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

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<ul><li><p>t,2</p><p>1Paratek Pharmaceuticals, 75 Kneeland Street, Boston, MA 02111, USA2</p><p>prevent infection. inhibiting compounds might be less likely to generate resistance is</p><p>chemical classes to obtain drugs with expanded spectrums</p><p>of antibacterial activity. Besides derivatives of other antibio-</p><p>to have a reduced propensity to select for resistance, as a</p><p>consequence of lacking intrinsic antibacterial activity, and</p><p>Drug Discovery Today: Therapeutic Strategies Vol. 1, No. 4 2004</p><p>hamtics, only two novel agents (a cyclic lipopeptide and an</p><p>oxazolidinone) and a new streptogramin combination have</p><p>reached clinical availability in the past two decades [1].</p><p>Current development efforts have made little headway in</p><p>resolving the resistance problem, and large pharmaceutical</p><p>companies are exiting the field.</p><p>must act on bacterial-specific targets. Although no one agent</p><p>boasts all of these qualifications, recent data suggest that the</p><p>time might be ripe for fully exploiting this new therapeutic</p><p>paradigm.</p><p>Here, we discuss small molecules or proteins that target</p><p>gene products involved in infection or VIRULENCE (see Glos-</p><p>sary), and those that use other strategies not involving</p><p>growth inhibition. Vaccines against specific microbial anti-</p><p>gens or toxins are not addressed.*Corresponding author: (M.N. Alekshun) malekshun@paratekpharm.com</p><p>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,</p><p>clinical microbiology add to the complexity of achieving success withthese novel interventions. However, as the clinical options for</p><p>treatment of infectious disease are eroded by resistance the time mightbe ripe for exploiting these strategies.</p><p>Introduction</p><p>The use of antibiotics to treat infectious diseases has provided</p><p>an immeasurable benefit to human health, but the wide-</p><p>spread emergence of bacteria that are resistant to these ther-</p><p>apeutics has raised grave concern about the future of an</p><p>antimicrobial approach. The pharmaceutical industry has,</p><p>for decades, responded by synthesizing derivatives of existing</p><p>As an alternative to antibiotics, targeting of VIRULENCE FAC-</p><p>TORS (see Glossary) has been viewed cautiously, but repeat-</p><p>edly. For this approach to be successful, the novel agents needTufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111</p><p>Targeting components of the infectious process as a</p><p>means to prevent infection has long been considered as</p><p>an alternative to classic antimicrobial therapies.</p><p>Although no clinically used drugs have yet emerged</p><p>from these efforts, the dwindling supply of anti-infec-</p><p>tive treatment options within the physicians arma-</p><p>mentarium has stoked a renewed interest in the</p><p>identification and development of novel strategies toof Molecular Biology and Microbiology and of Medicine,</p><p>SA</p><p>Section Editor:Gary Woodnutt Diversa Corp., San Diego, CA, USA</p><p>There are multiple virulence factors that are required to initiate andmaintain infection in the host. As many of these are external to the</p><p>infecting cell, they provide clear target opportunities that wouldcircumvent the need for cellular penetration. However, by definition,</p><p>compounds that inhibit these targets would not kill the organism andthus many of the screening processes currently used will not be</p><p>applicable. The difficulties associated with progressing targets of thistype are discussed with some key examples of areas that might become</p><p>clinical candidates in the near future. The suggestion that virulenceinfection: to kill or nThe Center for Adaptation Genetics and Drug Resistance, and the Departments</p><p>, UTHERAPEUTICSTRATEGIES</p><p>DRUG DISCOVERY</p><p>TODAY</p><p>Targeting virulence</p><p>Michael N. Alekshun1,*, Stuart B. Levy1</p><p>Editors-in-Chief</p><p>Raymond Baker formerly University of Sout</p><p>Eliot Ohlstein GlaxoSmithKline, USA</p><p>Infectious diseaseso preventot to kill?</p><p>pton, UK and Merck Sharp &amp; Dohme, UK</p></li><li><p>Broad-spectrum approaches</p><p>Strategies that target transcription factors</p><p>Given that infection is regulated primarily at the level of</p><p>transcription, SMALL-MOLECULE INHIBITORS (see Glossary) of bac-</p><p>terial transcription factors can be expected to exhibit pleio-</p><p>tropic effects on the virulence phenotype. Proteins within the</p><p>AraC (MarA) and MarR protein families are attractive targets</p><p>dramatically prevented these organisms from colonizing the</p><p>mouse kidney (M.N. Alekshun et al., unpublished). These</p><p>results prompted efforts to identify small molecule AraC</p><p>(MarA) inhibitors. Many of these newly identified agents</p><p>have activity in vitro against proteins from E. coli, Salmonella</p><p>enterica serovar Typhimurium, Proteus vulgaris and P. aerugi-</p><p>nosa and many exhibited infection prevention in the murine</p><p>UTI model (M.N. Alekshun et al., unpublished).</p><p>Members of the SarA protein family in Staphylococcus aureus</p><p>are MarR orthologs. Recent data have indicated a beneficial</p><p>effect of acetylsalicylate in an experimental rabbit model of S.</p><p>aureus endocarditis [7]. More current studies have shown that</p><p>salicylate attenuated virulence in both laboratory and clini-</p><p>cally derived S. aureus isolates by negatively affecting the</p><p>interaction of the pathogen with fibronectin and fibrinogen</p><p>and exotoxin production in vitro [8]. This effect was depen-</p><p>dent on the presence of SarA. Thus, the modulating effects of</p><p>Drug Discovery Today: Therapeutic Strategies | Infectious diseases Vol. 1, No. 4 2004</p><p>Glossary</p><p>Pathogens: organisms that are capable of causing disease, including</p><p>classical pathogens, opportunists and commensals.</p><p>Small-molecule inhibitors: low molecular weight organic chemicals</p><p>that target and inhibit the function of a virulence factor.</p><p>Virulence: the capacity of a bacterium to cause disease.</p><p>Virulence factors: traits expressed by bacteria that aid the infectious</p><p>process. Might encompass an antibiotic-resistance determinant, an</p><p>adhesin, an invasion, a toxin, biofilm formation and so on.</p><p>ar</p><p>ype</p><p>kid</p><p>co</p><p>e infor these efforts because they regulate virulence in many</p><p>medically important Gram-negative [2] and Gram-positive</p><p>[3] PATHOGENS (see Glossary). With regard to the former, inac-</p><p>tivation of the gene specifying an AraC (MarA) family</p><p>member in Pseudomonas aeruginosa [4], Vibrio cholerae [5]</p><p>and Yersinia pestis [6] renders these organisms avirulent</p><p>(Table 1).</p><p>Naturally occurring small molecule modulators are known</p><p>to affect many AraC (MarA) family members, for example,</p><p>Escherichia coli AraC (arabinose) and Rob (bile salts, fatty acids</p><p>and dipyridyl) and V. cholerae ToxT (TcpN) (bile salts). Thus, it</p><p>is reasonable to envisage additional medicinal chemistry</p><p>efforts that would exploit these natural scaffolds. Using a</p><p>murine model of ascending pyelonephritis (urinary tract</p><p>infection [UTI]), we have found that removal of MarA and</p><p>its paralogs from multi-drug resistant uropathogenic E. coli</p><p>Table 1. Effect(s) of deleting a gene(s) specifying an AraC (M</p><p>Organism Protein (alternative designation) Phenot</p><p>Escherichia coli MarA, SoxS, Rob Reduced</p><p>Proteus mirabilis UreR Reduced</p><p>and urinYersinia pestis LcrF Reduced let</p><p>Pseudomonas aeruginosa ExsA Reduced let</p><p>Vibrio cholerae ToxT Reduced let</p><p>Staphylococcus aureus Uncharacterized Reduced ab</p><p>Streptococcus pneumoniae rr09 (SPr0578 or SP0661) Reduced let</p><p>SP1433 Reduced CI</p><p>Mycobacterium tuberculosis Rv1395 Reduced lun</p><p>Rv1931c Reduced lun</p><p>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</p><p>relative to the wild-type organism the mutant is less virulent.</p><p>484 www.drugdiscoverytoday.comsalicylate on the virulence of S. aureus in vitro correlated with a</p><p>positive outcome in vivo.</p><p>Strategies that target quorum-sensing systems</p><p>Both Gram-negative and Gram-positive bacteria, including P.</p><p>aeruginosa, E. coli, S. aureus and the streptococci, use quorum</p><p>sensing (QS) to regulate the expression of many virulence</p><p>factors [9]. In Gram-negative bacteria, small molecules called</p><p>acylated homoserine lactones (AHLs) act as the QS signals,</p><p>whereas small peptides fill this role in Gram-positive organ-</p><p>isms. Some virulence factors regulated by QS include biofilm</p><p>formation, the development of competence, secretion of</p><p>exotoxins and enzymes and sporulation. There are several</p><p>recent developments that have documented the efficacy of</p><p>AHL antagonists in animal models of infection [1012].</p><p>Hentzer et al. [11] and Wu et al. [10] used a murine model of</p><p>P. aeruginosa pulmonary infection to show that treatment</p><p>A) protein in an animal model(s) of infection</p><p>of mutant organisms in infection models in vivo Refs</p><p>ney colonization in UTI model M.N. Alekshun</p><p>et al., unpublished</p><p>lonization of kidneys, bladder</p><p>a cochallenge UTI modela[42]</p><p>hality and CI in a model of bacteremiab [6]</p><p>hality in a pneumonia model [4]</p><p>hality following orogastric infection [43]</p><p>scess formation and systemic dissemination [44]</p><p>hality and bacteremia [45,46]b [47]</p><p>g burden in a model of bacteremia [48]</p><p>g and spleen burden [49]</p><p>t bacteria. Total CFU/g of tissue is then determined for each organism.</p><p>ost infected with a mixture of both organisms. A reduced competitive index indicates that</p></li><li><p>Vol. 1, No. 4 2004 Drug Discovery Today: Therapeutic Strategies | Infectious diseaseswith synthetic furanones (initially derived from the marine</p><p>alga Delisea pulchra) resulted in a three-log decrease in the</p><p>colony forming units (CFU) per gram of lung tissue. The</p><p>pathologic response of the lungs to the infection was less</p><p>severe and fewer abscesses were found in infected mice trea-</p><p>ted with these agents [10].</p><p>Initial studies with naturally occurring thiolactone-con-</p><p>taining peptides demonstrated infection prevention activity</p><p>in a subcutaneous S. aureus abscess mouse model of infection</p><p>[13]. More recently, DellAcqua et al. [12] used a vascular-graft</p><p>rat model of S. aureus and Staphylococcus epidermidis (includ-</p><p>ing both susceptible and multi-drug resistant strains) infec-</p><p>tion to show that the delivery of a QS-peptide antagonist,</p><p>either by local or parenteral administration, reduced signifi-</p><p>cantly the bacterial load on an implanted Dacron graft. The</p><p>combination of local and parenteral treatments was success-</p><p>ful in effecting complete protection in this model [12].</p><p>Narrow-spectrum approaches</p><p>Strategies that target toxins</p><p>The selective targeting of bacterial toxins has been viewed as a</p><p>precise alternative to classic antibiotic therapy and has</p><p>received renewed interest in light of the anthrax attacks in</p><p>the US in 2001. Two groups have demonstrated efficacy of</p><p>peptide-based inhibitors of anthrax toxin in a rat model of</p><p>toxicity (intoxication) [14,15]. In this model, rats are given a</p><p>mixture of the anthrax protective antigen and lethal factor</p><p>[PA and LF; the combination of which is referred to as lethal</p><p>toxin (LeTx)] and death of the host occurs within hours after</p><p>inoculation. More recently, three other groups have identi-</p><p>fied small-molecule inhibitors of the anthrax LF. Two of these</p><p>groups have solved the 3D structures of toxin-inhibitor co-</p><p>crystal complexes thereby paving the way for approaches in</p><p>structure based drug design (SBDD; see Ref. [16] and refer-</p><p>ences therein). Although some of these compounds offer</p><p>protection against LF-mediated cytotoxicity in vitro, only</p><p>one has shown small-molecule efficacy in the LeTx rat model</p><p>[17]. In this particular experiment, administration of the</p><p>small-molecule inhibitor in conjunction with, or separate</p><p>from, the LeTx demonstrated efficacy in vivo [17].</p><p>Like many Gram-negative pathogens, Y. pestis uses a type-</p><p>III secretion system (TTSS) to deliver host effector proteins</p><p>(Yops) into mammalian cells during infection. YopH is a</p><p>potent tyrosine phosphatase that interferes with phagocyto-</p><p>sis; Y. pestis mutants lacking yopH are avirulent. Liang et al.</p><p>[18] screened a small library of commercially available car-</p><p>boxylic acids and identified a potent YopH inhibitor that,</p><p>relative to other mammalian tyrosine phosphatases, exhib-</p><p>ited specificity for the bacterial protein. A recent YopH-inhi-</p><p>bitor co-crystal structure should facilitate SDBB in this area</p><p>[19] but the activity in vivo of these inhibitors remains to bedetermined.Strategies that target cell-surface modification</p><p>Using bacteriophage as an alternative anti-infective thera-</p><p>peutic strategy has received renewed interest but the use of</p><p>live bacteriophage, or phage-derived lysins, would ultimately</p><p>result in death (lysis) of the infecting organism and thereby</p><p>not fall within the anti-infection paradigm. Recent experi-</p><p>ments, however, have used a phage-derived endosialidase</p><p>(endoE), which specifically and selectively hydrolyzes the</p><p>E. coli K1 capsular polysaccharide and so alters pathogen</p><p>virulence without killing the organism [20]. Intraperitoneal</p><p>administration of endoE in a neonatal rat model of E. coli</p><p>bacteremia results in almost complete protection from bac-</p><p>teremia and blood samples were pathogen-free within 24 h</p><p>following the initiation of therapy [20].</p><p>Strategies that target surface proteins: pili, adhesins,</p><p>chaperons and sortase</p><p>E. coli type-I and type-P pili are virulence factors that have</p><p>important roles in cystitis and pyelonephritis, respectively.</p><p>Both possess domains (adhesins) that are responsible for the</p><p>binding to polysaccharide receptors that are located on the</p><p>surfaces of host cells. The adhesins are assembled onto the pili</p><p>by chaperones and efforts to identify small-molecule-chaper-</p><p>one inhibitors (pilicides) have been described using a cha-</p><p>peronadhesin co-crystal structure [21]. These compounds</p><p>(bicyclic b-lactams, 2-pyridones and N-substituted amino</p><p>acids [22]) appear to effectively dissociate the chaperone</p><p>adhesin complex in vitro by targeting the active site of the</p><p>chaperone [21]. Efficacy in vivo, however, has not been deter-</p><p>mined. Carbohydrate-based compounds that target the active</p><p>site of the adhesin have also been described [23,24]. These</p><p>agents exhibit modest activity in vitro, as measured using both</p><p>biophysical and hemagglutination assays [23,24]. The effi-</p><p>cacy of these particular compounds in an animal model of</p><p>UTI has not been shown, but an effect in vivo has been shown</p><p>with glycolipid analogs [25].</p><p>The Streptococcus mutans SpaA is an adhesin that functions</p><p>in the colonization of the oral cavity and the development of</p><p>dental caries. Recent studies have identified a synthetic pep-</p><p>tide (p1025) that inhibited binding of SpaA to salivary agglu-</p><p>tinin in vitro [26]. The efficacy of p1025 in human volunteers</p><p>was then tested in a small double-blind placebo controlled</p><p>trial [26]. The oral cavity of all volunteers was first deconta-</p><p>minated using chlorhexidine gluconate and, subsequently,</p><p>four patients were treated with p1025, four with a buffer</p><p>placebo and three with an inactive peptide control [26].</p><p>Bacterial recolonization was monitored for a period of 120</p><p>days and, although the...</p></li></ul>