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 establishmentof recalcitrant, biofilm-related infections, is a well-known and notoriousexample of a highly resistant micro-organism. Since resistance development isunavoidable with conventional antibiotics that target bacterial viability, it isvital to develop alternative treatment options on top. Strategies aimed at moresubtle manipulation of bacterial behavior have recently attracted attention.Here, we provide a literature overview of several small-molecule potentiatorsfor antibiotics, identified for the treatment of Staphylococcus aureus infection.Typically, these potentiators are not bactericidal by themselves and functionby reversing resistance mechanisms, by attenuating Staphylococcus aureusvirulence, and/or by interfering with quorum sensing.
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 scientificachievements of the twentieth century. With time, however,the utility of these drugs has become compromised by theemergence of antimicrobial resistance (AMR). Resistant micro-organisms are able to withstand the attack by antimicrobialdrugs, so that infections persist. Infections with, for instance,methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae, multidrug-resistant tuber-culosis (MDR-TB), or vancomycin-resistant Enterococcus areregularly making headline news: these “superbugs” are difficultto treat with existing medicines.The decreasing effectiveness of conventional antimicrobial
drugs is posing a serious problem to patients and health careproviders. The World Health Organization has recognizedantimicrobial resistance as a global problem since 2001.1 In arecently published report, commissioned by the Britishgovernment, economist O’Neill and coauthors estimate thatby 2050 10 million lives a year will be at risk due to the rise ofdrug-resistant infections.2 Even today, superbacteria cause theloss of 700 000 people worldwide, every year.2 Even more, thespread of antibiotic resistance could possibly shake thefoundations of modern healthcare in numerous aspects:without access to effective antibiotics, the slightest injurycould become life-threatening and key medical procedures (e.g.,surgery, caesarean section, transplantation, or chemotherapy forcancer) could become too dangerous to perform. Theeconomic impact is also already substantial and continues toclimb.3
In the not so distant past, antimicrobial discoveryexperienced an era of prosperity (the so-called “golden era”).
Systematic screening of soil-derived microbes for antibacterialactivity, a very successful platform introduced by SelmanWaksman in the 1940s, led to the discovery of nearly allantibiotic classes in use today.4 Streptomycin, chloramphenicol,and erythromycin A are only a few examples of drugs identifiedvia one of the many, large antibiotic screening programs afterthe Second World War. Indeed, chemotherapy for bacterialinfections has been dominated by natural products.Antibacterial drugs have been identified via a “classical”
approach, in which determination of the minimum inhibitoryconcentration (MIC) in whole-cell assays was the mainstay.Some antimicrobials, derived from nature, were marketed evenwithout modification. Tetracycline, vancomycin, penicillin G,and the recently marketed daptomycin are examples of drugsthat combine satisfactory in vitro potency (MIC) withacceptable physicochemical, pharmacological, and toxicologicalprofiles. Typical limitations of natural products are chemicallability, suboptimal pharmacokinetic characteristics, and toxicityissues. Hence, many natural substances became leads forchemical optimization programs (semisynthetic derivatives).5
In the 1960s, the returns from the once effective and “brute”screening efforts diminished, and although there have been anumber of antibacterial agents that had their origin in syntheticchemistry (e.g., fluoroquinolones and oxazolidinones), effortsto 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 isvery small. The majority of the most successful antibiotics
Received: June 19, 2017Published: September 11, 2017
essentially hit only a few major targets or pathways: cell wallsynthesis, the ribosome, DNA gyrase or topoisomerase, andfolate biosynthesis. Even if genomic analyses are revealingpotential targets in pathogens and target-based approaches mayoffer new opportunities, pharmaceutical companies have beenrather unsuccessful in identifying novel agents via thisparadigm.Consequently, pharma has mainly been investigating
structural variations of established antibacterial classes. Fordecades, in a multidisciplinary effort, medicinal chemists havebeen introducing additional properties (e.g., solubility,antibacterial spectrum, and tolerability) into natural antibiotics,without jeopardizing intrinsic activity. Toward this end, both denovo synthesis and semisynthetic approaches have been used.5
Development and marketing approval of new antibiotics havenot kept pace with the increasing public health threat of anunremitting emergence of resistance. The origin of thechemoresistance is multifactorial. Inappropriate prescriptionby clinicians and veterinarians, imprudent utilization, andextensive use of antimicrobials in bioindustry, together withglobalization and a lack of public awareness, have definitelycontributed to this pandemic health problem.2
A detailed analysis of the reasons for AMR is beyond thescope of this Review, but by reducing unnecessary con-sumption, improving hygiene, and preventing spread ofinfection, we can already have a powerful impact. However,the question remains whether such measures will suffice.Bacteria have demonstrated the ability to develop resistance tovirtually every antibiotic introduced by the medical community.Rapid bacterial selection by the use of conventional antibioticsinevitably leads to an acceleration of resistance development. Itis important to keep in mind that bacteria have always beenresistant to antibiotics. As described earlier, most antibiotics are(derived from) natural compounds produced by microbes. Foras long as antibiotics have existed, bacterial resistance hasexisted alongside. In a wide variety of environments, soilsamples have been found to contain resistance genes.6
Illustrating this aspect is the fact that, several years before theintroduction of penicillin as an antibiotic, “a substancedestroying the growth-inhibiting property of penicillin” wasalready identified.7
Moreover, the very short generation time of bacteria, theirlarge population size, and the ability to exchange DNA viahorizontal gene transfer gives the microbes a major advantagein the everlasting battle. An extra challenge of antibioticresistance is that bacteria have evolved several distinctmechanisms to cope with antibiotic therapy. Moreover, manypathogens harbor several mechanisms simultaneously.In such a scenario, where selective pressure from the
environment drives enrichment of specific genes that promotefitness and survival, it is imperative to continuously discoverfresh antimicrobials or new practices that are effective for thetreatment of infectious diseases.
■ Staphylococcus aureusStaphylococcus aureus (S. aureus) is a Gram-positive bacteriumthat appears as grape-like clusters of berry-shaped cells uponmicroscopic examination. S. aureus appears as a harmlesscommensal organism and colonizes the skin and mucosae ofhuman beings and several animal species.8 Approximately 30%of the human population carries this micro-organism in theanterior 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 appearsas a harmless commensal, it can cause disease as well. As a veryversatile pathogen, this micro-organism can cause a range ofinfections and syndromes, most notably skin and soft tissueinfections. When inoculated into an open wound, infections(e.g., boils, pimples, and impetigo) develop frequently. Many ofsuch infections cause the production of pus and are said to be“pyogenic” (pus-forming). Other clinical manifestations includeendocarditis, osteoarticular infection, and pneumonia, next totoxin-mediated disease (e.g., food poisoning, toxic shocksyndrome, and scaled skin syndrome), and prosthetic deviceand catheter infections.10 In animals, S. aureus can cause seriousinfections too, such as bumblefoot in poultry. It is also one ofthe major causal agents of mastitis in dairy cows, one of themost frequent and costly diseases in the dairy industry.11,12
Staphylococcal infections often occur when resistance of thehost is low because of debilitating illness, wounds, or treatmentwith steroids or other drugs that compromise immunity.S. aureus is an opportunistic pathogen that possesses an
extensive arsenal of virulence factors. Moreover, some strainshold a battery of resistance mechanisms against conventionalantibiotics. Compounding the problem even further is the factthat S. aureus is notorious for its ability to form biofilms. Theformation of such a biofilm, a surface-attached encasement ofcells in a polymer-based matrix, enables the superbug to persist,due to increased resistance to antibiotics and the host immunesystem. Bacteria in biofilm communities display significantlyhigher resistance to several kinds of stress than their planktonicbrethren. Thus, biofilm formation adds a further level ofcomplexity to the already existing problem of antimicrobialresistance.Microbes regulate genes in response to signals in the
environment. A potential signal that prokaryotes can respondto is the presence of other bacterial cells. The ability of bacteriato assess their population density and control geneticallymediated responses is a very dynamic and important aspect ofmicrobial physiology. It is of practical use to ensure thatsufficient cell numbers are present before a specific geneproduct is made. This type of control is called quorum sensing(QS).13 QS involves production and recognition of smallsignaling molecules. These “chemical words” are alwaysproduced at a certain level. When their extracellularconcentration (which is representative for their “quorum”)reaches a threshold, they bind to a specific receptor. Ultimately,transcription of certain genes is activated. Bacterial biofilmformation, virulence in general, and antibiotic resistance areoften mediated by this cell-to-cell communication system. Forexample, toxin production by one bacterial cell would have noeffect and hence be a waste of resources. However, acoordinated expression of the toxin by a sufficiently highpopulation of cells may successfully cause disease. In S. aureus,both biofilm formation and virulence in general are regulatedvia QS.It is increasingly important and necessary to find new
antimicrobial agents, but since resistance development isunavoidable with agents that target bacterial viability, it isimperative to devise alternative measures as well. Strategiesaimed at more subtle manipulation of bacterial behavior haverecently attracted attention. In this Review, we provide thereader with a literature overview of several potentiators forantibiotics identified for the treatment of S. aureus infection. Wewill concentrate on small molecule potentiators that are notbactericidal but, in combination with conventional antibiotics,
enhance the antimicrobial activity of the latter. The discussedpotentiators function by reversing resistance mechanisms, byattenuating S. aureus virulence, and/or by interfering with QS.In Potentiations of Antibiotics against S.aureus via Inhibition ofResistance Mechanisms, we will focus on “resistance-modifyingagents” (RMAs) that potentiate the effect of antibiotics viainhibition or modification of specific resistance mechanisms ofS. aureus. First, several examples of compounds targetingantibiotic resistance elements acquired by S. aureus will begiven. Second, efflux pump inhibitors (EPIs) will be described.Third, compounds specifically targeting S. aureus biofilms andsmall-colony variants (SCVs) will be discussed. In TargetingS.aureus Virulence as an Alternative Approach, a closer lookwill be taken at potentiators that target S. aureus virulencefactors. Finally, in Interference with S.aureus QS as anAlternative Strategy, important examples of potentiatorsspecifically modulating QS mechanisms will be given. Asbiofilm 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 Potentiation of Antibiotics against S.aureusvia Inhibition of Resistance Mechanisms). In each category,descriptive examples (especially of medicinal chemistryprograms) will be given to create a framework. Conventionalantibiotics, vaccines, bacteriophages, lysins, and therapeuticantibodies (which obviously also have a place in the control ofbacterial infections14) will not be discussed here.
■ POTENTIATION OF ANTIBIOTICS AGAINSTS. aureus VIA INHIBITION OF RESISTANCEMECHANISMS
Inhibition of Acquired Antibiotic Resistance Ele-ments. Potentiation of antibiotic activity can occur viainhibition of antibiotic resistance elements. The prototypeexample in this category is the antibiotic-potentiator combina-tion Augmentin. Peptidoglycan (PG), the most importantcomponent of the bacterial cell wall, is a polymer made of N-acetylmuramic acid (NAM) alternating with N-acetylglucos-amine (NAG), which are cross-linked by chains of four aminoacids. Synthesis of PG (and ultimately the cell wall) occurs in anumber of stages that take place in different locations in thecell. Glycosidic bonds are formed between the disaccharidesand cross-links between the neighboring peptides. Thetranspeptidation and carboxypeptidation reactions are mediatedby penicillin binding proteins (PBPs).15 Eventually, severallayers of PG are formed. The β-lactam structure, present in β-lactam antibiotics, is capable of binding the PBPs and blockstheir ability to function normally. The transpeptidases areserine hydrolases: attack of the serine hydroxyl function on theβ-lactam amide bond forms a highly stable penicilloyl−enzymeintermediate. The inhibition of the enzyme is irreversible andcompromises cross-linking (and bacterial cell wall synthesis ingeneral).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 theantibiotic has the chance to exert its effect. The destruction ofthe amide bond in the drug renders it incapable of binding toPBPs, and thus, the bacterium becomes resistant to that drug oreven class of drugs.The β-lactamase inhibitor clavulanic acid (1 in Figure 1)
“augments” the activity of the β-lactam antibiotic amoxicillin, byinhibiting β-lactamases. Clavulanate was identified in 1976, anduntil now, it has allowed the continued use of amoxicillin,
demonstrating the effectiveness of adding inhibitors ofantibiotic-degrading enzymes. Next to clavulanic acid alsosulbactam (2) and tazobactam (3) have been registered. Thedevelopment of new β-lactamase inhibitors has been the subjectof several studies.16,17 The combination of ceftazidime withavibactam (4), a non-β-lactam β-lactamase inhibitor, forexample, was approved by the FDA in 2015 for the treatmentof certain multidrug-resistant (MDR) Gram-negative infec-tions.18
Inactivation of β-lactamases is a successful strategy toovercome resistance. However, MRSA strains have developedresistance through the acquisition of a different PBP (PBP2A)with reduced affinity for β-lactams. The gene that encodes thePBP2A protein is mecA and is acquired through horizontal genetransfer of a mobile genetic element known as staphylococcalcassette chromosome mec (SCCmec). With PBP2A, the use of aβ-lactamase inhibitor does not give too much solace. Onestrategy to overcome β-lactam resistance in MRSA is to developanalogues that act as PBP2A inhibitors.19
Another strategy that has been explored only very recently isthe combination of a β-lactam antibiotic with wall teichoic acid(WTA) synthesis inhibitors. There is emerging evidence thatWTAs, long anionic polymers covalently attached to PG inGram-positive bacteria, play an important role in the expressionof β-lactam resistance in MRSA. In S. aureus, the synthesis ofWTA 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-1-phosphate from uridine diphosphate (UDP)-NAG to themembrane-anchored undecaprenyl-phosphate carrier lipid.Then, TarA adds an N-acetylmannosamine moiety. Ultimately,what follows is elongation of this product into long polymerswith ribitol phosphate repeats.20 The function of WTAs isincompletely understood, but they are not essential for S. aureussurvival in vitro, since tarO can be deleted. However, it has beensuggested that WTAs are virulence factors required forstaphylococcal adhesion to host tissue and, more recently, inthe expression of β-lactam resistance. Farha et al. providedevidence that “a strain devoid of WTA leads to the mislocalizationof PBP4, compromising its role as a transpeptidase in PG cross-linking and specif ically in the case of CA-MRSA USA300 results insensitivity to certain β-lactams”.21
Figure 1. Structures of β-lactamase inhibitors (1−4) and inhibitors ofwall teichoic acid synthesis (5−8).
The natural product tunicamycin (5) was shown to inhibitTarO and displays synergy with β-lactam antibiotics, decreasingthe MIC of oxacillin against 1 MRSA clinical isolate from 50 to0.4 μg/mL at a concentration of only 0.08 μg/mL.20 Becauseblocking WTA biogenesis was shown to restore the efficacy ofβ-lactams in MRSA and also because tunicamycin is anonselective glycosyltransferase inhibitor (that also inhibitisPG synthesis) and has significant eukaryotic toxicity, Farha etal. set out to identify new inhibitors of WTA synthesis. Using alibrary of 2080 previously approved drugs (PADs), theresearchers conducted a screen for compounds capable ofpotentiating the activity of cefuroxime. The antiplatelet agentticlopidine (6) showed strong synergistic interactions withcefuroxime against several MRSA strains, lowering MICs by upto 64-fold. The authors also assessed the in vivo efficacy of thecombination of ticlopidine−cefuroxime in a Galleria mellonellamodel of MRSA infection. A significantly higher fraction oflarvae survived infection following combined treatment,compared to that of cefuroxime and ticlopidine alone. Themolecular target of the thienopyridine was identified as TarO,and the molecule is devoid of antibiotic activity on its own.21
This meticulous chemical genetic study of Farha et al. hasprovided a promising new lead for further study, and the sameresearch group identified a ticlopidine derivative (7) that showsimproved activity and avoids cytochrome P-450 mediatedoxidation to less potent TarO inhibitors. Because strongsynergies of cefuroxime with clopidogrel (8), a commerciallyavailable analogue of ticlopidine, were observed as well, theauthors also synthesized clopidogrel analogues.22
In a similar way to Farha et al., Ejim and co-workersconducted a systematic screening of approved nonantibioticcompounds for potentiator activity. They explored combina-tions of minocycline with 1057 PADs. Disulfiram (9) (Figure
2), an inhibitor of acetaldehyde dehydrogenase used for thetreatment of alcoholism, showed strong synergy with theantibiotic on growth inhibition of S. aureus.23
Hu et al. unraveled that a series of “clicked” triazolylglycolipid derivatives had the ability to increase thesusceptibility of a panel of clinical isolates of MRSA to β-lactam antibiotics. The glycolipids showed weak antibacterialeffect when used alone (≥256 μg/mL) against MRSAATCC43300. The authors used “Glc12” (10), the compoundthat showed the best synergistic effect in this study, toinvestigate the mechanism of action. Using quantitative real-time PCR, the expression level of mecA was analyzed in theabsence and presence of oxacillin and/or Glc12. The antibioticalone enhanced expression of mecA while Glc12 alone did not.Excitingly, the gene expression was lower with the combinationtreatment, compared to oxacillin alone. This suggests that aPBP2A suppression pathway is involved, but the exactmechanism remains unknown.24
In S. aureus, exposure to cell wall-acting antibiotics (includingβ-lactams and glycopeptides) leads to the upregulation ofexpression of over 100 genes, which is referred to as the cellwall stress stimulon (CWSS). VraSR regulates the transcriptionof genes involved in cell wall biosynthesis.25,26 The group ofMelander reported on several 2-aminoimidazole-containingcompounds, derived from the marine natural products oroidin(11) and bromoageliferin (12) that suppress resistance inseveral MRSA clinical isolates. For the activity of the leadcompound from this series (13), they showed that the VraSRregulatory system plays an important role.27 Related series ofcompounds are able to inhibit and disperse biofilms (seebelow).The same group identified structurally very similar 1,4-
disubstituted 2-aminoimidazoletriazoles (2-AITs), one of which
Figure 2. Structures of disulfiram (9), Glc12 (10), and several 2-aminoimidazol-containing compounds (11−14).
(14) reduces the MIC of oxacillin in MRSA. Here as well,selected derivatives proved capable of inhibiting biofilmformation (see below).28
Next to that, it has been demonstrated that the antipsychoticdrug thioridazine (15) (Figure 3) reduces the oxacillin-inducedtranscription of mecA and expression of PBP2A. Thecombination of thioridazine and oxacillin also reducesexpression of BlaZ (encoding β-lactamase), as well astranscription of several genes of the VraSR regulon. Thephenothiazine increases the susceptibility of MRSA to β-lactams in several clinical isolates.29−31
Although the only antibiotic adjuvants that have been provenclinically useful are the β-lactamase inhibitors, Li et al. showthat the search for natural antibiotic potentiators is still aninteresting enterprise for the management of superbuginfections. They identified new erythromycin derivatives thatenhance the activity of β-lactams against MRSA in vitro, withouthaving anti-MRSA effect when used alone. Further research isrequired to ascertain the mechanism of action.32
Fukumoto et al. identified cyslabdan (16), an actinomycetemetabolite, as a potentiator for imipenem activity againstMRSA. The labdane-type diterpene did not inhibit growth butmarkedly reduced the MIC of the antibiotic from 16 to 0.015μg/mL. The activity of several other β-lactam antibiotics wasalso potentiated. Further studies on the mechanism of actionare in progress.33
Remarkably, MRSA has evolved an inducible mechanism forresistance against β-lactam antibiotics: the integral membraneprotein BlaR1 is a sensor/signal transducer, which isphosphorylated upon exposure to β-lactam antibiotics andcommunicates the presence of the latter to the cytoplasm. Theprocess is not completely understood, but signal transductionleads to activation of the cytoplasmic domain of BlaR1, a zincprotease. This ultimately leads to the derepression oftranscriptional 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 kinaseinhibitors for their ability to lower the MIC of oxacillin againstMRSA252.34 In this initial screening, compound 17 wasidentified. The known mammalian serine/threonine kinase
inhibitor gave a 4-fold decrease in the MIC of oxacillin at aconcentration of 7 μg/mL, while the MIC of the kinaseinhibitor alone was ≥64 μg/mL. In their efforts to optimize thestructure of this hit, 70 structural analogues of 17 weresynthesized and tested. One of the most potent derivatives (18)exhibited remarkable activity in lowering the oxacillin MICagainst 3 MRSA strains, without showing antibacterial activityon its own.Relief of antibiotic resistance is a strategy that is potentially
also applicable to other resistance enzymes than β-lactamasesalone. Resistance to aminoglycosides for example can occur viathe production of an aminoglycoside-modifying enzyme(AME) that chemically modifies the drug. Three types ofAMEs have been identified in clinical isolates of S. aureus:aminoglycoside-3″-O-phosphoryltransferase III [aph(3″)-III],aminoglycoside-4′-O-phosphoryltransferase I [ant(4′)-I], andaminoglycoside-6′-N-acetyltransferase/2″-O-phosphoryltrans-ferase [aac(6′)/aph(2″)]. Chemical modification of the amino-glycoside drugs by these enzymes decreases their ability to bindto the 30S ribosomal subunit.35 Resistance to lincosamides,macrolides, and streptogramins is mediated by a similarmechanism. Three related determinants, ermA, ermB, andermC confer resistance to this group of antibiotics. The genescode for erythromycin resistance methylases (ERMs), whichmethylate a site on the ribosome, resulting in a conformationalchange that ultimately leads to a decreased ability of the drugsto bind it.36 Inhibitors of AMEs37,38 and ERMs39,40 have beendescribed. Until now, however, none of these was sufficientlypotent for further development. An additional and more recentapproach includes the work of Hanessian and co-workers. Via asemisynthetic strategy, the researchers developed tetradeoxyaminoglycoside derivatives with improved activity against apanel of clinically relevant MRSA strains.41 Recently, ThambanChandrika et al. also synthesized modified aminoglycosideswith improved antibacterial properties and reduced sensitivityto inactivation by AMEs.42
Podoll et al. discovered a tricyclic indoline that selectivelypotentiates the effect of a variety of β-lactam antibiotics towardMRSA, without showing antiproliferative effect on its own. Bysynthesizing and screening a small-molecule library of 120
Figure 3. Structure of thioridazine (15), cyslabdan (16), kinase inhibitors 17 and 18, tricyclic indolines 19 and 20, and a tetracyclic indolenine (21).
polycyclic indolines, they discovered compound 19 (termedOf1 in the paper) as an RMA that reduced the MIC ofmethicillin from 128 to 8 μg/mL.43 The same group alsoreported a structure−activity relationship investigation of 19,with the identification of 20, showing improved synergisticaction with β-lactam antibiotics and reduced mammaliantoxicity. Further investigation of the mode of action andevaluation of in vivo efficacy is warranted.44
Inspired by the discovery of several polycyclic indolines asresensitizing molecules, the Wang Laboratory synthesized andevaluated a series of bridged tetracylic indolenines too. One oftheir compounds (21) is a selective potentiator of β-lactamactivity in a multidrug-resistant MRSA strain. Of note is thatthe compound shows low antibacterial activity on its own.Further studies (structure−activity relationship, mechanism ofaction, etc.) are ongoing.45
Efflux Pump Inhibition. Inhibition of efflux pumps alsoappears to be a promising strategy to sidestep antibioticresistance. Compounds that prevent antibiotics from beingpumped out are desirable potentiators. Reserpine (22) (Figure4) was an early example that demonstrated the potential ofefflux pump inhibition in S. aureus. The plant alkaloid, a knowninhibitor of mammalian MDR efflux pumps, was shown toreduce the ciprofloxacin MIC.46
Next to reserpine, various other natural products have beenshown to inhibit bacterial efflux pumps. It seems that a varietyof Berberis medicinal plants that produce berberine (23), acationic antimicrobial, also synthesize the flavone 5′-methox-yhydnocarpin (5′-MHC, 24), an inhibitor of the NorA effluxpump of S. aureus. Without 5′-MHC, the plant-derivedantimicrobial alkaloid gets readily extruded by S. aureus.47 Areview compiled by Abreu et al. gives the interested reader anoverview of plant-derived products with potentiating effects. Alarge part is dedicated to efflux pump inhibitors.48
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 toresensitize S. aureus to multiple antibiotics.49 Sabatini and co-workers confirmed the efflux pump inhibitor activity of theNSAID and identified a new class of analogues, acting asinhibitors of the S. aureus NorA efflux pump. The most activeinhibitor (26) showed only modest antistaphylococcal activityand was able to restore the antibacterial activity of ciprofloxacinin norA-overexpressing S. aureus strains.50
To date, several inhibitors targeting bacterial efflux pumpshave been discovered and patented.51,52 Yet, no efflux pumpinhibitor has been approved for therapeutic use.
Figure 4. Examples of efflux pump inhibitors.
Figure 5. Compounds that are able to disassemble biofilms or specifically target small colony variants.
Potentiators Specifically Targeting S. aureus Biofilmsand Small-Colony Variants. Not only does the persistence ofinfection depend on the acquisition of genetic elements thatconfer resistance to antibacterial treatment, but also biofilmformation results in obstinate infection and reduced sensitivityto antibiotics. Furthermore, in some cases, S. aureus can survivein a semidormant state, referred to as SCVs. The latter areknown to form biofilms. Hence, efforts have been made todisassemble biofilms and to specifically target SCVs.As outlined above, the group of Melander designed several 2-
AITs, based on marine natural products. Compound 27 (Figure5) was able to inhibit and disperse bacterial biofilms, includingalso S. aureus biofilms.53 Here as well, the activity was not dueto bactericidal effects. Via structure−activity relationship studiesand library screening using the core 2-aminoimidazole motif,the group identified more compounds with antibiofilmproperties.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
Abouelhassan and co-workers identified a series of quinolinesmall molecules that demonstrate antibiofilm activity. Startingfrom a bromophenazine (29) with antibacterial activity againstS. aureus, the authors discovered 21 structurally similarhalogenated quinolones via a scaffold hopping approach.Although several derivatives showed antibacterial activity, thenovel small molecule 30 possessed the ability to inhibitS. aureus ATCC 29213 biofilm formation at concentrations thatdo not inhibit planktonic growth.58 Furthermore, researchersfrom the same group discovered that combinations of gallic acid(31) with compounds from their library of halogenatedquinolines had potent antibacterial activities against a broadrange of pathogens, including MRSA. Notably, MRSA biofilmswere also effectively eradicated with this combination therapy.59
Furthermore, Garrison et al. published work on the synthesisand biological evaluation of halogenated phenazine derivativesof 29. Several analogues showed potent biofilm eradicationactivity.60
Lee et al. investigated 36 halogenated indole derivatives fortheir ability to inhibit biofilm formation by Escherichia coli andS. aureus.61 The most potent derivative, 5-iodoindole (32),effectively prevented biofilm formation by these bacteria and, asthe researchers’ observations indicate, could be used incombination with conventional antibiotics to eradicate biofilms.Moreover, 5-iodoindole was found to reduce the production ofthe virulence factor staphyloxanthin (STX) by S. aureus (seebelow).The steroidal glycoalkaloid tomatidine (33) specifically
inhibits the growth of S. aureus SCV strains. The MIC oftomatidine against SCVs was 0.12 μg/mL, whereas no clinicallysignificant MIC was measurable against normal strains. SinceSCVs are less susceptible to the effect of classical antibiotics likeaminoglycosides, Mitchell et al. investigated whether toma-
tidine could be used in combination with gentamicin to treatheterogeneous populations of S. aureus. The authors found thatgentamicin at 4 μg/mL was able to inhibit the growth of thenormal S. aureus strain CF04-L, while tomatidine at 0.12 μg/mL was not. Additionally, it was found that the aminoglycosideat 4 μg/mL did not inhibit the growth of SCV CF07-S, whereasthe saponine at 0.12 μg/mL did. Interestingly, tomatidinecomplements the antibacterial effect of the conventionalantibiotic.62 Furthermore, this research group found thattomatidine is a specific aminoglycoside potentiator: itpotentiated the inhibitory effect of other aminoglycosides(kanamycin, tobramycin, amikacin, and streptomycin) but notof other classes of antibiotics (vancomycin, oxacillin, cipro-floxacin, erythromycin, and tetracycline were tested). Thesynergy remains to be confirmed in vivo; however, no cellulartarget has been identified yet. The authors also demonstratedthat exposure of S. aureus to tomatidine repressed hemolyticactivity of the bacterium. The natural product even blocked theexpression of several genes that are normally influenced by theagr system (see below).63 Recently, the research group reportedthe first structure−activity study of the steroid alkaloid, isolatedfrom solanaceous plants.64
The examples described in Potentiation of Antibiotics againstS.aureus via Inhibition of Resistance Mechanisms demonstratethat phenotypical screening is well suited to uncover interestingpotentiators. However, unraveling the mechanism of actionseems challenging in many cases. Future efforts should bedirected to uncover the mechanism by which these compoundselicit their effect.As demonstrated by avibactam (4), the development of new
β-lactamase inhibitors seems to be a very successful strategy toovercome resistance. Another promising development programis that of the antiplatelet drug ticlopidine (6) as WTA synthesisinhibitor. Combinations of β-lactam antibiotics with ticlopidinelimit the growth of MRSA strains both in vitro and in vivo. Afurther advantage of this combination is the fact thatticlopidine, a well-known drug in clinical use, has demonstrateda strong record of examination in humans. It also provides anew probe for assessing the importance of WTA synthesis. Onthe other hand, repurposing candidates face their ownchallenges (see Discussion).
■ TARGETING S. AUREUS VIRULENCE AS ANALTERNATIVE APPROACH
Another alternative to killing bacteria or stopping their growthis to find compounds that target virulence.65 Instead of focusingon bacterial viability, this strategy intends to disarm bacteria.Several virulence pathways can be targeted, such as adhesionand secretion but also bacterial communication (i.e., QS),which is involved in the regulation of virulence in S. aureus.Below, several virulence factors currently being targeted inS. aureus will be highlighted.
Sortase Inhibitors. In order to initiate infection, S. aureusadheres to components of the host extracellular matrix. Themicrobe adheres to and invades host tissues using a variety ofcell wall-anchored (CWA) molecules, of which the “microbialsurface components recognizing adhesive matrix molecules”(MSCRAMMs) are the largest class. MSCRAMMs recognizehost extracellular proteins like fibrinogen, collagen, andfibronectin and are covalently linked to PG by the trans-peptidase sortase A (SrtA). SrtA is required for abscessformation and staphylococcal persistence in host tissues.66 Also,inhibition of SrtA causes reduction of biofilm formation insome staphylococcal strains.67
Hence, over the past decade, many studies have focused onagents that inhibit SrtA. Examples of natural products areisoaaptamine 34 (Figure 6), isolated from the marine spongeAaptos aaptos,68 and bis-indole alkaloid 35 from Spongosoritessp.69 Using HTS, Suree et al. identified several compounds thatinhibit SrtA. Structure−activity relationship analysis led to theidentification of potent pyridazinone and pyrazolethionederivatives (SrtA IC50 values in submicromolar range).70
More recently, Zhang et al. identified compound 36 andrelated compounds, which block sortase activity both in vitroand in vivo.71 Cascioferro et al. wrote a review on SrtAinhibitors.72
Interference with STX Biosynthesis. STX plays animportant role in S. aureus virulence. The golden carotenoidpigment, with numerous double bonds, shields the bacteriumfrom host oxidant killing.73 The first committed step in thebiosynthetic pathway of STX (Figure 7), catalyzed by theenzyme CrtM (or 4,4′-diapophytoene synthase or dehydros-qualene synthase), starts with the condensation of twomolecules of farnesyl diphosphate (FPP) to produce the C30species presqualene diphosphate.74 The latter then undergoesskeletal rearrangement and loss of diphosphate to form 4,4′-diapophytoene (or dehydrosqualene). The formation of
dehydrosqualene in S. aureus is very similar to the formationof squalene (via the enzyme squalene synthase, SQS) in plants,animals, and fungi. Squalene is an important precursor ofseveral sterols like cholesterol, ergosterol, and plant sterols. In2008, Oldfield and co-workers questioned whether S. aureusCrtM and the human squalene synthase possess structuralsimilarity. Although there is only modest sequence resem-blance, the researchers found that the overall fold of the twoenzymes shows clear similarity. Therefore, they investigatedwhether any of the many known squalene synthase inhibitorspreviously developed as cholesterol lowering drugs also haveactivity in inhibiting STX biosynthesis and hence S. aureusvirulence.75 They reported the first-generation CrtM inhibitorBPH-652 (37), which is now completing IND-enabling studiesby AuricX Pharmaceuticals Inc.Phosphonosulfonate BPH-652 did not affect S. aureus
growth, nor survival in vitro, but upon treatment with 37, theresulting nonpigmented bacteria were less able to survive infreshly isolated human whole blood than nontreated S. aureus(as expected because they contained no or less antioxidantcarotenoid pigment). Several CrtM inhibitors have subse-quently been identified by the same group.76,77
Further down the biosynthetic pathway of STX, dehydros-qualene is converted into 4,4′-diaponeurosporene via successivedehydrogenations, catalyzed by CrtN (or 4,4′-diapophytoenedesaturase). Very recently, Chen et al. screened a library ofapproximately 400 existing drugs against S. aureus infection andidentified naftifine (38) as a CrtN inhibitor. This allylamineantifungal drug was capable of prohibiting STX biosynthesis atnanomolar concentrations and attenuated the virulence of avariety of clinical S. aureus isolates (including MRSA strains) inmouse infection models. Naftifine did not function as anantibiotic against S. aureus.78 On the basis of this pioneeringwork, Wang et al. used a scaffold hopping approach to discovera new class of benzofuran-containing CrtN inhibitors with
Figure 7. Small molecules that interfere with staphyloxanthine biosynthesis.
enhanced oral bioavailability. The most potent analogueidentified in this study (39) is depicted in Figure 7.79
As indicated above, 5-iodoindole (32) was observed todecrease STX production in S. aureus as well.61 Since indoleand 7-benzyloxyindole were found to inhibit the production ofthe carotenoid pigment, the researchers investigated STXproduction in two S. aureus strains, including an MRSA strain.5-Iodoindole was found to be much more potent than indole. Amechanism of action was not described.Interference with Caseinolytic Protein Protease.
Another attractive target for antivirulence drugs is thecaseinolytic protein protease (ClpP). This serine proteaseplays a central role in S. aureus virulence.80 The absence ofClpP causes upregulation of several transcriptional repressors ofvirulence genes, such as the sarA family and also adownregulation of the Agr (accessory gene regulation) QSsystem (see below).81
Bottcher and Sieber demonstrated that inhibition of thisvirulence regulator by synthetic β-lactones decreased expressionof major virulence factors. Their most potent inhibitor, D3 (40)(Figure 8), was able to abolish hemolytic and proteolyticactivities in MRSA and also showed a decrease in lipase andDNase expression.82 On the basis of D3, several optimizedstructures have been published.83,84 Compound U1 (41), witha phenylethyl side chain, appeared as one of the most potentcandidates. Weinandy et al. showed that, although U1 exhibitslimited plasma stability (incubation in plasma led to rapidhydrolysis), local application of the β-lactone led to reductionof abscess development in mice. In this setup, no inhibition ofS. aureus growth was seen.85
Substantial progress has been made in antivirulenceapproaches. However, the small molecules described in thissection are still in preclinical development. Moreover, althoughthese compounds have demonstrated that S. aureus virulencefactors can be antagonized, it is currently not clear whethertheir potential use is restricted to prophylaxis or may also bebeneficial for treatment of S. aureus infections. Nonetheless,antivirulence is a promising and developing discipline.
■ INTERFERENCE WITH S. AUREUS QS AS ANALTERNATIVE STRATEGY
Accessory Gene Regulator System. In staphylococci, theaccessory gene regulator (agr) locus is part of the globalvirulence response (Figure 9).86,87 The operon comprises twotranscripts, RNAII and RNAIII (where RNA is ribonucleicacid), originating from the P2 and P3 promotors, respectively.RNAII encodes the constituents of the Agr system itself: AgrB,AgrD, AgrC, and AgrA. This autoactivation circuit is a two-component signal transduction system (TCS) and employscyclic thiolactone peptides as signaling molecules. Theseautoinducing peptides (AIPs) are produced as a pro-peptide(AgrD). AgrB, a transmembrane endopeptidase, subsequentlyprocesses the pro-peptides into active AIPs (7−9 amino acidpeptides that contain a thiolactone ring) and secretes them.The type I signal peptidase SpsB also plays a role in theprocessing of AgrD. The AIPs accumulate in the extracellularenvironment and, when reaching a threshold concentration,bind to AgrC, the sensor kinase of the TCS. AgrC, which isphosphorylated in an AIP-dependent manner, then activatesAgrA, the response regulator. The activated AgrA then binds
Figure 8. Structures that interfere with caseinolytic protein protease.
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 agr promotor regions P2 and P3, with a preference for P2 atlow concentrations, maintaining activation of its ownexpression.88
RNAIII, a regulatory mRNA, acts as the effector of the AgrQS system. The Agr system in S. aureus controls the expressionof 70 to 150 genes and is primarily implicated in theinvasiveness of S. aureus.89 It is responsible for the upregulationof both toxins and enzymes (proteases, nucleases, lipases, etc.).Almost all known virulence factors are upregulated by agr, whilecell surface proteins, such as protein A and fibronectin-bindingproteins, are downregulated. Surface-binding proteins such asMSCRAMMs are important for establishment of infection (seeabove), while nutrient acquisition becomes more important inlater stages of infection.87,90 Agr is important for acutevirulence, while the absence of Agr functionality may bebeneficial in specific types of S. aureus infection (chronic,biofilm-related infections, and bacteraemia, for example).Indeed, agr mutants exhibit reduced production of virulencefactors but are shown to produce extensively thick andunstructured biofilms.91−93
The structure of RNAIII, but not its sequence, is conservedamong staphylococcal species.94 RNAIII acts both as anmRNA, encoding δ-hemolysin, and as a regulator of virulence.Precise mechanisms of RNAIII action have been elucidated fora number of target genes. Both transcriptional and translationalregulation are involved.95 Agr belongs to a complex regulatorynetwork. Several regulatory genes (e.g., sarA, sarU, clpP, etc.)directly or indirectly up- or downregulate its expression. Thisintricate regulation of agr expression provides additionalregulation loops. A detailed description of this multifacetedregulation goes beyond the scope of this Review, and the readeris referred to Novick and Geisinger.86 Nevertheless, the highcomplexity of this regulatory network reinforces the fact thatS. aureus has the ability to very precisely adapt its physiologyaccording to environmental signals and changes. The use of QSmodulators as an alternative to or as potentiators of routine
antibiotics has been proposed in literature, and efficaciousinhibitors of Agr signaling have been reported.93,96,97
The secondary fungal metabolite ambuic acid (42) (Figure10), for instance, was shown to inhibit AIP biosynthesis.98
Allelic variations in the agr region have resulted in the existenceof four main classes of S. aureus AIPs (agr-I to agr-IV). With adifferent amino acid sequence, but conserved thiolactonemacrocycle, the specific AIPs of each group can induce theirown agr expression and competitively inhibit noncognate AgrCreceptors. This phenomenon is also sometimes referred to ascross-inhibition and offers therapeutic potential. Severalstructure−activity relationship studies have been set up withthe aim to develop inhibitors of S. aureus agr groups. Modifiedand/or truncated AIPs have been suggested as ligands capableof intercepting the AIP−AgrC interaction. Khan et al. andGordon 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 Staphylococcus epidermidis (S. epi-dermidis) and S. aureus and also between S. intermedius andS. aureus agr groups has been evaluated.93
Even nonstaphylococcal autoinducers have been gauged asvirulence attenuators. Qazi et al. found that the Pseudomonasaeruginosa QS molecule N-(3-oxododecanoyl)-L-homoserinelactone (or 3-oxo-C12-HSL, 43) elicits inhibitory effects againstagr at subgrowth inhibitory concentrations. The lactoneantagonizes the production of S. aureus exotoxins.99 Thesame group utilized 43 as a starting point for the synthesis of aseries of analogues to gain insight into the structural featuresinvolved, the mechanism of action, and in vivo efficacy.100 Inthis study, the authors identified two new related classes ofcompounds: tetramic acids (TMAs) and tetronic acids (TOAs).These molecules act as noncompetitive AgrC inhibitors and aremore potent than the parent compound 3-oxo-C12-HSL. Theirmost potent analogue identified (C14-TOA, 44) reducedcolonization of human nasal epithelial cells and also reducedarthritis in a murine infection model without showing toxicity.
Figure 10. Compounds that disturb accessory gene regulator quorum sensing signaling.
The cyclic dipeptides cyclo(L-Tyr-L-Pro) 45 and cyclo(L-Phe-L-Pro) 46 produced by the human vaginal isolate Lactobacillusreuteri RC-14 show antagonistic activity toward all four S. aureusagr groups; the molecules repress the expression of toxic shocksyndrome toxin-1 (TSST-1) via a yet unknown mechanism.101
The marine Photobacterium halotolerans produces a group ofmolecules, called solonamides, that display S. aureus Agrinhibitory activity as well. These cyclodepsipeptides stronglyreduce expression of RNAIII as competitive inhibitors of theAgrC receptor. Solonamide B (47) was found to inhibit all agrtypes and dramatically reduced the hemolytic activity andphenol-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 ofAIP to AgrC.Not only has AgrC been described as a target for
antivirulence molecules, but also ω-hydroxyemodin (OHM,49) antagonizes AgrA function. Daly et al. showed thispolyhydroxyanthraquinone to exert in vivo efficacy againstS. aureus QS-mediated virulence by direct binding to AgrA.104
Khodaverdian et al. recently described other agents that bindthe response regulator AgrA. Via in silico screening, a series ofnaphthalene and biaryl compounds were found to inhibit theproduction of α-hemolysin and PSMα. One of these smallmolecules is the FDA-approved NSAID diflunisal (50).105
Finally, savirin (Staphylococcus aureus virulence inhibitor, 51), aselective inhibitor of AgrA, was discovered by HTS andimpedes agr-mediated QS across all four agr groups.106 Savarinis a molecule that displays S. aureus-specific Agr inhibition.Indeed, drugs interfering with S. aureus Agr ideally do notinterfere with the important skin commensal S. epidermi-dis.93,106
RAP/TRAP System. An additional QS system in S. aureushas been proposed. This system operates via the autoinducerRNAIII-activating protein (RAP), which activates histidinephosphorylation of target of RNAIII-activating protein(TRAP), ultimately resulting in production of the regulatoryRNAIII (Figure 9).107 It is unclear how RAP secretion occurs.Additionally, the precise function and protein chemistry ofTRAP are not fully understood: it is unclear what enzyme isinvolved in the phosphorylation of TRAP and how the TRAPsignal is transferred to the genome.108,109 Also, it is not yetcompletely clear whether the agr and RAP/TRAP mechanismsact independently or in tandem.110−113 TRAP is a membrane-associated protein but has no transmembrane domain. Balabanet al. demonstrated that a TRAP-negative mutant formed verylittle biofilm compared to the parent strain S. aureus 8325-4.This implies that TRAP regulates genes involved in biofilmformation in S. aureus.114 TRAP expression is constitutive, butits phosphorylation is regulated by RAP. Since TRAP is highlyconserved among staphylococcal strains and because it isconstitutively expressed, it has been investigated in vaccine
development for preventing staphylococcal mastitis in dairycows.115
RNAIII-inhibiting peptide (RIP), originally isolated fromculture supernatants of coagulase negative staphylococci,competes with RAP for TRAP phosphorylation.116−119 Thesequence of synthetic RIP (H-Tyr-Ser-Pro-Trp-Thr-Asn-Phe-NH2) shows some degree of similarity with the sequence ofresidues 4−9 of RAP (Tyr-Lys-Pro-Ile-Thr-Asn). This suggeststhat both molecules bind to the same receptor, one as agonist(RAP) and one as antagonist (RIP). RIP was shown to inhibitS. aureus pathogenesis: the peptide inhibits agr activity(synthesis of both RNAII and RNAIII). Interestingly, RIPwas also found to reduce the ability of S. aureus to adhere topolystyrene and to mammalian human epithelial type 2 (HEp2)cells.117 This “dual activity” (inhibition of both virulence andadhesion) is in contrast to what is seen with AIPs. Indeed, asstated earlier, inhibition of the Agr system leads to increasedrather than decreased biofilm formation.Balaban et al. demonstrated that the heptapeptide RIP can
treat preformed device-associated staphylococcal infections in arat graft model. Also, RIP-teicoplanin combination treatmentresulted in a significant reduction of the bacterial load,compared to treatment with either agent alone.114 Via alaninescanning, the key residues for the activity of RIP were studiedby Baldasserre et al. For this structure−activity relationshipstudy, the researchers synthesized single alanine derivatives ofRIP, as well as truncated compounds. None of the derivativesshowed in vitro killing activities.120 Inter alia, an enhancedactivity 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 oftopical FS-10 and intraperitoneal tigecycline can be used totreat systemic MRSA infection in a murine model. The exactmechanism of action of the truncated RIP derivative has yet tobe elucidated, but the authors hypothesize that, analogous toRIP, 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 thebark and leaves of the American witch hazel (Hamamelisvirginiana L.).122 In a publication from 2008, Kiran et al.discovered that HAM acts as a nonpeptide analogue of RIP.Analogous to RIP, the natural tannin was shown to preventbiofilm formation and RNAIII production in vitro as well as invivo (in device-associated infections).123 Brackman et al. laterdemonstrated that HAM increases the susceptibility of S. aureusbiofilms toward vancomycin in vitro and also in vivo in aCaenorhabditis elegans (C. elegans) and a Galleria mellonellamodel of infection.124 Only very recently, the same groupcreated a framework for the presumed mechanism of action ofthe natural molecule.125 In this pioneering work, they provideevidence that HAM indeed affects S. aureus biofilmsusceptibility through the TRAP receptor. The authorsconclude that HAM affects a set of genes, which ultimatelyleads to a reduction in cell wall thickness and the amount of
Figure 11. Structure of hamamelitannin (52) and optimized analogue (53).
extracellular deoxyribonucleic acid (eDNA) in the biofilmmatrix. HAM increases the susceptibility of S. aureus biofilmstoward several classes of antibiotics and only affectssusceptibility of S. aureus biofilms (and not that of otherstaphylococci or other bacteria). Finally, the potential clinicalapplication of HAM was evaluated in a C. elegans and a mousemammary gland infection model of S. aureus. The moleculeaffected biofilm susceptibility in both models.HAM is an interesting hit, but its structure is not ideal.
Therefore, our group investigated the structure−activityrelationship of HAM in order to identify derivatives, whichare more active and metabolically more stable.126 Vermote et al.reported on the identification of a metabolically stablecompound (53) with potent in vitro activity and exceptionalactivity in a C. elegans infection model and a murine mastitismodel, while lacking cytotoxicity against MRC-5 (MedicalResearch Council cell strain 5) lung fibroblast cells. Severalother HAM derivatives that warrant further study have beenidentified as well.127−129
Inhibition of QS signaling in S. aureus seems to be apromising antivirulence approach. Many small molecules thatinterfere with S. aureus QS have been identified. However, withrespect to QS inhibition, a number of questions remainunanswered.QS systems are very complex, diverse, and sometimes even
“redundantly organized”, which implies that our currentknowledge of QS probably only scratches the surface. Aconcern when targeting Agr QS, for example, is whetherinhibitors of this system can possibly be used in biofilm-relatedinfections, since biofilm formation is negatively impacted byAgr. Yarwood et al. found that, in an established biofilm,pockets of agr-activated S. aureus cells detached under in vitroflow conditions, while agr inactive cells remained in thebiofilm.110 Boles and Horswill reported that exogenous AIPaddition reactivates the agr system in a mature biofilm andtriggered detachment. Maybe even more importantly, thisbiofilm detachment restored sensitivity of the dispersed cells tothe antibiotic rifampin.130 It seems that, in this context, thescientific community is caught between Scylla and Charybdis:either we choose for Agr inhibition with the chance of inducingmore chronic biofilm-related infections (but also less virulent)or we go for Agr activation, which might convert S. aureus into amore invasive pathogen (but also more amenable to treat-ment). Also, in order to interfere with the Agr system for thetreatment of infections, determination of the agr type will benecessary and care providers will need specific AIP signals on-hand for therapy (as each type of staphylococcal strainrecognizes unique AIP signals).131
When looking at the RAP/TRAP system, the precisefunction 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, howexactly? Conflicting results between authors110−113 calls forfurther research to improve our understanding of themechanism of action of the QS inhibitors. Nevertheless, severalresearch groups have provided very promising new leads forfurther study and have presented strong preclinical data,including sufficient in vivo efficacy using appropriate animalmodels.
■ DISCUSSION
Multidrug-resistant pathogens are a growing threat to humanhealth, and their worldwide spread presents formidabletherapeutic difficulties for clinicians. The scarcity of newantibiotics being introduced adds to this problem. The currentantibiotic pipeline is weak; over the past decade, the FDA hasapproved new antibiotics, but none of these belong to a newantibiotic class, with the exception of bedaquiline (for thetreatment of TB). Antibiotics have saved countless lives, andwithout access to such drugs, the slightest injury could becomelife-threatening, with increased mortality, morbidity, and cost ofpatient care. Moreover, exhaustion of efficacious antibioticsmay preclude one from performing common surgicalprocedures and immunosuppressing cancer therapy in thefuture. A prominent example of such a multidrug-resistantpathogen is MRSA. Doom-and-gloom reports about thissuperbug, which is notorious for its biofilm formation andresistance to conventional antibiotics, are published regularly.The development of new antibiotics is one important
approach for the treatment of (multidrug-resistant) bacterialinfections. However, given the difficulty of antibiotic drugdiscovery and the unrelenting challenge of resistance,alternative approaches should be considered as well. Onesuch strategy is the use of combinations of drugs, a paradigmthat is clinically proven in many areas of medicine. The use ofdrug cocktails is important in the treatment of many cancersand viral infections (e.g., with human immunodeficiency virus[HIV]). Also for the treatment of bacterial infections,combination therapy is well-established. Patients with TB, forinstance, are treated with combinations of up to four drugs.Another well-known example is the combination of sulfame-thoxazole and trimethoprim, two antibiotics that inhibitsuccessive steps in the folate biosynthesis. Furthermore, thestreptogramins quinupristin/dalfopristin are used in combina-tion for the treatment of infections by staphylococci andvancomycin-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 haveprimarily been done in vitro), the combination of several β-lactam antibiotics with either vancomycin or daptomycin seemsan attractive alternative to monotherapy. Davis et al. reviewedcombination antibiotic treatment for serious MRSA infec-tions.132
Not only have antibiotic−antibiotic combinations have beenstudied, but also in this Review, we focused on the pairing of anantibiotic with a nonantibiotic molecule, which potentiates theactivity of the former. Although not possessing growth-inhibitory activity by themselves, small-molecule “potentiators”can reduce antibiotic doses. The potentiators discussed hereinfunction by reversing resistance mechanisms, by attenuatingS. aureus virulence, and/or by interfering with QS.Such a strategy potentially has a few advantages over
“conventional” approaches.133,134 First, an increased number ofpharmacological targets could be addressed. Second, since mostvirulence factors are not essential for bacterial viability,antivirulence drugs would exert less selective pressure onbacteria. Gerdt and Blackwell demonstrated a “robustness” ofQS inhibitors (QSIs) against resistance. The authors expectthat QSI-resistant mutants will arise, but hypothesize that, incontrast to organisms that are resistant toward traditionalantibiotics, such mutants would struggle to overtake the
population.135 Third, gut microbiota would potentially bepreserved because antivirulence compounds exert a veryspecific effect.However, development of antivirulence drugs presents
challenges as well. Established screening systems withdetermination of MICs can no longer be used; to screensuch compounds, specialized assays (both in vitro and in vivo)need to be developed. Also, in this particular field, in additionto proving the interference with a certain target (e.g., aresistance element, virulence factor, or QS component), it isalso essential to prove that such interference leads topotentiation of the activity of a simultaneously administeredantibiotic. Moreover, specific disarming drugs will very likelyhave a narrow spectrum of activity. This also means that theirpotential success in the clinic will depend on rapid diagnosticsto identify the causative agent.133 Another question is whethersuch drugs will be able to attenuate already existing infectionsin humans.96,97 Moreover, it remains to be seen whetherbacteria will be able to find a way around antivirulence therapywith their complex, diverse, and even “redundantly organized”regulatory systems that control virulence. Another importantissue with the deployment of drug combinations is the fact thatdrug−drug interactions (DDIs) can occur. ADME properties ofeach compound can be altered by coadministration withanother drug (e.g., plasma protein binding and drugmetabolism). Hence, searching for optimized drug ratios willbe required.136,137
In the myriad of recently reported potentiators for antibioticsagainst S. aureus, it is difficult to benchmark compounds. Only afew potentiators have been evaluated in vivo, and althoughsome small molecules perform well in animal models ofS. aureus infection, it would be very interesting to see how thedifferent potentiators described in literature relate to eachother. Now, different animal models, conditions, drugconcentrations, and S. aureus strains are used to test potentiatoractivity. It must come as no surprise that comparison of thepotentiating ability of small molecules is challenging whendifferent 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 otherpurposes. Indeed, the process of identifying new indications forexisting drugs outside the scope of the original medical use is anincreasingly popular strategy. As such repositioning candidateshave survived several stages of clinical testing, their safety andpharmacokinetic profile is well-known and the risk of failuredue to toxicology reasons is reduced. The strategy can possiblylead to an acceleration of the R&D process and is economicallyattractive when compared with the cost of de novo drugdiscovery and development. However, repositioning strategiesface some unique challenges as well. As prior art might alreadyexist for the repositioning candidates, intellectual propertyissues may be complex. Also, from a commercial point of view,it is of utmost importance to evaluate the candidate’s potentialfor attaining a competitive profile in the market.138
Taking into account all of the above-mentioned concerns, thechallenge of resistant staphylococcal infections is complex andmultidisciplinary and we believe that the potentiators high-lighted herein illustrate important starting points for futuretherapeutic options.
■ AUTHOR INFORMATIONCorresponding Author*Tel: +32 (0)9 264 81 24. Fax: +32 (0)9 264 81 46. E-mail:[email protected] Van Calenbergh: 0000-0002-4201-1264Author ContributionsAll authors have given approval to the final version of themanuscript.NotesThe authors declare no competing financial interest.
■ REFERENCES(1) WHO. (2001) WHO Global Strategy for Containment ofAntimicrobial Reistance, WHO/CDS/CSR/DRS/2001.2, available athttp://www.who.int/drugresistance/WHO_Global_Strategy_English.pdf.(2) O’Neill, J. (2016) Tackling drug-resistant infections globally: finalreport and recommendations, available at http://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf.
(3) Huttner, A., Harbarth, S., Carlet, J., Cosgrove, S., Goossens, H.,Holmes, A., Jarlier, V., Voss, A., and Pittet, D. (2013) Antimicrobialresistance: a global view from the 2013 World Healthcare-AssociatedInfections Forum. Antimicrob. Resist. In. 2, 31.(4) Lewis, K. (2013) Platforms for antibiotic discovery. Nat. Rev.Drug Discovery 12, 371−387.(5) von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., andHabich, D. (2006) Antibacterial Natural Products in MedicinalChemistryExodus or Revival? Angew. Chem., Int. Ed. 45, 5072−5129.(6) D’Costa, V. M., McGrann, K. M., Hughes, D. W., and Wright, G.D. (2006) Sampling the Antibiotic Resistome. Science 311, 374−377.(7) Abraham, E. P., and Chain, E. (1940) An Enzyme from Bacteriaable to Destroy Penicillin. Nature 146, 837−837.(8) Williams, R. E. O. (1963) Healthy carriage of Staphylococcusaureus: its prevalence and importance. Bacteriol. Rev. 27, 56−71.(9) Wertheim, H. F. L., Melles, D. C., Vos, M. C., van Leeuwen, W.,van Belkum, A., Verbrugh, H. A., and Nouwen, J. L. (2005) The role ofnasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis. 5,751−762.(10) Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L., andFowler, V. G. (2015) Staphylococcus aureus Infections: Epidemiology,Pathophysiology, Clinical Manifestations, and Management. Clin.Microbiol. Rev. 28, 603−661.(11) McDougall, S. (2002) Bovine mastitis: epidemiology, treatmentand control. N. Z. Vet. J. 50, 81−84.(12) Halasa, T., Huijps, K., Østeras, O., and Hogeveen, H. (2007)Economic effects of bovine mastitis and mastitis management: Areview. Vet. Q. 29, 18−31.(13) Waters, C. M., and Bassler, B. L. (2005) QUORUM SENSING:Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol. 21,319−346.(14) Assis, L. M., Nedeljkovic, M., and Dessen, A. (2017) Newstrategies for targeting and treatment of multi-drug resistantStaphylococcus aureus. Drug Resist. Updates 31, 1−14.(15) Pinho, M. G., Kjos, M., and Veening, J.-W. (2013) How to get(a)round: mechanisms controlling growth and division of coccoidbacteria. Nat. Rev. Microbiol. 11, 601−614.(16) De Pascale, G., and Wright, G. D. (2010) Antibiotic Resistanceby Enzyme Inactivation: From Mechanisms to Solutions. Chem-BioChem 11, 1325−1334.(17) Drawz, S. M., and Bonomo, R. A. (2010) Three Decades of β-Lactamase Inhibitors. Clin. Microbiol. Rev. 23, 160−201.(18) Ehmann, D. E., Jahic, H., Ross, P. L., Gu, R.-F., Hu, J., Kern, G.,Walkup, G. K., and Fisher, S. L. (2012) Avibactam is a covalent,reversible, non−β-lactam β-lactamase inhibitor. Proc. Natl. Acad. Sci. U.S. A. 109, 11663−11668.(19) Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) MolecularBasis and Phenotype of Methicillin Resistance in Staphylococcusaureus and Insights into New β-Lactams That Meet the Challenge.Antimicrob. Agents Chemother. 53, 4051−4063.(20) Campbell, J., Singh, A. K., Santa Maria, J. P., Kim, Y., Brown, S.,Swoboda, J. G., Mylonakis, E., Wilkinson, B. J., and Walker, S. (2011)Synthetic Lethal Compound Combinations Reveal a FundamentalConnection between Wall Teichoic Acid and PeptidoglycanBiosyntheses in Staphylococcus aureus. ACS Chem. Biol. 6, 106−116.(21) Farha, M. A., Leung, A., Sewell, E. W., D’Elia, M. A., Allison, S.E., Ejim, L., Pereira, P. M., Pinho, M. G., Wright, G. D., and Brown, E.D. (2013) Inhibition of WTA Synthesis Blocks the Cooperative Actionof PBPs and Sensitizes MRSA to β-Lactams. ACS Chem. Biol. 8, 226−233.(22) Farha, M. A., Koteva, K., Gale, R. T., Sewell, E. W., Wright, G.D., and Brown, E. D. (2014) Designing analogs of ticlopidine, a wallteichoic acid inhibitor, to avoid formation of its oxidative metabolites.Bioorg. Med. Chem. Lett. 24, 905−910.(23) Ejim, L., Farha, M. A., Falconer, S. B., Wildenhain, J., Coombes,B. K., Tyers, M., Brown, E. D., and Wright, G. D. (2011)Combinations of antibiotics and nonantibiotic drugs enhanceantimicrobial efficacy. Nat. Chem. Biol. 7, 348−350.
(24) Hu, X.-L., Li, D., Shao, L., Dong, X., He, X.-P., Chen, G.-R., andChen, D. (2015) Triazole-Linked Glycolipids Enhance the Suscept-ibility of MRSA to β-Lactam Antibiotics. ACS Med. Chem. Lett. 6,793−797.(25) Gardete, S., Wu, S. W., Gill, S., and Tomasz, A. (2006) Role ofVraSR in Antibiotic Resistance and Antibiotic-Induced StressResponse in Staphylococcus aureus. Antimicrob. Agents Chemother.50, 3424−3434.(26) Jo, D. S., Montgomery, C. P., Yin, S., Boyle-Vavra, S., and Daum,R. S. (2011) Improved Oxacillin Treatment Outcomes inExperimental Skin and Lung Infection by a Methicillin-ResistantStaphylococcus aureus Isolate with a vraSR Operon Deletion.Antimicrob. Agents Chemother. 55, 2818−2823.(27) Harris, T. L., Worthington, R. J., and Melander, C. (2012)Potent Small-Molecule Suppression of Oxacillin Resistance inMethicillin-Resistant Staphylococcus aureus. Angew. Chem., Int. Ed.51, 11254−11257.(28) Furlani, R. E., Yeagley, A. A., and Melander, C. (2013) A flexibleapproach to 1,4-di-substituted 2-aminoimidazoles that inhibit anddisperse biofilms and potentiate the effects of beta-lactams againstmulti-drug resistant bacteria. Eur. J. Med. Chem. 62, 59−70.(29) Bonde, M., Hojland, D. H., Kolmos, H. J., Kallipolitis, B. H., andKlitgaard, J. K. (2011) Thioridazine affects transcription of genesinvolved in cell wall biosynthesis in methicillin-resistant Staph-ylococcus aureus. FEMS Microbiol. Lett. 318, 168−176.(30) Klitgaard, J. K., Skov, M. N., Kallipolitis, B. H., and Kolmos, H. J.(2008) Reversal of methicillin resistance in Staphylococcus aureus bythioridazine. J. Antimicrob. Chemother. 62, 1215−1221.(31) Poulsen, M. O., Jacobsen, K., Thorsing, M., Kristensen, N. R. D.,Clasen, J., Lillebaek, E. M. S., Skov, M. N., Kallipolitis, B. H., Kolmos,H. J., and Klitgaard, J. K. (2013) Thioridazine potentiates the effect ofa beta-lactam antibiotic against Staphylococcus aureus independentlyof mecA expression. Res. Microbiol. 164, 181−188.(32) Li, Z., He, M., Dong, X., Lin, H., Ge, H., Shen, S., Li, J., Ye, R.D., and Chen, D. (2015) New erythromycin derivatives enhance beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus.Lett. Appl. Microbiol. 60, 352−358.(33) Fukumoto, A., Kim, Y. P., Hanaki, H., Shiomi, K., Tomoda, H.,and Omura, S. (2008) Cyslabdan, a new potentiator of imipenemactivity against methicillin-resistant Staphylococcus aureus, producedby Streptomyces sp K04−0144 - II. Biological activities. J. Antibiot. 61,7−10.(34) Boudreau, M. A., Fishovitz, J., Llarrull, L. I., Xiao, Q., andMobashery, S. (2015) Phosphorylation of BlaR1 in Manifestation ofAntibiotic Resistance in Methicillin-Resistant Staphylococcus aureusand Its Abrogation by Small Molecules. ACS Infect. Dis. 1, 454−459.(35) Chandrakanth, R. K., Raju, S., and Patil, S. A. (2008)Aminoglycoside-resistance mechanisms in multidrug-resistant Staph-ylococcus aureus clinical isolates. Curr. Microbiol. 56, 558−562.(36) Lina, G., Quaglia, A., Reverdy, M.-E., Leclercq, R., Vandenesch,F., and Etienne, J. (1999) Distribution of Genes Encoding Resistanceto Macrolides, Lincosamides, and Streptogramins among Staph-ylococci. Antimicrob. Agents Ch. 43, 1062−1066.(37) Daigle, D. M., McKay, G. A., and Wright, G. D. (1997)Inhibition of Aminoglycoside Antibiotic Resistance Enzymes byProtein Kinase Inhibitors. J. Biol. Chem. 272, 24755−24758.(38) Vong, K., Tam, I. S., Yan, X., and Auclair, K. (2012) Inhibitorsof Aminoglycoside Resistance Activated in Cells. ACS Chem. Biol. 7,470−475.(39) Hajduk, P. J., Dinges, J., Schkeryantz, J. M., Janowick, D.,Kaminski, M., Tufano, M., Augeri, D. J., Petros, A., Nienaber, V.,Zhong, P., Hammond, R., Coen, M., Beutel, B., Katz, L., and Fesik, S.W. (1999) Novel Inhibitors of Erm Methyltransferases from NMR andParallel Synthesis. J. Med. Chem. 42, 3852−3859.(40) Feder, M., Purta, E., Koscinski, L., Cubrilo, S., MaravicVlahovicek, G., and Bujnicki, J. M. (2008) Virtual Screening andExperimental Verification to Identify Potential Inhibitors of the ErmCMethyltransferase Responsible for Bacterial Resistance against Macro-lide Antibiotics. ChemMedChem 3, 316−322.
(41) Hanessian, S., Giguere, A., Grzyb, J., Maianti, J. P., Saavedra, O.M., Aggen, J. B., Linsell, M. S., Goldblum, A. A., Hildebrandt, D. J.,Kane, T. R., Dozzo, P., Gliedt, M. J., Matias, R. D., Feeney, L. A., andArmstrong, E. S. (2011) Toward Overcoming Staphylococcus aureusAminoglycoside Resistance Mechanisms with a Functionally DesignedNeomycin Analogue. ACS Med. Chem. Lett. 2, 924−928.(42) Thamban Chandrika, N., Green, K. D., Houghton, J. L., andGarneau-Tsodikova, S. (2015) Synthesis and Biological Activity ofMono- and Di-N-acylated Aminoglycosides. ACS Med. Chem. Lett. 6,1134−1139.(43) Podoll, J. D., Liu, Y. X., Chang, L., Walls, S., Wang, W., andWang, X. (2013) Bio-inspired synthesis yields a tricyclic indoline thatselectively resensitizes methicillin-resistant Staphylococcus aureus(MRSA) to beta-lactam antibiotics. Proc. Natl. Acad. Sci. U. S. A.110, 15573−15578.(44) Chang, L., Podoll, J. D., Wang, W., Walls, S., O’Rourke, C. P.,and Wang, X. (2014) Structure−Activity Relationship Studies of theTricyclic Indoline Resistance-Modifying Agent. J. Med. Chem. 57,3803−3817.(45) Xu, W. Q., Wang, W., and Wang, X. (2015) Gold-CatalyzedCyclization Leads to a Bridged Tetracyclic Indolenine that Repressesbeta-Lactam Resistance. Angew. Chem., Int. Ed. 54, 9546−9549.(46) Schmitz, F. J., Fluit, A. C., Luckefahr, M., Engler, B., Hofmann,B., Verhoef, J., Heinz, H. P., Hadding, U., and Jones, M. E. (1998) Theeffect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin againstclinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 42,807−810.(47) Stermitz, F. R., Lorenz, P., Tawara, J. N., Zenewicz, L. A., andLewis, K. (2000) Synergy in a medicinal plant: Antimicrobial action ofberberine potentiated by 5 ′-methoxyhydnocarpin, a multidrug pumpinhibitor. Proc. Natl. Acad. Sci. U. S. A. 97, 1433−1437.(48) Abreu, A. C., McBain, A. J., and Simoes, M. (2012) Plants assources of new antimicrobials and resistance-modifying agents. Nat.Prod. Rep. 29, 1007−1021.(49) Kalle, A. M., and Rizvi, A. (2011) Inhibition of BacterialMultidrug Resistance by Celecoxib, a Cyclooxygenase-2 Inhibitor.Antimicrob. Agents Chemother. 55, 439−442.(50) Sabatini, S., Gosetto, F., Serritella, S., Manfroni, G., Tabarrini,O., Iraci, N., Brincat, J. P., Carosati, E., Villarini, M., Kaatz, G. W., andCecchetti, V. (2012) Pyrazolo[4,3-c][1,2]benzothiazines 5,5-Dioxide:A Promising New Class of Staphylococcus aureus NorA Efflux PumpInhibitors. J. Med. Chem. 55, 3568−3572.(51) Van Bambeke, F., and Lee, J. M. P. (2010) Inhibitors of bacterialefflux pumps as adjuvants in antibacterial therapy and diagnostic toolsfor detection of resistance by efflux, in Frontiers in Anti-Infective DrugDiscovery (Atta-ur-Rahman, M. I. C., Ed.), Bentham SciencePublishers, Emirate of Sharjah.(52) Bhardwaj, A. K., and Mohanty, P. (2012) Bacterial Efflux PumpsInvolved in Multidrug Resistance and their Inhibitors: Rejuvinating theAntimicrobial Chemotherapy. Recent Pat. Anti-Infect. Drug Discovery 7,73−89.(53) Rogers, S. A., and Melander, C. (2008) Construction andScreening of a 2-Aminoimidazole Library Identifies a Small MoleculeCapable of Inhibiting and Dispersing Bacterial Biofilms across Order,Class, and Phylum. Angew. Chem. 120, 5307−5309.(54) Rogers, S. A., Huigens Iii, R. W., and Melander, C. (2009) A 2-Aminobenzimidazole That Inhibits and Disperses Gram-PositiveBiofilms through a Zinc-Dependent Mechanism. J. Am. Chem. Soc.131, 9868−9869.(55) Rogers, S. A., Bero, J. D., and Melander, C. (2010) ChemicalSynthesis and Biological Screening of 2-Aminoimidazole-BasedBacterial and Fungal Antibiofilm Agents. ChemBioChem 11, 396−410.(56) Su, Z., Peng, L., Worthington, R. J., and Melander, C. (2011)Evaluation of 4,5-Disubstituted-2-Aminoimidazole−Triazole Conju-gates for Antibiofilm/Antibiotic Resensitization Activity AgainstMRSA and Acinetobacter baumannii. ChemMedChem 6, 2243−2251.(57) Olson, K. M., Starks, C. M., Williams, R. B., O’Neil-Johnson, M.,Huang, Z., Ellis, M., Reilly, J. E., and Eldridge, G. R. (2011) Novel
Pentadecenyl Tetrazole Enhances Susceptibility of Methicillin-Resistant Staphylococcus aureus Biofilms to Gentamicin. Antimicrob.Agents Chemother. 55, 3691−3695.(58) Abouelhassan, Y., Garrison, A. T., Burch, G. M., Wong, W.,Norwood, V. M., IV, and Huigens, R. W., III (2014) Discovery ofquinoline small molecules with potent dispersal activity againstmethicillin-resistant Staphylococcus aureus and Staphylococcusepidermidis biofilms using a scaffold hopping strategy. Bioorg. Med.Chem. Lett. 24, 5076−5080.(59) Abouelhassan, Y., Garrison, A. T., Bai, F., Norwood, V. M.,Nguyen, M. T., Jin, S. G., and Huigens, R. W. (2015) APhytochemical-Halogenated Quinoline Combination Therapy Strategyfor the Treatment of Pathogenic Bacteria. ChemMedChem 10, 1157−1162.(60) Garrison, A. T., Abouelhassan, Y., Kallifidas, D., Bai, F.,Ukhanova, M., Mai, V., Jin, S., Luesch, H., and Huigens, R. W. (2015)Halogenated Phenazines that Potently Eradicate Biofilms, MRSAPersister Cells in Non-Biofilm Cultures, and Mycobacterium tuber-culosis. Angew. Chem., Int. Ed. 54, 14819−14823.(61) Lee, J.-H., Kim, Y.-G., Gwon, G., Wood, T. K., and Lee, J.(2016) Halogenated indoles eradicate bacterial persister cells andbiofilms. AMB Express 6, 123.(62) Mitchell, G., Gattuso, M., Grondin, G., Marsault, E., Bouarab,K., and Malouin, F. (2011) Tomatidine Inhibits Replication ofStaphylococcus aureus Small-Colony Variants in Cystic FibrosisAirway Epithelial Cells. Antimicrob. Agents Chemother. 55, 1937−1945.(63) Mitchell, G., Lafrance, M., Boulanger, S., Seguin, D. L., Guay, I.,Gattuso, M., Marsault, E., Bouarab, K., and Malouin, F. (2012)Tomatidine acts in synergy with aminoglycoside antibiotics againstmultiresistant Staphylococcus aureus and prevents virulence geneexpression. J. Antimicrob. Chemother. 67, 559−568.(64) Chagnon, F., Guay, I., Bonin, M. A., Mitchell, G., Bouarab, K.,Malouin, F., and Marsault, E. (2014) Unraveling the structure-activityrelationship of tomatidine, a steroid alkaloid with unique antibioticproperties against persistent forms of Staphylococcus aureus. Eur. J.Med. Chem. 80, 605−620.(65) Dickey, S. W., Cheung, G. Y. C., and Otto, M. (2017) Differentdrugs for bad bugs: antivirulence strategies in the age of antibioticresistance. Nat. Rev. Drug Discovery 16, 457−471.(66) Cheng, A. G., Kim, H. K., Burts, M. L., Krausz, T., Schneewind,O., and Missiakas, D. M. (2009) Genetic requirements for Staph-ylococcus aureus abscess formation and persistence in host tissues.FASEB J. 23, 3393−3404.(67) Sibbald, M. J., Yang, X. M., Tsompanidou, E., Qu, D., Hecker,M., Becher, D., Buist, G., and van Dijl, J. M. (2012) Partiallyoverlapping substrate specificities of staphylococcal group A sortases.Proteomics 12, 3049−3062.(68) Jang, K. H., Chung, S.-C., Shin, J., Lee, S.-H., Kim, T.-I., Lee, H.-S., and Oh, K.-B. (2007) Aaptamines as sortase A inhibitors from thetropical sponge Aaptos aaptos. Bioorg. Med. Chem. Lett. 17, 5366−5369.(69) Oh, K.-B., Mar, W., Kim, S., Kim, J.-Y., Oh, M.-N., Kim, J.-G.,Shin, D., Sim, C. J., and Shin, J. (2005) Bis(indole) alkaloids as sortaseA inhibitors from the sponge Spongosorites sp. Bioorg. Med. Chem.Lett. 15, 4927−4931.(70) Suree, N., Yi, S. W., Thieu, W., Marohn, M., Damoiseaux, R.,Chan, A., Jung, M. E., and Clubb, R. T. (2009) Discovery andstructure−activity relationship analysis of Staphylococcus aureussortase A inhibitors. Bioorg. Med. Chem. 17, 7174−7185.(71) Zhang, J., Liu, H., Zhu, K., Gong, S., Dramsi, S., Wang, Y.-T., Li,J., Chen, F., Zhang, R., Zhou, L., Lan, L., Jiang, H., Schneewind, O.,Luo, C., and Yang, C.-G. (2014) Antiinfective therapy with a smallmolecule inhibitor of Staphylococcus aureus sortase. Proc. Natl. Acad.Sci. U. S. A. 111, 13517−13522.(72) Cascioferro, S., Raffa, D., Maggio, B., Raimondi, M. V., Schillaci,D., and Daidone, G. (2015) Sortase A Inhibitors: Recent Advancesand Future Perspectives. J. Med. Chem. 58, 9108−9123.(73) Liu, G. Y., Essex, A., Buchanan, J. T., Datta, V., Hoffman, H. M.,Bastian, J. F., Fierer, J., and Nizet, V. (2005) Staphylococcus aureus
golden pigment impairs neutrophil killing and promotes virulencethrough its antioxidant activity. J. Exp. Med. 202, 209−215.(74) Clauditz, A., Resch, A., Wieland, K.-P., Peschel, A., and Gotz, F.(2006) Staphyloxanthin Plays a Role in the Fitness of Staphylococcusaureus and Its Ability To Cope with Oxidative Stress. Infect. Immun.74, 4950−4953.(75) Liu, C.-I., Liu, G. Y., Song, Y., Yin, F., Hensler, M. E., Jeng, W.-Y., Nizet, V., Wang, A. H.-J., and Oldfield, E. (2008) A CholesterolBiosynthesis Inhibitor Blocks Staphylococcus aureus Virulence. Science319, 1391−1394.(76) Song, Y., Lin, F.-Y., Yin, F., Hensler, M., Rodrígues Poveda, C.A., Mukkamala, D., Cao, R., Wang, H., Morita, C. T., GonzalezPacanowska, D., Nizet, V., and Oldfield, E. (2009) Phosphonosulfo-nates Are Potent, Selective Inhibitors of Dehydrosqualene Synthaseand Staphyloxanthin Biosynthesis in Staphylococcus aureus. J. Med.Chem. 52, 976−988.(77) Song, Y., Liu, C.-I., Lin, F.-Y., No, J. H., Hensler, M., Liu, Y.-L.,Jeng, W.-Y., Low, J., Liu, G. Y., Nizet, V., Wang, A. H. J., and Oldfield,E. (2009) Inhibition of Staphyloxanthin Virulence Factor Biosynthesisin Staphylococcus aureus: In Vitro, in Vivo, and CrystallographicResults. J. Med. Chem. 52, 3869−3880.(78) Chen, F., Di, H., Wang, Y., Cao, Q., Xu, B., Zhang, X., Yang, N.,Liu, G., Yang, C.-G., Xu, Y., Jiang, H., Lian, F., Zhang, N., Li, J., andLan, L. (2016) Small-molecule targeting of a diapophytoene desaturaseinhibits S. aureus virulence. Nat. Chem. Biol. 12, 174−179.(79) Wang, Y., Chen, F., Di, H., Xu, Y., Xiao, Q., Wang, X., Wei, H.,Lu, Y., Zhang, L., Zhu, J., Sheng, C., Lan, L., and Li, J. (2016)Discovery of Potent Benzofuran-Derived Diapophytoene Desaturase(CrtN) Inhibitors with Enhanced Oral Bioavailability for theTreatment of Methicillin-Resistant Staphylococcus aureus (MRSA)Infections. J. Med. Chem. 59, 3215−3230.(80) Frees, D., Qazi, S. N. A., Hill, P. J., and Ingmer, H. (2003)Alternative roles of ClpX and ClpP in Staphylococcus aureus stresstolerance and virulence. Mol. Microbiol. 48, 1565−1578.(81) Michel, A., Agerer, F., Hauck, C. R., Herrmann, M., Ullrich, J.,Hacker, J., and Ohlsen, K. (2006) Global Regulatory Impact of ClpPProtease of Staphylococcus aureus on Regulons Involved in Virulence,Oxidative Stress Response, Autolysis, and DNA Repair. J. Bacteriol.188, 5783−5796.(82) Bottcher, T., and Sieber, S. A. (2008) β-Lactones as SpecificInhibitors of ClpP Attenuate the Production of Extracellular VirulenceFactors of Staphylococcus aureus. J. Am. Chem. Soc. 130, 14400−14401.(83) Bottcher, T., and Sieber, S. A. (2009) Structurally Refined β-Lactones as Potent Inhibitors of Devastating Bacterial VirulenceFactors. ChemBioChem 10, 663−666.(84) Zeiler, E., Korotkov, V. S., Lorenz-Baath, K., Bottcher, T., andSieber, S. A. (2012) Development and characterization of improved β-lactone-based anti-virulence drugs targeting ClpP. Bioorg. Med. Chem.20, 583−591.(85) Weinandy, F., Lorenz-Baath, K., Korotkov, V. S., Bottcher, T.,Sethi, S., Chakraborty, T., and Sieber, S. A. (2014) A β-Lactone-BasedAntivirulence Drug Ameliorates Staphylococcus aureus Skin Infectionsin Mice. ChemMedChem 9, 710−713.(86) Novick, R. P., and Geisinger, E. (2008) Quorum Sensing inStaphylococci. Annu. Rev. Genet. 42, 541−564.(87) Singh, R., and Ray, P. (2014) Quorum sensing-mediatedregulation of staphylococcal virulence and antibiotic resistance. FutureMicrobiol. 9, 669−681.(88) Koenig, R. L., Ray, J. L., Maleki, S. J., Smeltzer, M. S., andHurlburt, B. K. (2004) Staphylococcus aureus AgrA Binding to theRNAIII-agr Regulatory Region. J. Bacteriol. 186, 7549−7555.(89) Dunman, P. M., Murphy, E., Haney, S., Palacios, D., Tucker-Kellogg, G., Wu, S., Brown, E. L., Zagursky, R. J., Shlaes, D., andProjan, S. J. (2001) Transcription Profiling-Based Identification ofStaphylococcus aureus Genes Regulated by the agrand/or sarA Loci. J.Bacteriol. 183, 7341−7353.
(90) Otto, M. (2004) Quorum-sensing control in Staphylococci – atarget for antimicrobial drug therapy? FEMS Microbiol. Lett. 241, 135−141.(91) Periasamy, S., Joo, H.-S., Duong, A. C., Bach, T.-H. L., Tan, V.Y., Chatterjee, S. S., Cheung, G. Y. C., and Otto, M. (2012) HowStaphylococcus aureus biofilms develop their characteristic structure.Proc. Natl. Acad. Sci. U. S. A. 109, 1281−1286.(92) Somerville, G. A., Beres, S. B., Fitzgerald, J. R., DeLeo, F. R.,Cole, R. L., Hoff, J. S., and Musser, J. M. (2002) In Vitro Serial Passageof Staphylococcus aureus: Changes in Physiology, Virulence FactorProduction, and agr Nucleotide Sequence. J. Bacteriol. 184, 1430−1437.(93) Khan, B. A., Yeh, A. J., Cheung, G. Y., and Otto, M. (2015)Investigational therapies targeting quorum-sensing for the treatment ofStaphylococcus aureus infections. Expert Opin. Invest. Drugs 24, 689−704.(94) Benito, Y., Kolb, F. A., Romby, P., Lina, G., Etienne, J., andVandenesch, F. (2000) Probing the structure of RNAIII, theStaphylococcus aureus agr regulatory RNA, and identification of theRNA domain involved in repression of protein A expression. RNA 6,668−679.(95) Felden, B., Vandenesch, F., Bouloc, P., and Romby, P. (2011)The Staphylococcus aureus RNome and Its Commitment toVirulence. PLoS Pathog. 7, e1002006.(96) Gordon, C. P., Williams, P., and Chan, W. C. (2013)Attenuating Staphylococcus aureus Virulence Gene Regulation: AMedicinal Chemistry Perspective. J. Med. Chem. 56, 1389−1404.(97) Brackman, G., and Coenye, T. (2015) Inhibition of QuorumSensing in Staphylococcus spp. Curr. Pharm. Des. 21, 2101−2108.(98) Nakayama, J., Uemura, Y., Nishiguchi, K., Yoshimura, N.,Igarashi, Y., and Sonomoto, K. (2009) Ambuic Acid Inhibits theBiosynthesis of Cyclic Peptide Quormones in Gram-Positive Bacteria.Antimicrob. Agents Chemother. 53, 580−586.(99) Qazi, S., Middleton, B., Muharram, S. H., Cockayne, A., Hill, P.,O’Shea, P., Chhabra, S. R., Camara, M., and Williams, P. (2006) N-Acylhomoserine Lactones Antagonize Virulence Gene Expression andQuorum Sensing in Staphylococcus aureus. Infect. Immun. 74, 910−919.(100) Murray, E. J., Crowley, R. C., Truman, A., Clarke, S. R.,Cottam, J. A., Jadhav, G. P., Steele, V. R., O’Shea, P., Lindholm, C.,Cockayne, A., Chhabra, S. R., Chan, W. C., and Williams, P. (2014)Targeting Staphylococcus aureus Quorum Sensing with NonpeptidicSmall Molecule Inhibitors. J. Med. Chem. 57, 2813−2819.(101) Li, J., Wang, W., Xu, S. X., Magarvey, N. A., and McCormick, J.K. (2011) Lactobacillus reuteri-produced cyclic dipeptides quench agr-mediated expression of toxic shock syndrome toxin-1 in staphylococci.Proc. Natl. Acad. Sci. U. S. A. 108, 3360−3365.(102) Mansson, M., Nielsen, A., Kjaerulff, L., Gotfredsen, C. H.,Wietz, M., Ingmer, H., Gram, L., and Larsen, T. O. (2011) Inhibitionof virulence gene expression in Staphylococcus aureus by noveldepsipeptides from a marine photobacterium. Mar. Drugs 9, 2537−2552.(103) Nielsen, A., Mansson, M., Bojer, M. S., Gram, L., Larsen, T. O.,Novick, R. P., Frees, D., Frøkiær, H., and Ingmer, H. (2014)Solonamide B Inhibits Quorum Sensing and Reduces Staphylococcusaureus Mediated Killing of Human Neutrophils. PLoS One 9, e84992.(104) Daly, S. M., Elmore, B. O., Kavanaugh, J. S., Triplett, K. D.,Figueroa, M., Raja, H. A., El-Elimat, T., Crosby, H. A., Femling, J. K.,Cech, N. B., Horswill, A. R., Oberlies, N. H., and Hall, P. R. (2015)omega-Hydroxyemodin Limits Staphylococcus aureus QuorumSensing-Mediated Pathogenesis and Inflammation. Antimicrob. AgentsChemother. 59, 2223−2235.(105) Khodaverdian, V., Pesho, M., Truitt, B., Bollinger, L., Patel, P.,Nithianantham, S., Yu, G., Delaney, E., Jankowsky, E., and Shoham, M.(2013) Discovery of Antivirulence Agents against Methicillin-ResistantStaphylococcus aureus. Antimicrob. Agents Chemother. 57, 3645−3652.(106) Sully, E. K., Malachowa, N., Elmore, B. O., Alexander, S. M.,Femling, J. K., Gray, B. M., DeLeo, F. R., Otto, M., Cheung, A. L.,Edwards, B. S., Sklar, L. A., Horswill, A. R., Hall, P. R., and Gresham,
H. D. (2014) Selective chemical inhibition of agr quorum sensing inStaphylococcus aureus promotes host defense with minimal impact onresistance. PLoS Pathog. 10, e1004174.(107) Gov, Y., Borovok, I., Korem, M., Singh, V. K., Jayaswal, R. K.,Wilkinson, B. J., Rich, S. M., and Balaban, N. (2004) Quorum Sensingin Staphylococci Is Regulated via Phosphorylation of Three ConservedHistidine Residues. J. Biol. Chem. 279, 14665−14672.(108) Han, Y.-H., Kim, Y.-G., Kim, D. Y., Ha, S. C., Lokanath, N. K.,and Kim, K. K. (2005) The target of RNAIII-activating protein(TRAP) from Staphylococcus aureus: purification, crystallization andpreliminary X-ray analysis. Biochim. Biophys. Acta, Proteins Proteomics1748, 134−136.(109) Henrick, K., and Hirshberg, M. (2012) Structure of the signaltransduction protein TRAP (target of RNAIII-activating protein). ActaCrystallogr., Sect. F: Struct. Biol. Cryst. Commun. 68, 744−750.(110) Yarwood, J. M., Bartels, D. J., Volper, E. M., and Greenberg, E.P. (2004) Quorum Sensing in Staphylococcus aureus Biofilms. J.Bacteriol. 186, 1838−1850.(111) Shaw, L. N., Jonsson, I.-M., Singh, V. K., Tarkowski, A., andStewart, G. C. (2007) Inactivation of traP Has No Effect on the AgrQuorum-Sensing System or Virulence of Staphylococcus aureus. Infect.Immun. 75, 4519−4527.(112) Tsang, L. H., Daily, S. T., Weiss, E. C., and Smeltzer, M. S.(2007) Mutation of traP in Staphylococcus aureus Has No Impact onExpression of agr or Biofilm Formation. Infect. Immun. 75, 4528−4533.(113) Adhikari, R. P., Arvidson, S., and Novick, R. P. (2007) ANonsense Mutation in agrA Accounts for the Defect in agr Expressionand the Avirulence of Staphylococcus aureus 8325−4 traP::kan. Infect.Immun. 75, 4534−4540.(114) Balaban, N., Cirioni, O., Giacometti, A., Ghiselli, R.,Braunstein, J. B., Silvestri, C., Mocchegiani, F., Saba, V., and Scalise,G. (2007) Treatment of Staphylococcus aureus Biofilm Infection bythe Quorum-Sensing Inhibitor RIP. Antimicrob. Agents Chemother. 51,2226−2229.(115) Leitner, G., Krifucks, O., Kiran, M. D., and Balaban, N. (2011)Vaccine development for the prevention of staphylococcal mastitis indairy cows. Vet. Immunol. Immunopathol. 142, 25−35.(116) Balaban, N., Goldkorn, T., Gov, Y., Hirshberg, M., Koyfman,N., Matthews, H. R., Nhan, R. T., Singh, B., and Uziel, O. (2001)Regulation of Staphylococcus aureus Pathogenesis via Target ofRNAIII-activating Protein (TRAP). J. Biol. Chem. 276, 2658−2667.(117) Gov, Y., Bitler, A., Dell’Acqua, G., Torres, J. V., and Balaban, N.(2001) RNAIII inhibiting peptide (RIP), a global inhibitor ofStaphylococcus aureus pathogenesis: structure and function analysis.Peptides 22, 1609−1620.(118) Balaban, N., and Novick, R. P. (1995) Autocrine regulation oftoxin synthesis by Staphylococcus aureus. Proc. Natl. Acad. Sci. U. S. A.92, 1619−1623.(119) Korem, M., Sheoran, A. S., Gov, Y., Tzipori, S., Borovok, I., andBalaban, N. (2003) Characterization of RAP, a quorum sensingactivator of Staphylococcus aureus. FEMS Microbiol. Lett. 223, 167−175.(120) Baldassarre, L., Fornasari, E., Cornacchia, C., Cirioni, O.,Silvestri, C., Castelli, P., Giocometti, A., and Cacciatore, I. (2013)Discovery of novel RIP derivatives by alanine scanning for thetreatment of S. aureus infections. MedChemComm 4, 1114−1117.(121) Simonetti, O., Cirioni, O., Cacciatore, I., Baldassarre, L.,Orlando, F., Pierpaoli, E., Lucarini, G., Orsetti, E., Provinciali, M.,Fornasari, E., Di Stefano, A., Giacometti, A., and Offidani, A. (2016)Efficacy of the Quorum Sensing Inhibitor FS10 Alone and inCombination with Tigecycline in an Animal Model of StaphylococcalInfected Wound. PLoS One 11, e0151956.(122) Gruttner, F. (1898) Beitrage zur Chemie der Rinde vonHamamelis virginica L. Arch. Pharm. 236, 278−320.(123) Kiran, M. D., Adikesavan, N. V., Cirioni, O., Giacometti, A.,Silvestri, C., Scalise, G., Ghiselli, R., Saba, V., Orlando, F., Shoham, M.,and Balaban, N. (2008) Discovery of a quorum-sensing inhibitor ofdrug-resistant staphylococcal infections by structure-based virtualscreening. Mol. Pharmacol. 73, 1578−1586.
(124) Brackman, G., Cos, P., Maes, L., Nelis, H. J., and Coenye, T.(2011) Quorum sensing inhibitors increase the susceptibility ofbacterial biofilms to antibiotics in vitro and in vivo. Antimicrob. AgentsChemother. 55, 2655−2661.(125) Brackman, G., Breyne, K., De Rycke, R., Vermote, A., VanNieuwerburgh, F., Meyer, E., Van Calenbergh, S., and Coenye, T.(2016) The Quorum Sensing Inhibitor Hamamelitannin IncreasesAntibiotic Susceptibility of Staphylococcus aureus Biofilms byAffecting Peptidoglycan Biosynthesis and eDNA Release. Sci. Rep. 6,20321.(126) Vermote, A., Brackman, G., Risseeuw, M. D. P., Vanhoutte, B.,Cos, P., Van Hecke, K., Breyne, K., Meyer, E., Coenye, T., and VanCalenbergh, S. (2016) Hamamelitannin Analogues that ModulateQuorum Sensing as Potentiators of Antibiotics against Staphylococcusaureus. Angew. Chem., Int. Ed. 55, 6551−6555.(127) Vermote, A., Brackman, G., Risseeuw, M. D. P., Coenye, T.,and Van Calenbergh, S. (2016) Design, synthesis and biologicalevaluation of novel hamamelitannin analogues as potentiators forvancomycin in the treatment of biofilm related Staphylococcus aureusinfections. Bioorg. Med. Chem. 24, 4563−4575.(128) Vermote, A., Brackman, G., Risseeuw, M. D. P., Cappoen, D.,Cos, P., Coenye, T., and Van Calenbergh, S. (2017) NovelPotentiators for Vancomycin in the Treatment of Biofilm-RelatedMRSA Infections via a Mix and Match Approach. ACS Med. Chem.Lett. 8, 38−42.(129) Vermote, A., Brackman, G., Risseeuw, M. D. P., Coenye, T.,and Van Calenbergh, S. (2017) Novel hamamelitannin analogues forthe treatment of biofilm related MRSA infections−A scaffold hoppingapproach. Eur. J. Med. Chem. 127, 757−770.(130) Boles, B. R., and Horswill, A. R. (2008) agr-Mediated Dispersalof Staphylococcus aureus Biofilms. PLoS Pathog. 4, e1000052.(131) Kiedrowski, M. R., and Horswill, A. R. (2011) New approachesfor treating staphylococcal biofilm infections. Ann. N. Y. Acad. Sci.1241, 104−121.(132) Davis, J. S., Van Hal, S., and Tong, S. Y. C. (2015)Combination Antibiotic Treatment of Serious Methicillin-ResistantStaphylococcus aureus Infections. Sem. Resp. Crit. Care M. 36, 3−16.(133) Heras, B., Scanlon, M. J., and Martin, J. L. (2015) Targetingvirulence not viability in the search for future antibacterials. Br. J. Clin.Pharmacol. 79, 208−215.(134) Escaich, S. (2008) Antivirulence as a new antibacterialapproach for chemotherapy. Curr. Opin. Chem. Biol. 12, 400−408.(135) Gerdt, J. P., and Blackwell, H. E. (2014) Competition StudiesConfirm Two Major Barriers That Can Preclude the Spread ofResistance to Quorum-Sensing Inhibitors in Bacteria. ACS Chem. Biol.9, 2291−2299.(136) Kalan, L., and Wright, G. D. (2011) Antibiotic adjuvants:multicomponent anti-infective strategies. Expert Rev. Mol. Med. 13, e5.(137) Worthington, R. J., and Melander, C. (2013) Combinationapproaches to combat multidrug-resistant bacteria. Trends Biotechnol.31, 177−184.(138) Ashburn, T. T., and Thor, K. B. (2004) Drug repositioning:identifying and developing new uses for existing drugs. Nat. Rev. DrugDiscovery 3, 673−683.
