targeting virulence to prevent infection: to kill or not to kill?

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    1Paratek Pharmaceuticals, 75 Kneeland Street, Boston, MA 02111, USA2

    prevent infection. inhibiting compounds might be less likely to generate resistance is

    chemical classes to obtain drugs with expanded spectrums

    of antibacterial activity. Besides derivatives of other antibio-

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

    consequence of lacking intrinsic antibacterial activity, and

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

    hamtics, 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.

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

    1740-6773/$ 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddstr.2004.10.006 www.drugdiscoverytoday.com 483intriguing 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.

    Introduction

    The use of antibiotics to treat infectious diseases has provided

    an immeasurable benefit to human health, but the wide-

    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

    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 needTufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111

    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 physicians arma-

    mentarium has stoked a renewed interest in the

    identification and development of novel strategies toof Molecular Biology and Microbiology and of Medicine,

    SA

    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 virulenceinfection: to kill or nThe Center for Adaptation Genetics and Drug Resistance, and the Departments

    , UTHERAPEUTICSTRATEGIES

    DRUG DISCOVERY

    TODAY

    Targeting virulence

    Michael N. Alekshun1,*, Stuart B. Levy1

    Editors-in-Chief

    Raymond Baker formerly University of Sout

    Eliot Ohlstein GlaxoSmithKline, USA

    Infectious diseaseso preventot to kill?

    pton, UK and Merck Sharp & Dohme, UK

  • 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

    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

    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.

    ar

    ype

    kid

    co

    e infor 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 (M

    Organism Protein (alternative designation) Phenot

    Escherichia coli MarA, SoxS, Rob Reduced

    Proteus mirabilis UreR Reduced

    and urinYersinia 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.comsalicylate 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 [1012].

    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