1. Introduction

2. Interference in quorum

sensing pathways

3. Conclusion

4. Expert opinion

Review

Quorum sensing inhibitors:a patent reviewTianyu Jiang & Minyong Li†

Shandong University, School of Pharmacy, Department of Medicinal Chemistry, Key Laboratory of

Chemical Biology of Natural Products (MOE), Shandong, China

Introduction: Quorum sensing (QS) is a cell-to-cell communication that regu-

lates gene expression and coordinates their behavior in accordance with the

cell population density as a result of discerning molecules termed autoin-

ducers (AIs). The processes that QS governed include biofilm formation, bacte-

rial virulence, antibiotic production, competence, conjugation, swarming

motility and sporulation. Three main QS AIs are acyl-homoserine lactones,

AI-2 and AI peptides. The attractive study of QS leads to an expansive number

of QS inhibitors and approaches interfering with QS appearing.

Areas covered: This review summarized the recent QS inhibitor-related patents

published from 2009 to 2012. The authors have analyzed these patents and

have provided an overview of QS inhibitors and their application.

Expert opinion: The main strategies for QS inhibition related to the patents

are disruption of the AI synthase, inactivation of the signal molecule, antago-

nism of the receptor and promotion of immune response to AI. Some of the

natural or synthetic QS inhibitors display excellent activity to manipulate bac-

terial pathogenicity to offer significant potential in clinical therapy. However,

more efforts are needed to be conducted to determine this form of communi-

cation to guide the development of QS inhibitors. Overall, QS is a suitable tar-

get for antimicrobial therapy and QS inhibitors are likely to lead to a

renaissance of anti-virulence drugs without tolerance, which is the ultimate

goal expected to achieve in this field.

Keywords: acyl-homoserine lactones, antibacterial, autoinducer-2, autoinducer peptides,

biofilm, inhibitor, quorum sensing

Expert Opin. Ther. Patents [Early Online]

1. Introduction

It is well known that quorum sensing (QS) is a mechanism of bacterial communi-cation (a cell-to-cell communication) that regulates gene expression and coordinatestheir behavior in accordance with the cell population density [1,2]. QS bacteria reg-ulate gene expression by creating, releasing and detecting chemical signaling mole-cules called autoinducers (AIs) in response to fluctuations in the surroundingenvironment [3,4]. The phenomenon of QS is first represented in the bioluminescentmarine bacterium Vibrio fischeri [5]. Moreover, in 1994, it is pointed out that QS isa newly discovered environmental sensing system, which is different from (shouldnot be confused with) autoregulation or autorepression [6]. We now recognize thata range of different categories of chemical signals are employed as QS signal mole-cules, such as acyl-homoserine lactones (AHL or AI-1), AI-2, AI-3, choleraeAI-1 (CAI-1), pseudomonas quinolone signal (PQS) and AI peptides [7-13]. Further-more, some species of bacteria utilize more than one chemical signal and/or morethan one kind of signal to intercommunicate. In general, Gram-negative bacteriause AHL and Gram-positive bacteria process oligopeptides as AIs to communicatewithin species, while AI-2, which exists in both Gram-positive and Gram-negativebacteria, performs a role of AIs for interspecies communication (Table 1). Typically,

10.1517/13543776.2013.779674 © 2013 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674 1All rights reserved: reproduction in whole or in part not permitted

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an enhancing concentration of AIs in the extracellular sur-rounding is concomitant with an increasing cell populationdensity. Some evidence indicates that when AIs reach theminimal threshold stimulatory level that is required for detec-tion, the activation of a signal transduction cascade coulddirect a modification in target gene expression that controlsdiverse biological functions [2,3]. These processes includebiofilm formation, bacterial virulence, antibiotic produc-tion, competence, conjugation, swarming motility andsporulation [14-18]. It is critical for bacteria to take advantageof intraspecies and interspecies cell--cell communication viaAIs to survive in nature. In addition, alternative hypotheseshave been presented for QS, which meant that QS may beincluded in diffusion sensing [19] and efficiency sensing [20].It seems that more work needs to be done to explore themost appropriate explanation for QS.Recent advances in the field indicate that the inhibition of

QS in pathogenic bacterium could result in a valuable strategyfor the development of antimicrobial agents. Compared withthe conventional antibiotics, QS inhibitors suppress their viru-lence through the particular interference with a signaling sys-tem instead of killing pathogens directly. Though a few oflatest study pointed that QS inhibitors may disrupt the growthof bacteria under certain conditions [21], numerous researchesheld the view that these compounds would be less likely toengender the evolution of bacterial resistance [22-24]. There are

more and more inhibitors of QS and approaches interferingwith QS appearing with the recent explosion of progress inthe field. Typically, inhibition of QS can be accomplished byinfluencing in the biosynthesis of signals, inactivating the AIsor inhibiting their receptors [25]. In this review, we wouldlike to summarize the QS inhibitors published from 2009to 2012 and their application for the antibacterial purpose(Table 2).

2. Interference in quorum sensing pathways

2.1.1 Interference in AI peptide-mediated QSGenerally, some specific oligopeptides are functional AIs inGram-positive bacteria, which are also known as AI peptides(AIP) (Figure 1). These AIPs are peptides ranging from 5 to17 amino acids and generally include a highly conservedcysteine residue, which forms a thioester linkage with theC-terminal carboxyl group to yield the cyclic peptide. Boththe cyclic structure of the peptide and the other half of theAIP known as the ‘tail’ are important for binding selectivityand signaling molecule specificity in Gram-positive bacteria(Table 2) [26,27]. We select AIP system of Staphylococcus aureusas an example to introduce AIP-mediated QS (Scheme 1).These oligopeptides are detected by a classical of two-component system, including a sensing receptor histidinekinase (HK) and a response regulator (RR) protein [28]. Afterbinding signal molecules, the HK receptor activates the RRprotein to induce expression of the target genes [25].

This AIP-based QS system, that is encoded by the accessorygene regulator (agr) locus, plays an important role in the path-ogenesis of S. aureus [29-31], The agr system contributes topathogenesis through controlling cell density-dependentexpression of bacterial virulence genes, which encode cellsurface proteins, such as protein A, coagulase and fibronectin-binding proteins, as well as secreted proteins including pro-teases, hemolysins, toxic shock syndrome toxin 1 (TSST-1)and enterotoxin B [32,33].

2.1.2 Inhibitors that disrupt AIP-mediated QSThe Janda laboratory recently provided an immunogenicmolecular entity comprising a cyclic peptide or analog thereofcovalently bonded to a macromolecular carrier, optionally viaa linker moiety, which can be used to induce the creation ofan immune response against a native QS cyclic signaling pep-tide produced by a Gram-positive bacterium so as to modu-late the expression of virulence factors [34]. The chemicalstructures of these synthetic haptens are depicted in Figure 2.Besides, they revealed a neutralizing antibody that can befacilitated to block Gram-positive bacterial QS, and henceto prevent infection or development of a disease correlativelyin a mammal. The mechanism of its action is to specificallybind to a cyclic peptide signaling molecule. An example relat-ing to the discovery is an antibody specific for the S. aureusAIP-4, which can disturb QS and prevent Staphylococcusinfection in mice.

Article highlights.

. This review summarizes the recent quorum sensing (QS)inhibitor-related patents and provided an overview ofQS inhibitors and their application. These inhibitors orapproaches could interfere at least one of the QSpathways including the AI peptide-mediated,AHL-mediated and AI-2-mediated quorum sensingpathway.

. The main strategies for QS inhibition are disruption ofthe autoinducer synthase, inactivation of the signalmolecule, antagonism of the receptor and promotion ofimmune response to autoinducer.

. A large number of patents declare inhibitors that coulddisturb the AHL-mediated QS because of AHLs’ curialrole in the pathogenicity of bacteria to some extent.

. More and more attention is paid to the development ofAI-2 inhibitors because AI-2 is functional in bothGram-positive and Gram-negative bacteria, which arelikely to achieve broad-spectrum modulation of QScontrols phenotypes.

. There are a lot of difficulties needing to be overcome inthe development of QS inhibitors. The mechanisms ofsome inhibitors are still unclear and a few of inhibitorsmay have potential toxicity towards host cells.Undoubtedly more efforts needed to be conducted tothe detailed molecular basis of QS manipulation toguide the development of QS inhibitors.

This box summarizes key points contained in the article.

T. Jiang & M. Li

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2.2 Interference in AHL-mediated QS2.2.1 AHL QS pathwayTypically, Gram-negative QS bacteria employ AHLs as AIs [6].Most of AHL QS systems reported to date employ AHL syn-thase encoded by homologs of gene luxI, which is initiallyidentified in V. fischeri. The AHL signal molecules bind to atranscriptional activator protein encoded by a homologousgene of luxR of V. fischeri [35]. These bacteria may utilize oneor more kinds of AHL signal molecules. Generally, theseAHLs are composed of a fatty acyl chain linked to a lactonizedhomoserine through an amide bond (Figure 3) [27].

AHL-mediated QS occurs via two mechanisms. In the firstmechanism (Scheme 2), the cytoplasmic LuxR family of RRproteins detects the intracellular AHLs [6]. When the cell den-sity and AHL concentrations are low, the LuxR-type proteinswithout ligands are insoluble and will be degraded [36,37]. TheAHLs accumulate gradually in the surroundings along withthe cell density increase. At a certain threshold concentrationof AHL, AHL binds to the cognate LuxR-type proteins toform the receptor--signal complexes. The complexes thenbecome dimers or multimers with each other in turn tobind DNA and control expression of QS target genes [38]. Inthe second mechanism (as shown in Scheme 3), accumulatedAHLs out of the cell are detected by the membrane-bound LuxN-type receptors which have two components his-tidine and kinase [37,39-41]. When AHL is bound by the LuxNprotein, its autophosphorylation and phosphotransfer activi-ties change. In other words, the LuxN--AHL complexes causea variation in the phosphorylation state of a downstream

DNA-binding transcription factor, which modifies its activityand then directs a QS gene expression [37,42-44].

2.2.2 Inhibitors that target the AHL synthesisBlockage of AHL production is a conceptually simple methodfor inhibiting QS pathways -- no production of signal mole-cule, no activation of QS. However, there are relatively fewreports on the inhibition of AHL synthase: LuxI-type pro-teins. In general, a sequentially reaction of AHLs synthesis iscatalyzed by LuxI-type synthases, which utilize S-adenosylme-thionine (SAM) as the amino donor for the formation of thehomoserine lactone ring moiety and an acylated carrierprotein as the precursor to develop the acyl side chain [25,45].

5¢-Methylthioadenosine/S-adenosylhomocysteine nucleosi-dase (MTANs) perform a crucial role in the biosynthesis ofAIs in QS bacteria. MTANs are tightly involved in maintain-ing homeostasis in bacteria. The proposed mechanism ofMTAN-catalyzed reaction is exhibited in Scheme 4. MTANis a bacterial enzyme encoded by the pfs gene and catalyzesthe irreversible hydrolytic deadenylation of methylthioadeno-sine (MTA) and S-adenosylhomocysteine (SAH) by utilizingSAM. In such a biosynthetic route, AI-1 and AI-2 are twokinds of AIs synthesized from SAM.

AI-1 remains with a family of AHLs, responsible for intra-species communication. In the synthesis of AI-1 molecules,the amino acid moiety of SAM is transferred to an acyl accep-tor to produce homoserine lactones and MTA is a by-product.AI-2 includes derivatives of 4,5-dihydroxy-2,3-pentanedione(DPD) and plays a role in interspecies communication. Inthe synthesis of DPD, MTAN produces S-ribosylhomocys-teine (SRH) from SAH, and SRH is subsequently convertedto homocysteine and DPD by LuxS. According to the biosyn-thesis of AIs, the accumulation of MTA is caused by blockingMTAN, thus resulting in suppression of the AI-1 formation.In addition, the inhibition of MTAN can directly obstructthe production of SRH, the precursor of AI-2. Consequently,MTAN offers a way to block the biosynthesis of these AIs soas to disrupt QS.

Recently, Schramm provided methods for treating bacterialinfections by making use of MTAN inhibitor with a sub-growth inhibiting dosage [46]. They further supplied pharma-ceutical compositions comprising a sub-bacterial-growthinhibiting amount of a MTAN inhibitor and a pharmaceuti-cally acceptable carrier. The Schramm laboratory designedand synthesized two different types of transition state

Table 1. Different QS pathway in bacteria.

Pathway AIs Bacteria

AIP pathway Various oligopeptides Gram-positiveAHL (AI-1) pathway Various AHLs Gram-negativeAI-2 pathway DPD derivatives Gram-negative and Gram-positiveAI-3 pathway Epinephrine or norepinephrine Gram-negativePQS pathway PQS Gram-negativeCAI-1 Hydroxyketones Gram-negative (V. cholerae)

OMet

S IleCys

AspPheThr

SerTyr

O

PheS LeuCys

SerSerAlaAsn

Val

OLeu

S LeuCys

AspPheAsn

Ile

Gly

OMet

S IleCys

TyrPheThrSer

Tyr

AIP-1 AIP-2

AIP-3 AIP-4

Figure 1. Structures of AIP signals employed by S. aureus.

QS inhibitors

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analogs: one is 5¢-thio-substituted immucillin that mimics anearly transition state with partial bond order between the ribo-syl and adenine groups and another is 5¢-thio-substitutedDADMe-immucillin, resembling a late transition state wherethe ribosyl cation is totally set apart from the adenine leavinggroup [47,48].

Several immucillin A and DADMe-immucillin A deriva-tives, including 5, 6 and 7 (Figure 4), were tested againstMTANs and exhibited high affinities for noncovalent enzy-me--inhibitor interactions. These compounds could inhibitthe cellular MTAN activity by giving IC50 values of 27,31 and 6 nM for 5, 6 and 7 in Vibrio cholerae N16961without growth inhibition, respectively. Furthermore, if theconcentrations of these inhibitors were increased, the AIresponse would become progressively weaker. The IC50 forsuppression of light induction in Vibrio harveyi BB170 wasdetermined to be 0.94, 11 and 1.4 nM with 5, 6 and 7, mean-while in V. harveyi BB120, the inhibitiory IC50 values were10.5, 14 and 1 nM for the same inhibitors, respectively.V. harveyi BB170 responds to AI-2 alone, while V. harveyiBB120 responds to AI-1 and AI-2. In addition, compound 7

led to a dose-dependent inhibition of AI-2 induction with anIC50 of 125 nM in wild-type Escherichia coli. These evidencesindicate that MTAN would play a crucial role in the QS as apotential target for bacterial anti-infective drug design.

2.2.3 Inactivation of AHL signal moleculeIf the generated signal molecules can be inactivated orcompletely degraded, the QS system also can be inhibited.This can be achieved by different methods and one of themis enzymatic destruction. For example, AHL lactonases, oneclass of AHL-degrading enzymes that have been identified,lead to hydrolyzation of the homoserine lactone ring withno influence on the rest of the molecular structure [49]. Themechanism of AHL lactonase is described in Scheme 3.

Kim et al. have screened a few QS inhibitors taking advan-tage of the screening method they invented [50]. The preferredQS inhibitors that they selected are the mutant lactonases

Table 2. QS bacteria mentioned in this review.

Organism Signal Phenotype

Gram-positive S. aureus AIP-1, AIP-2,AIP-3, AIP-4

Production of protein A,proteases hemolysins,enterotoxin B, TSST-1, etc.

A. naeslundii AI-2 Biofilm formation and virulenceB. anthracis AI-2 Toxin expressionS. epidermidis AI-2 Biofilm formation

Gram-negative V. cholerae CAI-1AI-2

Biofilm formation, virulence factor productionand protease production

V. harveyi 3-hydroxy-C4-HSLAI-2 (S-THMF-borate)CAI-1

Bioluminescence, metalloprotease productionand type III secretion

V. fischeri 3-oxo-C6-HSL BioluminescenceC. violaceum C6-HSL Exoenzymes, antibiotics, cyanideA. tumefaciens 3-oxo-C8-HSL Ti plasmid conjugationP. aeruginosa C3-oxo-C12-HSL

C4-HSLPQS

Multiple extracellular enzymes, biofilm formationand secondary metabolites

P. putida 3-oxo-C12-HSL Biofilm formationB. glumae C8-HSL Virulence and motilityE. coli AI-2 Biofilm formation and virulenceS. typhimurium AI-2 (R-THMF) Virulence

Metabolic genes,phenol solube

modulins

RNAIII Exotoxinssurface proteins

AIP

AgrC

AgrAP

PAgrB

AgrD

Scheme 1. AIP-mediated QS in S. aureus [2]. The AIP activates

AgrC (the transmembrane receptor HK) and then causes

the phosphotransfer from AgrC to AgrA (the response

regulator), which leads to transcription of RNAIII-

dependent and -independent genes. AIP is generated by

the posttranslational processing of AgrD that is imparted by

the membrane-bound AgrB.

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V69L and V69L/I190F, which possess enhanced activity con-trast with ordinary lactonase. Thus, they can interfere theAHL-mediated QS owing to their capability of degradingAHL. Accordingly, the mutant lactonase V69L of SEQ IDNO. 13 has amino acid substitution of Val with Leu at position69 of the known wild-type Bacillus thuringiensis lactonase, andthe mutant lactonase V69L/I190F of SEQ ID NO. 14 hasamino acid substitution of Val with Leu at position 69 andamino acid substitution of Ile with Phe at position 190 of theknown wild-type B. thuringiensis lactonase.

In addition, their invention provided a simple procedurethat more QS inhibitors can be screened by following few

procedures: first, determine the suitable concentration ofAHL, at which the growth of the host cells is affected depend-ing on the presence or absence of inhibitor. And then select astrain that is capable of growing at the correspondingconcentration.

Casal and Bernardez have found a strain of a new species ofa-Proteobacteria capable of degrading N-acyl-homoserine,which could be accommodated to control bacterial infectionsrelated to AHL-QS [51]. This bacterium displays a differentenzymatic activity from lactonase because it cannot be observ-ably recovered by acidification after having been exposed tothe action of the bacteria.

H2N

H2N

H2N

HN

OO

NH

HN

NH

HN

NH

HN

HNHN

O

O

O

OH

O

OS

O

O

O

OHO

OHO

OH

O

O

S

NH

OO

HN

NH

HN

NH

HN

NH

HN

HNHN

O

O

O

OH

O

O

O

O

O

O

OOH

O

NH2

NH2

O

O

SH

1

NH

O O

HN

NH

HN

NH

HN

HNHN

O

O

O

OH

O

O

O

O

O

O

O

O

O

SH

H2N

HN

OO N

H

HN

NH

HN

NH

HN

HNHN

O

O

O

OS

O

O

O

OHO

OHO

OtBu

O

O

SH

OH

2

3

4

Figure 2. A series of synthetic haptens (1 -- 4) for immunization and elicitation of an immune response of monoclonal

antibodies to AIP-1, AIP-2, AIP-3 and AIP-4, which are provided by the Janda laboratory.

QS inhibitors

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2.2.4 Inhibitors that target the AHL receptorThe majority of work carried out on inhibition of AHL QShas focused on seeking out receptor antagonists that canbind to AHL receptor but would not lead to eliciting the sub-sequent biological response [27]. There is no wonder that var-ious receptors/regulators respond to specific AHLs (Figure 3).Generally, structural modification of AHLs gains moreattention and efforts in search of AHL antagonists.

Blackwell et al. designed and synthesized a series of N-AHLanalogs comprised of a wide range of acyl groups [52,53]. Someof these compounds are capable of acting as antagonists, whichmay compete against native AHL ligands to reduce the produc-tion of biofilm, virulence factors and enzymes in QS bacteria.For example, three compounds (Figure 5) displayed effectiveactivity against TraR in Agrobacterium tumefaciens and LasRin Pseudomonas aeruginosa. Moreover, compounds 8 and 10

strongly inhibit P. aeruginosa biofilm formation on glass slidesafter 48 h in the presence of synthetic ligands at 50 µM.

In their further study, they designed, optimized and synthe-sized more compounds as nonnative AHL ligands to probethe role of key features of the AHL structure on QS activity [53].Several focused collections of compounds on nonnative AHLsare systematically evaluated against the R proteins fromA. tumefaciens (TraR), P. aeruginosa (LasR), and V. fischeri

OO

NH

OR

n

O

OHN

OO

3-oxo-C6-HSL (LuxR) 3-OH-C4-HSL (LuxN)

V. harveyiV. fischeri

O

OHN

OO

A. tumefaciens

3-oxo-C8-HSL (TraR)

P. aeruginosa

O

OHN

OO

3-oxo-C12-HSL (LasR)

O

OHN

O

C4-HSL (RhlR)

O

OHN

O

C6-HSL (CviR)

C. violaceum

P. aeruginosa

General structure of AHL

O

OHN

O

C6-HSL (SwrR)

S. liquefaciens

O

OHN

O

C4-HSL (SwrR)

S. liquefaciens

O

OHN

OOH

Figure 3. General structure of the AHL AI and the examples of natural AHLs and their corresponding regulators.

LuxILuxR

LuxR

luxCDABE

luxR luxI

AHL

Scheme 2. The LuxI/LuxR QS circuit in V. fischeri. This scheme

was referred to Miller and Bassler’s review [1].

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(LuxR) to determine their ability to modulate R proteinfunction.

Among the compounds (Figure 6), compound 11 showedinhibition both for LasR and TraR, and compound 13, withlonger acyl chain, displayed strong inhibitory activity inLuxR. Compound 25 displayed inhibitory activity in TraR(93%) and compound 26 exhibited highest level of inhibition

in LuxR (81%); however, the sulfony HLs behaved weak tomoderate inhibition against LasR. Both the 4-azido PHL(15) and 4-phenyl PHL (16) were able to be strong inhibitorsof TraR and LuxR. As one of the new and potent syntheticinhibitors, compound 18 displayed powerful inhibition inTraR (90%), and decent inhibition in LuxR (68%). More-over, compound 21 is a strong inhibitor of LuxR (60%) and

Scheme 3. Role of MTAN in bacteria [47].

O

HN

O

ORn

HN

O

ORn

OH

OH

AHL lactonase

H2O

Scheme 4. Mechanism of AHL lactonase [49].

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a modest inhibitor of LasR. Molecule 19 exhibited antagonis-tic activity against TraR (90%) and LasR (49%). It is notablethat compound 17 is the most active inhibitor against thethree R proteins. More examples of compounds as potentinhibitors are showed in Figure 6. In addition, we get informa-tion from the invention that functional acyl group, stereo-chemistry and acyl chain structure, play a multifaceted rolein AHL analogs -- R proteins binding system, resulting inactivation and inhibition of QS.P. aeruginosa, the common environmental microorganism,

is an important example because of its opportunistic pathoge-nicity for human being. Biofilm formation and its specificarchitecture are the key factor to contribute to the pathogen-esis and antibiotic resistance of P. aeruginosa, which areregulated by various QS systems in general.Iyer et al. designed and synthesized some novel analogs

(27 -- 29, Figure 7) of an AI of Pseudomonas QS system, butanolhomoserine lactone (AHL-2) [54]. It is worth noting that theseanalogs may be used to enhance the uptake of antibiotics bydelivering antibiotic-AHL analog to its target. The syntheticQS molecule is chemically linked to a suitable antibioticcovalently to form a conjugate that is then introduced into abiological system. The antibiotic may be ciprofloxacin,gentamicin, tobramycin, clarithromycin, piperacillin, etc. Forexample, the nascent (24 h) biofilms were exposed to 25 µMcompound 29 -- ciprofloxacin conjugate for 48 h and freeciprofloxacin. Compared with free ciprofloxacin, obvious inhi-bition was observed in the culture that treated with conjugate.The efficacy of conjugate in disrupting mature biofilms wasalso better than free ciprofloxacin. P. aeruginosa PAO1 wasemployed in the biofilm disruption assessment. This resultindicated that the QS molecule antibiotic was more efficient

on inhibiting nascent and mature biofilms of P. aeruginosathan the free antibiotic [55]. However, the mechanism of actionof these compounds is still unknown.

Recently, Meijler et al. synthesized a series of compoundswhich could directly act to inhibit bacterial QS [56]. Suchinhibitors preferably have the formula A-B, in which A is anelectrophilic functional group and B is the natural ligand ofthe target protein or a portion. The inhibitors interact withthe target protein in such a mode that the A functional group,is able to covalently bind to a residue of target protein so as toblock binding of the natural ligand. It has been shown that atleast some of these inhibitory compounds that have a suffi-ciently similar structure of 3-oxo-C12-HSL, are able to targetthe P. aeruginosa QS regulator, LasR.

They evaluated the influence of the nine synthetic inhibitors(Figure 8) on P. aeruginosaQS-related gene expression and henceisothiocyanate analogs display inhibition of QS at low concentra-tions in all assays. For example, compound 32 showed significantinhibition of QS-controlled biofilm formation with the wild-type P. aeruginosa PAO1 strain. However, the strongest inhibitorof luminescence of the wild-type PAO1 reporter strain appearedto be molecule 33 (IC50: 45.2 ± 0.7 mM). Compounds 38, 32and 31 were followed (IC50: 100 ± 7, 113 ± 19, ~ 300 mM).

It is likely that the isothiocyanate analogs could covalentlybound Cys79 that locates in the LasR-binding pocket.

In addition, in the assessment of antagonism on E. coli-based LasR, the chloroacetamide compound 38 was the bestantagonist (IC50: 1.1 ± 0.5 mM), and the isothiocyanatescompound 31, 32, 33 seem less effective (IC50: 39.1 ± 9.4,29.8 ± 0.5, 19.2 ± 3.9 mM).

Handelsman and Borlee made use of QS antagonists(40 -- 48, Figure 9) to inhibit AHL QS controlled phenotypes

HN

NH

OO

OH8

NH

OO

OH NH

BrO

O

H O9 10

A. tumefaciensWCF47 IC50: 1.1 μM IC50: 1.0 μM IC50: 0.25 μM

P. aeruginosaPAO-JP2 IC50: 14.8 μM IC50: 33.1 μM IC50:1 6.1 μM

Figure 5. Compounds synthesized in the Blackwell laboratory with significant activity against TraR in A. tumefaciens and

LasR in P. aeruginosa.

HN

N

N

N

HO

S

HN

N

N

N

HO

S

HN

N

N

N

NH2NH2NH2

HO

S

5 6 7

Figure 4. Chemical structures of MTAN inhibitors provided by the Schramm laboratory.

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of pathogens, such as expression of virulence, swarming motil-ity and biofilm formation [57]. Hence, bacteria will be more sus-ceptible to the host immune response or to treatment withtraditional antibacterial agents. Some selected compoundsseemed to be specific for antagonism of the interaction betweenLasR and 3-oxo-C12-HSL (LasR transcriptional activator thatcontrols virulence gene expression in P. aeruginosa).

Recently, Blackwell and McInnis selected a series of thiolac-tone compounds that can inhibit QS in various bacteria [58]. Inparticular, these compounds are effective for regulation of det-rimental bacterial biofilm formation and virulence productionthrough disturbing the interaction between AHL and LasR.For example, those compounds described in Table 3 contributeparticularly to disruption of bacterial QS and biofilm dis-ruption in E. coli, V. fischeri and/or A. tumefaciens. These

compounds have the potential to be put into the use of admin-istration in the pharmaceutically acceptable formation or act asprodrug derivatives.

It is important to note that their results suggest that thiolac-tone derivatives could remain biological activity for longertime than the natural lactone analogs, which is beneficialfor therapeutics.

There are three QS pathways (Scheme 5) in V. harveyi,mediating by AI-1, AI-2 and CAI-1, accordingly. AI-1, alsocalled 3-OH-C4-HSL (as shown in Figure 3), is an AHLtype AI and it is the strongest of the three V. harveyi signals.All three AIs in V. harveyi, including AI-1, are explored bymembrane-bound sensor-kinase proteins [39,59]. After releasedfrom V. harveyi, AI-1 stores up in the extracellular environ-ment, and subsequently interacts with LuxN to trigger the

ONH

O

O

ONH

O

O

ONH

O

OO

ONH

O

OCl

ONH

O

ON3

ONH

O

O

14

15

16

ONH

O

O

O

18

ONH

O

O

21

6

ONH

O

OO

F3CO19

ONH

O

O

Cl 20

ONH

O

O

NN

F3C

22

ONH

O

OO

O

O23

ONH

O

O

Br 17

13

12

11

ONH

S

O

O

O

ONH

S

O

O

O

ONH

S

O

O

O

24

25

26

Figure 6. Examples of compounds as potent inhibitors provided by the Blackwell laboratory.

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QS gene cascade. This mechanism greatly differs from the for-merly described LuxR-AHL signaling mechanism.Bassler and Swem identified 15 novel small molecules

(Figure 10) that are capable of disrupting AHL-mediatedQS [60]. The molecules identified as antagonists are phenox-yacetamides with no similarity in structure to AI-1 (3-OH-C4-HSL, Figure 3). These antagonists can be applied tomanipulate QS-induced activities to control bacteria behavior,by targeting AHL-type receptor LuxN, wherein the behavior ispathogenicity, type III secretion, bioluminescence, siderophoreproduction or metalloprotease production.These molecules are the first antagonists that aim at an

AHL membrane-bound sensor kinase. Some examples ofantagonists are depicted in Figure 11.Moreover, the Bassler laboratory has synthesized a novel

series of small molecules that could inhibit two classes ofAHL receptors: the membrane-bound sensor kinase, LuxNand the cytoplasmic transcriptional regulator, CviR [61]. Typ-ically, LuxN binds the strongest AI AI-1 (3-OH-C4-HSL) inV. harveyi, and another AI C6-HSL is detected by LuxR-type receptor CviR from Chromobacterium violaceum. It isimportant to note that LuxN-type and LuxR-type receptorshave no evident sequence homology and their signal transduc-tion mechanisms are also different though both receptors bindAHLs with similar structures. Bassler’s results indicate thatthere are some similarities in AHL-binding pockets betweenLuxN-type and LuxR-type receptors, which lead to the con-clusion that LuxN-type receptor antagonists would reasonablyalso inhibit a LuxR-type receptor. In their further studies, theyexplored the antagonism function of these compounds. Themechanism of CviR inhibition is involved in the breaking ofDNA binding or the interference of the interactions withRNA polymerase. As for LuxN, the antagonists competewith the native signaling molecules for binding to the

periplasmic domain of LuxN to prevent LuxN from trans-forming from kinase-mode into phosphatase-mode.

The most potent molecules 74 -- 76 are displayedin Figure 12. These brand compounds can act as promisingantagonists to interfere QS controlled activities such as viru-lence factor and problematic bacterial biofilm, which arebeneficial for developing new antimicrobial therapeutics.

2.2.5 Other classes of inhibitorsSuga recently found that a specific pyrimidinone compound isable to inhibit the QS of specific bacteria [62]. The general for-mula of a QS inhibitor comprising a pyrimidinone com-pound as shown in Figure 13, wherein R1 represents aC1-5 alkyl group or a phenyl group and R2 represents ahydrogen atom or a C1-5 alkyl group, provided that whenR2 represents a hydrogen atom, R1 cannot be a methyl group.Accordingly, the pyrimidinone compound inhibits the toxinproduction, disrupts the biofilm formation and strips offand removes an already formed biofilm, to control diseasecaused by bacteria that belong to the Erwinia species, particu-larly effective for controlling Burkholderia glumae. Further-more, the pyrimidinone compound is suitably used as abacterial disease control agent for agriculture and horticulture,due to its excellent control activity against intractable bacterialplant diseases. For example, the compound 77 remarkablyinhibits the production of the QS signal molecules, C6-HSLand C8-HSL that belong to AHLs family, as well as the pro-duction amount of toxin by increasing the concentration ofcompound 77 in B. glumae. Moreover, the results indicatedthat the compound 78 can interfere the deposit of biofilms,or remove deposited biofilm in B. glumae. However, the pyr-imidinone compounds 77 -- 82 can kill almost all B. glumaeafter 36 h of cultivation, exhibiting a significant effect onthe survival of B. glumae. It is controversial that thesecompounds could be appropriate QS inhibitors.

Mathee et al. provided several methods of inhibiting QS inpathogenic bacteria using ellagitannins as inhibitors of bacte-rial QS [35]. It has been previously reported that Conocarpuserectus, a medicinal plant, was useful in restraining the QS sys-tem of P. aeruginosa with unknown active components [63-65].Their patent data presented that identified two hydrolysabletannins, vescalagin and castalagin (Figure 14), to be responsiblefor anti-QS activity in C. erectus.

Bioassays include effect of crude extract of C. erectus (Com-bretaceae) and purified ellagitannins on AHL production,QS-related genes expression and virulence factors production,respectively. Compared with control, there was 91% decreasein LasA activity when the P. aeruginosa PAO1 was incubatedwith C. erectus crude extract at 1 mg/ml. Vescalagin and cas-talagin, two purified ellagitannins, led to 73 and 80% reduc-tion in LasA activity, respectively. LasB elastase activity wasalso effectively inhibited in the presence of C. erectus crudeextract (40 µg/ml) and purified vescalagin and castalaginwith the decrease of 60, 67 and 63%. Moreover, there were20 and 21% reductions in C4-AHL levels when the strain

Analogs of AHL-2

A quorum-sensing molecule-antibiotic conjugate

NN

N

O

O O

F

O

NHS

O

30

O

O

NH

Se

S

O

NH

S

S

O

NH

O

27 28 29

Figure 7. Synthetic analogs of AHL-2 that reduce bacterial

pathogenicity and one non-limiting example of a QS

molecule--antibiotic conjugate provided by the Iyer

laboratory.

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was grown in the presence of C. erectus crude extract (1 mg/ml)and purified vescalagin. Significant reductions were observedin the activity of some or all tested QS genes (lasI, lasR, rhlIand rhlR) with the C. erectus crude extract or purifiedellagitannins. In addition, the C. erectus crude extract and puri-fied ellagitannins did not affect the bacteria growth. Theresults indicated that both the crude extract and the purifiedcompounds are able to inhibit QS though the mode of actionis still unclear.

Duan et al. discovered that diphylloside A and its deriva-tives have the ability to reduce the pathogenicity of the path-ogenic bacteria through disrupting the QS system withoutgrowth inhibition [66]. They are selected by a QS-related anti-bacterial drug screening system composing P. aeruginosa.Diphylloside A (Figure 15) and its derivatives belong to com-ponents of flavonoids that derived from epimedium Chineseherbal medicines. When the concentration of diphylloside Areached the 40 µg/mL, this compound led to inhibition ofLasI/R QS system. Besides, the optimal inhibition concentra-tion was 166.67 µg/mL. Moreover, diphylloside A and itsderivatives, thereof, are easy extracted and prepared from theraw materials. The diphylloside A and the derivatives could

be taken as lead compounds of antibacterial drugs, whichthereby promote the development of novel antibacterial drugsand their application in clinical and agricultural production.

Yu et al. recently separated and extracted a monomericcompound (87, Figure 16) from marine Penicillium sp.QY013 [67] and another compound (88, Figure 16) from thefusarium QY010 [68]. Both of them were evaluated by takingadvantage of QSIS2 selector which was constructed byRasmussen et al. [69]. These compounds could only inhibitQS-dependent pathogenic behaviors without influencing thegrowth of bacteria, which lead to remote possibility of drugresistance of the pathogenic bacteria. For example, they dis-rupt the QS-system regulated pathogenicity of P. aeruginosaand Chromobacterium, which can markedly reduce produc-tion of virulence factors of the P. aeruginosa and yield of themycetin. There were 75.9 and 97.8% decrease in mycetinproduction when C. violaceum CV026 was grown with com-pound 88 at 200 and 300 mg/ml, respectively. Compound 88

also caused 40.8 and 66.5% reduction in elastase level whenthe concentration reached 300 and 500 mg/ml in P. aerugi-nosa PAO1 respectively. There were 20.3, 56.5 and 91.4%reduction in elastase production when P. aeruginosa PAO1

O

O

NH

N

OO

C nS

Isothiocyanates31 n = 732 n = 833 n = 9

O

O

NH

HN

OO

nBr

O

Bromoacetamides34 n = 735 n = 836 n = 9

O

O

NH

HN

OO

nCl

O

Chloroacetamindes37 n = 738 n = 839 n = 9

Figure 8. Nine synthetic analogs classified as isothiocyanates, bromoacetamides or chloroacetamides provided by

Meijler et al.

N

O

O

N+N+

OO–

SO

OHN OH

Cl

O

–OHN

Br

O

NHN

O

O

NH

SH2N

O

OHO Cl

NH SO

NH2

O

OO

NH

O

SO

NH2

N

O

O

O

F N OH

N OH

O

O

Br

40 41 42

43 44 45

46 47 48

Figure 9. Chemical structures of AHL QS antagonists selected from the Handelsman laboratory.

QS inhibitors

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was grown with compound 87 at 250, 500 and 1,000 µg/mL,respectively. Compound 87 also had the ability to disruptthe generation of mycetin in C. violaceum. Moreover, 27.5,44.6 and 87.2% of biofilms could be cleared when thePAO1 strain was incubated with compound 87 at 250,500 and 1,000 µg/mL, respectively.In addition, Gong et al. obtained a compound (89, Figure 17)

by fermenting the marine penicillium [70]. The compound caninhibit the QS of P. aeruginosa, in which such an inhibitioneffect is gradually enhanced by the increase of the concentra-tion. Compound 89 was estimated by use of QSIS2 selector [69].

There was decrease in expression of QS-related gene (lasB, lasI,lasR, rhlI rhlR and toxA) when PAO1 strain was grown in thepresence of compound 89 (50 mg/ml).

The raw materials of the three compounds are easy to obtainand the preparation of them are easy to control. The com-pounds and derivatives can be put into antibacterial use, makingmuch sense to human health and agricultural production.

Steggles described that the antibacterial compositions,including a QS inhibitor, can be used to treat multispeciesinfections, preferably dental diseases, including gingivitis, peri-odontitis, pericoronitis, peri-implantitis and inflammation [71].

Table 3. Examples of most active thiolactone compounds and their IC50, which are selected by Blackwell et al.

Compound E. coli

DH5a

IC50 (mM)

V. fischeri

ESI 114

IC50 (mM)

A. tumefaciens

WCF

IC50 (mM)

49

S

HN

O

O O8

- 0.45 1.8

50

S

HN

O

OBr

0.40 0.77 -

51

S

HN

O

OO

7.2 - -

52

S

HN

O

O

2.5 - -

53

S

HN

O

O

2.9 0.35 -

54

S

HN

O

O O4

- 0.35 -

55

S

HN

O

O6

0.14 0.13 2.8

56

S

HN

O

O5

0.79 0.31 10

57

S

HN

O

O4

1.1 0.84 -

58

S

HN

O

O O

- - 3.2

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The QS inhibitor involved in the compositions is an extract ofgarlic, containing S-allyl cysteine that may block the AHL sig-naling pathway so as to interfere the biofilm formation. Fur-thermore, 83 patients who had advanced periodontal disease,and who had been unsuccessfully treated with conventionaltreatment, were treated with the same kind of the composition.The result shows that 54% of them excellently responded tothe treatment with stable full mouth healing. Previously,Bjarnsholt et al. described a single bacterial infection can betreated with very large doses of garlic extract [72]. However,most preferably composition mixtures described in the inven-tion can be made with about 2 mg of the garlic extract andabout 250 mg of the antibiotic, which can treat more complexinfections and make it more practical for real treatment.

Ren and Pan recently found that brominated furanones(Figure 18), which are known as QS inhibitors, can be usedto revert the antibiotic tolerance of persister cells and enhancetheir susceptibility to antibiotics by modulating transcriptionof specific membrane genes [73].

They also provide a method for eliminating a bacterialinfection including bacterial persister cells. First, a bromi-nated furanone is administered to the bacterial infection, inwhich the compound reverts the antibiotic tolerance of thebacterial persister cells. And second, one or more antimicro-bial agents are administered to the bacterial infection. Theantimicrobial agents would be more effective since the bromi-nated furanone has enhanced the persister cells’ susceptibility.(The antimicrobial agent is chosen from the group including ab-lactam, an aminoglycoside, a quinolone, a tetracycline and acephalosporin.)

Persister cells are non-growing dormant cells in the pres-ence of antibiotics. However, persister cells resume growthwhen the antibiotic treatment is stopped, which result indrug tolerance. It has been recently demonstrated that QSpromotes persister formation in P. aeruginosa PAO1. If weperturb the relative QS system with QS inhibitors, the per-sister formation would be controlled and their susceptibilityto antibiotics would be restored. Compound 90 is the firstnon-metabolite small molecule to be capable of reverting anti-biotic tolerance of persister cells, which suggests that other QSinhibitors may also make contribution to solve the problem ofantibiotic tolerance.

Scheie et al. synthesized a series of thiophenone compoundsand some of them showed the capability of inhibiting AI-1 andAI-2 QS microbial communication and disrupting biofilm for-mation [74]. V. harveyi BB886 (AI-1 reporter) and V. harveyiBB170 (AI-2 reporter) were employed, respectively, in theAI-1 and AI-2 QS inhibition assessments that were measuredby inhibition of bioluminescence. The results indicated thatthe thiophenone compound displayed strong inhibition towardQS. (Light units of thiophenone compound is about 13, fura-none is about 500 and control is about 66,000 in AI-1 QS inhi-bition, while the light units of thiophenone is about 3,furanone is about 1,600 and control is about 7,500 inAI-2 QS inhibition.) Moreover, some thiophenones exhibitedgood inhibitory activity against biofilm formation by variousbacteria such as Staphylococcus epidermidis, Enterococcus faeca-lis, V. harveyi and Pseudoalteromonas. For example, after incu-bation with thiophenones (Figure 19), the biofilm reductionwas > 50% in static biofilm model or shaking biofilm model.

LuxP

AI-2

AI-2

LuxULuxU

LuxO LuxO

P

P

LuxR

LuxR

Periplasm

Inner membrane

LuxQ

LuxP

LuxULuxU

LuxO LuxO

P

P

LuxQ

+

sRNAs

+ Hfq

sRNAs

Low cell population density High cell population density

LuxN LuxNCqsS CqsS

AI-1

CAI-1AI-1

CAI-1

BioluminescenceMetalloproteaseSiderophoreExopolysaccharideOther genes

Outer membrane

Periplasm

Inner membrane

Outer membrane

δ54

Scheme 5. QS pathways in V. harveyi. This scheme was adapted to the Milton’s review [82].

QS inhibitors

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In their opinions, thiophenone compounds interfere the AI-1or AI-2 QS so as to inhibit biofilm formation. And the thio-phenone is more effective than furanone which is known asQS inhibitor. Compounds 102 -- 105 performed strong inhibi-tion on biofilm formation of V. harveyi (the biofilm reductionwere 70% at 50 µM, 95% at 15 µM, 70% at 50 µM and90% at 25 µM, respectively). However, the four compoundsalso showed planktonic bacterial growth inhibition.The Gerwick laboratory found that the Honaucin A com-

pounds, and variants and analogs (Figure 20), thereof, can beused as bacterial QS inhibitors [75]. They tested the com-pounds of Honaucin A family with V. harveyi BB120, thebioluminescent strain and E. coli JB 525 that produces an

unstable green fluorescent protein in response to C6 -- C8

AHLs [76-78]. Compounds 106 -- 114 have the ability to signif-icantly inhibit QS in V. harveyi with IC50 < 30 µM. And someof them also show QS inhibition in E. coli.

It’s no doubt that the QS system plays a significant role inregulating the biofilm formation that makes a contributionto the antibiotic tolerance. Recently, Ammendola et al.synthesized a series of compounds that could block QS-dependant biofilm formation of several pathogens, includingGram-positive and Gram-negative bacteria, such as P. aeru-ginosa, S. aureus and S. epidermidis, more preferablymethicillin-resistant S. aureus strains, without inhibitingbacterial growth [79]. Some effective compounds (Figure 21)

SNH

O

O S

O

OS

O

O

Cl

O

ClN

NH

Cl

NSN

SNH

O

O

O

NH

FFF N

H

SO

NN

N

N

HN

S

O

O

ONH

OHO

N N

S

OO

NH

NH

Cl

OHN

O

HN

ClO

S

O

SO

O HN

O

NH

N

S

O

72

Cl

HN O

NH

SO

ON

O

N

O

S

N

N

O

NH

O

NHS

O

O

S

N

59 6160

62 63 64

65 66

68

67

69

73

7071

Figure 10. Fifteen molecules that are identified can act as AHL-mediated QS inhibitors.

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to inhibit biofilm formation of P. aeruginosa PAO1 at acompound concentration of 100 µM are exhibited inFigure 21 (> 50%).

Kyd and Cooley provided immunogenic conjugate mole-cules as a therapeutic or prophylactic vaccine comprising anAHL and catalase to treat P. aeruginosa infections by inhib-iting a chain of QS regulatory virulence factors and biofilmsformation [80]. Preferably the AHL is N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL, also called 3-oxo-C12-HSL,as shown in Figure 3) or butyryl L-homoserine lactone (BHL,also called C4-HSL, as shown in Figure 3) and the catalase isthe protein KatA from P. aeruginosa. The immunogenicAHL--catalase conjugation could induce an immuneresponse to AHL to limit the biofilms formation of P.

aeruginosa, and meanwhile induce an immune response tothe conjugated protein KatA to weaken the bacterial catalasefunctions, which would make P. aeruginosa more susceptibleto host immune defenses. In their human immunity experi-ments, 10 of the 36 patients with cystic fibrosis (27.8%) and5 of 9 healthy adult controls exhibited antibody reactivity toOdDHL. The results suggest that the vaccine construct willinduce an antibody response, which is useful for treatmentof P. aeruginosa infections.

A number of QS blocking compounds were utilized fortreatment of diseases of compromised skin integrity [81]. It iseasy for human pathogens, such as P. aeruginosa, to colonizein the area of compromised skin integrity and result in infec-tion. A porcine model was employed in the assessment of

Cl

HN O

NH

SO

ON

O

73IC50 = 2.7 μM

71IC50 = 2.9 μM

61IC50 = 2.6 μM

68IC50 = 2.4 μM

64IC50 = 6.23 μM

O

HN

O

HNS

O

O

S

N

SO

O

Cl

O

Cl

OO

NH

N

HN

S

O

O

Figure 11. Structures and IC50 values of five molecules that inhibit LuxN-type receptor (V. harveyi strain JMH624 was

employed in identification of LuxN antagonists), provided by the Bassler laboratory.

IC50 = 302 μMIC50 = 3.0 μMIC50 = 1.2 μM

IC50 = 28 μMIC50 = 150 μM

IC50 = 6.0 μMIC50 = 723 nM

IC50 = 14 μMIC50 = 40 μM

IC50 = 873 μMIC50 = 295 nM

OO

HN

S

O

OO

HN

S

O

Cl Cl Cl

OO

HN

O

O

74 75 76

V. harveyi BB120 −V. harveyi BB960V. harveyi JMH624C. violaceum wild type

Figure 12. Structures and IC50 values of the three potent compounds which are capable of inhibiting both LuxN- and LuxR-

type receptors, discovered by the Bassler laboratory.

QS inhibitors

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compositions that the invention provided on P. aeruginosa-infected bums. The compositions comprising compoundsthat could inhibit AHL-mediated QS were capable of reduc-ing bacterial colonization and disrupting the biofilm forma-tion in areas of compromised skin integrity without toxic

effect. Examples of QS blocking compounds are depictedin Figure 22.

2.3 Interference in AI-2-mediated QS2.3.1 AI-2 QS pathwayIt has been proposed that AI-2 QS systems exist in bothGram-positive and Gram-negative strains, while AI-2 isreferred to as a universal AI that mediates intraspecies andinterspecies communication [82-91]. And indeed, the synthasefor the AI-2 precursor DPD, designated LuxS, has sincebeen found in a wide variety of bacteria [12,92]. Some examplesof species of bacteria-possessed LuxS are shown in Table 4

[27,47,93]. And more and more species will join the list of bac-teria with the further study of AI-2 QS. Moreover, fromanother point of view, the LuxS and AI-2 also make contribu-tions to bacterial metabolism, which influences bacterial fit-ness to some extent [94]. The AI-2 system is known to

N

NOH

O

R2

R1

N

NOH

O

N

NOH

O

N

NOH

O

N

NOH

O

N

NOH

O

N

NOH

O

N

NOH

O

77 78 79

8380 81 82

The general structure of a pyrimidinone compound

Figure 13. General formula of a pyrimidinone compound and examples of active pyrimidinone compounds, discovered by

Suga et al.

HO

HO

OH

OH

O OO O

O

O

OO

OOHO

HO

OHHOOH

OH

OH

OH

OH

HOH

OHHO HO

HO

OH

OH

O OO O

O

O

OO

OOHO

HOOHHO

OH

OH

OH

OH

OH

OHH

OH HO

84Castalagin

85Vescalagin

Figure 14. Chemical structures of castalagin and vescalagin that were identified by the Mathee laboratory.

OR2O

OH O

OR3

OR1

86

Figure 15. General structure of diphylloside A, provided by

the Duan laboratory.

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manipulate a range of microbial processes, including viru-lence, biofilm formation, cell motility, bacterial conjugationand bioluminescence [12,94-97].

AI-2 is synthesized starting from SAM, which yieldsSAH by transmethylation. Then, SAH is hydrolyzed toSRH and adenine by SAHN or MTAN. SRH is transformedto L-homocysteine (Hcy) and DPD through the reaction cat-alyzed by LuxS. The DPD is the precursor of AI-2 [88,98,99]. Ithas been found that the AI-2 precursor DPD can spontane-ously rearrange to form a variety of DPD derivatives thatinterconvert and exist in equilibrium, which is known as theAI-2 pool. Cyclization of DPD generates a couple of biologi-cally active AI-2 forms; for instance, DPD will be convertedinto a hydrated form R-THMF, the AI-2 signal in Salmonella

typhimurium [100]. Moreover, the other hydration of DPD,S-DHMF is consequently incorporated with boric acid to bringabout AI-2 signal molecule, S-THMF-borate, in V. harveyi [99].It is probably that bacteria could answer to their ownAI-2 and also to AI-2 created by other species of bacteriadue to the interconversion of AI-2 molecules [101]. Becausethe chemical nature of the signaling molecule varies betweenspecies, there is no doubt that the nature of the AI-2 receptorfor these signals is also variable. Till date, three types of recep-tors that respond to AI-2 have been characterized [45]. In 2002,the crystal structure of the receptor protein involved inAI-2 signaling in V. harveyi, LuxP, has been characterized,forming complex with its ligand determined as S-THMF-borate, which plays a significant role in bioluminescence [99].However, in the bacteria S. typhimurium, the protein LsrB isthe AI-2 signaling molecule-binding protein and the relevantAI-2 molecule is R-THMF [102]. In 2006, the AI-2 receptor,RbsB, was discovered in the bacterium Actinobacillus actinomy-cetemcomitans, though the active AI-2 signaling molecule hasnot been identified (Scheme 6) [103].

We discuss the LuxP-based system in V. harveyi as the typ-ical example of the AI-2 QS pathway. However, in fact, thebioluminescence in V. harveyi is actually managed by the mul-tichannel QS system [104]. One of them is the AI-2 pathway,the other is a Gram-negative-like system that utilizes anAHL (3-OH-C4-HSL) as the AI that binds to the LuxNreceptor. In addition, there is a third AI CAI-1 in V. harveyi,although the mechanism of its action is less well defined [8].

In bacterium V. harveyi, the respond to AI-2 needs twoproteins, LuxP, a periplasmic-binding protein, and LuxQ, atwo-component hybrid sensor kinase [99]. LuxP andLuxQ exist in a complex known as LuxPQ [105]. As the con-centration of AI-2 arrives at a threshold level, AI-2 binds toLuxP and alters the activity of the LuxPQ binders to initiatea transition from kinase to phosphatase, which leads to thedephosphorylation of the downstream proteins LuxU andLuxO. Dephosphorylated LuxO becomes inactive and does notarouse the production of small regulatory RNAs, ultimately lead-ing to the production of LuxR, the QS RR that regulates tran-scription of target genes, including a few virulence genes [105].

Moreover, two strains of V. harveyi, BB170 and MM32,are widely used in the identification of QSIs due to emittingluminescence in response to AI-2 stimulation. V. harveyiBB170 lacks the native LuxN receptor but contains theLuxP receptor to detect AI-2. V. harveyi MM32 (lacksLuxN and LuxS) has only the AI-2 passway without theability to sense AI-1. However, V. harveyi MM32 does notproduce AI-2 molecules itself.

2.3.2 Inhibitors that target AI-2 synthesisAs mentioned earlier, MTANs play a crucial role in the bio-synthesis of AI-1 and AI-2 in QS bacteria. Therefore,MTAN offers a means to block the biosynthesis of AI-2 soas to disrupt QS. The Schramm laboratory designed and syn-thesized two different types of transition state analogs as

O

O

NO

HO

87 88

Figure 16. The two compounds that can act as potential QS

inhibitors separated by the Yu laboratory.

HO

OOH

OHO

H

H

89

Figure 17. Compound that can inhibit QS in P. aeruginosa

separated by Gong et al.

O

Br

OBr

OOBr

Br

OOBr

Br

BrBr

OOBr

Br

Br

OOBr

Br

Br

90 91 92

93 94

Figure 18. Examples of brominated furanones provided by

Ren et al.

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mentioned above [46]: 5¢-thio-substituted immucillin and 5¢-thio-substituted DADMe-immucillin. Some of them, 5, 6

and 7 (Figure 4) are capable of inhibiting AI-2 QS.Recently, the Jones laboratory has took advantage of furanone

analogs to inhibit AI-2-mediated QS and hence to inhibitanthrax toxin production, especially the expression of protectiveantigen, and the growth of B. anthracis infection [106]. The struc-tures of furanones are shown in Figure 23. They also providedmethods for preventing B. anthracis infection by restraining theactivity of the B. anthracis LuxS polypeptide that synthesizes afunctional AI-2 molecule. The B. anthracis LuxS polypeptide isinhibited by mutating the luxS gene, which is replaced with anucleotide sequence conferring antibiotic resistance.

2.3.3 Inhibitors that target the AI-2 receptorWang et al. discovered compounds (Figure 24) comprising aboronic acid moiety, which would form a complex in bind-ing with LuxP and hence can competitively antagonizeAI-2-mediated QS through monitoring the inhibition ofluminescence production in V. harveyi MM32 strain [107].Furthermore, other structural scaffolds pyrogallol and pheno-thiazine (Figure 24) display especially potent inhibitory activi-ties after a random screening against AI-2-induced QS.Pyrogallol’s (compound 143) mechanism of action is possiblethrough its complexation with boric acids, mimicking theDPD--boric acid complex. And phenothiazine (compound 140)most likely realized its effect through direct binding to LuxP.

SO

Br

SO

Cl

SO

O O

SO

Br

Br

SO

Br

BrSO

Br

Br

Br

SO

Ph

Br

SO

Br

OO

OOH

SO

IS

O BrS

O

SN

95

102 103 104 105

96 97

98 99 100 101

Figure 19. Examples of thiophenones which could cause > 50% biofilm reduction.

OO

ClO

O

OO

O OH

OCl O

OCl

O OH

O

HOBr

HOI

O O

OO

Br

OO

OO

I

OO

OO

Cl

OO

O

OCl

O

106IC50: 5.6 μM

107IC50: 17.6 μM

108IC50: 14.6 μM

109IC50: 10.5 μM

110IC50: 1.2 μM

113IC50: 0.24 μM

114IC50: 0.13 μM

111IC50: 22.1 μM

112IC50: 15.9 μM

O

Figure 20. Examples of active Honaucin A compounds that can be used as QS inhibitors, provided by the Gerwick laboratory.

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The result suggested that the best active compounds in theboronic acid series are para-substituted phenylboronic acidswithout additional ionizable functional groups [108,109]. More-over, pyrogallols and phenothiazine appear to show best activityduring pyrogallols and analogs [108-110].

The AI-2 antagonists according to their disclosure havetherapeutic benefits because of their capability to suppressvirulence factors production, drug resistance and/or biofilmformation. These AI-2 antagonists may compete withAI-2 inducers such as DPD resulting in inhibition of QS.

NH

N

O O

NN

O O

N

N

O O

NN

O

N

O

FN

O

F

N

OF

N

O

F

F

NH

O

O OH

NH

O

NN

114 116

117118

119 120

121 122

123 124

Figure 21. Some examples of effective QS inhibitors provided by the Ammendola laboratory.

SClNH

HN

O

O

NN

NS

NH

OO

N

FOO

SNH

HN

O

O

NN

SNH

HN

O

O

S

SNH

HN

O

O

S

Br

125 126

127 128

129 130

Figure 22. Examples of quorum sensing blocking compounds that could be utilized for treatment of diseases of

compromised skin integrity.

Table 4. Some examples of LuxS-possessing bacteria species [47].

Species of bacteria

Gram-positive bacteria B. subtilis, B. anthracis, B. halodurans, B. burgdorferi, C. botulinum, C. perfringens, C. difficile,E. faecalis, L. monocytogenes, M. tuberculosis, S. aureus, S. pyogenes, S. pneumoniae

Gram-negative bacteria H. influenzae, N. meningitidis, V. cholera, V. harveyi, E. coli, S. typhimurium, Y. pestis, C. jejuni, H. pylori

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Otherwise, these AI-2 antagonists may be capable of probingbacterial AI-2 functions.The Li laboratory designed and synthesized two series of

triazole and furan derivatives that could target LuxPQ proteinso as to disturb native AI-2 molecule binding to the receptor,which interfered with the subsequent gene expression [111,112].They took full advantage of computer-aided drug design andthe structural information of LuxPQ. As abovementioned,LuxP and LuxQ are thought to associate to form LuxPQ com-plex, which causes a conformational change. Moreover,LuxPQ protein only exists in AI-2 QS bacteria and the corre-sponding homologous proteins are not present in the mam-mal. Hence, it is more appropriate and accurate to designAI-2 QS inhibitors based on LuxPQ protein than LuxPprotein. Some triazole and furan compounds with goodinhibitory activities are displayed in Figures 25 and 26.The inhibition of AI-2 QS of all synthesized compounds

was evaluated through screening the decrease in biolumines-cence in the MM32 strain of V. harveyi. The results of theirbiological test showed that most of these molecules performinhibitory activities. It is noted that inhibitory activities offuran compounds are better than triazole compounds, andcompound 157 exhibited the best AI-2 inhibition effect withIC50 value of 7.4 µM. These novel structural inhibitors, tria-zole and furan derivatives would be of great benefit to thedevelopment of new therapeutic antibacterial agents.

2.3.4 DPD analogsSintim et al. designed and synthesized a series of phosphory-lated and branched DPD analogs which could act as efficientQS inhibitors in bacteria, such as E. coli, S. typhimurium,V. harveyi, P. aeruginosa, etc. [113].

These compounds were evaluated for their QS modula-tion in E. coli and S. typhimurium. Both E. coli LW7 andS. typhimurium MET715, which produce b-galactosidasein response to AI-2, were employed in the bioassays. Com-pound 174 (isobutyl--DPD) was the most potent inhibitorwith IC50 value at the nanomolar range. Some examples,including the most effective compound isobutyl--DPD withIC50, are described in Figure 27.

In E. coli, AI-2 is transported into the cells by the ATP-binding cassette (ABC) transporter and then phosphorylatedby the processing enzyme, kinase LsrK. The transcriptionalregulator LsrR is released from the lsr operon after bindingto AI-2 phosphate, which could regulate the expression oftarget genes [114]. The AI-2 uptake mechanism is describedin Scheme 7.

They made a hypothesis that these DPD analogs may bephosphorylated by the kinase LsrK instead of native AI-2 signal-ing molecules and thus competitive bind to the LsrR transcrip-tional regulator. For the alkyl analogs there was at least threecarbon lengths for inhibiting LsrR protein. However, if thecyclic compounds are larger than cyclophenyl--DPD, they maynot be tolerated and phosphorylated by LsrK, leading to lessantagonism on LsrR.

In addition, compound 174 (isobutyl-DPD) was able tomarkedly promote the clearance of preformed E. coli biofilmsgrown in vitro, used in the combination with the antibioticgentamicin. Compound 170 (phenyl-DPD) was likewiseeffective in removing preexisting P. aeruginosa biofilms whenit was used with the antibiotic gentamicin. These promisingDPD analogs with the capability of modulating QS could bebeneficial for preventing or curing bacterial infections.

2.3.5 Other class inhibitorActinomyces naeslundii, one of the important Gram-positivebacteria colonizing the oral cavity [115,116], employ AI-2 as

O

BrH

BrO O

BrBr

BrO

O BrO O Br

O

Br

O BrO

Br

131 132

133 134 135

Figure 23. Furanone analogs that can inhibit AI-2 QS,

provided by the Jones laboratory.

HO

O

OOH

O

O

OHHOO

OHHO

OHHOH2O

H2O

O

OOH

HO

O

OH

HO

OHHO

O

O

HO

OHO B OHHO

–2H2O

+B(OH)4–4,5-dihydroxyl-2,3-

pentanedione(DPD)

(2R, 4S)-DHMF

(2S, 4S)-THMF(2S, 4S)-DHMF

R-THMF

S-THMF-borate

Scheme 6. Equilibrium of the biological forms of DPD. R-THMF is the AI-2 signal employed by S. typhimurium, S-THMF-borate

is the AI-2 signal employed by V. harveyi.

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B(OH)2 B(OH)2

B(OH)2

B(OH)2

O

O

O

F

NC

S

HN

HOOH

OH HOOH

OHOH

HO

HO

OH

139IC50: 5.7 ± 3.5 μM

142IC50: 61.3 ± 15.6 μM

147IC50: 3.2 ± 1.0 μM

146IC50: 4.0 ± 2.3 μM

145IC50: 2.9 ± 0.9 μM

144IC50: 3.7 ± 1.2 μM

143IC50: 2.0 ± 1.2 μM

141IC50: 47.1 ± 4.6 μM

140IC50: 10.7 ± 3.7 μM

138IC50: 3.6 ± 0.4 μM

137IC50: 4.1 ± 1.3 μM

136IC50: 4.8 ± 2.0 μM

Boronic acid compounds

Pyrogallols and analogues

Phenothiazine and analogues

S

NS

CH3

S

N

NHCl

OH

O

O2N NO2OH

O

OH

OHOH

N

N

Figure 24. Structures and IC50 values of examples which have QS inhibitory effects, discovered in the Wang laboratory.

149IC50: 65.1 μM

151IC50: 50.2 μM

153IC50: 62.2 μM

152IC50: 42.3 μM

154IC50: 30.0 μM

150IC50: 62.6 μM

148IC50: 49.5 μM

N NN

NH

N NN

NH

O F Cl

Cl

N NN

NH

F O

N NN

NH

Cl

Cl

N NN

NH

O

OO

N NN

NH

O O

N

N NN

NH

O

ON

Figure 25. Examples of triazole compounds with AI-2 QS inhibitory activities, provided by the Li laboratory.

QS inhibitors

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QS signaling molecule and therefore control the incorporationof foreign DNA, acid tolerance, virulence and biofilm forma-tion. It is important that A. naeslundii regulate their behaviorto adapt to environmental stimuli resulting in better

colonization on tooth surfaces. In fact, the AI-2 is producedby Streptococcus oralis that grow with A. naeslundii in oral pla-que, which suggest the interspecies signaling AI-2 play a keyrole in the formation of oral biofilms [92].

157IC50: 7.4 μM

160IC50: 19.0 μM

159IC50: 16.9 μM

158IC50: 13.0 μM

161IC50: 17.8 μM

164IC50: 20.9 μM

165IC50: 21.0 μM

162IC50: 20.6 μM

163IC50: 26.0 μM

156IC50: 13.0 μM

155IC50: 8.8 μM

OHN

O

OCl

ClO

HN

O

O

Cl OHN

OCl

N

OHN

OBr

N

OHN

Cl

Cl

OHN

Cl

Cl F

OHN

ClCl

OHN

Cl

Cl Cl

OHN

Cl

Cl

OHN

Cl

ClO

OHN

Br

O

Figure 26. Examples of furan compounds with AI-2 QS inhibitory activities, provided by the Li laboratory.

OHOH

O

OOH

OH

O

O

OHOH

O

O

OHOH

O

O

OHOH

O

OOH

OH

O

O

OHOH

O

O

OHOH

O

O

OHOH

O

O

E. coli LW7 IC50: 54 nM

IC50: 2000 nMS. typhimuriumMET715

166 167 168 169

170 171 172 173

174

Figure 27. Examples of DPD analogs and IC50 of compound 174, provided by Herman laboratory.

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Recently, Trivedi et al. provide some oral care composi-tions including carbonate compounds (Figure 28), which arecapable of inhibiting growth and formation of oral biofilmsthrough QS inhibition [117]. If the quantities of carbonate com-pounds are high enough, a therapeutically effective treatmentwould be provided. For example, these oral care compositionscan inhibit oral biofilm formation and attachment to solid sur-faces caused by A. naeslundii, one of the dental plaque-associatedbacteria, leading to some oral healthcare benefit.

3. Conclusion

The four main strategies have been explored for the develop-ment of QS inhibitors. First, it is interference with the AI syn-thase, another is inactivation of the signal molecule, the thirdis the antagonism of the receptor and the last is promotion ofimmune response to AI. In search of QS inhibitors, target onreceptors is a common approach because ligand binding is theprior condition of the succeeding QS procedure. Some of QSinhibitors discussed above, including synthetic compoundsand natural substances, act as antagonists to bind to the cor-relative receptors competitively. Furthermore, some of themcan inhibit more than one type of receptor so as to have theability to become broad-spectrum QS inhibitors.

The inhibitors of MTAN often draw our attention. Theevidence that MTAN are response for the biosynthesis oftwo types of AIs, AI-1 and AI-2, indicates that MTAN wouldbe a potential target for antibacterial drug design. In brief,when there is no signaling molecules production, there is noQS occurrence.

It is significant to note that some synthetic AI analogscan not only lead to QS inhibition but also be accommo-dated in the targeted delivery of antibiotics with covalentlyor noncovalently linked to a suitable antibiotic. This phe-nomenon provides another method to reduce bacterialpathogenicity.

O O

O OH

O O OH

O

O OOH

O

175

176

177

Figure 28. Structural information for a set of three

compounds containing a carbonate moiety, provided by

the Trivedi et al.

LsrC

LsrDLsrB

LsrA

LsrA

AI-2

AI-2 AI-2

LsrK

ATP ADP

P

LsrR

lsr A lsr C lsr D lsr B lsr F lsr Glsr RlsrK

LuxS AI-2

AI-2

AI-2

AI-2

AI-2

AI-2

AI-2

AI-2

AI-2 AI-2

AI-2 AI-2

AI-2

Scheme 7. Transport and processing of AI-2 in E. coli. This scheme was duplicated from Pereira’s review [114]. AI-2 accumulates

in the extracellular surroundings. The signaling molecule is recognized by LsrB and then internalized by the ABC-

like transporter. Afterward, AI-2 is phosphorylated by the processing enzyme, kinase LsrK. The negative regulator LsrR is

released from the lsr operon after binding to the phosphorylated AI-2, which results in the depression of lsr expression.

QS inhibitors

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As described at the outset, lots of compounds provided bydisclosure can be used in the conjunction with antibacterialcompositions, which may be beneficial for treatment of bacte-rial infection. Furthermore, one of the inventions provides anti-bodies that specifically bind with AIPs in order to inhibit QS inmammals. Another invention provides a method of vaccineconstruct to induce an immune response to AHL to limit thepathogenicity of bacteria. Such immunological approach maybecome a useful tool to cure bacterial infections.The reason why QS draws our attention is that QS play a

crucial part in regulation of bacterial virulence, drug resistanceand biofilm formation, which can be clinically significantbecause these problems seem to be hard to resolve with cur-rently available antibiotics [47]. However, we should graspmore knowledge of the mechanism of QS inhibition so as tofind powerful QS inhibitors.As we discussed the patents published from 2009 to

2012 above, we found that most of them provide inhibitorsthat disturb the AHL-mediated QS perhaps because of AHLs’curial role in the pathogenicity of bacteria to some extent.However, AI-2-mediated QS is especially important

because it is functional in both Gram-positive and Gram-negative bacteria. AI-2 signaling operates within a range ofbacterial species. More attention is paid to the developmentof AI-2 inhibitors, which are likely to achieve broad-spectrummodulation of QS-controlled phenotypes. Therefore, we areespecially interested in searching for active, specific andbroad-spectrum AI-2 inhibitors with a deeper understandingof the molecular basis behind AI-2 QS.

4. Expert opinion

Since the discovery of the first QS systems, QS research hasattracted significant interest because of the potential to manipu-late bacterial virulence and other pathogenetic properties. Weare intrigued by the prospect that QS inhibitors interfere theQS system without impacting cell growth or killing the celland, therefore, leading to no or less promotion to drug resis-tance, which is beneficial for the development of antimicrobials.Chemists have been exploring QS antagonists ranging from

natural products to the high-throughput screening of diversechemical libraries. And many compounds are selected to actas inhibitors with good activity and stability at nanomolarlevel, showing their potential to manipulate bacterialvirulence and pathogenicity.The rapid advancement in our knowledge of the character and

extent of intercellular QS signaling in bacteria indicated that QSinhibitors have many advantages over conventional antimicrobialagents. They do not kill bacteria directly, which results in lessgrowth pressure for bacteria to promote their evolution. In addi-tion, an immunological approach such as vaccine developmentof QS AIs can be and have been applied to clinical therapy.Though lots of inhibitors display markedly inhibition of

QS in biological activity experiments, it is uncertain that QSinhibitors can become effective antibacterial agents in human

beings. There are a lot of difficulties that need to be overcomein the development of QS inhibitors. Some QS studies indi-cate that QS inhibitors may suppress future biofilm formationwithout influence on already formed/existing biofilm and sodoes virulence factor secretion. Furthermore, the mechanismof action of some QS inhibitors is still not clear. And a fewof QS inhibitors may have potential toxicity toward host cells.For instance, the electrophilic substances, furanones mightreact with proteins to form covalent bonds via a Michaeladdition, thus resulting in cytotoxicity [118,119].

It is worth noting that AI-2-mediated QS has gained moreattention in recent years due to modulation of interspecies com-munication in a wide range of bacteria. It has been obtained thatsome native and synthesized molecules can act as AI-2 QSinhibitors together with valuable SAR data. The LsrB-based sys-tem of S. typhimurium and the LuxP-based system of V. harveyiare the typical and only well-defined AI-2 QS pathways [45].Thus, there is a definite need to understand the functional rolesof AI-2 signaling pathway in other bacterial species especiallypathogenic bacteria. We expect that more active or broad-spectrum AI-2 QS inhibitors would be discovered with thefurther studies of the molecular basis behind AI-2 QS.

Various QS pathways constitute microorganism communi-cation network but exact functional roles of them are not veryclear. And it should be mentioned that several classes of QSmolecules exist in bacteria. If we want to develop broad-spectrum antimicrobial agents by the approach of inhibitionof QS, more work is required to be done to grasp the detailedmolecular basis of QS manipulation in order to fully under-stand the microbial communication network. A large amountof structurally diverse agonists and antagonists that have beendiscovered to date can be used as chemical tools to study thisform of communication. To some extent, such informationwould also contribute to the rational de novo design of new anti-microbial agents with improved efficacy and selectivity. Accord-ingly, it will make contribution to the practical therapeutics, ifwe grasp the knowledge of intertwining relationships amongall kinds of QS pathways, including their biological meaning,compensatory mechanisms and cause--consequence relation-ships [27]. In conclusion, QS is a suitable target for antimicrobialtherapies and QS inhibitors are likely to lead to a renaissance ofantimicrobials without tolerance, which is the ultimate goalexpected to achieve in this field.

Declaration of interest

This project was funded by the National Natural ScienceFoundation of China (No 81001362), the PhD ProgramFoundation of Ministry of Education of China (No20090131120080), the Doctoral Fund of Shandong Province(No BS2009SW011), the Shandong Natural Science Founda-tion (No JQ201019) and the Independent Innovation Foun-dation of Shandong University, IIFSDU (No 2010JQ005).The authors state no other conflicts of interest.

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BibliographyPapers of special note have been highlighted as

either of interest (�) or of considerable interest(��) to readers.

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2. Atkinson S, Williams P. Quorum sensing

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3. Ng W-L, Bassler BL. Bacterial

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Ann Rev Genet 2009;43:197-222. An interesting report that discusses the

fundamental mechanisms involved in

V. harveyi and V. cholerae QS systems.

4. Waters CM, Bassler BL. Quorum

sensing: cell-to-cell communication in

bacteria. Annu Rev Cell Dev Biol

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bioluminescence. Annu Rev Microbiol

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6. Fuqua WC, Winans SC, Greenberg EP.

Quorum sensing in bacteria: the

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V. fischeri.

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AffiliationTianyu Jiang & Minyong Li†

†Author for correspondence

Shandong University,

School of Pharmacy,

Department of Medicinal Chemistry,

Key Laboratory of Chemical Biology of

Natural Products (MOE),

Jinan, Shandong 250012, China

Fax: +86 531 8838 2076;

E-mail: mli@sdu.edu.cn

T. Jiang & M. Li

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