THERAPEUTICSTRATEGIES

DRUG DISCOVERY

TODAY

Drug Discovery Today: Therapeutic Strategies Vol. 1, No. 4 2004

Editors-in-Chief

Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK

Eliot Ohlstein – GlaxoSmithKline, USA

Infectious diseases

Targeting virulence to preventinfection: to kill or not to kill?Michael N. Alekshun1,*, Stuart B. Levy1,2

1Paratek Pharmaceuticals, 75 Kneeland Street, Boston, MA 02111, USA2The Center for Adaptation Genetics and Drug Resistance, and the Departments of Molecular Biology and Microbiology and of Medicine,

Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA

Targeting components of the infectious process as a

means to prevent infection has long been considered as

an alternative to classic antimicrobial therapies.

Although no clinically used drugs have yet emerged

from these efforts, the dwindling supply of anti-infec-

tive treatment options within the physician’s arma-

mentarium has stoked a renewed interest in the

identification and development of novel strategies to

prevent infection.

*Corresponding author: (M.N. Alekshun) malekshun@paratekpharm.com

1740-6773/$ � 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddstr.2004.10.006

Section Editor:Gary Woodnutt – Diversa Corp., San Diego, CA, USA

There are multiple virulence factors that are required to initiate andmaintain infection in the host. As many of these are external to the

infecting cell, they provide clear target opportunities that wouldcircumvent the need for cellular penetration. However, by definition,

compounds that inhibit these targets would not kill the organism andthus many of the screening processes currently used will not be

applicable. The difficulties associated with progressing targets of thistype are discussed with some key examples of areas that might become

clinical candidates in the near future. The suggestion that virulenceinhibiting compounds might be less likely to generate resistance is

intriguing but unproven. The lack of a clear path through regulatorybodies and the inability to test activity by traditional, and well accepted,

clinical microbiology add to the complexity of achieving success withthese novel interventions. However, as the clinical options for

treatment of infectious disease are eroded by resistance the time mightbe ripe for exploiting these strategies.

an immeasurable benefit to human health, but the wide-

Introduction

The use of antibiotics to treat infectious diseases has provided

spread emergence of bacteria that are resistant to these ther-

apeutics has raised grave concern about the future of an

antimicrobial approach. The pharmaceutical industry has,

for decades, responded by synthesizing derivatives of existing

chemical classes to obtain drugs with ‘expanded’ spectrums

of antibacterial activity. Besides derivatives of other antibio-

tics, only two novel agents (a cyclic lipopeptide and an

oxazolidinone) and a new streptogramin combination have

reached clinical availability in the past two decades [1].

Current development efforts have made little headway in

resolving the resistance problem, and large pharmaceutical

companies are exiting the field.

As an alternative to antibiotics, targeting of VIRULENCE FAC-

TORS (see Glossary) has been viewed cautiously, but repeat-

edly. For this approach to be successful, the novel agents need

to have a reduced propensity to select for resistance, as a

consequence of lacking intrinsic antibacterial activity, and

must act on bacterial-specific targets. Although no one agent

boasts all of these qualifications, recent data suggest that the

time might be ripe for fully exploiting this new therapeutic

paradigm.

Here, we discuss small molecules or proteins that target

gene products involved in infection or VIRULENCE (see Glos-

sary), and those that use other strategies not involving

growth inhibition. Vaccines against specific microbial anti-

gens or toxins are not addressed.

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Drug Discovery Today: Therapeutic Strategies | Infectious diseases Vol. 1, No. 4 2004

Glossary

Pathogens: organisms that are capable of causing disease, including

classical pathogens, opportunists and commensals.

Small-molecule inhibitors: low molecular weight organic chemicals

that target and inhibit the function of a virulence factor.

Virulence: the capacity of a bacterium to cause disease.

Virulence factors: traits expressed by bacteria that aid the infectious

process. Might encompass an antibiotic-resistance determinant, an

adhesin, an invasion, a toxin, biofilm formation and so on.

‘Broad-spectrum’ approaches

Strategies that target transcription factors

Given that infection is regulated primarily at the level of

transcription, SMALL-MOLECULE INHIBITORS (see Glossary) of bac-

terial transcription factors can be expected to exhibit pleio-

tropic effects on the virulence phenotype. Proteins within the

AraC (MarA) and MarR protein families are attractive targets

for these efforts because they regulate virulence in many

medically important Gram-negative [2] and Gram-positive

[3] PATHOGENS (see Glossary). With regard to the former, inac-

tivation of the gene specifying an AraC (MarA) family

member in Pseudomonas aeruginosa [4], Vibrio cholerae [5]

and Yersinia pestis [6] renders these organisms avirulent

(Table 1).

Naturally occurring small molecule modulators are known

to affect many AraC (MarA) family members, for example,

Escherichia coli AraC (arabinose) and Rob (bile salts, fatty acids

and dipyridyl) and V. cholerae ToxT (TcpN) (bile salts). Thus, it

is reasonable to envisage additional medicinal chemistry

efforts that would exploit these natural scaffolds. Using a

murine model of ascending pyelonephritis (urinary tract

infection [UTI]), we have found that removal of MarA and

its paralogs from multi-drug resistant uropathogenic E. coli

Table 1. Effect(s) of deleting a gene(s) specifying an AraC (Mar

Organism Protein (alternative designation) Phenotype

Escherichia coli MarA, SoxS, Rob Reduced kid

Proteus mirabilis UreR Reduced co

and urine in

Yersinia pestis LcrF Reduced let

Pseudomonas aeruginosa ExsA Reduced let

Vibrio cholerae ToxT Reduced let

Staphylococcus aureus Uncharacterized Reduced ab

Streptococcus pneumoniae rr09 (SPr0578 or SP0661) Reduced let

SP1433 Reduced CI

Mycobacterium tuberculosis Rv1395 Reduced lun

Rv1931c Reduced lun

Abbreviations: CI, competitive index; UTI, urinary tract infection.a In the cochallenge experiment, mice are infected with an �1:1 ratio of wild-type and mutanb The competitive index represents the ratio of wild-type to mutant bacteria recovered from a h

relative to the wild-type organism the mutant is less virulent.

484 www.drugdiscoverytoday.com

dramatically prevented these organisms from colonizing the

mouse kidney (M.N. Alekshun et al., unpublished). These

results prompted efforts to identify small molecule AraC

(MarA) inhibitors. Many of these newly identified agents

have activity in vitro against proteins from E. coli, Salmonella

enterica serovar Typhimurium, Proteus vulgaris and P. aerugi-

nosa and many exhibited infection prevention in the murine

UTI model (M.N. Alekshun et al., unpublished).

Members of the SarA protein family in Staphylococcus aureus

are MarR orthologs. Recent data have indicated a beneficial

effect of acetylsalicylate in an experimental rabbit model of S.

aureus endocarditis [7]. More current studies have shown that

salicylate attenuated virulence in both laboratory and clini-

cally derived S. aureus isolates by negatively affecting the

interaction of the pathogen with fibronectin and fibrinogen

and exotoxin production in vitro [8]. This effect was depen-

dent on the presence of SarA. Thus, the modulating effects of

salicylate on the virulence of S. aureus in vitro correlated with a

positive outcome in vivo.

Strategies that target quorum-sensing systems

Both Gram-negative and Gram-positive bacteria, including P.

aeruginosa, E. coli, S. aureus and the streptococci, use quorum

sensing (QS) to regulate the expression of many virulence

factors [9]. In Gram-negative bacteria, small molecules called

acylated homoserine lactones (AHLs) act as the QS signals,

whereas small peptides fill this role in Gram-positive organ-

isms. Some virulence factors regulated by QS include biofilm

formation, the development of competence, secretion of

exotoxins and enzymes and sporulation. There are several

recent developments that have documented the efficacy of

AHL antagonists in animal models of infection [10–12].

Hentzer et al. [11] and Wu et al. [10] used a murine model of

P. aeruginosa pulmonary infection to show that treatment

A) protein in an animal model(s) of infection

of mutant organisms in infection models in vivo Refs

ney colonization in UTI model M.N. Alekshun

et al., unpublished

lonization of kidneys, bladder

a cochallenge UTI modela[42]

hality and CI in a model of bacteremiab [6]

hality in a pneumonia model [4]

hality following orogastric infection [43]

scess formation and systemic dissemination [44]

hality and bacteremia [45,46]b [47]

g burden in a model of bacteremia [48]

g and spleen burden [49]

t bacteria. Total CFU/g of tissue is then determined for each organism.

ost infected with a mixture of both organisms. A reduced competitive index indicates that

Vol. 1, No. 4 2004 Drug Discovery Today: Therapeutic Strategies | Infectious diseases

with synthetic furanones (initially derived from the marine

alga Delisea pulchra) resulted in a three-log decrease in the

colony forming units (CFU) per gram of lung tissue. The

pathologic response of the lungs to the infection was less

severe and fewer abscesses were found in infected mice trea-

ted with these agents [10].

Initial studies with naturally occurring thiolactone-con-

taining peptides demonstrated infection prevention activity

in a subcutaneous S. aureus abscess mouse model of infection

[13]. More recently, Dell’Acqua et al. [12] used a vascular-graft

rat model of S. aureus and Staphylococcus epidermidis (includ-

ing both susceptible and multi-drug resistant strains) infec-

tion to show that the delivery of a QS-peptide antagonist,

either by local or parenteral administration, reduced signifi-

cantly the bacterial load on an implanted Dacron graft. The

combination of local and parenteral treatments was success-

ful in effecting complete protection in this model [12].

‘Narrow-spectrum’ approaches

Strategies that target toxins

The selective targeting of bacterial toxins has been viewed as a

precise alternative to classic antibiotic therapy and has

received renewed interest in light of the anthrax attacks in

the US in 2001. Two groups have demonstrated efficacy of

peptide-based inhibitors of anthrax toxin in a rat model of

toxicity (intoxication) [14,15]. In this model, rats are given a

mixture of the anthrax protective antigen and lethal factor

[PA and LF; the combination of which is referred to as lethal

toxin (LeTx)] and death of the host occurs within hours after

inoculation. More recently, three other groups have identi-

fied small-molecule inhibitors of the anthrax LF. Two of these

groups have solved the 3D structures of toxin-inhibitor co-

crystal complexes thereby paving the way for approaches in

structure based drug design (SBDD; see Ref. [16] and refer-

ences therein). Although some of these compounds offer

protection against LF-mediated cytotoxicity in vitro, only

one has shown small-molecule efficacy in the LeTx rat model

[17]. In this particular experiment, administration of the

small-molecule inhibitor in conjunction with, or separate

from, the LeTx demonstrated efficacy in vivo [17].

Like many Gram-negative pathogens, Y. pestis uses a type-

III secretion system (TTSS) to deliver host effector proteins

(Yops) into mammalian cells during infection. YopH is a

potent tyrosine phosphatase that interferes with phagocyto-

sis; Y. pestis mutants lacking yopH are avirulent. Liang et al.

[18] screened a small library of commercially available car-

boxylic acids and identified a potent YopH inhibitor that,

relative to other mammalian tyrosine phosphatases, exhib-

ited specificity for the bacterial protein. A recent YopH-inhi-

bitor co-crystal structure should facilitate SDBB in this area

[19] but the activity in vivo of these inhibitors remains to be

determined.

Strategies that target cell-surface modification

Using bacteriophage as an alternative anti-infective thera-

peutic strategy has received renewed interest but the use of

live bacteriophage, or phage-derived lysins, would ultimately

result in death (lysis) of the infecting organism and thereby

not fall within the anti-infection paradigm. Recent experi-

ments, however, have used a phage-derived endosialidase

(endoE), which specifically and selectively hydrolyzes the

E. coli K1 capsular polysaccharide and so alters pathogen

virulence without killing the organism [20]. Intraperitoneal

administration of endoE in a neonatal rat model of E. coli

bacteremia results in almost complete protection from bac-

teremia and blood samples were pathogen-free within 24 h

following the initiation of therapy [20].

Strategies that target surface proteins: pili, adhesins,

chaperons and sortase

E. coli type-I and type-P pili are virulence factors that have

important roles in cystitis and pyelonephritis, respectively.

Both possess domains (adhesins) that are responsible for the

binding to polysaccharide receptors that are located on the

surfaces of host cells. The adhesins are assembled onto the pili

by chaperones and efforts to identify small-molecule-chaper-

one inhibitors (pilicides) have been described using a cha-

peron–adhesin co-crystal structure [21]. These compounds

(bicyclic b-lactams, 2-pyridones and N-substituted amino

acids [22]) appear to effectively dissociate the chaperone–

adhesin complex in vitro by targeting the active site of the

chaperone [21]. Efficacy in vivo, however, has not been deter-

mined. Carbohydrate-based compounds that target the active

site of the adhesin have also been described [23,24]. These

agents exhibit modest activity in vitro, as measured using both

biophysical and hemagglutination assays [23,24]. The effi-

cacy of these particular compounds in an animal model of

UTI has not been shown, but an effect in vivo has been shown

with glycolipid analogs [25].

The Streptococcus mutans SpaA is an adhesin that functions

in the colonization of the oral cavity and the development of

dental caries. Recent studies have identified a synthetic pep-

tide (p1025) that inhibited binding of SpaA to salivary agglu-

tinin in vitro [26]. The efficacy of p1025 in human volunteers

was then tested in a small double-blind placebo controlled

trial [26]. The oral cavity of all volunteers was first deconta-

minated using chlorhexidine gluconate and, subsequently,

four patients were treated with p1025, four with a buffer

placebo and three with an inactive peptide control [26].

Bacterial recolonization was monitored for a period of 120

days and, although the presence of S. mutans was evident in

patients receiving either the placebo (at 21 days) or the

inactive peptide control (at 58 days), 75% of the individuals

in the p1025 treatment group remained free of S. mutans [26].

Importantly, recolonization of Actinomyces naeslundii, a

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Drug Discovery Today: Therapeutic Strategies | Infectious diseases Vol. 1, No. 4 2004

Gram-positive oral commensal, was unaffected by the control

peptide and the therapeutic peptide [26].

The sortase enzymes (SrtA and SrtB) are cysteine protease-

transpeptidase Gram-positive virulence factors that are

responsible for the covalent attachment of surface-exposed

proteins, including protein A, fibronectin and collagen-bind-

ing proteins. Compared with wild-type strains, S. aureus that

lack srtA are less lethal, produce a reduced inflammatory

response, and are attenuated in mouse kidney and rat endo-

carditis infection models [27,28]. Peptide- [29], carbohydrate-

[30] and small-molecule- [31–33] based SrtA inhibitors have

been described. Although compounds that have evolved

from these efforts possess activity in vitro, their efficacy in

vivo awaits experimental verification.

Probiotics

Bacterial interference by competitive exclusion

Probiotics [beneficial (commensal) bacteria, for example,

Lactobacillus spp., Bifidobacterium spp. and E. coli Nissle

1917] have long been touted as an alterative to pharmaco-

logic intervention for the treatment of many diseases includ-

ing inflammatory bowel disease, UTI, wound infections,

atopic eczema, vaginitis, arthritis and travellers’ diarrhea.

Probiotics are most commonly administered by oral con-

sumption and data from many recent randomized, double-

blind and comparative or placebo-controlled clinical trials are

very encouraging [34].

From the clinical perspective, Lactobacillus rhamnosus strain

GG (LGG) is probably the most extensively studied probiotic

[35]. LGG is ‘non-pathogenic’ and the beneficial effects of this

and other probiotic organisms are multifactorial. LGG adheres

strongly to tissue culture cells in vitro, and colonizes the human

gastrointestinal tract. Experiments in vivo suggest that it com-

petes with the normal flora and pathogenic bacteria for host

colonization and has positive effects on intestinal and sys-

temic immunity [35]. Notably, LGG produce antimicrobial

compounds that exhibit a broad spectrum of activity in vitro

[36], but their possible role in the multifaceted competitive

exclusion activity of the organism is unclear.

In a fascinating set of recent experiments, subcutaneous

delivery of Lactobacillus salivarius 118 to interleukin-10 (IL-

10) deficient mice had a statistically significant anti-inflam-

matory effect on the development of colitis and arthritis in

this species [37]. Lactobacillus fermentum RC-14 has been

shown to prevent abscess formation in a rat model of surgical

implant S. aureus infection [38]. The protective effect was

subsequently attributed to a secreted biosurfactant lacking

intrinsic antimicrobial activity and containing many col-

lagen-binding proteins [38].

Agents in clinical development

The vast majority of anti-virulence therapies are yet to be

studied in the clinical setting, but there is hope that one will

486 www.drugdiscoverytoday.com

make it. Clostridium difficile is an important cause of

nosocomial diarrhea and the treatment of infected patients

usually requires therapy with either vancomycin or metro-

nidazole, two drugs that have profound effects on the intest-

inal flora. GT160-246 (Tolevamer1) is a high molecular

weight anionic polymer that lacks antimicrobial activity

and targets the A and B toxins of C. difficile [39]. This drug

has recently completed a randomized, double-blind phase-II

clinical trial in which the efficacy of two separate doses of

Tolevamer1 was compared with oral vancomycin. Interest-

ingly, the time to resolution of diarrhea and rate of recurrence

was similar for both therapies, that is, one antibacterial and

the other not. A larger phase-III clinical trial is currently being

planned.

Conclusions

The concept of targeting virulence as a new therapeutic

strategy to prevent (or treat) infection has relied on the

premise that affecting the ability of an organism to cause

disease, for example, preventing the function of a virulence

factor(s), would render it non-infectious. The approach, in

theory, seems easily achievable but in reality there are several

outstanding issues.

Because virulence factors are generally required for growth

in vivo, the in vitro whole-cell screens that would normally be

employed in classical antibiotic discovery programs are of

limited use. The adoption of this new preventative (or ther-

apeutic) concept, therefore, requires a paradigm shift in the

research and development (R&D) infrastructure of an orga-

nization. Standard minimal inhibitory concentrations (MIC)

determinations will need to be replaced with other assays that

are predictive of virulence factor expression (e.g. toxin excre-

tion and cytotoxicity towards tissue culture cells). Also, a

change in the mindset of the regulatory authorities (e.g. the

Food and Drug Administration and the European Medicines

Agency) will be needed. These factors ultimately affect the

economic considerations that drive modern-day R&D efforts

and, collectively, are major reasons why the discovery and

development of these new agents has been so difficult to

achieve.

Many of the approaches described herein have relied on

peptides to demonstrate feasibility. Although these agents are

suitable from the basic science perspective, the production

costs, stability in vivo, pharmacokinetic considerations, for

example, of small-peptide-derived therapeutics it is unlikely

to garner enthusiastic support for real-life treatment scenar-

ios. Fortunately, many researchers are moving in the

direction of small-molecule therapeutics for anthrax toxin

(Table 2).

The development of resistance to these new approaches

has also been the subject of discussion. History has taught

that the overuse (and misuse) of any (small-molecule) anti-

infective will ultimately lead to resistance [40]. Because the

Vol. 1, No. 4 2004 Drug Discovery Today: Therapeutic Strategies | Infectious diseases

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Links

� Paratek Pharmaceuticals: http://www.paratekpharm.com

� Tolevamer1 (Genzyme): http://www.genzyme.ie/corp/news/all_

news/GENZ%20PR-050304b.asp

� Biosignal: http://www.biosignal.com.au/

� 4SC: http://www.4sc.de/

� Cengenet Therapeutics: http://www.strubix.com

examples that are described in this review are not

inhibitory to the growth of the bacterium, the direct selec-

tive pressure by the prophylactic molecule should be

reduced, and any selection occurring by the presence of

the drug outside the therapeutic area (e.g. in the environ-

ment) would be unlikely. It could still be argued that a

selective pressure could occur in vivo. In this scenario, how-

ever, even the emergence of several hundred ‘resistant’

mutants might not be sufficient to cause or maintain infec-

tion.

Ultimately, the efficacy of these new therapies must be

proven in human clinical trials. With classic antibiotics,

multiple (large) active comparator-controlled and non-

inferiority pivotal studies are generally required for approval.

Will it be more appropriate to test initially the therapeutic

efficacy of new antivirulence agents in instances where pla-

cebo-controlled trials can be conducted, such as for traveller’s

diarrhea or post-surgical UTI? As no current antibacterial

agent has a label indication for the prevention of infection,

what clinical model will be used to demonstrate preventative

therapy?

Efforts in the fields of cancer chemotherapy [41] and

chronic diseases, such as atherosclerosis, inflammation and

diabetes, are actively seeking new treatment modalities,

which include the design of agents that target regulatory

systems and pathways. Not surprisingly, several products

from these efforts have reached clinical use. Thus, one would

hope that the ‘mature’ antibacterial field could adapt to the

paradigms that have already been accepted in these other

therapeutic areas.

Related articles

Alksne, L.E. and Projan, S.J. (2000) Bacterial virulence as a target for

antimicrobial chemotherapy. Curr. Opin. Biotechnol. 11, 625–636

Lee, Y.M., et al. (2003) Targeting virulence for antimicrobial chemother-

apy. Curr. Opin. Pharmacol. 3, 513–519

Ebert, D. and J.J. Bull (2003) Challenging the trade-off model for the

evolution of virulence: is virulence management feasible? Trends Microbiol.

11, 15–20

Projan, S.J. (2002) New (and not so new) antibacterial targets – from

where and when will the novel drugs come? Curr. Opin. Pharmacol. 2, 513–

522

Shlaes, D.M. (2003) The abandonment of antibacterials: why and where-

fore? Curr. Opin. Pharmacol. 3, 470–473

www.drugdiscoverytoday.com 487

Drug Discovery Today: Therapeutic Strategies | Infectious diseases Vol. 1, No. 4 2004

Outstanding questions

� Will the stand-alone efficacy of these novel agents be sufficient, or will

they need to be used in conjunction with antibiotics?

� If approved, could these agents be used in areas in which infection is

thought to be linked to chronic disease, for example, atherosclerosis,

Alzheimer’s disease and ulcer disease?

� What effect(s) would this new therapeutic approach have on the

colonization of protective commensal organisms?

� Will small-molecule inhibitors of virulence factors work in immuno-

compromised patients?

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