Small molecules as regulators of bacterial Quorum … · therapeutic potential The objective of this chapter is to show and discuss the ... attractive targets for developing new antibacterial

Small molecules as regulators of bacterial Quorum Sensing. New strategy

in the development of antimicrobial agents

Carlos Mario Meléndez Gómez1 and Vladimir V. Kouznetsov

2*

1 Grupo de Investigación en Química Orgánica y Biomédica, Programa de Química, Facultad de Ciencias Básicas,

Universidad del Atlántico, A.A.1890, Barranquilla, Colombia 2 Laboratorio de Química Orgánica y Biomolecular, CMN, Universidad Industrial de Santander, Parque Tecnológico

Guatiguara, Km 2 vía refugio, Piedecuesta, A.A. 681011, Colombia

The cell-to-cell communication process (quorum sensing, QS) is an important signaling phenomenon used by bacteria and

relies on small, secreted signaling molecules. Because bacterial QS circuits regulates the expression of virulence genes

through intercellular communication, any mode of their disruption is emerged as an anti-virulence strategy with enormous

therapeutic potential The objective of this chapter is to show and discuss the advances in the bacterial QS networks,

evaluating this complex process through an analysis of the structure and function of natural and synthetic signal molecules

(autoinducers), the molecular diversification tactics and strategies and structure-activity relationship (SAR), as well as the

chemical methods controlling the regulatory activities. Discussion is divided into diverse parts: 1) Bacterial QS and the

development of new antimicrobial agents; 2) Backgrounds. Signaling molecules in the regulations of bacterial QS

network; 3) Enzymatic autoinducer inactivation as a strategy in the modulation of bacterial quorum sensing; 4) Molecular

modifications of signal molecules for LasR/RhlR regulatory activity; 5) Perspectives and conclusions.

Keywords: Quorum sensing; autoinducers; quorum quenching; small molecules; structural diversification

1. Bacterial QS and the development of new antimicrobial agents

Development of antibiotic resistance in pathogenic microorganisms is an ongoing public health threat. Emerging drug

resistance problem, which conducts to global economic and healthcare crisis, is a major and serious task of chemistry,

biology and medicine [1]. Among various approaches that can be undertaken in order to better control the emergence

and spread of drug-resistant pathogenic microorganisms, quorum sensing (QS) stands out relatively new attractive tactic

of combat against this problem. QS is a widespread phenomenon of cell-to-cell communication in several

microorganisms that is associated with the coordination of the expression of beneficial phenotypes, regulation of local

population densities and multiple virulence factors [2]. This coordination consists of the producing, releasing and

detecting small diffusible signaling molecules known as “autoinducers” (AIs). At a threshold signal concentration and

population, these molecules bind to a receptor protein and initiate changes in gene expression.

This mechanism was first discovered in 1970 during a bioluminescence study using Vibrio fischeri, a marine

bacterium associated with Hawaiian squid [3]. In that time, it was thought to be restricted to only a limited species.

Later on, extensive studies in this area have been performed with pathogenic bacteria. Nowadays, similar systems are

recognized in many organisms, including animal and plant pathogens. Over 100 species of bacteria are identified to

produce AIs in a cell density dependent manner similar to V. fischeri. It is well known that while controlling

synchronously the cell-to-cell communication process, Gram-negative, opportunistic pathogens as Bacillus subtilis,

Pseudomonas aeruginosa, Staphylococcus aureus and others can regulate biofilm formation, group motility, an arsenal

of excreted virulence factors and initiate chronic and severe infections. Several lines of evidence indicate that QS

enhances virulence of bacterial pathogens in animal models as well as in human infections hence, due to their critical

role in regulating virulence, disruption of bacterial QS is considered as a new, perspective anti-infective strategy.

Noteworthy that in contrast to traditional bacteriocidal or bacteriostatic antibiotics, disrupting QS does not cause

lethality but rather inhibits pathogen virulence.

Functional QS circuits are particularly attractive therapeutic targets for the development of new antibacterial agents,

as well as a starting point for new biochemical investigations on bacterial interactions. Actually, there is renewed

interest in drugs, which attenuate virulence rather than bacterial growth. Thus, some recent progress in exploiting this

information through the design of anti-virulence deception strategies that disrupt QS through signal molecule

inactivation, inhibition of signal molecule biosynthesis or the blockade of signal transduction are very significant and

vital to resolve drug resistance problem [4,5].

2. Backgrounds. Signaling molecules in the regulations of bacterial QS network

Many of bacterial pathogens are found to control virulence factor expression by a cell-to-cell communication system, in

which a signal molecule is generated and secreted into the surrounding environment. While the bacterial population

grows, the concentration of the signal molecule (molecules, AIs) increases until it reaches the threshold concentration at

which it binds and activates the related receptor protein. In this manner, all members of the population receive a

Antimicrobial research: Novel bioknowledge and educational programs (A. Méndez-Vilas, Ed.)

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physiological call to reprogram gene expression throughout the population. Thus, QS regulates a wide variety of

physiological processes including bioluminescence, competence, root nodulation, sporulation, antibiotic biosynthesis,

motility, plasmid conjugal transfer, biofilm maturation, and the expression of key virulence factors in plant, animal and

human pathogens. Among these characteristic and beneficial phenotypes, biofilm, a biological architecture of

aggregated microbes on a surface, is closely associated with virulence which task consists of overwhelm host defenses.

As result, biofilm infections tend to be chronic and difficult to eradicate [6,7].

In order to control these processes mentioned above, bacteria use three classes of AIs (Fig. 1). The first identified

molecule of the most common class of AIs is N-acetyl-L-homoserine lactone (AHL). This latter molecule and its

analogs are usually used by Gram-negative bacteria, whereas oligopeptides are mainly found in Gram-positive bacteria

[8]. The N-acyl-L-homoserine lactone and its analogues, acylated homoserine lactones (AHLs), are a class of small

neutral lipid molecules composed of a homoserine lactone ring with an acyl chain, which often contains 4 to 18 carbon

atoms [9].

Fig. 1 General structures of known main autoinducers.

The third class of AIs comprises by various alifatic bifunctional compounds. Studying QS in Vibrio cholerae, the

causative agent of the disease cholera, it was found that a precursor based on the 4,5-dihydroxy-2,3-pentanedione

(DPD) structure spontaneously cyclizes into a signal molecule called as autoinducer-2 (AI-2), which chelates borate to

give a new signal molecule, identified as (2S,4S)-2-methyl-2,3,4-tetrahydroxytetrahydrofuran-borate, termed (AI-2-

borate) [10,11]. The AI-2 has been also proposed to be of biological significance in Salmonella typhimurium [11] and in

Vibrio harveyi [12]. Another signal molecule of this class was identified to be (S)-3-hydroxytridecan-4-one and called

as cholera autoinducer-1 (CAI-1) [13-15] (Fig. 1). It seems to be that autoinducers of the third class are non-species

specific signals, which mediate interspecies communication among Gram-negative and Gram-positive bacteria.

2.1 Signaling molecules used by Gram-negative bacteria

The QS signaling system in Gram-negative bacteria usually corresponds to the biosynthesis of N-acyl-L-homoserine

lactones, which are synthetized by AHL synthases. As AHLs are small neutral molecules, they can diffuse in and out of

cells by different mechanisms and when their concentration eventually reaches a sufficiently high concentration, can

bind to specific receptors that belong to a large class of DNA-binding transcription factors called “R-proteins,” which

are the second component of the QS system. This binding (complex Receptor-AHLs) regulates the transcription of

target genes required for bacterial group behavior [16] (Fig. 2).

A lot of the basic information on the bacterial QS systems has been learned from the first study of QS systems in the

light-producing Gram-negative bacteria Vibrio fischeri, which are living in light organ of the Hawaiian bobtail squid

Euprymna scolopes. When the bacteria density is sufficiently high, genes involved in bioluminescence are expressed

and light is produced [3].

Fig. 2 Simplified scheme of AHLs quorum sensing system in Gram-negative bacteria.


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It was found that there are three elements: a signal generator LuxI (synthase), the cognate receptor LuxR (“R-

proteins”) and acyl-L-homoserine lactone, in this intraspecial bacterial cell-to-cell communication. Thus, an inducer

synthase (LuxI–type protein in the lux operon in V. fischeri) produces a special small AHLs molecule, which is

accumulated while the bacteria population grows. This molecule can be detected by its related cytoplasmic receptor

(LuxR–type protein). The protein complex AHL–LuxR often homodimerizes and binds adjacent to QS promoters („„lux

boxes‟‟), which can initiate the transcription of target genes required for bacterial group behavior.

2.1.1 QS circuits in Vibrio fischeri

The QS circuits (AHL synthases/receptors) of V. fischeri produce the N-3-oxohexanoyl-L-homoserine lactone (3-oxo-

C6-HSL) as main autoinducer molecule. The marine bacterium V. fischeri is a bioluminescent specie, which produces

light depending of the cellular density [16]. This fact is attributed to QS network where the autoinducer, 3-oxo-C6-HSL,

is biosynthesized through the LuxI proteins and is recognized by LuxR proteins to form complex LuxR-(3-oxo-C6-

HSL) that regulates bioluminescence process via genic expression. It means that the 3-oxo-C6-HSL molecule and

similar AHLs are internalized by diffusion and bind to an intracellular receptor molecule to activate the response (Fig.

3).

Fig. 3 Simplified (3-oxo-AHL)-dependent quorum sensing (LuxI/LuxR) system in V. fischeri.

AHL synthesis by LuxI-type synthases generally proceeds through a sequentially reaction mechanism using S-

adenosylmethionine (SAM) as an amino donor in the biosynthesis of the homoserine lactone (HL) ring core, whereas a

charged (acylated) carrier protein (ACP) acting as the precursor of the acyl side chain). The acyl-ACP binds to the AHL

synthase (a LuxI-type synthase), while the acylation and lactonization reactions occur through the S-

adenosylmethionine (SAM), which permit the intramolecular lactonization reaction. The 3-oxo-C6-HSL is then

released, along with the byproduct holo-ACP and 5-methyl-thioadenosine (Fig. 4).

Therefore, the basic behavior of a Gram-negative QS system corresponds to: i) The biosynthesis of the signal (AHL

autoinducer); ii) Interaction (binding) of the AHL to a LuxR-type protein (receptor) at a threshold concentration, and iii)

Regulation of gene transcription by the receptor complex (Fig. 2). AHL molecules are used as the primary QS

molecules in Gram-negative bacteria. These AHL–(LuxI/LuxR)-based QS systems and related bacterial QS systems

play a key role in regulating virulence and are particularly attractive targets for developing new antibacterial agents

[18,19]. However, several Gram-negative bacteria have been shown to use different two or more AHL signals, besides

other ligands and receptors to „„regulate‟‟ their QS systems. For example, besides the production of an AHL, V. harveyi

also produces AI-2, which is believed to involve in the regulation of bioluminescence.

Fig. 4 Biosynthetic pathway of primary AHL molecule in V. fischeri.


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Due to the emerging threat of multidrug resistance in relevant pathogens such as Pseudomonas aeruginosa,

Klebsiella pneumoniae and others, the research trends are directed to develop new molecules, which alter bacterial QS

system and, thus could mitigate virulence without having risks of resistance development, since interference with

virulence generally does not affect the growth and fitness of the bacteria [17].

Particularly, there is a wide interest in inhibiting AHL-mediated QS in some pathogenic species like P. aeruginosa,

due to the clinical importance of this opportunistic bacteria in life threatening hospital-acquired infections [20]. P.

aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections

and commonly infects immunocompromised patients [22]. P. aeruginosa infection is often aggravated by the formation

of biofilm, a mode of bacterial growth associated with antibiotic tolerance. The treatment of its infection confronts

major challenges due to the constant emergence of antibiotic-resistant variants. Antibiotic resistance to this bacterium

increases the rate of disease occurrence and mortality. With these problems, there is an urgent need to develop novel

antibiotic and anti-virulence strategies, which may be facilitated by an approach that explores more QS networks in P.

aeruginosa.

2.1.2 QS circuits in Pseudomonas aeruginosa

QS circuits of this bacterium is more complex than system in V. fischeri. P. aeruginosa use first N-(3-oxododecanoyl)-

L-homoserine lactone (3-oxo-C12-HSL) as primary AHL signal molecule for their QS. This AHL signal is

biosynthesized by a AHL synthase type protein called LasI and recognized by an intracellular LasR-type receptor

producing complex (3-oxo-C12-HSL)–LasR (Fig. 5) [21].

Fig. 5 Scheme of QS network in Pseudomonas aeruginosa.

While population density is increasing and QS is advancing, the dominant AHL synthase (LasI) comes to be RhlI

enzyme, which makes another autoinducer, N-butyryl-L-homoserine lactone (C4-HSL) and regulates cognate protein

RhlR, to which the C4-HSL is binding. Once activated by their native autoinducers, LasR and RhlR homodimerize and

trigger or repress transcription factors to regulate a specific set of beneficial phenotypes genes. These systems directly

or indirectly regulate over 10% of the P. aeruginosa genome. In addition, P. aeruginosa also develops a third different

QS system named PqsR, which does not respond to AHLs autoinducers. For its own regulation, the protein PqsA−D

and PqsE biosynthesize the 2-heptyl-4(1H)-quinolone (HHQ) [25], which is transformed into the autoinducer called 2-

heptyl-3-hydroxy-4(1H)-quinolone (PQS) by the action of the protein PqsH, this QS system is known as Pseudomonas

quinolone signal (Fig. 5) [22-24].

The (3-oxo-C12-HSL)–LasR complex induces expression of LasI in addition to the expression of RhlR (regulates

positively both AHL QS circuits), while the RhlR-BHL complex controls its own auto-induction but has no effect on

the LasR system. In addition, LasR and RhlR have positively and negatively regulate effects respectively, in the

expression of genes involved in PqsR. While PQS regulates its own production, increase the RhlR expression, without

direct regulatory activity on the LasR system [2].


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Pseudomonas aeruginosa is a complex organism with multiple QS modules and signals. There are three types of

signaling molecules that function in a growth stage-dependent manner. Because of this design and development of new

selective antibacterial agents against this bacterium is difficult problem.

2.2 QS in Gram-positive bacteria

The Gram-positive bacteria QS network is different to QS circuitry of Gram-negative bacteria. First of all, the Gram-

positive bacterium does not harbor LuxI or LuxR homologues and primarily uses modified oligopeptides as

autoinducers (autoinducer peptides, AIP or QS peptides) [26,27]. These peptides are genetically encoded and are

generated ribosomally within the cell.

Noteworthy, as the oligopeptide molecules are encoded by genes, each species of bacteria is capable of producing a

peptide signal with a unique sequence. In general, the secretion of the AIP is facilitated by a membrane associated ATP-

binding cassette (ABC) transporter. Due to the physico-chemical parameters, these peptides are permeable to biological

membranes; therefore, secretion of QS peptides is mediated by specialized transporters, which are not capable to

permeate the cell membrane. As the population density increases, the AIPs accumulate in the environment. When a

certain threshold level is reached, binding of an AIP to a receptor occurs.

While the LuxR-type receptors are cytoplasmic, the sensors (receptors) for oligopeptide autoinducers in Gram-

positive bacteria are membrane-bound. Thus, there is two-component signaling proteins (membrane-bound receptors)

system, which transduce information via a series of phosphorylation events. A typical system consists of a membrane-

bound histidine kinase receptor and a cognate cytoplasmic response regulator, which functions as a transcriptional

regulator. The binding process between a signal peptide molecules and a membrane-bound sensor kinase leads to its

autophosporylation, resulting in ATP- guided phosphorylation of a conserved histidine residue (H) in the cytoplasm.

The phosphate group is transferred to the conserved aspartate residue (D) of a cognate response regulator (Fig. 6)

[28].

Fig. 6 Simplified scheme of QS network in Gram-positive bacteria.

As results, the activated response regulator effects the transcription of target genes, including the AIP, the ABC

transporter and genes for the receptor kinase and response regulator [27]. Taking into consideration that the bacterial

species are quite different, the nomenclature of the QS mechanisms can be diverse, due to the involved genes and

receptor(s). Examples of Gram-positive bacteria, which use quorum sensing peptide-based systems, include the

Streptococcus pneumonia, which use ComD/ComE to control competence development [29] or the QS system

AgrC/AgrA in the Staphylococcus aureus, which regulate the pathogenesis process [30].

3. Enzymatic autoinducer inactivation as a strategy in the modulation of bacterial

quorum sensing

As QS networks of several important bacterial pathogens, like P. aeruginosa, S. aureus, V. cholerae, and others play a

key role in the expression profile of diverse genes, including antibiotic tolerance and virulence determinants, their QS

circuits are logical, potential targets for antimicrobial chemotherapy. Disruption of bacterial quorum sensing systems

can be realized through various general approaches: 1) Inhibition of signal autoinducer production, 2) Degrading


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signals, 3) Antagonizing signal binding to LuxR-family receptor, 4) Trapping signals, and 5) Suppression of synthase

and receptor activities, stabilities or production. Among them, the first three methods stand out as more important and

more developed. All these processes interfere with QS and could be coined as “quorum quenching” (QQ).

QQ enzymes mechanism aims to degrade or inactivate the AHL autoinducers. There are three known classes of

enzymes which target AHL signals: lactonases, acylases, and oxidoreductases. All these enzymes can modify the AHLs

structure. Any change in the structure of AHLs will significantly lower their affinity for their response regulators,

diminishing their ability to affect gene regulation.

3.1 AHL Lactonases

The first AHL lactonase was a 250-amino acid protein characterized as a metalloprotease, identified in treated soil

samples, encoded by the gene aiiA (autoinducer inactivation gene) from a Gram-positive Bacillus sp. 240B1 [32]. The

enzymes catalyze the hydrolysis of the ester bond in the lactone ring of a wide variety of AHLs [33]. Subsequently,

diverse AiiA-like enzymes were found and isolated from various Bacillus species such as B. thuringiensis, B. cereus, B.

anthracis, and B. mycoides, which are sharing more that 90% sequence homology in many cases [32].

The AHL lactonase enzyme family possesses hydrolytic activity toward a broad spectrum of AHLs, acting on the

acyl chain length and the oxidation state at the C3 position of the acyl chain that leads to the generation of acyl

homoserine [33]. The catalytic mechanism proposed has been based on the two independent crystal structures of an

AHL lactonase of the B. thuringiensis, and suggesting that two zinc ions are present in the binding site, coordinated by

five histidine and two aspartate residues.

Another family of AHL-lactonases was identified in diverse species as Agrobacterium tumefaciens (attM) [34],

Klebsiella pneumoniae (ahlK) and Arthrobacter sp. (ahlD) [35], which share about 30-58% homology in amino acid

sequence. These have the same conserved zinc-binding motif; therefore, it is likely that the catalytic mechanism is quite

similar. Recently, a novel group of lactonases encoded by BpiB genes (for biofilm phenotype inhibiting genes) was

discovered [36]. It was found in three BpiB genes, originating from Nitrobacter sp. strain Nb-311A, Pseudomonas

fluorescence, and Xanthomonas campestris, encode BpiB01, BpiB04, and BpiB07, respectively. Their activity was

characterized as lactonase protein due to its role in degradation of 3-oxo-C12-HSL (Fig. 7A).

Fig. 7. Enzymatic degradation of AHL autoinducers.

3.2 AHL Acylases

The acylases protein are AHL-degrading enzymes, targeted the amide bond that connects the fatty acyl chain to the

homoserine lactone (Fig. 7B). Acylases were first identified in strains of Variovorax sp. and Rhodococcus erythropolis

[37], that are able to degrade and utilize multiple AHLs as a source of both energy and nitrogen [31,38]. Degradation

required an AHL acylase, which forms HSL and the corresponding fatty acid (Fig. 7B). The fatty acid supported

growth, whereas the HSL only served as nitrogen source.

The first enzymes characterized as HSL acylases were isolated in betaproteobacterium Ralstonia strain XJ12B and

named AiiD. This protein shares amino acid sequence homology with members of the N-terminal nucleophile hydrolase

(Ntn-hydrolase, aminohydrolase) superfamily, which can catalyze the hydrolysis of amide function, so-called post-

translational amide cleavage [39]. Recombinant expression of AiiD protein in P. aeruginosa proved to prevent AHL

accumulation in the culture medium and reduce virulence. Thus, it was concluded that AHL acylases catalyze the

complete and irreversible degradation of the AHLs through the hydrolysis of their amide bond. However, six genes that

encode AHL-acylase have been characterized. These AHL-acylases degrade long-chain AHLs more efficiently than

short-chain forms.

For example, it was found that the PA2385 protein in P. aeruginosa, previously labeled as pVdQ played a role in QQ

emzyme process. Its overexpression in P. aeruginosa PAO1 inhibited accumulation of 3-oxo-C12-HSL. However, a

PvdQ knockout strain was still able to utilize HSL as a sole source of carbon, implying that another enzyme is involved


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in the degradation process. That way, a second AHL acylase was found and was named QuiP (quorum signal utilization

and inactivation). QuiP enzyme shares 21% amino acid sequence homology with PvdQ and 23% homology with AiiD

from Ralstonia spp., and it is another member of the Ntn-hydrolase family, with substrate preference for long-chain

AHLs. In a related experiment, expression of PvdQ in P. aeruginosa was shown to abolish not only accumulation of 3-

oxo-C12-HSL, but also accumulation of Pseudomonas quinolone signal [40]. Noteworthy, certain enzymes, such as

PvdQ and QuiP appear to be unable to degrade AHL that has an acyl chain shorter than eight carbons.

4. Molecular modifications of signal molecules for LasR/RhlR regulatory activity

A decisive role in the regulation of bacterial QS network belongs to the small signal molecules (AHLs, AI-2, AIPs,

PQS, etc.) [41-44], which regulate cell–cell signaling process both in Gram-negative and Gram-positive bacteria. Their

binding to the cognate protein receptors does not only depend on their critic concentration, but also on their molecular

structure. Thus, in order to modulate QS circuits, many molecular signal-like small molecules have been obtained using

synthetic methods [45]. Generally, this strategy would work affecting more the LuxR-type receptor protein of QS

communication circuit. Homologues of LuxI/ LuxR have been identified in diverse bacterial genomes with a variety of

different AHLs regulating a range of physiological functions. However, now it is known that each bacterial species

responds specifically to its own unique AHL autoinducer; in general, the same molecular core is maintained, with

changes only in the size of the side chain (Fig. 8) [46].

Fig. 8 AHL core with different alkyl chain in diverse bacteria. Molecular specificity vs biological specifity.

Synthetic molecules capable of modulating diverse QS pathway (regulation of LuxR-type protein) have been

discovered through a design and high-throughput synthesis process, emulating and diversifying the molecular structure

of the natural AHL signaling molecule as a template, exhibit valuables structure-activity trends [47].

Non-native AHLs represent the most extensively studied class of synthetic QS modulators, being decisive in

identifying relevant results on SAR analysis. Critical structural aspects for the non-native synthetic molecules derived

from natural AHL corresponds to: i) The length of the lateral acyl chain; ii) The modification at the 3-carbon of the acyl

chain (is important, but not essential for activity); iii) The stereochemistry of the lactone ring (the L-stereoisomer is

needed for activity); iv) Molecular modifications to the lactone ring (Not in all cases result in active compounds); v)

The incorporation of aromatic functionality into AHLs (in the lactone ring or side lateral chain), generally yields

analogues with inhibitory activity [48].

4.1 General approaches looking for new QS modular

Structural aspects denote the basis of the focused molecular libraries that can guide the SAR studies, related to the

diverse structural modifications that must be realized. Based in the AHL native structure (for example, 3-oxo-C6-HSL,

3-oxo-C12-HSL and C4-HSL) three levels of molecular complexity are presented in which synthetic modifications are

made on the lateral acyl chain (Levels I and II), as well as changes on the lactam core (Level III) (Fig. 9).


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Fig. 9 Chemical modifications of AHL signal molecules.

Several structural modifications are focused on the carbonyl-alkyl chain (4–14 carbons) (Level I), diverse C-4 phenyl

substituted N-acyl-L-homoserine lactone analogues (1) were evaluated for both their inducing activity and their ability

to competitively inhibit the action of AHL, 3-oxo-C6-HSL, responsible for the bioluminescence in V. fischeri [49].

Almost of these phenyls substituted analogues displayed significant antagonist activity, possibly due to the interaction

between the aryl group and aromatic amino acids of the LuxR receptor that prevents it from adopting the active dimeric

form. On the other hand, replacement of the β-methylene group to the ketoamide in the acyl chain of 3-oxo-C12-HSL

autoinducers (2) with functions containing heteroatoms as an NH or sulfonyl combined with alkyl chains of diverse

length, resulted in a strong QS antagonist activity of these designed molecules in P. aeruginosa bioluminescent assay

(Fig. 9) [50].

The structural level II consists in the complete modification of the side alkyl chain of AHL autoinducer. These

modifications showed that the alkyl chain length is a very significant factor in the antagonist activity, evidenced in the

QS inhibition of α-(N-alkyl-carboxamide)-γ-butyrolactones (3) evaluated in bacteria V. fischeri. These studies revealed

that the tested compounds with slightly shorter chain resulted be less active as antogonists (Fig. 9) [51]. On the other

hand, it was also found that the decrease in the LasA production in P. aeruginosa caused by the C-14 substituted

alkaloid (R)-norbgugaine ((R)-2-tetradecylpyrrolidine) (8), natural molecules [52]. The realized incorporation of

alkylthiomethyl substituent in γ-lactame derivatives of AHL showed important effects on the P. aeruginosa Las and Rhl

QS pathways. The synthetized 5-((alkylthio)methyl) pyrrolidin-2-one and its (propyl-, hexyl-) derivatives (5) were

found to strongly inhibit both QS networks [53]. Meanwhile, the alkyl sulfonamide chain in N-sulfonyl-HSL (6)

displayed antagonist activity of the QS transcriptional regulator LuxR in V. fischeri [54].

The molecular level III is related to the modification of lactone ring [55]. Introduction of simple (hetero)aromatic and

alicyclic replacements for the lactone, as nonchiral 2-aminothiazole, 2-cyclohexanol, pyridine ring system to construct

new small heterocyclic molecules (7) among others (Fig. 9) abolished LasR-induction ability or did not show any

improvement over native AHL autoinducer in P. aeruginosa. These results suggested special chemical features in the

core, which are related with the antagonist activity [50]. In order to analyze the inhibition or activation ability of an

aromatic ring for the R-protein - DNA binding, it was developed a set of substituted aniline derivatives with hidroxyl or

carboxyamide (substituted in orhto- or meta-positions) (8), which can act as H bond acceptors leading to the potent

antagonists of LasR in P. aeruginosa [56,57]. The new structural features identified in this study for both agonists and

antagonists are currently being used to design new focused libraries of analogs that should contain more potent

antagonists.

Diverse structural modifications as alkyl side chains, H donor groups or substituted phenyl groups defined the lines

may be denoted in the construction of focused libraries based on the AHL core, in the three-diverse level of focused


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molecular library, based on AHL autoinducers structures: non-native AHL (level I-II), non-lactone modulators (level

III).

4.2 Further development of new modulators (agonists and antogonists) of native AHL autoinducers

This general tendency is completely observed in the work done by Blackwell and co-workers [55], with a strong

tendency to generate changes in the native AHL core and non-lactone modifications (Fig. 10). Focused modification on

the lateral chain (Level I-II) of known native AHLs from Gram-negative bacteria and structural phenyl analogues (9)

were evaluated as antagonist over Lax R-type receptors (LasR and TraR proteins) in two bacterial strain: P. aeruginosa

and A. tumefaciens, respectively. The compounds (9a-c) showed antagonism significant activity against TraR in A.

tumefaciens and were 1-2 orders of magnitude more active than the previously reported LuxR-type protein antagonists

examined as controls at 10 µM [51]. The same three compounds were also identified as potent antagonist against LasR

in P. aureginosa [55].

Fig. 10 Structural diversification of regulators of bacterial QS.

The substitution with aromatic phenyl groups in the side chain generated phenyl AHL (PHL) molecules (10) with

important antagonistic activities on V. fischeri, which is extremely dependent on the substituents (and their locations) on

the phenyl-acetanoyl group. The bioassays with the 4-bromo-PHL molecule (9b) showed 79% inhibition at 5µM, while

the one of the 4-iodo-PHL compound (10d) exhibed the highest activity (85%) (Fig. 10). Moreover, compounds with

very large and electronic density attractor groups such as 4-phenyl-PHL (10e) and 4-trifluoromethyl-PHL (10f)

displayed high activities (80%). Otherwise, replacement of the 4-halogen substituent with hydrogen or bond-donating

substituents (4-amino-PHL, and 4-hydroxy-PHL) decreased considerably the inhibitory activity [56]. Hence,

hydrophobic and bulky groups are critical structural features that confer antagonistic activity in Gram-negative bacteria

evidenced in the remarkable antagonist activities of 4-bromo-PHL (11a), 4-iodo PHL (11b), heptyl (11c), nonyl (11d)

derivatives that modulate three R-proteins TraR, LasR, and LuxR of A. tumefaciens, P. aureginose and V. Fischeri,

respectively [44] (Fig. 10).

Azido groups coupled to AHL derivatives were also evaluated for the ability to antagonize the receptors LasR and

AbaR in P. aureginosa and A. baumannii. The study showed that the azido-AHL compounds (12a-e) exhibited potent

antagonist activities. Interestingly, the potent inhibitor (12d) was converted to a strong LasR agonist upon the inclusion

of a single methylene unit between the triazole and cyclohexyl groups (12f) [57], evidencing that small changes in

structure of the derivatives may lead to drastic response in the activity. For example, the pent-4-enamide and

cyclopentane substituted derivatives (13d,e) showed a strong RhlR agonist activity in P. aeruginosa, while PHL

inhibitor molecules (13a-c) exhibited both significant RhlR agonist and antagonist properties. Dual modulator effects of

small molecules on global QS network in P. aeruginosa can indicate that both agonism and antagonism of RhlR should

have large effects on important QS-controlled virulence phenotypes [18,58].


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Although the non-native-AHL derivatives have proven their efficiency widely as agonists and antagonists in a range

of species as P. aeruginosa LasR and QscR [59], Vibrio fischeri (LuxR) [60], Agrobacterium tumefaciens (TraR) [44],

Pectobacterium carotovora (ExpR1/ExpR2) [61], and Chromobacterium violaceum (CviR) [62], the AHL head group is

prone to hydrolysis at pH values of 7, but above this pH, the hydrolyzed compounds are inactive. The focused

modifications correspond to the construction of non-lactone AHL libraries (Level III) for evaluation of their activities as

agonists and antagonists of Pseudomonas aeruginosa (LasR), Vibrio fischeri (LuxR), and Agrobacterium tumefaciens

(TraR). The synthetized 3-oxo-C12-HSL acyl chain derivatives with head groups aniline (14a), cyclopentyl (14b) and

glycine ethyl ester (14c) resulted be the most potent selective LasR modulator, while the glycine ethyl ester analogs

(14d) and (14e) (Fig. 10) were a strong LasR agonist and LuxR antagonist, respectively [63]. Thiolactones (non-native

AHLs tio-analogues) were also assessed for both antagonistic and agonistic activities in LuxR-type receptors (LasR,

LuxR, and TraR). Analysis of their multireceptor activity revealed that the thiolactones (15a-e) work as potent LuxR

antagonists in V. fischeri possessing nanomolar IC50 values, the thiolactone (15b) is also a strong LuxR antagonist, and

simultaneously a non-native TraR agonist, whereas the analogues (15d) and (15e) also displayed nanomolar IC50 values

in the E. coli LasR protein [64].

The design of the LasR modulators has been focused on synthesizing compounds that maintain the homoserine

lactone (HSL) head group, while changing the acyl tail represented in the level I and II of structural modifications or

making variations in the head group using phenyl compounds (level III). These focused strategies allowed the discovery

of numerous non-native AHL antagonists and agonists [65]. Although these AHLs derivatives can be used as potent

regulators of QS in Gram-negative species, they are beset by a number of shortcomings like the hydrolysis of HSL head

group in aqueous media [66] or the degradation by bacterial and host lactonases and acylases [67]. To design new LasR

antagonists that would avoid the limitations of designed and synthetized AHL leads mentioned above, a new diversity

strategy aimed at the molecular modifications in which the alterations of amide and phenyl moieties of AHL natural

autoinducers were employed.

Using this strategy, triphenyl derivative (16a) (TP-1P) (Fig. 10) was found as a potent agonist that maximally

activated LasR in P. aeruginosa assays, displaying an EC50 of 71 nM (~2-fold lower than 3-oxo-C12-HSL). In addition,

the compound (16b) was also shown as a moderate LasR inhibitor [68]. Study of reported X-ray crystal structures of the

LasR N-terminal ligand binding domain with nitro-triphenyl compound (17a) showed that the 2-nitrophenyl ring of this

molecule closely mimics the HSL head group in native 3-oxo-C12-HSL molecule [69]. Thus, a small library based on

nitrophenyl fragment was developed demonstrating that the obtained nitro-compounds (17b, c) work well as LasR

agonists in E. coli strain, whereas the nitro-triphenyl compounds (17d-g) can act as potent LasR antagonists at 50 μM E.

coli strain [70].

The relative structural simplicity of the key “actors” (AIs) in QS circuits has inspired the design and synthesis of

analogues of these signals for use as non-native QS agonists and antagonists. High-throughput synthesis has proven to

be a particularly valuable tool in identifying biologically active synthetic AHL mimics and the elucidation of structure-

activity trends, as libraries of AHL analogues with systematic modifications can be synthesized and tested in a time-

and cost-efficient fashion. These focused synthetic approaches have generated sets of compounds that have been

screened for QS modulatory activity. Many studies have focused on developing analogues of the native AHL signal

molecule, in which the acyl side chain or the lactone moiety was modified by organic synthesis (Fig. 11).

The “molecular language” of QS have inspired diverse chemical approaches aimed at elucidating mechanistic aspects

of QS, the relative structural simplicity of these signals, especially the AHLs, has inspired to design and synthesize

analogues of these signals for use as nonnative QS agonists and antagonists with a potential value as chemical probes.

In addition to these targeted analogue studies, a variety of other chemical strategies have been applied to QS research as

combinatorial approaches. Use of small molecule QS modulators,[55] affinity chromatography for the isolation of QS

receptors [71], reagents and fluorescent probes [72], antibodies as quorum “quenchers” [73], abiotic polymeric “sinks”

and “pools” for QS signals, and electrochemical sensing of QS signals.


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Fig. 11 Synthetic efforts directed towards novel QS agonists and antagonists.

5. Perspectives and conclusions

The generation of diverse focused molecular libraries based on natural autoinducers to regulate bacterial QS systems

has attracted an enormous interest in recent years. A large number of structurally diverse nonnative agonists and

antagonists has been discovered, providing researchers with a comprehensive set of chemical tools to study and

remarkable structure activity relationships, identifying molecular characteristics necessary for regulation of bacterial QS

systems.

The construction of lead compounds, which allow a greater regulation of cellular communication systems for

efficient chemotherapeutic applications, is one of the challenges corresponding to this chemical biology study area.

These investigations must be supplemented by more efficient and versatile synthetic strategies that allow a greater

molecular diversification by extending the range of study in the chemical space as well as the need to standardize the

methods of study used for the evaluation of the modulation relationships between the small molecules and the bacterial

QS.

Both the efficient synthetic diversification strategies and the biochemical analysis methods must be further developed

and complemented by a deepening in fundamental studies into the molecular basis of quorum sensing modulation in

terms of the fundamental bonding interactions, covered by spectroscopic and computational studies that show the

process of protein-receptor interaction and its relationship with the regulation of the complex QS circuits, as well as the

analysis of these data in diverse bacterial strains, allowing the generation of a general context for the ideal molecular

characteristics for the regulation of bacterial QS.

Acknowledgements. C.M.M.G. thanks la Vicerrectoría de investigaciones de la Universidad del Antlántico for financial support.

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Small molecules as regulators of bacterial Quorum … · therapeutic potential The objective of this chapter is to show and discuss the ... attractive targets for developing new antibacterial