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Small-molecule potentiators for conventional antibiotics against Staphylococcus aureus Arno Vermote, and Serge Van Calenbergh ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00084 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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ACS Infectious Diseases

Small-molecule

potentiators

for

conventional

antibiotics

against

Staphylococcus aureus Arno Vermotea, Serge Van Calenbergh*,a a Laboratory

for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium *Corresponding Author, e-mail: [email protected] ORCID Serge Van Calenbergh: 0000-0002-4201-1264

ABSTRACT Antimicrobial resistance constitutes a global health problem, while the discovery and development of novel antibiotics is stagnating. Methicillin-resistant Staphylococcus aureus, responsible for the establishment of recalcitrant, biofilm-related infections, is a well-known and notorious example of a highly resistant micro-organism. Since resistance development is unavoidable with conventional antibiotics that target bacterial viability, it is vital to develop alternative treatment options on top. Strategies aimed at more subtle manipulation of bacterial behavior have recently attracted attention. Here, we provide a literature overview of several small-molecule potentiators for antibiotics, identified for the treatment of Staphylococcus aureus infection. Typically, these potentiators are not bactericidal by themselves and function either by reversing resistance mechanisms, by attenuating Staphylococcus aureus virulence, and/or by interfering with quorum sensing.

KEYWORDS Antibiotics, Potentiators, Staphylococcus aureus, MRSA, Resistance, Virulence

ACS Paragon Plus Environment


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INTRODUCTION The advent of antimicrobial drugs for treating infections, such as those caused by bacteria (antibiotics), fungi (antifungals), viruses (antivirals), and parasites (antiparasitics), is considered as one of the most important scientific achievements of the twentieth century. With time, however, the utility of these drugs has become compromised by the emergence of antimicrobial resistance (AMR). Resistant micro-organisms are able to withstand the attack by antimicrobial drugs, so that infections persist. Infections with, for instance, methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae,

multidrug-resistant

tuberculosis

(MDR-TB),

or

vancomycin-resistant

Enterococcus are regularly making headline news: these ‘superbugs’ are difficult to treat with existing medicines. The decreasing effectiveness of conventional antimicrobial drugs is posing a serious problem to patients and health care providers. The World Health Organization has recognized antimicrobial resistance as a global problem since 2001. 1 In a recently published report, commissioned by the British government, economist O’Neill and co-authors estimate that by 2050 10 million lives a year will be at risk due to the rise of drug-resistant infections.2 Even today, superbacteria cause the loss of 700,000 people worldwide, every year.2 Even more, the spread of antibiotic resistance could possibly shake the foundations of modern healthcare in numerous aspects: without access to effective antibiotics, the slightest injury could become life-threatening and key medical procedures (e.g., surgery, caesarean section, transplantation, or chemotherapy for cancer) too dangerous to perform. The economic impact is also already substantial and continues to climb.3 In the not so distant past, antimicrobial discovery experienced an era of prosperity (the so-called ‘golden era’). Systematic screening of soil-derived microbes for antibacterial activity, a very successful platform introduced by Selman Waksman in the 1940s, led to the


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discovery of nearly all antibiotic classes in use today.4 Streptomycin, chloramphenicol, and erythromycin A are only a few examples of drugs identified via one of the many, large antibiotic screening programs after the Second World War. Indeed, chemotherapy for bacterial infections has been dominated by natural products. Antibacterial drugs have been identified via a ‘classical’ approach, in which determination of the minimum inhibitory concentration (MIC) in whole-cell assays was the mainstay. Some antimicrobials, derived from nature, were marketed even without modification. Tetracycline, vancomycin, penicillin G, and the recently marketed daptomycin are examples of drugs that combine

satisfactory

in

vitro

potency

(MIC)

with

acceptable

physicochemical,

pharmacological, and toxicological profiles. Typical limitations of natural products are chemical lability, suboptimal pharmacokinetic characteristics, and toxicity issues. Hence, many natural substances became leads for chemical optimization programs (semi-synthetic derivatives).5 In the 1960s, the returns from the once effective and ‘brute’ screening efforts diminished and although there have been a number of antibacterial agents that had their origin in synthetic chemistry (e.g., fluoroquinolones and oxazolidinones), efforts to develop antibacterial discovery platforms based on genomics, combinatorial chemistry, high-throughput screening (HTS), and rational drug design were rather unproductive.4 The number of currently exploited antibacterial targets is very small. The majority of the most successful antibiotics essentially hit only a few major targets or pathways: cell wall synthesis, the ribosome, DNA gyrase or topoisomerase, and folate biosynthesis. Even if genomic analyses are revealing potential targets in pathogens and target-based approaches may offer new opportunities, pharmaceutical companies have been rather unsuccessful in identifying novel agents via this paradigm.



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Consequently, pharma has mainly been investigating structural variations of established antibacterial classes. For decades, in a multidisciplinary effort, medicinal chemists have been introducing additional properties (e.g., solubility, antibacterial spectrum, and tolerability) into natural antibiotics, without jeopardizing intrinsic activity. Towards this end, both de novo synthesis and semisynthetic approaches have been used. 5 Development and marketing approval of new antibiotics have not kept pace with the increasing public health threat of an unremitting emergence of resistance. The origin of the chemoresistance is multifactorial. Inappropriate prescription by clinicians and veterinarians, imprudent utilization, and extensive use of antimicrobials in bio-industry, together with globalization and a lack of public awareness, have definitely contributed to this pandemic health problem.2 A detailed analysis of the reasons for AMR is beyond the scope of this review, but by reducing unnecessary consumption, improving hygiene, and preventing spread of infection we can already have a powerful impact. However, the question remains whether such measures will suffice. Bacteria have demonstrated the ability to develop resistance to virtually every antibiotic introduced by the medical community. Rapid bacterial selection by the use of conventional antibiotics inevitably leads to an acceleration of resistance development. It is important to keep in mind that bacteria have always been resistant to antibiotics. As described earlier, most antibiotics are (derived from) natural compounds produced by microbes. For as long as antibiotics have existed, bacterial resistance has existed alongside. In a wide variety of environments, soil samples have been found to contain resistance genes. 6 Illustrating this aspect, is the fact that several years before the introduction of penicillin as an antibiotic, “a substance destroying the growth-inhibiting property of penicillin” was already identified.7 Moreover, the very short generation time of bacteria, their large population size, and the ability to exchange DNA via horizontal gene transfer gives the microbes a major advantage in


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the everlasting battle. An extra challenge of antibiotic resistance is that bacteria have evolved several distinct mechanisms to cope with antibiotic therapy. Moreover, many pathogens harbor several mechanisms simultaneously. In such a scenario, where selective pressure from the environment drives enrichment of specific genes that promote fitness and survival, it is imperative to continuously discover fresh antimicrobials or new practices that are effective for the treatment of infectious diseases.

STAPHYLOCOCCUS AUREUS Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that appears as grape-like clusters of berry-shaped cells upon microscopic examination. S. aureus appears as a harmless commensal organism and colonizes the skin and mucosae of human beings and several animal species.8 Approximately 30% of the human population carries this micro-organism in the anterior nares. The nose is the most frequent carriage site for ‘golden staph’, although multiple body sites (e.g., skin, axillae, perineum, pharynx) can be colonized.9 While S. aureus appears as a harmless commensal, it can cause disease as well. As a very versatile pathogen, this micro-organism can cause a range of infections and syndromes, most notably skin and soft tissue infections. When inoculated into an open wound, infections (e.g., boils, pimples, and impetigo) develop frequently. Many of such infections cause the production of pus and are said to be ‘pyogenic’ (pus-forming). Other clinical manifestations include endocarditis, osteoarticular infection, and pneumonia, next to toxin-mediated disease (e.g., food poisoning, toxic shock syndrome, and scaled skin syndrome), and prosthetic device and catheter infections.10 In animals, S. aureus can cause serious infections too, such as bumblefoot in poultry. It is also one of the major causal agents of mastitis in dairy cows, one of the most frequent and costly diseases in the dairy industry. 11, 12 Staphylococcal infections often occur



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when resistance of the host is low because of debilitating illness, wounds, or treatment with steroids or other drugs that compromise immunity. S. aureus is an opportunistic pathogen that possesses an extensive arsenal of virulence factors. Moreover, some strains hold a battery of resistance mechanisms against conventional antibiotics. Compounding the problem even further is the fact that S. aureus is notorious for its ability to form biofilms. The formation of such a biofilm, a surface-attached encasement of cells in a polymer-based matrix, enables the superbug to persist, due to increased resistance to antibiotics and the host immune system. Bacteria in biofilm communities display significantly higher resistance to several kinds of stress than their planktonic brethren. Thus, biofilm formation adds a further level of complexity to the already existing problem of antimicrobial resistance. Microbes regulate genes in response to signals in the environment. A potential signal that prokaryotes can respond to is the presence of other bacterial cells. The ability of bacteria to assess their population density and control genetically mediated responses is a very dynamic and important aspect of microbial physiology. It is of practical use to ensure that sufficient cell numbers are present before a specific gene product is made. This type of control is called quorum sensing (QS).13 QS involves production and recognition of small signaling molecules. These ‘chemical words’ are always produced at a certain level. When their extracellular concentration (which is representative for their ‘quorum’) reaches a threshold, they bind to a specific receptor. Ultimately, transcription of certain genes is activated. Bacterial biofilm formation, virulence in general, and antibiotic resistance are often mediated by this cell-to-cell communication system. For example, toxin production by one bacterial cell would have no effect and hence be a waste of resources. However, a coordinated expression of the toxin by a sufficiently high population of cells, may successfully cause disease. In S. aureus, both biofilm formation and virulence in general are regulated via QS.


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It is increasingly important and necessary to find new antimicrobial agents, but since resistance development is unavoidable with agents that target bacterial viability, it is imperative to devise alternative measures as well. Strategies aimed at more subtle manipulation of bacterial behavior have recently attracted attention. In this review, we provide the reader with a literature overview of several potentiators for antibiotics identified for the treatment of S. aureus infection. We will concentrate on small molecule potentiators that are not bactericidal, but in combination with conventional antibiotics, enhance the antimicrobial activity of the latter. The discussed potentiators function either by reversing resistance mechanisms, by attenuating S. aureus virulence, and/or by interfering with QS. In Chapter 3, we will focus on ‘resistance-modifying agents’ (RMAs) that potentiate the effect of antibiotics via inhibition or modification of specific resistance mechanisms of S. aureus. Firstly, several examples of compounds targeting antibiotic resistance elements acquired by S. aureus will be given. Secondly, efflux pump inhibitors will be described. Thirdly, compounds specifically targeting S. aureus biofilms and small-colony variants (SCVs) will be discussed. In Chapter 4, a closer look will be taken at potentiators that target S. aureus virulence factors. Finally, in Chapter 5, important examples of potentiators specifically modulating QS mechanisms will be given. As biofilm formation and virulence in general are regulated via QS, it is possible that biofilm as a target appears in different sections (i.e., not solely in Chapter 3). In each category, descriptive examples (especially of medicinal chemistry programs) will be given to create a framework. Conventional antibiotics, vaccines, bacteriophages, lysins, and therapeutic antibodies (which obviously also have a place in the control of bacterial infections14) will not be discussed here.

POTENTIATIONS OF ANTIBIOTICS AGAINST S. AUREUS VIA INHIBITION OF RESISTANCE MECHANISMS Inhibition of acquired antibiotic resistance elements



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Potentiation of antibiotic activity can occur via inhibition of antibiotic resistance elements. The prototype example in this category is the antibiotic-potentiator combination Augmentin®. Peptidoglycan (PG), the most important component of the bacterial cell wall, is a polymer made of N-acetylmuramic acid (NAM), alternating with N-acetylglucosamine (NAG), which are cross-linked by chains of four amino acids. Synthesis of PG (and ultimately the cell wall) occurs in a number of stages that take place in different locations in the cell. Glycosidic bonds are formed between the disaccharides and cross-links between the neighboring peptides. The transpeptidation and carboxypeptidation reactions are mediated by penicillin binding proteins (PBPs).15 Eventually, several layers of PG are formed. The β-lactam structure, present in β-lactam antibiotics, is capable of binding the PBPs and blocks their ability to function normally. The transpeptidases are serine hydrolases: attack of the serine hydroxyl function on the β-lactam amide bond forms a highly stable penicilloyl-enzyme intermediate. The inhibition of the enzyme is irreversible and compromises cross-linking (and bacterial cell wall synthesis in general). An important mechanism by which S. aureus has become resistant to β-lactam agents, is the production of β-lactamases (or penicillinases) that hydrolyze the β-lactam ring before the antibiotic has the chance to exert its effect. The destruction of the amide bond in the drug renders it incapable of binding to PBPs and thus, the bacterium becomes resistant to that drug or even class of drugs. The β-lactamase inhibitor clavulanic acid (1 in Figure 1) ‘augments’ the activity of the β-lactam antibiotic amoxicillin, by inhibiting β-lactamases. Clavulanate was identified in 1976 and until now it has allowed the continued use of amoxicillin, demonstrating the effectiveness of adding inhibitors of antibiotic-degrading enzymes. Next to clavulanic acid, also sulbactam (2) and tazobactam (3) have been registered. The development of new β-lactamase inhibitors has been the subject of several studies.16,


17

The combination of

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ceftazidime with avibactam (4), a non-β-lactam β-lactamase inhibitor, for example, was approved by the FDA in 2015 for the treatment of certain MDR Gram-negative infections.18 Inactivation of β-lactamases is a successful strategy to overcome resistance. However, MRSA strains have developed resistance through the acquisition of a different PBP (PBP2A) with reduced affinity for β-lactams. The gene that encodes the PBP2A protein is mecA and is acquired through horizontal gene transfer of a mobile genetic element known as staphylococcal cassette chromosome mec (SCCmec). With PBP2A, the use of a β-lactamase inhibitor doesn’t give too much solace. One strategy to overcome β-lactam resistance in MRSA is to develop analogues that act as PBP2A inhibitors. 19

Figure 1: Structures of β-lactamase inhibitors (1-4) and inhibitors of wall teichoic acid synthesis (5-8).

Another strategy that has been explored only very recently, is the combination of a β-lactam antibiotic with wall teichoic acid (WTA) synthesis inhibitors. There is emerging evidence that WTAs, long anionic polymers covalently attached to PG in Gram-positive bacteria, play an important role in the expression of β-lactam resistance in MRSA. In S. aureus, the synthesis



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of WTA is mediated via the consecutive action of Tar enzymes. Specifically, the TarO protein (encoded in the tarO gene) initiates the assembly with the transfer of N-acetylglucosamine-1phosphate from UDP-NAG to the membrane-anchored undecaprenyl-phosphate carrier lipid. Then, TarA adds an N-acetylmannosamine moiety. Ultimately, what follows is elongation of this product into long polymers with ribitol phosphate repeats. 20 The function of WTAs is incompletely understood but they are not essential for S. aureus survival in vitro, since tarO can be deleted. However, it has been suggested that WTAs are virulence factors required for staphylococcal adhesion to host tissue and, more recently, in the expression of β-lactam resistance. Farha et al. provided evidence that “a strain devoid of WTA leads to the mislocalization of PBP4, compromising its role as a transpeptidase in PG cross-linking and specifically in the case of CA-MRSA USA300 results in sensitivity to certain β-lactams”.21 The natural product tunicamycin (5) was shown to inhibit TarO and displays synergy with β-lactam antibiotics, decreasing the MIC of oxacillin against 1 MRSA clinical isolate from 50 to 0.4 µg/mL at a concentration of only 0.08 µg/mL.20 Because blocking WTA biogenesis was shown to restore the efficacy of β-lactams in MRSA and also because tunicamycin is a non-selective glycosyltransferase inhibitor (that also inhibitis PG synthesis) and has significant eukaryotic toxicity, Farha et al. set out to identify new inhibitors of WTA synthesis. Using a library of 2,080 previously approved drugs (PADs), the researchers conducted a screen for compounds capable of potentiating the activity of cefuroxime. The antiplatelet agent ticlopidine (6) showed strong synergistic interactions with cefuroxime against several MRSA strains, lowering MICs by up to 64-fold. The authors also assessed the in vivo efficacy of the combination ticlopidine-cefuroxime in a Galleria mellonella model of MRSA infection. A significantly higher fraction of larvae survived infection following combined treatment, compared to that of cefuroxime and ticlopidine alone. The molecular target of the thienopyridine was identified as TarO and the molecule is devoid of antibiotic


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activity on its own.21 This meticulous chemical genetic study of Farha et al. has provided a promising new lead for further study and the same research group identified a ticlopidine derivative (7) that shows improved activity and avoids cytochrome P-450 mediated oxidation to less potent TarO inhibitors. Because strong synergies of cefuroxime with clopidogrel (8), a commercially available analogue of ticlopidine, were observed as well, the authors also synthesized clopidogrel analogues.22 In a similar way to Farha et al., Ejim and coworkers conducted a systematic screening of approved non-antibiotic compounds for potentiator activity. They explored combinations of minocycline with 1,057 PADs. Disulfiram (9) (Figure 2), an inhibitor of acetaldehyde dehydrogenase used for the treatment of alcoholism, showed strong synergy with the antibiotic on growth inhibition of S. aureus.23 Hu et al. unraveled that a series of ‘clicked’ triazolyl glycolipid derivatives had the ability to increase the susceptibility of a panel of clinical isolates of MRSA to β-lactam antibiotics. The glycolipids showed weak antibacterial effect when used alone (≥256 µg/mL) against MRSA ATCC43300. The authors used ‘Glc12’ (10), the compound that showed the best synergistic effect in this study, to investigate the mechanism of action. Using quantitative real-time PCR, the expression level of mecA was analyzed in the absence and presence of oxacillin and/or Glc12. The antibiotic alone enhanced expression of mecA, Glc12 alone did not. Excitingly, the gene expression was lower with the combination treatment, compared to oxacillin alone. This suggests that a PBP2A suppression pathway is involved, but the exact mechanism remains unknown.24 In S. aureus, exposure to cell wall-acting antibiotics (including β-lactams and glycopeptides) leads to the upregulation of expression of over 100 genes, which is referred to as the cell wall stress stimulon (CWSS). VraSR regulates the transcription of genes involved in cell wall biosynthesis.25,

26

The group of Melander reported on several 2-aminoimidazole-containing



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compounds, derived from the marine natural products oroidin (11) and bromoageliferin (12) that suppress resistance in several MRSA clinical isolates. For the activity of the lead compound from this series (13), they showed that the VraSR regulatory system plays an important role.27 Related series of compounds are able to inhibit and disperse biofilms (see below).

Figure 2: Structures of disulfiram (9), Glc12 (10), and several 2-aminoimidazol-containing compounds (11-14).

The

same

group

identified

structurally

very

similar

1,4-di-substituted

2-aminoimidazoletriazoles (2-AIT), one of which (14) reduces the MIC of oxacillin in MRSA. Here as well, selected derivatives proved capable of inhibiting biofilm formation (see below).28


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Next to that, it has been demonstrated that the antipsychotic drug thioridazine (15) (Figure 3) reduces the oxacillin-induced transcription of mecA and expression of PBP2A. The combination of thioridazine and oxacillin also reduces expression of BlaZ (encoding β-lactamase), as well as transcription of several genes of the VraSR regulon. The phenothiazine increases the susceptibility of MRSA to β-lactams in several clinical isolates.2931

Although the only antibiotic adjuvants that have been proven clinically useful are the β-lactamase inhibitors, Li et al. (2015) show that the search for natural antibiotic potentiators is still an interesting enterprise for the management of superbug infections. They identified new erythromycin derivatives that enhance the activity of β-lactams against MRSA in vitro, without having anti-MRSA effect when used alone. Further research is required to ascertain the mechanism of action.32 Fukumoto et al. identified cyslabdan (16), an actinomycete metabolite, as a potentiator for imipenem activity against MRSA. The labdane-type diterpene did not inhibit growth, but markedly reduced the MIC of the antibiotic from 16 to 0.015 µg/mL. The activity of several other β-lactam antibiotics was also potentiated. Further studies on the mechanism of action are in progress.33 Remarkably, MRSA has evolved an inducible mechanism for resistance against β-lactam antibiotics: the integral membrane protein BlaR1 is a sensor/signal transducer, which is phosphorylated upon exposure to β-lactam antibiotics and communicates the presence of the latter to the cytoplasm. The process is not completely understood but signal transduction leads to activation of the cytoplasmic domain of BlaR1, a zinc protease. This ultimately leads to the derepression of transcriptional events that result in expression of antibiotic-resistance determinants, such as β-lactamase and PBP2A. Boudreau et al. tested a library of 80 known protein kinase inhibitors for their ability to lower the MIC of oxacillin against MRSA252.34 In



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this initial screening, compound 17 was identified. The known mammalian serine/threonine kinase inhibitor gave a 4-fold decrease in the MIC of oxacillin at a concentration of 7 µg/mL, while the MIC of the kinase inhibitor alone was ≥64 µg/mL. In their efforts to optimize the structure of this hit, 70 structural analogues of 17 were synthesized and tested. One of the most potent derivatives (18) exhibited remarkable activity in lowering the oxacillin MIC against 3 MRSA strains, without showing antibacterial activity on its own. Relief of antibiotic resistance is a strategy that is potentially also applicable to other resistance enzymes than β-lactamases alone. Resistance to aminoglycosides for example can occur via the production of an aminoglycoside-modifying enzyme (AME) that chemically modifies the drug. Three types of AMEs have been identified in clinical isolates of S. aureus: aminoglycoside-3”-O-phosphoryltransferase

III

phosphoryltransferase

aminoglycoside-6‘-N-acetyltransferase/2”-O-

I

[ant(4’)-I],

and

[aph(3”)-III],

aminoglycoside-4’-O-

phosphoryltransferase [aac(6’)/aph(2”)]. Chemical modification of the aminoglycoside drugs by these enzymes decreases their ability to bind to the 30S ribosomal subunit.35 Resistance to lincosamides, macrolides, and streptogramins is mediated by a similar mechanism. Three related determinants, ermA, ermB, and ermC confer resistance to this group of antibiotics. The genes code for erythromycin resistance methylases (ERMs), which methylate a site on the ribosome, resulting in a conformational change that ultimately leads to a decreased ability of the drugs to bind it.36 Inhibitors of AMEs37, 38 and ERMs39, 40 have been described. Until now, however, none of these was sufficiently potent for further development. An additional and more recent approach includes the work of Hanessian and coworkers. Via a semisynthetic strategy, the researchers developed tetradeoxy aminoglycoside derivatives with improved activity against a panel of clinically relevant MRSA strains.41 Recently, Thamban Chandrika et al. also synthesized modified aminoglycosides with improved antibacterial properties and reduced sensitivity to inactivation by AMEs.42


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Figure 3: Structure of thioridazine (15), cyslabdan (16), kinase inhibitors 17 and 18, tricyclic indolines 19 and 20, and a tetracyclic indolenine (21).

Podoll et al. discovered a tricyclic indoline that selectively potentiates the effect of a variety of β-lactam antibiotics towards MRSA, without showing antiproliferative effect on its own. By synthesizing and screening a small-molecule library of 120 polycyclic indolines, they discovered compound 19 (termed Of1 in the paper) as an RMA that reduced the MIC of methicillin from 128 to 8 µg/mL.43 The same group also reported a structure-activity relationship investigation of 19, with the identification of 20, showing improved synergistic action with β-lactam antibiotics and reduced mammalian toxicity. Further investigation of the mode of action and evaluation of in vivo efficacy is warranted.44 Inspired by the discovery of several polycyclic indolines as resensitizing molecules, the Wang Laboratory synthesized and evaluated a series of bridged tetracylic indolenines too. One of their compounds (21) is a selective potentiator of β-lactam activity in a multidrug-resistant MRSA strain. Of note is that the compound shows low antibacterial activity on its own. Further studies (structure-activity relationship, mechanism of action, etc.) are ongoing.45



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Efflux pump inhibition Inhibition of efflux pumps also appears to be a promising strategy to sidestep antibiotic resistance. Compounds that prevent antibiotics from being pumped out are desirable potentiators. Reserpine (22) (Figure 4) was an early example that demonstrated the potential of efflux pump inhibition in S. aureus. The plant alkaloid, a known inhibitor of mammalian MDR efflux pumps, was shown to reduce the ciprofloxacin MIC. 46

Figure 4: Examples of efflux pump inhibitors.

Next to reserpine, various other natural products have been shown to inhibit bacterial efflux pumps. It seems that a variety of Berberis medicinal plants that produce berberine (23), a cationic antimicrobial, also synthesize the flavone 5’-methoxyhydnocarpin (5’-MHC, 24), an inhibitor of the NorA efflux pump of S. aureus. Without 5’-MHC, the plant-derived antimicrobial alkaloid gets readily extruded by S. aureus.47 A review compiled by Abreu et al. gives the interested reader an overview of plant-derived products with potentiating effects. A large part is dedicated to efflux pump inhibitors.48


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Efflux pump inhibitors are not restricted to natural products. To give an example, the nonsteroidal anti-inflammatory drug (NSAID) celecoxib (25) has recently been demonstrated to resensitize S. aureus to multiple antibiotics.49 Sabatini and coworkers confirmed the efflux pump inhibitor activity of the NSAID and identified a new class of analogues, acting as inhibitors of the S. aureus NorA efflux pump. The most active inhibitor (26) showed only modest antistaphylococcal activity and was able to restore the antibacterial activity of ciprofloxacin in norA-overexpressing S. aureus strains.50 To date, several inhibitors targeting bacterial efflux pumps have been discovered and patented.51, 52 Yet, no efflux pump inhibitor has been approved for therapeutic use.

Potentiators specifically targeting S. aureus biofilms and small-colony variants

Persistence of infection does not only depend on the acquisition of genetic elements that confer resistance to antibacterial treatment. Also biofilm formation, for instance, results in obstinate infection and reduced sensitivity to antibiotics. Next to that, in some cases, S. aureus can survive in a semi-dormant state, referred to as SCVs. The latter are known to form biofilms. Hence, efforts have been made to disassemble biofilms and to specifically target SCVs. As outlined above, the group of Melander designed several 2-AITs, based on marine natural products. Compound 27 (Figure 5) was able to inhibit and disperse bacterial biofilms, including also S. aureus biofilms.53 Here as well, the activity was not due to bactericidal effects. Via structure-activity relationship studies and library screening using the core 2-aminoimidazole motif, the group identified more compounds with antibiofilm properties. 28, 54-56

Another example is the pentadecenyl tetrazole SEQ-914 (28) that acts as a potentiator of gentamicin against MRSA biofilms. Further lead optimization is warranted. 57



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Figure 5: Compounds that are able to disassemble biofilms or specifically target small colony variants.

Abouelhassan and coworkers identified a series of quinoline small molecules that demonstrate antibiofilm activity. Starting from a bromophenazine (29) with antibacterial activity against S. aureus, the authors discovered 21 structurally similar halogenated quinolones via a scaffold hopping approach. Although several derivatives showed antibacterial activity, the novel small molecule 30 possessed the ability to inhibit S. aureus ATCC 29213 biofilm formation at concentrations that do not inhibit planktonic growth.58 Furthermore, researchers from the same group discovered that combinations of gallic acid (31) with compounds from their library of halogenated quinolines had potent antibacterial activities against a broad range of pathogens, including MRSA. Notably, MRSA biofilms were also effectively eradicated with this combination therapy.59 Furthermore, Garrison et al. published work on the synthesis and biological evaluation of halogenated phenazine derivatives of 29. Several analogues showed potent biofilm eradication activity.60 Lee et al. investigated 36 halogenated indole derivatives for their ability to inhibit biofilm formation by Escherichia coli and S. aureus.61 The most potent derivative, 5-iodoindole (32), effectively prevented biofilm formation by these bacteria and, as the researchers’ observations indicate, could be used in combination with conventional antibiotics to eradicate biofilms.


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Moreover, 5-iodoindole was found to reduce the production of the virulence factor staphyloxanthin (STX) by S. aureus (see below). The steroidal glycoalkaloid tomatidine (33) specifically inhibits the growth of S. aureus SCV strains. The MIC of tomatidine against SCVs was 0.12 µg/mL, whereas no clinically significant MIC was measurable against normal strains. Since SCVs are less susceptible to the effect of classical antibiotics like aminoglycosides, Mitchell et al. investigated whether tomatidine could be used in combination with gentamicin to treat heterogeneous populations of S. aureus. The authors found that gentamicin at 4 µg/mL was able to inhibit the growth of the normal S. aureus strain CF04-L, while tomatidine at 0.12 µg/mL was not. Additionally, it was found that the aminoglycoside at 4 µg/mL did not inhibit the growth of SCV CF07-S, whereas the saponine at 0.12 µg/mL did. Interestingly, tomatidine complements the antibacterial effect of the conventional antibiotic. 62 Furthermore, this research group found that tomatidine is a specific aminoglycoside potentiator: it potentiated the inhibitory effect of other aminoglycosides (kanamycin, tobramycin, amikacin, and streptomycin) but not of other classes of antibiotics (vancomycin, oxacillin, ciprofloxacin, erythromycin, and tetracycline were tested). The synergy remains to be confirmed in vivo, however, and no cellular target has been identified yet. The authors also demonstrated that exposure of S. aureus to tomatidine repressed haemolytic activity of the bacterium. The natural product even blocked the expression of several genes that are normally influenced by the agr system (see below).63 Recently, the research group reported the first structure-activity study of the steroid alkaloid, isolated from solanaceous plants.64

The examples described in Chapter 3 demonstrate that phenotypical screening is well suited to uncover interesting potentiators. However, unraveling the mechanism of action seems



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challenging in many cases. Future efforts should be directed to uncover the mechanism by which these compounds elicit their effect. As demonstrated by avibactam (4), the development of new β-lactamase inhibitors seems a very successful strategy to overcome resistance. Another promising development program is that of the antiplatelet drug ticlopidine (6) as WTA synthesis inhibitor. Combinations of β-lactam antibiotics with ticlopidine limit the growth of MRSA strains both in vitro and in vivo. A further advantage of this combination is the fact that ticlopidine, a well-known drug in clinical use, has demonstrated a strong record of examination in humans. It also provides a new probe for assessing the importance of WTA synthesis. On the other hand, repurposing candidates face their own challenges (see Discussion).

Targeting S. aureus virulence as an alternative approach Another alternative to killing bacteria or stopping their growth, is to find compounds that target virulence.65 Instead of focusing on bacterial viability, this strategy intends to disarm bacteria. Several virulence pathways can be targeted, such as adhesion and secretion, but also bacterial communication (i.e., QS), which is involved in the regulation of virulence in S. aureus. In what’s next, several virulence factors currently being targeted in S. aureus, will be highlighted.

Sortase inhibitors In order to initiate infection, S. aureus adheres to components of the host extracellular matrix. The microbe adheres to and invades host tissues using a variety of cell wall-anchored (CWA) molecules, of which the ‘microbial surface components recognizing adhesive matrix molecules’ (MSCRAMMs) are the largest class. MSCRAMMs recognize host extracellular proteins like fibrinogen, collagen, and fibronectin and are covalently linked to PG by the transpeptidase sortase A (SrtA). SrtA is required for abscess formation and staphylococcal


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persistence in host tissues.66 Also, inhibition of SrtA causes reduction of biofilm formation in some staphylococcal strains.67

Figure 6: Examples of sortase inhibitors.

Hence, over the past decade, many studies have focused on agents that inhibit SrtA. Examples of natural products are isoaaptamine 34 (Figure 6), isolated from the marine sponge Aaptos aaptos68 and bis-indole alkaloid 35 from Spongosorites sp.69 Using HTS, Suree et al. identified several compounds that inhibit SrtA. Structure-activity relationship analysis led to the identification of potent pyridazinone and pyrazolethione derivatives (SrtA IC 50 values in submicromolar range).70 More recently, Zhang et al. identified compound 36 and related compounds, which block sortase activity both in vitro and in vivo.71 Cascioferro et al. wrote a review on SrtA inhibitors.72

Interference with STX biosynthesis STX plays an important role in S. aureus virulence. The golden carotenoid pigment, with numerous double bonds, shields the bacterium from host oxidant killing. 73 The first committed step in the biosynthetic pathway of STX (Figure 7), catalyzed by the enzyme CrtM (or 4,4’-diapophytoene synthase or dehydrosqualene synthase), starts with the condensation of two molecules of farnesyl diphosphate (FPP) to produce the C30 species presqualene diphosphate.74 The latter then undergoes skeletal rearrangement and loss of diphosphate to form 4,4’-diapophytoene (or dehydrosqualene). The formation of dehydrosqualene in S. aureus is very similar to the formation of squalene (via the enzyme squalene synthase,



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SQS) in plants, animals, and fungi. Squalene is an important precursor of several sterols like cholesterol, ergosterol, and plant sterols. In 2008, Oldfield and coworkers questioned whether S. aureus CrtM and the human squalene synthase possess structural similarity. Although there is only modest sequence resemblance, the researchers found that the overall fold of the two enzymes shows clear similarity. Therefore, they investigated whether any of the many known squalene synthase inhibitors previously developed as cholesterol lowering drugs also have activity in inhibiting STX biosynthesis and hence S. aureus virulence.75 They reported the first-generation CrtM inhibitor BPH-652 (37), which is now completing IND-enabling studies by AuricX Pharmaceuticals Inc.

Figure 7: Small molecules that interfere with staphyloxanthine biosynthesis.

Phosphonosulfonate BPH-652 did not affect S. aureus growth, nor survival in vitro, but upon treatment with 37, the resulting non-pigmented bacteria were less able to survive in freshly


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isolated human whole blood than non-treated S. aureus (as expected because they contained no or less antioxidant carotenoid pigment). Several CrtM inhibitors have subsequently been identified by the same group.76, 77 Further down the biosynthetic pathway of STX, dehydrosqualene is converted into 4,4’-diaponeurosporene

via

successive

dehydrogenations,

catalyzed

by

CrtN

(or

4,4’-diapophytoene desaturase). Very recently, Chen et al. screened a library of approximately 400 existing drugs against S. aureus infection and identified naftifine (38) as a CrtN inhibitor. This allylamine antifungal drug was capable of prohibiting STX biosynthesis at nanomolar concentrations and attenuated the virulence of a variety of clinical S. aureus isolates (including MRSA strains) in mouse infection models. Naftifine did not function as an antibiotic against S. aureus.78 Based on this pioneering work, Wang et al. used a scaffold hopping approach to discover a new class of benzofuran-containing CrtN inhibitors with enhanced oral bioavailability. The most potent analogue identified in this study (39) is depicted in Figure 7.79 As indicated above, 5-iodoindole (32) was observed to decrease STX production in S. aureus as well.61 Since indole and 7-benzyloxyindole were found to inhibit the production of the carotenoid pigment, the researchers investigated STX production in two S. aureus strains, including an MRSA strain. 5-Iodoindole was found much more potent than indole. A mechanism of action was not described.

Interference with caseinolytic protein protease Another attractive target for antivirulence drugs is the caseinolytic protein protease (ClpP). This serine protease plays a central role in S. aureus virulence.80 The absence of ClpP causes upregulation of several transcriptional repressors of virulence genes, such as the sarA family and also a downregulation of the Agr QS system (see below). 81



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Figure 8: Structures that interfere with caseinolytic protein protease.

Böttcher and Sieber demonstrated that inhibition of this virulence regulator by synthetic β-lactones decreased expression of major virulence factors. Their most potent inhibitor, D3 (40) (Figure 8), was able to abolish hemolytic and proteolytic activities in MRSA and also showed a decrease in lipase and DNase expression. 82 Based on D3, several optimized structures have been published.83,

84

Compound U1 (41), with a phenylethyl side chain

appeared as one of the most potent candidates. Weinandy et al. showed that although U1 exhibits limited plasma stability (incubation in plasma led to rapid hydrolysis), local application of the β-lactone led to reduction of abscess development in mice. In this setup, no inhibition of S. aureus growth was seen.85

Substantial progress has been made in antivirulence approaches. However, the small molecules described in this chapter are still in preclinical development. Moreover, although these compounds have demonstrated that S. aureus virulence factors can be antagonized, it is currently not clear whether their potential use is restricted to prophylaxis or may also be beneficial for treatment of S. aureus infections. Nonetheless, antivirulence is a promising and developing discipline.

Interference with S aureus QS as an alternative strategy The accessory gene regulator system In staphylococci, the accessory gene regulator (agr) locus is part of the global virulence response (Figure 9).86,

87

The operon comprises two transcripts, RNAII and RNAIII,


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originating from the P2 and P3 promotors, respectively. RNAII encodes the constituents of the Agr system itself: AgrB, AgrD, AgrC, and AgrA. This auto-activation circuit is a two-component signal transduction system (TCS) and employs cyclic thiolactone peptides as signaling molecules. These auto-inducing peptides (AIP) are produced as a pro-peptide (AgrD). AgrB, a transmembrane endopeptidase, subsequently processes the pro-peptides into active AIPs (7-9 amino acid peptides that contain a thiolactone ring) and secretes them. The type I signal peptidase SpsB also plays a role in the processing of AgrD. The AIPs accumulate in the extracellular environment and, when reaching a threshold concentration, bind to AgrC, the sensor kinase of the TCS. AgrC, which is phosphorylated in an AIP-dependent manner, then activates AgrA, the response regulator. The activated AgrA then binds the agr promotor regions P2 and P3, with a preference for P2 at low concentrations, maintaining activation of its own expression.88 RNAIII, a regulatory mRNA, acts as the effector of the Agr QS system. The Agr system in S. aureus controls the expression of 70 to 150 genes and is primarily implicated in the invasiveness of S. aureus.89 It is responsible for the upregulation of both toxins and enzymes (proteases, nucleases, lipases, etc.). Almost all known virulence factors are upregulated by agr, while cell surface proteins, such as protein A and fibronectin-binding proteins are downregulated. Surface-binding proteins such as MSCRAMMs are important for establishment of infection (see above), while nutrient acquisition becomes more important in later stages of infection.87, 90 Agr is important for acute virulence, while the absence of Agr functionality may be beneficial in specific types of S. aureus infection (chronic, biofilm-related infections and bacteraemia for example). Indeed, agr mutants exhibit reduced production of virulence factors, but are shown to produce extensively thick and unstructured biofilms.91-93



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Figure 9: Cartoon of the Agr quorum sensing system (left) and the RAP/TRAP system (right) in S. aureus. The exact protein chemistry of the RAP/TRAP system is incompletely understood. It is also unclear whether the two systems are linked.

The structure of RNAIII, but not its sequence, is conserved among staphylococcal species. 94 RNAIII acts both as an mRNA, encoding δ-hemolysin, and as a regulator of virulence. Precise mechanisms of RNAIII action have been elucidated for a number of target genes. Both transcriptional and translational regulation are involved.95 Agr belongs to a complex regulatory network. Several regulatory genes (e.g., sarA, sarU, clpP, etc.) directly or indirectly up- or downregulate its expression. This intricate regulation of agr expression provides additional regulation loops. A detailed description of this multifaceted regulation goes beyond the scope of this review and the reader is referred to Novick & Geisinger.86 Nevertheless, the high complexity of this regulatory network reinforces the fact that S. aureus has the ability to very precisely adapt its physiology according to environmental signals and


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changes. The use of QS modulators as an alternative to or as potentiators of routine antibiotics has been proposed in literature and efficacious inhibitors of Agr signaling have been reported.93, 96, 97 The secondary fungal metabolite ambuic acid (42) (Figure 10) for instance, was shown to inhibit AIP biosynthesis.98 Allelic variations in the agr region have resulted in the existence of four main classes of S. aureus AIPs (agr-I to agr-IV). With a different amino acid sequence, but conserved thiolactone macrocycle, the specific AIPs of each group can induce their own agr expression and competitively inhibit non-cognate AgrC receptors. This phenomenon is also sometimes referred to as cross-inhibition and offers therapeutic potential. Several structure-activity relationship studies have been set up with the aim to develop inhibitors of S. aureus agr groups. Modified and/or truncated AIPs have been suggested as ligands capable of intercepting AIP:AgrC interaction. Khan et al. and Gordon et al. reviewed the efforts in this area of research.93, 96 Also non-S. aureus AIPs have been investigated. For example, cross interference between S. epidermidis and S. aureus and also between S. intermedius and S. aureus agr groups has been evaluated.93 Even non-staphylococcal autoinducers have been gauged as virulence attenuators. Qazi et al. found that the Pseudomonas aeruginosa QS molecule N-(3-oxododecanoyl)-L-homoserine lactone (or 3-oxo-C12-HSL, 43) elicits inhibitory effects against agr at subgrowth inhibitory concentrations. The lactone antagonizes the production of S. aureus exotoxins.99 The same group utilized 43 as a starting point for the synthesis of a series of analogues to gain insight into the structural features involved, the mechanism of action, and in vivo efficacy.100 In this study, the authors identified two new related classes of compounds: tetramic acids (TMA) and tetronic acids (TOA). These molecules act as noncompetitive AgrC inhibitors and are more potent than the parent compound 3-oxo-C12-HSL. Their most potent analogue identified



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(C14-TOA, 44) reduced colonization of human nasal epithelial cells and also reduced arthritis in a murine infection model without showing toxicity. The cyclic dipeptides cyclo( L-Tyr-L-Pro) 45 and cyclo(L-Phe-L-Pro) 46 produced by the human vaginal isolate Lactobacillus reuteri RC-14 show antagonistic activity towards all four S. aureus agr groups; the molecules repress the expression of toxic shock syndrome toxin-1 (TSST-1) via a yet unknown mechanism.101 The marine Photobacterium halotolerans produces a group of molecules, called solonamides, that display S. aureus Agr inhibitory activity as well. These cyclodepsipeptides strongly reduce expression of RNAIII as competitive inhibitors of the AgrC receptor. Solonamide B (47) was found to inhibit all agr types and dramatically reduced the hemolytic activity and phenol-soluble

modulin

(PSM)

production

of

community-associated

(CA)-MRSA

USA300.102, 103 Blockage of AIP-mediated QS is not restricted to peptides. Receptor binding studies indicate that benzbromarone (48), traditionally used as gout medication, may inhibit binding of AIP to AgrC.


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Figure 10: Compounds that disturb accessory gene regulator quorum sensing signaling.

Not only AgrC has been described as a target for antivirulence molecules. ω-Hydroxyemodin (OHM,

49),

for

instance,

antagonizes

AgrA

function.

Daly et al.

showed

this

polyhydroxyanthraquinone to exert in vivo efficacy against S. aureus QS-mediated virulence by direct binding to AgrA.104 Khodaverdian et al. recently described other agents that bind the response regulator AgrA. Via in silico screening, a series of naphthalene and biaryl compounds were found to inhibit the production of α-hemolysin and PSMα. One of these small molecules is the FDA-approved NSAID diflunisal (50).105 Finally, savirin (Staphylococcus aureus virulence inhibitor, 51), a selective inhibitor of AgrA, was discovered by HTS and impedes agr-mediated QS across all four agr groups.106 Savarin is a molecule that displays S. aureus-specific Agr inhibition. Indeed, drugs interfering with S. aureus Agr ideally not interfere with the important skin commensal S. epidermidis.93, 106



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The RAP/TRAP system An additional QS system in S. aureus has been proposed. This system operates via the autoinducer RNAIII-activating protein (RAP), which activates histidine phosphorylation of Target of RNAIII-activating protein (TRAP), ultimately resulting in production of the regulatory RNAIII (Figure 9).107 It is unclear how RAP secretion occurs. Next to that, the precise function and protein chemistry of TRAP are not fully understood: it is unclear what enzyme is involved in the phosphorylation of TRAP and how the TRAP signal is transferred to the genome.108,

109

Also, it is not yet completely clear whether the agr and RAP/TRAP

mechanisms act independently or in tandem.110-113 TRAP is a membrane-associated protein but has no transmembrane domain. Balaban et al. demonstrated that a TRAP-negative mutant formed very little biofilm compared to the parent strain S. aureus 8325-4. This implies that TRAP regulates genes involved in biofilm formation in S. aureus.114 TRAP expression is constitutive, but its phosphorylation is regulated by RAP. Since TRAP is highly conserved among staphylococcal strains and because it is constitutively expressed, it has been investigated in vaccine development for preventing staphylococcal mastitis in dairy cows.115 RNAIII-inhibiting Peptide (RIP), originally isolated from culture supernatants of coagulase negative staphylococci, competes with RAP for TRAP phosphorylation. 116-119 The sequence of synthetic RIP (H-Tyr-Ser-Pro-Trp-Thr-Asn-Phe-NH2) shows some degree of similarity with the sequence of residues 4-9 of RAP (Tyr-Lys-Pro-Ile-Thr-Asn). This suggests that both molecules bind to the same receptor, one as agonist (RAP) and one as antagonist (RIP). RIP was shown to inhibit S. aureus pathogenesis: the peptide inhibits agr activity (synthesis of both RNAII and RNAIII). Interestingly, RIP was also found to reduce the ability of S. aureus to adhere to polystyrene and to mammalian HEp2-cells.117 This ‘dual activity’ (inhibition of both virulence and adhesion) is in contrast to what is seen with AIPs. Indeed, as stated earlier, inhibition of the Agr system leads to increased rather than decreased biofilm formation.


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Balaban et al. demonstrated that the heptapeptide RIP can treat preformed device-associated staphylococcal infections in a rat graft model. Also, RIP-teicoplanin combination treatment resulted in a significant reduction of the bacterial load, compared to treatment with either agent alone.114 Via alanine scanning, the key residues for the activity of RIP were studied by Baldasserre et al. For this structure-activity relationship study, the researchers synthesized single alanine derivatives of RIP, as well as truncated compounds. None of the derivatives showed in vitro killing activities.120 Inter alia, an enhanced activity was seen for tetrapeptide FS-10 (H-Ser-Pro-Trp-Thr-NH2) in a rat model of vascular graft infection. Very recently, Simonetti et al. showed that the combined administration of topical FS-10 and intraperitoneal tigecycline can be used to treat systemic MRSA infection in a murine model. The exact mechanism of action of the truncated RIP derivative has yet to be elucidated, but the authors hypothesize that, analogous to RIP, FS-10 could compete with RAP for activation of TRAP.121 Hamamelitannin (HAM, 52) (Figure 11) or 2’,5-di-O-galloyl-D-hamamelose, is a natural molecule isolated from the bark and leaves of the American witch hazel (Hamamelis virginiana L.).122 In a publication from 2008, Kiran et al. discovered that HAM acts as a non-peptide analogue of RIP. Analogous to RIP, the natural tannin was shown to prevent biofilm formation and RNAIII production in vitro as well as in vivo (in device-associated infections).123 Brackman et al. later demonstrated that HAM increases the susceptibility of S. aureus biofilms towards vancomycin in vitro and also in vivo in a Caenorhabditis elegans (C. elegans) and a Galleria mellonella model of infection.124 Only very recently, the same group created a framework for the presumed mechanism of action of the natural molecule. 125 In this pioneering work, they provide evidence that HAM indeed affects S. aureus biofilm susceptibility through the TRAP receptor. The authors conclude that HAM affects a set of genes, which ultimately leads to a reduction in cell wall thickness and the amount of



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extracellular DNA (eDNA) in the biofilm matrix. HAM increases the susceptibility of S. aureus biofilms towards several classes of antibiotics and only affects susceptibility of S. aureus biofilms (and not that of other staphylococci or other bacteria). Finally, the potential clinical application of HAM was evaluated in a C. elegans and a mouse mammary gland infection model of S. aureus. The molecule affected biofilm susceptibility in both models.

Figure 11: Structure of hamamelitannin (52) and optimized analogue (53).

HAM is an interesting hit, but its structure is not ideal. Therefore, our group investigated the structure-activity relationship of HAM in order to identify derivatives, which are more active and metabolically more stable.126 Vermote et al. reported on the identification of a metabolically stable compound (53) with potent in vitro activity and exceptional activity in a C. elegans infection model and a murine mastitis model, while lacking cytotoxicity against MRC-5 lung fibroblast cells. Several other HAM derivatives that warrant further study have been identified as well.127-129

Inhibition of QS signaling in S. aureus seems a promising antivirulence approach. Many small molecules that interfere with S. aureus QS have been identified. However, with respect to QS inhibition, a number of questions remain unanswered. QS systems are very complex, diverse, and sometimes even ‘redundantly organized’, which implies that our current knowledge of QS probably only scratches the surface. A concern when targeting Agr QS, for example, is whether inhibitors of this system can possibly be used in biofilm-related infections, since biofilm formation is negatively impacted by Agr. Yarwood et al. found that in an established biofilm, pockets of agr-activated S. aureus cells


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detached under in vitro flow conditions, while agr inactive cells remained in the biofilm.110 Boles and Horswill (2008) reported that exogenous AIP addition reactivates the agr system in a mature biofilm and triggered detachment. Maybe even more importantly, this biofilm detachment restored sensitivity of the dispersed cells to the antibiotic rifampin. 130 It seems that, in this context, the scientific community is caught between Scylla and Charybdis: either we choose for Agr inhibition with the chance of inducing more chronic biofilm-related infections (but also less virulent) or we go for Agr activation, which might convert S. aureus into a more invasive pathogen (but also more amenable to treatment). Also, in order to interfere with the Agr system for the treatment of infections, determination of the agr type will be necessary and care providers will need specific AIP signals on-hand for therapy (as each type of staphylococcal strain recognizes unique AIP signals). 131 When looking at the RAP/TRAP system, the precise function and protein chemistry is not completely understood. How does TRAP get phosphorylated upon binding with RAP? How is the signal transferred onto the genome? Is the RAP/TRAP system connected to the Agr system, and if so, how exactly? Conflicting results between authors110,111,112,113 calls for further research to improve our understanding of the mechanism of action of the QS inhibitors. Nevertheless, several research groups have provided very promising new leads for further study and have presented strong preclinical data, including sufficient in vivo efficacy using appropriate animal models.

Discussion Multidrug-resistant pathogens are a growing threat to human health and their worldwide spread presents formidable therapeutic difficulties for clinicians. The scarcity of new antibiotics being introduced adds to this problem. The current antibiotic pipeline is weak; over the last decade, the FDA has approved new antibiotics, but none of these belongs to a new



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antibiotic class, with the exception of bedaquiline (for the treatment of TB). Antibiotics have saved countless lives and without access to such drugs, the slightest injury could become life-threatening, with increased mortality, morbidity, and cost of patient care. Moreover, exhaustion of efficacious antibiotics may preclude to perform common surgical procedures and immunosuppressing cancer therapy in the future. A prominent example of such a multidrug-resistant pathogen is MRSA. Doom-and-gloom reports about this superbug, which is notorious for its biofilm formation and resistance to conventional antibiotics, are published regularly. The development of new antibiotics is one important approach for the treatment of (multidrug-resistant) bacterial infections. However, given the difficulty of antibiotic drug discovery and the unrelenting challenge of resistance, alternative approaches should be considered as well. One such strategy is the use of combinations of drugs, a paradigm that is clinically proven in many areas of medicine. The use of drug cocktails is important in the treatment of many cancers and viral infections (e.g., with human immunodeficiency virus [HIV]). Also for the treatment of bacterial infections, combination therapy is well-established. Patients with TB, for instance, are treated with combinations of up to four drugs. Another well-known example is the combination of sulfamethoxazole and trimethoprim, two antibiotics that inhibit successive steps in the folate biosynthesis. Furthermore, the streptogramins quinupristin/dalfopristin are used in combination for the treatment of infections by staphylococci and vancomycin-resistant Enterococcus faecalis. For the treatment of severe MRSA infections, antibiotic combination therapy has been investigated extensively. Although limited clinical data are available (and tests have primarily been done in vitro), the combination of several β-lactam antibiotics with either vancomycin or daptomycin seems an attractive alternative to monotherapy. Davis et al. reviewed combination antibiotic treatment for serious MRSA infections.132


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Not only antibiotic-antibiotic combinations have been studied. In this review, we focused on the pairing of an antibiotic with a non-antibiotic molecule, which potentiates the activity of the former. Although not possessing growth-inhibitory activity by themselves, small-molecule ‘potentiators’ can reduce antibiotic doses. The potentiators discussed herein function either by reversing resistance mechanisms, by attenuating S. aureus virulence, and/or by interfering with QS. Such a strategy potentially has a few advantages over ‘conventional’ approaches. 133,

134

Firstly, an increased number of pharmacological targets could be addressed. Secondly, since most virulence factors are not essential for bacterial viability, antivirulence drugs would exert less selective pressure on bacteria. Gerdt and Blackwell demonstrated a ‘robustness’ of QS inhibitors (QSI) against resistance. The authors expect that QSI-resistant mutants will arise, but hypothesize that, in contrast to organisms that are resistant towards traditional antibiotics, such mutants would struggle to overtake the population. 135 Thirdly, gut microbiota would potentially be preserved because antivirulence compounds exert a very specific effect. However, development of antivirulence drugs presents challenges as well. Established screening systems with determination of MICs can no longer be used; to screen such compounds, specialized assays (both in vitro and in vivo) need to be developed. Also, in this particular field, in addition to proving the interference with a certain target (e.g., a resistance element, virulence factor, or QS component), it is also essential to prove that such interference leads to potentiation of the activity of a simultaneously administered antibiotic. Moreover, specific disarming drugs will very likely have a narrow spectrum of activity. This also means that their potential success in the clinic will depend on rapid diagnostics to identify the causative agent.133 Another question is whether such drugs will be able to attenuate already existing infections in humans.96, 97 Moreover, it remains to be seen whether bacteria will be able to find a way around antivirulence therapy with their complex, diverse, and even



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‘redundantly organized’ regulatory systems that control virulence. Another important issue with the deployment of drug combinations, is the fact that drug-drug interactions (DDIs) can occur. ADME properties of each compound can be altered by co-administration with another drug (e.g., plasma protein binding and drug metabolism). Hence, searching for optimized drug ratios will be required.136, 137 In the myriad of recently reported potentiators for antibiotics against S. aureus, it is difficult to benchmark compounds. Only a few potentiators have been evaluated in vivo and although some small molecules perform well in animal models of S. aureus infection, it would be very interesting to see how the different potentiators described in literature relate to each other. Now, different animal models, conditions, drug concentrations, and S. aureus strains are used to test potentiator activity. It must come as no surprise that comparison of the potentiating ability of small molecules is challenging when different measures for outcome are used in literature. Some of the examples described in this review, including e.g., ticlopidine (6), clopidogrel (8), disulfiram (9), celecoxib (25), and naftifine (38), are currently in clinical use for other purposes. Indeed, the process of identifying new indications for existing drugs outside the scope of the original medical use is an increasingly popular strategy. As such repositioning candidates have survived several stages of clinical testing, their safety and pharmacokinetic profile is well known and the risk of failure due to toxicology reasons is reduced. The strategy can possibly lead to an acceleration of the R&D process and is economically attractive when compared with the cost of de novo drug discovery and development. However, repositioning strategies face some unique challenges as well. As prior art might already exist for the repositioning candidates, intellectual property issues may be complex. Also, from a commercial point of view, it is of utmost importance to evaluate the candidate’s potential for attaining a competitive profile in the market.138


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Taking into account all of the abovementioned, the challenge of resistant staphylococcal infections is complex and multidisciplinary and we believe that the potentiators highlighted herein illustrate important starting points for future therapeutic options.



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Abbreviations 2-AIT 3-oxo-C12-HSL 5’-MHC aac(6’)/aph(2”) Agr AIP AME AMR ant(4’)-I aph(3”)-III C. elegans CA-MRSA ccr ClpP CrtM CrtN CWA DDI eDNA EPI ERM FPP HAM HEp2 HIV HTS L. MDR MDR-TB MIC MRC-5 MRSA MSCRAMM NAG NAM NSAID OHM PAD PBP PG PSM QS QSI RAP RIP RMA RNA S. aureus S. epidermidis SarA/U

2-aminoimidazole/triazole N-(3-oxododecanoyl)-L-homoserine lactone 5’-methoxyhydnocarpin Aminoglycoside-6‘-N-acetyltransferase/2”-O-phosphoryltransferase Accessory gene regulation Auto-inducing peptide Aminoglycoside-modifying enzyme Antimicrobial resistance Aminoglycoside-4’-O-phosphoryltransferase I Aminoglycoside-3”-O-phosphoryltransferase III Caenorhabditis elegans Community-acquired MRSA Cassette chromosome recombinase Caseinolytic protein protease 4,4’-diapophytoene synthase or dehydrosqualene synthase 4,4’-diapophytoene desaturase Cell wall-anchored Drug-drug interaction Extracellular deoxyribonucleic acid Efflux pump inhibitor Erythromycin resistance methylase Farnesyl diphosphate Hamamelitannin Human Epithelial type 2 Human immunodeficiency virus High-throughput screening Linnaeus Multidrug-resistant Multidrug-resistant tuberculosis Minimum inhibitory concentration Medical Research Council cell strain 5 Methicillin-resistant Staphylococcus aureus Microbial surface components recognizing adhesive matrix molecule N-acetylglucosamine N-acetylmuramic acid Non-steroidal non-inflammatory drug ω-Hydroxyemodin Previously approved drug Penicillin binding protein Peptidoglycan Phenol-soluble modulin Quorum sensing Quorum sensing inhibitor RNAIII-activating protein RNAIII-inhibiting protein Resistance-modifying agent Ribonucleic acid Staphylococcus aureus Staphylococcus epidermidis Staphylococcal accessory regulator A/U


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Savarin SCC SCV SpsB SQS SrtA STX TB TCS TMA TOA TRAP TSST-1 UDP WHO WTA


Staphylococcus aureus virulence inhibitor Staphylococcal chromosome cassettes Small colony variant Staphylococcus aureus signal peptidase Squalene synthase Sortase A Staphyloxanthine Tuberculosis Two-component signal transduction system Tetramic acid Tetronic acid Target of RNAIII-activating protein Toxic shock syndrome toxin-1 Uridine diphosphate World Health Organization Wall teichoic acid

Author information Corresponding Author Tel: +32 (0)9 264 81 24. Fax: +32 (0)9 264 81 46. E-mail: [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.



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