Antibiotic resistance is a serious public health concern at the global level. Available antibiotics have saved millions of lives, but are progressively losing their efficacy against many bacterial pathogens, and very few new antibiotics are being developed by the pharmaceutical industry. Over the last few decades, progress in understanding the pathogenic process of bacterial infections has led researchers to focus on bacterial virulence factors as potential targets for ‘antivirulence' drugs, i.e. compounds which inhibit the ability of bacteria to cause damage to the host, as opposed to inhibition of bacterial growth which is typical of antibiotics. Hundreds of virulence inhibitors have been examined to date in vitro and/or in animal models, but only a few were entered into clinical trials and none were approved, thus hindering the clinical validation of antivirulence therapy. To breathe new life into antivirulence research and speed-up its transfer to the clinic, antivirulence activities have also been sought in drugs already approved for different therapeutic purposes in humans. If effective, these drugs could be repositioned for antivirulence therapy and have an easier and faster transfer to the clinic. In this work we summarize the approaches which have led to the identification of repurposing candidates with antivirulence activities, and discuss the challenges and opportunities related to antivirulence therapy and drug repurposing. While this approach undoubtedly holds promise for boosting antivirulence drug research, some important issues remain to be addressed in order to make antivirulence drugs viable alternatives to traditional antibacterials.

Towards the post-antibiotic era

The second half of the 20th century witnessed huge strides in the fight against infectious diseases, thanks to the discovery and development of vaccines and antibiotics. During this ‘Golden Age of Antibiotics', impressive research efforts were made in the area of antibiotic discovery, leading to the identification of almost all antibiotic classes known so far [1].

Antibiotics are by definition natural substances produced by microorganisms to compete with other microorganisms in natural environments, implying that antibiotic-producing microorganisms are programmed to resist the antibiotics they produce. Thus, long before antibiotics were discovered, microorganisms evolved molecular tools to drive resistance [2] and it was clear at the beginning of the antibiotic era that resistance could emerge among antibiotic-susceptible microorganisms [3]. While antibiotic resistance represents an unavoidable process of natural selection, the massive use of antibiotics in some settings, such as hospitals and intensive animal farming, has accelerated this process, driving the evolution of bacteria towards evermore antibiotic-resistant phenotypes. Previous surveillance studies have reported an alarmingly high level of resistance to antibiotics for most common bacterial pathogens worldwide, with some species showing a clear trend towards multidrug- or even pan-resistance [4]. This holds true especially for the so-called ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) that cause significant morbidity and mortality to humans because of their impressive level of antibiotic resistance [5].

While novel antibiotics are urgently needed to halt this trend, the pipeline of antibiotic discovery is almost dry. The poor return on investment from antibiotics accounts for the alarmingly slow progress in industrial research in this field [6]. De novo antibiotic discovery is a risky pursuit, since (i) relevant investments are required in the long term, (ii) the chance to identify new compounds is low, (iii) the lifetime of antibiotics is short due to rapid emergence of resistance and (iv) the use of new antibiotics are appropriately restricted, thus lowering sales. This explains why only two new systemic antibacterial agents have been approved by the US Food and Drug Administration (FDA) between 2008 and 2012 [5], and only a few more molecules are in late clinical development [6].

Antivirulence approaches to control infections

Since the end of the 20th century, awareness regarding the risk of failure of antibiotic therapies has increased along with huge progress in the understanding of the fundamental factors that microbes use to cause infection. Hence, researchers started looking at virulence mechanisms as molecular targets for the development of novel anti-infective drugs aimed at inhibiting the infection process rather than bacterial growth [7]. Such molecules are currently referred to as ‘antivirulence' drugs [8].

Bacteria have evolved virulence factors expressed upon the control of complex regulatory mechanisms in order to adapt to a challenging environment, such as the mammalian host, and the majority of them share basic strategies to cause infection. The first step is usually to bind host cells, thanks to the production of adhesins. This is followed by actions aimed at escaping the host immune system, such as invasion of the host cell, biofilm formation and other immune evasion mechanisms. During infection, bacterial growth and restraint of the immune system are ensured by the deployment of factors such as toxins, tissue-degrading enzymes and micronutrient scavenging factors [7].

Given the importance of adhesion in the early phase of infection, several compounds inhibiting this process have been identified so far. As an example, mannosides are able to reduce the bacterial burden in mice infected with uro-pathogenic and adherent-invasive Escherichia coli, by targeting the adhesion protein FimH of type-1 fimbriae [9].

While this strategy is aimed at the direct inactivation of a specific virulence factor, other strategies focused on inhibiting the mechanisms controlling the coordinated expression of different virulence factors. A well-known example is virstatin, which blocks the transcriptional regulator ToxT, hence simultaneously abrogating the expression of two major virulence factors of Vibrio cholerae, the pilus (an adhesion factor) and cholera toxin [10]. Another example is LED209, an inhibitor of QseC, a histidine sensor kinase present in many enterobacteria, which is activated upon detection of microbiota-generated autoinducer-3 or host stress signals (e.g. catecholamines). LED209 exhibits anti-infectious activity in mice infected with Salmonella enterica ser. Typhimurium, Francisella turalensis and adherent-invasive E. coli [9,11].

Quorum sensing (QS) is a chemical communication system which allows many bacteria to coordinate virulence at the population level, and is undoubtedly the regulatory system most studied as a target for antivirulence drugs [12]. In P. aeruginosa, QS positively controls biofilm formation and the expression of a wide array of secreted virulence factors contributing to pathogenesis. The best known molecule inhibiting P. aeruginosa QS is furanone C-30, which showed significant efficacy against P. aeruginosa in a murine lung infection model [13].

Pros and cons of antivirulence drugs

In the comparison between antivirulence drugs and conventional antibiotics, a key issue is that traditional antibiotics hit a small number of cellular processes widely conserved among bacteria, such as envelope biogenesis/stability, replication, transcription or translation [14]. The limited number of targeted functions is consistent with the low rate of antibiotic discovery [15]. Conversely, there is a multiplicity of molecular mechanisms governing bacterial virulence that constitute suitable targets for antivirulence drugs as they are pathogen specific and not present in host cells. Moreover, unlike antibiotics, antivirulence drugs are not expected to damage the beneficial microbiota. The selective targeting of pathogens would be more successful if antivirulence drugs interfere with the expression of phenotypes strictly related to virulence, such as adhesion factors and toxins. Conversely, the possible effect of molecules hampering global regulatory circuits widespread among pathogenic and non-pathogenic bacteria is more difficult to predict. For instance, since the sensor kinase QseC is widespread in Gram-negative bacteria, the possibility that LED209 [11] could affect natural functionality of the resident microbiota cannot be excluded. Therefore, besides the specific activity against the target pathogen, any effect of antivirulence drugs on the beneficial human microbiota should be investigated.

Another important issue sustaining the search for antivirulence drugs is the pillar concept that disarming bacterial pathogens rather than killing them would decrease the evolution rate towards resistance [8]. However, different molecular mechanisms conferring resistance to antivirulence compounds can be envisaged and some of them have been documented. These include: (i) modification of the target, exemplified by the L113P amino acid variant of the transcriptional regulator ToxT, conferring resistance to the cholera toxin inhibitor virstatin [10], (ii) overexpression of the target, as shown in Agrobacterium tumefaciens in which overproduction of the QS receptor TraR decreases the anti-QS activity of analogues of the autoinducer [16] and (iii) overexpression of efflux pumps extruding the antivirulence drug outside the cell, as in the case of P. aeruginosa clinical isolates unaffected by the QS inhibitor furanone C-30 [17].

The existence of mechanisms of resistance does not imply that they will be positively selected during infection. Indeed, resistance mechanisms might emerge but they are only expected to establish if they confer increased fitness to the resistant strain. In principle, since virulence factors are required for the proficient establishment of infection, it is likely that their abrogation reduces bacterial fitness in the host. Examples are given by the decreased P. aeruginosa load in lungs of infected mice treated with furanone C-30 [13], and by the antivirulence compound BPH-652, which improves clearance of S. aureus in mice by inhibiting the production of staphyloxanthin, a bacterial antioxidant pigment conferring protection from reactive oxygen species and neutrophil-based killing [18]. Although the detrimental effect of antivirulence drugs on bacterial fitness in the host can be envisaged, in vitro evolution experiments suggest that drug-resistant variants will not emerge if the therapy targets ‘public' goods, i.e. virulence factors that are secreted and shared between individuals [19]. This is probably due to the fact that when a small fraction of the population produces and secretes virulence factors, these are not sufficient to sustain the growth of the whole population. Moreover, mutants that are resistant to inhibitors of secreted virulence factors experience the metabolic burden for their production with no private benefit in return; this possibly imposes a negative selective pressure for the maintenance of resistant strains in the population [19,20]. Accordingly, a study on P. aeruginosa reported no increase in the fitness of a mutant strain mimicking a genetic variant resistant to an inhibitor of the QS response in a population of sensitive cells [21].

Overall, resistant strains are probably prone to positive selection in vivo only if they gain a ‘private' advantage over the susceptible population. This could be the case for inhibitors of adhesion factors. Conversely, antivirulence drugs should limit the emergence of resistant variants if they target ‘public’ goods shared with susceptible clones, such as secreted virulence factors or communication systems (i.e. QS) governing their expression [19].

Additional issues that deserve consideration are the impact of reduced virulence factor production on the immune system, and the possible selection for hyper-virulent phenotypes. Virulence factors are known to stimulate the immune response, hence decreased expression of virulence could reduce the clearance of pathogens by the immune system. A recent study reported that P. aeruginosa load in infected larvae of Galleria mellonella does not linearly correlate with the availability of pyoverdine [22], a secreted siderophore that acts as an iron scavenger and an inducer of virulence gene expression [23], suggesting that reduced production of the pyoverdine virulence factor could ultimately lead to a decreased stimulation of the immune system [22]. It has also been suggested that antivirulence drugs could select for mutants overproducing virulence factors, hence endowed with increasing virulence potential in the absence of antivirulence treatment [24]. However, to the best of our knowledge, no experimental evidence supporting this hypothesis has been provided so far. Thus, while additional studies are needed to assess the real benefits of antivirulence drugs in vivo, available experimental evidence supports the antivirulence approach as a promising therapeutic option.

Overview of repurposing candidates for antivirulence therapy

Searching for off-target activities in drugs already approved for use in humans represents a potential shortcut to develop new therapeutic options. As compared with de novo drug discovery, drug repurposing (or repositioning) has a higher probability of yielding bioavailable and safe compounds, which can move straight into clinical trials or serve as leads for drug optimization programmes. Drug repurposing is expected to reduce the time and costs generally associated with standard drug discovery processes. Accordingly, UK and US funders have launched programmes to re-evaluate deprioritized drugs for new therapeutic uses [25]. Drug repositioning opportunities can derive from (i) targeted (or rational) repurposing, that could be inspired by novel molecular insights into pathogenic mechanisms of diseases, (ii) dedicated platforms to screen available drug libraries for new activities of interest or (iii) serendipitous observations, as was the case for the blockbuster drug sildenafil (Viagra™), which was originally developed to treat cardiovascular disorders [26].

In the few last years an increasing number of studies have identified some antibacterial activity in several drugs approved for different purposes, including anticancer, antifungal, antipsychotic and cardiovascular therapies. The mode of action and range of antibacterial activity of these repurposing candidates have been reviewed in two recent articles [27,28]. Herein, we will focus on those drugs that showed remarkable and specific antivirulence activity against opportunistic bacterial pathogens in vitro and/or in animal models of infection, and will examine their potential as repurposing candidates for antivirulence therapy. Given the focus of this review on antivirulence strategies, repurposed drugs with bacteriostatic/bactericidal activity (e.g. gallium nitrate) [29] or that affect the infectivity of intracellular pathogens by interfering with host cell pathways (e.g. pimozide) [30] will not be discussed.

The first candidate for antivirulence repositioning was identified through a rational repurposing approach, upon the observation that genetic disruption of uracil metabolism in P. aeruginosa strongly reduced the expression of several virulence determinants, including QS, motility and biofilm formation [31]. This led the researchers to screen uracil analogues for biofilm-inhibitory activity against P. aeruginosa [31]. Results highlighted a potent anti-biofilm effect of 5-fluorouracil, a drug currently used in the therapy of solid tumours. 5-fluorouracil was also effective in reducing the production of QS-dependent virulence factors in P. aeruginosa [31], as well as biofilm formation in E. coli [32], in agreement with previous work showing the anti-biofilm effects of sub-MIC 5-fluorouracil on Staphylococcus epidermidis [33]. The antivirulence potential of fluorinated pyrimidines was further confirmed by the observation that the antimycotic drug flucytosine (5-fluorocytosine) and 5-fluorouridine were able to suppress P. aeruginosa lethality in a mouse model of lung infection [34] and in Caenorhabditis elegans [35], respectively. Notably, 5-fluorouracil has a general inhibitory effect on several virulence phenotypes [31], while 5-fluorocytosine and 5-fluorouridine appear to exert their antivirulence activity by specifically targeting the production of the pyoverdine siderophore and pyoverdine-regulated virulence factors [34,35], which are essential for P. aeruginosa infectivity in several animal models [3638]. Unlike 5-fluorouracil, which has a bacteriostatic effect on P. aeruginosa, 5-fluorocytosine and 5-fluorouridine do not affect P. aeruginosa growth even at high concentrations [34,35,39], in line with the finding that flucytosine treatment at clinically meaningful doses reduced P. aeruginosa toxicity without affecting bacterial load in mouse lungs [34]. Thus, fluoropyrimidine drugs hold promise for antivirulence therapy against P. aeruginosa, though the specific molecular mechanism(s) underlying their activity and specificity, as well as their possible effect(s) on other bacterial pathogens, remains elusive.

The majority of drugs with antivirulence activity have been identified by screening libraries of approved drugs, by using either experimental (wet lab) approaches based on suitable screening systems and commercially available drug libraries, or in silico approaches based on the prediction of drug–target interactions by bioinformatics tools. The wet approach led to the identification of anti-QS activities in the anthelmintic drug niclosamide [40] and in the immunosuppressive drug cyclosporine A [41], anti-biofilm activity in the anti-inflammatory drug azathioprine [42] and antivirulence activity in pentetic acid, a contrast agent for magnetic resonance imaging [43]. Niclosamide was identified as an inhibitor of the Las QS system of P. aeruginosa through a whole-cell screening platform, and proved to be effective in reducing the expression of several Las-dependent virulence factors and P. aeruginosa infectivity in the G. mellonella insect model [40]. While the specific molecular target(s) of niclosamide in P. aeruginosa cells is still unknown, this drug was also demonstrated to inhibit cancer cell proliferation [44], replication of several different viruses [45], and growth of Mycobacterium tuberculosis and S. aureus [46,47], suggesting that niclosamide may be endowed with a wide range of side properties in addition to its primary anthelmintic activity. However, considering that niclosamide targets mitochondrial oxidative phosphorylation in tapeworms [48], it cannot be excluded that at least some of the activities of this drug could be indirect effects related to impaired energy metabolism, both in eukaryotic and prokaryotic cells. The anti-QS activity of cyclosporine A was discovered by means of a fluorescence assay to detect inhibitors of the interaction between the purified Streptococcus pyogenes Rgg3 receptor and its cognate signal molecule [41]. The binding of cyclosporine A to Rgg3 was then characterized at the structural level [49], and cyclosporine A was confirmed to block Rgg3-dependent transcription in several streptococcal species and to inhibit biofilm formation in S. pyogenes [41]. However, the efficacy of cyclosporine A in infection models has not been assessed yet. Azathioprine was investigated as an anti-biofilm agent due to the finding that it was able to repress cyclic diguanylate (c-di-GMP) biosynthesis in an E. coli reporter strain expressing the diguanylate cyclase WspR of P. aeruginosa [42]. Notably, azathioprine appeared to affect c-di-GMP levels without direct inhibition of diguanylate cyclases, and significantly reduced biofilm formation in E. coli but not P. aeruginosa [42]. Whether this selectivity relies on the different c-di-GMP effectors of the two bacteria or on the inability of azathioprine to cross the highly impervious cell envelope of P. aeruginosa has not been determined. Another screening assay based on a P. aeruginosa biosensor identified anti-c-di-GMP activity in the anticancer drug doxorubicin. However, while doxorubicin significantly reduced c-di-GMP levels and expression of several biofilm-related genes, it actually resulted in increased biofilm formation by P. aeruginosa, probably because of enhanced release of extracellular DNA from doxorubicin-treated bacteria [50]. FDA-approved drugs were also screened in the search for inhibitors of P. aeruginosa elastase [43]. The hit compound, pentetic acid, not only inhibited elastase expression, but also reduced biofilm formation, cytotoxicity in cultured epithelial cells and P. aeruginosa lethality in a mouse airway infection model, with a mechanism of action that could at least partially involve inhibition of the PQS QS system [43].

A couple of repurposing candidates have been identified through computational molecular docking. A virtual screening of FDA-approved drugs able to interact with nine virulence determinants of P. aeruginosa predicted the binding of the selective oestrogen receptor modulator raloxifene to the pyocyanin biosynthetic enzyme PhzB2. Raloxifene was indeed demonstrated to reduce pyocyanin production in vitro and P. aeruginosa pathogenicity in C. elegans [51]. A similar approach led to the identification of the anti-inflammatory drug diflunisal as a potential inhibitor of the phosphoryl-binding pocket of the S. aureus QS regulator AgrA [52]. Diflunisal efficiently reduced QS-dependent haemolysis in vitro [52], and was recently demonstrated to prevent skeletal cell death and bone destruction during S. aureus osteomyelitis in mice [53]. An overview of all the drugs proposed for antivirulence repositioning is provided in Table 1, and their chemical structures are shown in Figure 1.

Figure 1.

Chemical structures of the repurposing candidates for antivirulence therapy discussed in this review.

Figure 1.

Chemical structures of the repurposing candidates for antivirulence therapy discussed in this review.

Table 1
Repurposing candidates with antivirulence activity against opportunistic bacterial pathogens
Drug Therapeutic use(s) Repurposing approach Bacterial species Anti-virulence target(s) Animal model References 
5-fluorouracil Anticancer Rational P. aeruginosa, E. coli QS, virulence factors, motility and/or biofilm  [29,30
Flucytosine Antifungal Wet screening P. aeruginosa Pyoverdine Murine lung infection [32
Niclosamide Anthelmintic Wet screening P. aeruginosa QS, virulence factors, motility, biofilm G. mellonella insect model [38
Cyclosporine A Immunosuppressive Wet screening S. pyogenes QS  [39,47
Azathioprine Anti-inflammatory Wet screening E. coli Biofilm formation, c-di-GMP  [40
Pentetic acid Contrast agent for magnetic resonance; chelating agent for heavy-metal poisoning Wet screening P. aeruginosa QS, virulence factors, biofilm Murine lung infection [41
Raloxifene Selective-oestrogen receptor modulator In silico screening P. aeruginosa Pyocyanin C. elegans [49
Diflunisal Anti-inflammatory In silico screening S. aureus QS Murine osteomyelitis model [50,51
Drug Therapeutic use(s) Repurposing approach Bacterial species Anti-virulence target(s) Animal model References 
5-fluorouracil Anticancer Rational P. aeruginosa, E. coli QS, virulence factors, motility and/or biofilm  [29,30
Flucytosine Antifungal Wet screening P. aeruginosa Pyoverdine Murine lung infection [32
Niclosamide Anthelmintic Wet screening P. aeruginosa QS, virulence factors, motility, biofilm G. mellonella insect model [38
Cyclosporine A Immunosuppressive Wet screening S. pyogenes QS  [39,47
Azathioprine Anti-inflammatory Wet screening E. coli Biofilm formation, c-di-GMP  [40
Pentetic acid Contrast agent for magnetic resonance; chelating agent for heavy-metal poisoning Wet screening P. aeruginosa QS, virulence factors, biofilm Murine lung infection [41
Raloxifene Selective-oestrogen receptor modulator In silico screening P. aeruginosa Pyocyanin C. elegans [49
Diflunisal Anti-inflammatory In silico screening S. aureus QS Murine osteomyelitis model [50,51

To our knowledge, there are no antivirulence drug candidates discovered by serendipity, with the exception of azithromycin. This macrolide antibiotic has been used since the 1980s to treat diffuse panbronchiolitis in patients with chronic P. aeruginosa lung infection, even if P. aeruginosa is considered clinically resistant to azithromycin. Some clinical trials then reported a small but consistent improvement in respiratory function in azithromycin-treated cystic fibrosis patients [54]. In vitro studies suggested that the clinical efficacy of azithromycin against P. aeruginosa could partly derive from its ability to inhibit a plethora of virulence traits [55]. Although azithromycin represents the only drug with antivirulence activity currently used in the clinic for antibacterial therapy, the actual contribution of the virulence inhibitory properties to azithromycin efficacy in humans is hardly predictable because of the concomitant anti-inflammatory properties and bactericidal effects of azithromycin under certain conditions [55].

Conclusions and perspectives

Hundreds of antivirulence compounds have been identified in the last 15 years. However, none of them entered into clinical practice, probably because of poor activity, unfavourable pharmacological properties and/or high toxicity. Efforts have been made in recent years to circumvent some of these limitations by repurposing existing drugs for antivirulence therapy. Although the identification of several antivirulence activities in currently available drugs supports this approach, there are still some issues that need to be addressed to make antivirulence drugs a viable alternative to current antibacterial therapies (Table 2).

Table 2
Possible advantages and drawbacks inherent to antivirulence and drug repurposing strategies
Advantages Drawbacks 
Antivirulence issues 
 Availability of new pathogen-specific molecular targets Necessity of adequate screening systems 
 Reduced impact on the resident microbiota Necessity of suitable virulence models and appropriate infection markers 
 Limited selective pressure for resistance, especially if targeting public goods Outcomes of clinical trials should be redefined to meet the specific mode of action of antivirulence drugs 
 Restricted spread of resistance mechanisms in the resident microbiota Reduced activation of the immune system and/or selection of hyper-virulent variants 
Drug repurposing issues 
 ADMET tests already available, if the administration route is maintained ADMET tests required, if the administration route is changed 
 Already approved for use in humans Need for new formulations, if the administration route is changed 
 Reduced time and costs for therapeutic use The primary activity of repurposing candidates could be an issue during treatment 
 Commercial drug libraries available for screening campaigns Challenge for intellectual property rights 
 Off-label use of FDA-approved drugs  
Advantages Drawbacks 
Antivirulence issues 
 Availability of new pathogen-specific molecular targets Necessity of adequate screening systems 
 Reduced impact on the resident microbiota Necessity of suitable virulence models and appropriate infection markers 
 Limited selective pressure for resistance, especially if targeting public goods Outcomes of clinical trials should be redefined to meet the specific mode of action of antivirulence drugs 
 Restricted spread of resistance mechanisms in the resident microbiota Reduced activation of the immune system and/or selection of hyper-virulent variants 
Drug repurposing issues 
 ADMET tests already available, if the administration route is maintained ADMET tests required, if the administration route is changed 
 Already approved for use in humans Need for new formulations, if the administration route is changed 
 Reduced time and costs for therapeutic use The primary activity of repurposing candidates could be an issue during treatment 
 Commercial drug libraries available for screening campaigns Challenge for intellectual property rights 
 Off-label use of FDA-approved drugs  

First, compared with the widespread and constitutively expressed antibiotic targets, the presence, expression levels and relevance to infection of virulence factors are species- and even strain-dependent. Thus, it is essential to evaluate the range of activity of antivirulence drugs on large panels of strains, both in vitro and in animal models. Among repurposing candidates, only fluoropyrimidines have been tested on panels of clinical isolates. This showed that sensitivity to the antivirulence activity of 5-fluorouracil is quite variable among P. aeruginosa strains [56], while sensitivity to the anti-pyoverdine activity of flucytosine and 5-fluorouridine appeared to be conserved [34,35]. On the other hand, pyoverdine-deficient strains are occasionally isolated from infected patients [57,58], suggesting that this virulence factor could be dispensable for some strains and/or infections, implying that pyoverdine-targeting drugs could be ineffective in some patients.

Second, the effect on the host of treatment with the repurposing candidate should be carefully considered, in order to evaluate the sustainability of the antivirulence therapy. Indeed, some repurposing candidates are toxic anticancer drugs, or anti-inflammatory drugs with potential adverse effects on immune cells or are endowed with many collateral activities other than antivirulence, as in the case of niclosamide. Also, the activity of the repurposing candidates on the host microbiota needs to be analysed. For instance, flucytosine is an antifungal agent, while 5-fluorouracil and niclosamide have growth-inhibitory activity against several bacteria [46,47,59]. Treatments with these drugs could thus cause dysbiosis and/or select for resistance determinants among commensal bacteria.

Third, some repurposing candidates have poor pharmacological properties, as is the case for niclosamide, an oral anthelmintic drug with very low intestinal absorption and short half-life in the bloodstream. While these features are useful to kill tapeworms in the gut, they represent drawbacks for the systemic treatment of bacterial infections. The development of suitable formulations for the repositioning of antivirulence drugs for antibacterial therapy could overcome these limitations [60,61]. However, the reverse of the coin is that the new formulation generally requires new absorption, distribution, metabolism, excretion and toxicity (ADMET) validation, thus partly compromising the advantage of the repurposing approach.

Finally, the most relevant weakness of antivirulence research is that a clear clinical validation of this antibacterial approach has not yet been provided, possibly due to the paucity of compounds that have entered clinical testing to date [62]. As discussed above, drug repurposing for antivirulence therapy is a young area of investigation, which has been mainly limited to academic research. Nevertheless, the increasing effort in searching for antivirulence activities in FDA-approved drugs raises the hope that the number of candidates for antivirulence therapies will increase in the near future, and this could facilitate the clinical validation of the antivirulence approach. This achievement could boost the interest of pharmaceutical companies for antivirulence drugs, plausibly attracted by the promise of developing antibacterials with reduced side effects and/or extended clinical lifespan as compared with traditional antibiotics. Moreover, pipelines for the industrial production of repurposed drugs already exist, thus further increasing profits of their therapeutic off-label use. Whether these potential benefits would be sufficient to counteract the inherent low reward of antivirulence drugs, related to their narrow range of activity, is hard to predict at present.

Summary
  • The development of new therapeutic approaches is sorely needed to counteract the increasing spread of antibiotic-resistant bacterial pathogens and the reduced rate of antibiotic discovery.

  • Antivirulence drugs are aimed at inhibiting the infection process rather than bacterial growth. The main advantages of the antivirulence approach are: (i) availability of new pathogen-specific molecular targets, (ii) reduced impact on the resident microbiota and (iii) reduced selective pressure for resistance.

  • Searching for secondary antivirulence activity in drugs already approved for use in humans for different purposes represents a shortcut to develop new therapeutic agents active against antibiotic-resistant pathogens. The drug repurposing approach is expected to reduce the time and costs generally associated with standard drug discovery processes.

  • Despite the inherent advantages of drug repurposing for antivirulence therapy, this approach has seldom been exploited, mainly due to the lack of clinical evidence and paucity of economic efforts in the field.

Abbreviations

     
  • ADMET

    absorption, distribution, metabolism, excretion and toxicity

  •  
  • c-di-GMP

    cyclic diguanylate

  •  
  • FDA

    US Food and Drug Administration

  •  
  • MIC

    minimum inhibitory concentration

  •  
  • QS

    quorum sensing

Author Contribution

All authors have contributed equally to this work.

Funding

Research on antivirulence drugs in our laboratories has been supported by the Italian Cystic Fibrosis Foundation [grants FFC #10/2013 and FFC #21/2015].

Acknowledgments

We acknowledge all the researchers who have contributed to antivirulence research over the years, and apologize to those colleagues whose work could not be reviewed due to space limitation.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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