,29,39; they are
thus likely to be suboptimal for therapeutic use. Halogenated furanones
are also too reactive to be used therapeutically. For this reason, the recent
identification of a triphenyl compound that is a potent agonist of quorum
sensing in P. aeruginosa is promising in that this compound is structurally
unrelated to AHL molecules and therefore may be useful as a scaffold for
developing future quorum sensing inhibitors that are more stable and
perhaps less reactive in vivo40.
Quorum sensing is not a phenomenon that is unique to Gram-nega-
tive pathogens. Staphylococcus aureus strains make autoinducing peptides
(AIP) that function as quorum sensing molecules in these bacteria. In gene-
ral, Gram-positive bacteria synthesize a precursor protein that is proteo-
lytically cleaved to form the processed quorum sensing signaling peptide.

544 VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY

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a Host cell
Figure 3 Bacterial protein functions that can be targeted to inhibit virulence
and examples of virulence inhibitors. (a) Virulence inhibitors could target:
Endosome toxin function (for example, B. anthracis LF catalytic activity or translocation
Inhibition of through PA); toxin delivery, by inhibiting various bacterial systems such as
toxin function LF
type II or type III secretion (T3SS); virulence gene regulation (for example,
PA AHL-mediated quorum sensing circuitry (LuxI or LuxR homologs) or
Inhibition of transcriptional regulators that control virulence gene expression); or bacterial
bacterial adhesion LF adhesion to host cells (for example, inhibition of the formation of pili by
pilicides). (b–e) Examples of virulence factor inhibitors include: B. anthracis
lethal factor inhibitor9 (b), the T3SS inhibitor INP0400 (ref. 20) (c), the
quorum sensing inhibitor furanone C-30 (ref. 37) (d), and a pilicide (a
Inhibition of bicyclic 2-pyridone compound)48 (e).
toxin delivery

AHL T3SS
AHL Apart from quorum sensing, transcriptional regulators that coordinate
AHL the expression of genes involved in adhesion, toxin production and secre-
AHL
Type II secretion
tion are themselves potential targets for future antimicrobials. A recent
AHL LuxI example is the small molecule virstatin (Fig. 3d), which was identified
AHL
in a screen for small-molecule inhibitors of the V. cholerae cholera toxin
LuxR
promoter44. Virstatin inhibits the transcriptional regulator ToxT, thereby
preventing the expression of two critical virulence factors in V. cholerae:
Inhibition of virulence regulation the toxin-coregulated pilus, which is involved in attachment to the intes-
tinal epithelium, and cholera toxin. In vivo, administration of virstatin
Virulence genes protected infant mice from intestinal colonization with V. cholerae44. This
Bacterial cell work also demonstrated that late administration of virstatin (12 h after
infection) still results in a 3-log drop in the ability to recover bacteria from
the mouse intestine, which shows that inhibition of virulence expression,
even in established infection, can still have potential therapeutic value.
Another example of inhibition of virulence regulation and expression
b O c was found in a screen for inhibitors of the T3SS in enteropathogenic
HO
E. coli (EPEC)45. In this study, the imine adduct from the condensation
O of a halogenated salicylaldehyde and 3-aminoacetophenone seemed to
HN OH
O N decrease the transcription of T3SS effectors and components of the T3SS
S NH N
O H machinery without affecting growth or motility. Thus, toxin delivery can
O
Cl be inhibited by novel therapeutics targeting not only structural compo-
nents of the secretion apparatus but also transcriptional regulators of
F secretory systems.

d e
COOH
O N O S
N lining epithelial cell surfaces of the respiratory, intestinal and repro-
N O
O surfaces by antibody molecules that line these tracts and perfuse host
This peptide is then actively transported extracellularly and can then
be recognized by a histidine-sensor kinase protein of a two-component
regulatory cascade. Upon recognition of the peptide autoinducer, the
two-component regulator activates a phosphorelay response, ultimately
resulting in activation of a quorum sensing transcriptional regulator.
Different S. aureus strains produce peptides whose sequences vary slightly
from those produced by other strains. Thus, a given peptide sequence can
activate its own histidine kinase two-component regulatory cascade while
antagonizing the histidine kinase two-component regulatory cascades in
other strains of S. aureus41. This has been exploited in a mouse model
of S. aureus–mediated abscess formation, in which administration of
inhibitory AIP to mice during the first 3 h of S. aureus infection inhibited
the pathology42,43. Clearly, such a therapeutic approach would require
a small molecule that could inhibit the quorum sensing systems of all
S. aureus strains.
Inhibition of adhesion
Equally important for establishing infection is adhesion of the bacte-
rial cell to the host. The human body has evolved multiple lines of
defense to prevent the majority of bacteria from adhering to host tis-
sues, including continual shedding of epithelial cell surfaces, mucus
Li ductive tracts, and direct inhibition of adhesion of bacteria to host
O
tissues. These multiple lines of defense erected by the body under-
score the importance of preventing bacterial adhesion to the host.
Moreover, because proteins involved in bacterial adhesion are specific
to prokaryotes, like bacterial secretion systems, they are rational tar-
gets for novel antimicrobials.
A potentially broad class of antimicrobials that target adhesion are
called ‘pilicides’. Pilicides are aimed at inhibiting the formation of pili
or fimbriae (Fig. 3a), which are hair-like projections protruding from
bacterial cells that facilitate adhesion. Pili consist of repeating subunits
of immunoglobulin-like domains wherein the N terminus of one sub-
unit is donated to complete the immunoglobulin fold of its neighbor
subunit; these structures are formed by donor-strand complementation
via the chaperone usher pathway46. In this pathway, the chaperone pro-
tein binds pilin subunits by donating its edge β-strand to complete the
folding of the pilin subunit’s immunoglobulin fold. Chaperone–pilin
complexes then traffic to outer-membrane usher channels where the
pilin fiber is formed by donor strand exchange between the chaperone

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and the adjacent subunit in the growing pilin fiber. So far, efforts at
developing pilicides have focused on inhibiting the periplasmic chape-
rone proteins of the chaperone usher pathway in uropathogenic E. coli
(UPEC). Bicyclic 2-pyridones (Fig. 3e) and N-substituted amino acid
derivatives have been shown to competitively inhibit binding of chaper-
ones to pilin subunits by surface plasmon resonance47. In vitro, bicyclic
2-pyridones have also been shown to inhibit hemagglutination and
biofilm formation in laboratory and clinical E. coli strains, and ex vivo
they have been shown to inhibit adhesion of the bacteria to bladder
carcinoma cells by ~90%48. It has been suggested that pilicides may have
broad-spectrum activity due to the conservation of both chaperone
structure and the chaperone-usher pathway49.
Virulence inhibitors as therapeutic candidates
Novel therapeutics that target virulence rather than simply in vitro cell
growth would both supplement and add diversity to our current antimi-
crobial armamentarium, with the caveat that this type of strategy may not
be effective in immunocompromised individuals who lack the ability to
clear the ‘disarmed’ pathogen. However, for the appropriate patient popu-
lation, a tantalizing advantage of targeting virulence is the potential of
this approach to impose weaker selective pressure that would be less likely
to foster the growth of antibiotic resistance, in contrast to therapeutics
targeted at straightforward viability. If the function of a given virulence
factor is unrelated to its viability within the host, there should be no in
vivo selection for the rare, resistant mutant to an inhibitor of that factor.
The difficulty is that it is unclear which virulence factors fit this criterion,
and thus each individual case will need to be examined carefully.
Even if an inhibitor targets a virulence factor required for in vivo via-
bility, the circumstances under which resistance could arise against the
specific inhibitor relative to conventional antibiotics are more limited,
because antivirulence inhibitors are often pathogen-specific and selec-
tion must occur in vivo. Thus, the window of opportunity during which
resistance could be selected for is more narrow, equaling the length of
an individual infectious cycle. Additionally, use of antivirulence drugs
could also have an impact on the development of resistance to our cur-
rent broad-spectrum antibiotics by offering an alternative therapy in
certain cases, thus reserving our current broad-spectrum agents to a more
limited set of cases in which they are most needed. Finally, because of
their narrow spectrum and different mechanisms of action, therapeutics
that act to inhibit virulence rather than cell growth also have a signifi-
cant advantage in helping to preserve normal and potentially beneficial
members of the normal human microbiota. This type of strategy would
likely have a significant impact on the morbidity associated with shifts
in host normal flora after antibiotic treatment.
The exact therapeutic role of antimicrobials that target virulence is as
yet unclear. Antivirulence therapies have the potential to function effec-
tively when used alone, when used in combination therapy with antibi-
otics, or simply as a prophylactic treatment. It is premature to rule out
the use of antivirulence drugs simply because they are not bacteriocidal,
as this would underestimate the role of host immunity. A long-standing
dogma suggests a preference for bacteriocidal over bacteriostatic anti-
biotics. However, there is actually little clinical evidence to suggest that
this dogma has significant bearing on the resolution of infection, with
other variables such as tissue penetration and bioavailability potentially
having a greater impact. Thus, a new paradigm for antimicrobial therapy
redefines the goal to be simply the tipping of the balance in favor of the
host, thus enabling it to control infection, rather than complete in vivo
killing of a pathogen by the drug itself.
Antivirulence drugs could have a role in prophylactic treatment during
an epidemic, a bioterrorist threat, or in select populations (for example,
travelers) for several reasons. They would not engender resistance in the
546
population toward conventional antibiotics, they would not alter the
natural microbiome of the host, and they may work optimally to pre-
vent infection, as evidenced by cases in which the drugs work best when
administered with the pathogen rather than in established infection. It is
likely that the unique mechanism of action for each antivirulence thera-
peutic will ultimately determine its utility as a prophylactic, mono- or
combination therapeutic agent.
Because virulence factors are required at different times during infec-
tion, the definition of the time window in which a given inhibitor will
be effective will likely be mechanism- and thus inhibitor-specific. For
example, as described above, in V. cholerae infection, inhibition of the
transcriptional regulator ToxT, which results in the downstream inhibi-
tion of cholera toxin synthesis, has therapeutic efficacy in established
infant mouse colonization 12 h after infection44. On the other hand,
cisplatin inhibition of LF translocation into the host cytosol by blocking
PA heptamer assembly is completely ineffective in mice if the inhibitor is
administered after anthrax toxin inoculation14. In contrast, if the mecha-
nism of protection is due to inhibition of the proteolytic activity of LF,
delayed administration of LFI is effective (granted, in combination with
ciprofloxacin) in rescuing infected mice9. Thus, the therapeutic efficacy
of inhibiting virulence factors needs to be evaluated on an individualized,
mechanistic basis, and efforts will need to be invested in determining
the in vivo time window in which a protein involved in virulence may be
effectively targeted. This analysis would provide insight into the timing
of different virulence programs and their requirement throughout the
course of infection.
Though some virulence inhibitors such as pilicides and T3SS inhibitors
have the potential to target a wider spectrum of bacteria, other virulence
inhibitors that disrupt mechanisms specific only to single pathogens or
that target bacterial proteins that do not have structurally similar ortho-
logs in many clinically relevant bacterial species will be exquisitely nar-
row-spectrum by definition. Although this new paradigm could be the
solution to the resistance crisis we currently face, it raises two problems
that are not insurmountable but that need to be addressed. One problem
is diagnostic; the other is economic.
The utility of therapeutic intervention with narrow-spectrum anti-
virulence antimicrobials will be very dependent on the clinician’s ability
to precisely diagnose the genus if not the species of bacterial infection in
a patient in order to select the correct antimicrobial therapeutic agent.
Thus, antimicrobials that target virulence will need to be developed hand
in glove with new diagnostic tests that will allow the clinician real-time
diagnosis. Certainly these technological problems are challenging, but
they are currently the focus of great interest, with efforts intensified par-
ticularly in the area of biodefense. In addition to real-time identification
of the culprit pathogen, a related issue is determining the susceptibility of
the culprit pathogen to virulence inhibitors. Definition of conventional
antibiotic susceptibilities has become a well-tuned science that includes
Kirby-Bauer (disc diffusion) tests and broth dilution measurements. In
contrast, determining susceptibilities to a virulence inhibitor cannot
use simple cell growth as a sensitivity indicator. Assays exist and can be
developed for determining susceptibility in vitro, such as the screening
assays used to identify these inhibitors or ones engineered to include
various gene reporter readouts. However, these assays too will need to
be individualized for each specific inhibitor.
Perhaps the greatest challenge of changing to a new paradigm is not
technological but economic. If compounds that target virulence are effec-
tive against a much more narrow range of bacteria, the economic incen-
tive for a pharmaceutical company to develop that compound into a drug,
with the accompanying diagnostic and susceptibility tools, is even lower
than current economic incentives to develop wide-spectrum antibiotics.
If we are to avoid plummeting into a postantibiotic era in the near future,
VOLUME 3 NUMBER 9 SEPTEMBER 2007 NATURE CHEMICAL BIOLOGY

clearly we must attack not only the technological hurdles facing new
antimicrobial development but also the economic hurdles.
Future approaches to new antimicrobial development
To supplement our dwindling antimicrobial arsenal, a broader range of
targets than the set of in vitro essential genes that are typically targeted
by conventional antibiotics must be considered. We propose that a new
paradigm for antimicrobial development should be based on targeting
gene functions that are required in vivo to cause disease. This strategy
includes targeting genes that are essential for causing virulence as well
as those essential for in vivo viability. As the ability of pathogenic bac-
teria to both cause damage and survive within the host is required for
causing disease, disrupting either of these processes could be exploited
therapeutically.
In vitro and in vivo bacterial essential gene functions are distinct. The
growth environment within a host is unlikely to be the same as the arti-
ficial ones induced in a laboratory, and therefore the genes required for
viability will likely also differ. This concept was illustrated in a trans-
poson site hybridization study conducted in Mycobacterium tuberculo-
sis–infected mice. The study revealed that a different set of M. tuberculosis
genes, representing ~5% of the M. tuberculosis genome, were required
for in vivo survival than were required for in vitro growth50. Current
antibiotic discovery approaches have focused only on in vitro essentials,
and thus the realm of in vivo essentials that could be targeted has not yet
been sufficiently explored.
One approach that has the capacity to identify new therapeutics that
target either bacterial in vivo essential gene functions or the host itself is
chemical screening of whole-organism infection models. Whole organ-
ism–based screening has the advantage of being able to identify small
molecules that are more likely to be permeable to the cell, effective at elici-
ting the desired phenotype (such as attenuation of infection), unlikely
to have gross toxic side effects, and have acceptable pharmacokinetic
profiles, at least in the model host, which may or may not translate from
host to host51. Finally, whole organism–based screening has the capac-
ity to define which proteins are, in fact, ‘drug-targetable’. Thus, whole-
organism screening has the potential to leapfrog over some of the major
hurdles commonly encountered after typical in vitro target-based screens.
Practically, of course, in order to identify candidate novel antimicrobials
in a whole-organism infection model, the model host must be of a size
suitable for chemical screening. The zebrafish (Danio rerio) has success-
fully been used in high-throughput chemical screens52,53 and has also
been used to model mycobacterial, streptococcal and Salmonella infec-
tions54–56. Thus, the marriage of chemical screening to zebrafish infection
models may be a new way of rapidly identifying compounds that target
in vivo essential gene functions in a vertebrate model host57.
The utility of whole organism–based chemical screening for new anti-
microbial candidates has already been demonstrated using Caenorhabditis
elegans as the model host. In this study, Moy and colleagues screened 6,000
synthetic compounds and 1,136 natural product extracts for compounds
that promote nematode survival of Enterococcus fecalis infection58. They
identified several compounds that rescue nematodes from the lethality of
infection at therapeutic concentrations that are well below the minimal
inhibitory concentrations (MICs) of the compounds against E. fecalis.
Unless one posits that C. elegans is able to concentrate the compounds
above the therapeutic index, one must assume that these compounds are
rescuing the nematodes either by targeting an E. fecalis in vivo essential
gene function, inhibiting virulence, or targeting the nematode immune
defense. Though the targets of these compounds have not yet been identi-
fied, and though it is unclear as yet whether these compounds target the
worm or the bacterium, this method highlights a novel approach for iden-
tifying compounds that target proteins essential for virulence in vivo.
NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 9 SEPTEMBER 2007
REVIEW
Inhibitors of in vivo essential genes as therapeutics
Inhibitors of in vivo essential gene functions can target (i) functions
required for viability in vitro (including conventional antibiotic tar-
gets), (ii) functions required for viability only in the host, (iii) functions
required for virulence or (iv) some combination of the above. The advan-
tages and disadvantages of a therapeutic will vary based on the functional
class or classes to which the target belongs. Advantages and disadvantages
are manifested in the spectrum of activity against different pathogens,
the window of time in which a drug is effective, and the level of selective
pressure for resistance.
Therapeutics that target gene functions essential for viability both in
vivo and in vitro (that is, conventional antibiotics) often target proteins
that are well conserved among species. Thus, this functional class has
the potential to be broad-spectrum, and the time window during which
it should be effective is relatively large. The disadvantage, of course,
is that this class of therapeutics can impose strong selective pressure
that fosters the growth of antibiotic resistance. At the other end of
the extreme are therapeutics that target gene functions required for
virulence. The advantages and disadvantages of this functional class
have been discussed above, and they include the imposition of less
selective pressure and a narrower window of efficacy and spectrum of
activity. Therapeutics that target in vivo essential gene functions will
have advantages and disadvantages that fall between these two extremes.
They should exert some selective pressure, but only within the context
of the host, and they may have a larger efficacy window and (poten-
tially) a broader spectrum of activity compared to antivirulence drugs.
Therapeutics that fall into a combination of these functional classes will
have even more complex profiles.
Conclusion
This review highlights a number of novel strategies for developing new
therapeutics against infection. There have been very few new classes of
antibiotics discovered in the past 40 years, with efforts dwindling in large
pharmaceutical companies. The effort that has been invested has been
disappointing, resulting in a rather grim picture for the current state of
antibiotic development. GlaxoSmithKline recently undertook a genomic,
target and whole cell–based approach over a period of seven years to iden-
tify compounds that inhibit genes thought to be essential for viability in
a number of pathogens59. Disappointingly, only five leads were identified
from a total of 70 high-throughput screens using the GSK compound col-
lection, which suggests that in vitro target-based screening of traditional
compound libraries biased to follow Lipinski’s ‘rule of five’60 is an inef-
fective way to identify new antimicrobials. However, GlaxoSmithKline’s
experience does suggest that future efforts at antimicrobial identification
will need to include screening of libraries that sample a wider diversity
of chemical space with potentially differing physicochemical properties
(possibly diversity oriented synthesis libraries61) and screening of natural
products, which as yet have not been exhaustively mined for new antimi-
crobials. In addition, the GlaxoSmithKline experience also suggests that
it is easier to find the target of a given lead compound than to engineer a
lead compound to have greater permeability; thus identification of new
antimicrobials is likely to be more fruitful in whole cell–based screens
rather than in in vitro target-based screens. We suggest that this approach
should be expanded to include whole-organism screening, which has the
advantage that one can identify small molecules that target either the host
or in vivo essential gene functions of the pathogen. Given the dearth of
antimicrobials with novel modes of action against Gram-negative hospi-
tal pathogens currently in phase 1 clinical trials, it is estimated that it may
be 10 to 15 years before antimicrobials against this class of pathogen are
available for therapeutic use59. We believe that this makes the argument
for pursuing therapeutics that target in vivo essential gene functions all
547
REVIEW
the more compelling, as antibiotic resistance continues to evolve and the
need for new antimicrobials continues to grow.
ACKNOWLEDGMENTS
We thank E.J. Rubin, J.E. Gomez and S.A. Stanley for helpful discussions and critical
reading of this manuscript and J.S.W. Lee for assistance in preparing figures.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturechemicalbiology
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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