The Molecular Architecture of Pseudomonas aeruginosa Quorum-Sensing Inhibitors

The survival selection pressure caused by antibiotic-mediated bactericidal and bacteriostatic activity is one of the important inducements for bacteria to develop drug resistance. Bacteria gain drug resistance through spontaneous mutation so as to achieve the goals of survival and reproduction. Quorum sensing (QS) is an intercellular communication system based on cell density that can regulate bacterial virulence and biofilm formation. The secretion of more than 30 virulence factors of P. aeruginosa is controlled by QS, and the formation and diffusion of biofilm is an important mechanism causing the multidrug resistance of P. aeruginosa, which is also closely related to the QS system. There are three main QS systems in P. aeruginosa: las system, rhl system, and pqs system. Quorum-sensing inhibitors (QSIs) can reduce the toxicity of bacteria without affecting the growth and enhance the sensitivity of bacterial biofilms to antibiotic treatment. These characteristics make QSIs a popular topic for research and development in the field of anti-infection. This paper reviews the research progress of the P. aeruginosa quorum-sensing system and QSIs, targeting three QS systems, which will provide help for the future research and development of novel quorum-sensing inhibitors.


Introduction
To battle against infection, antibiotics are often used to kill pathogenic bacteria. However, with the abuse and misuse of antibiotics, bacteria increasingly exhibit resistance to available antimicrobial drugs [1,2]. Hence, it is urgent to find novel strategies to tackle the new generation of drug-resistant pathogens. Quorum sensing (QS) is a well-known cell-to-cell signal communication system that allows bacteria to monitor their cell density by releasing signaling molecules called autoinducers (AIs) to cope with the changes in society and environment [3,4]. QS conducts and regulates the expression of related genes through signal molecules, such as bioluminescence, pigment production, formation of biofilm, and the secretion of virulence factors [5].
As the core of the QS system, signal molecules play a key role in the signal transmission process. It has been reported that blocking the QS system of bacteria can significantly reduce the pathogenicity of pathogens without affecting their growth. Therefore, targeting the QS system to treat bacterial infections provides a new direction for effectively slowing down the development of bacterial resistance. The initial demonstration of quorum-sensing inhibitors (QSIs) can be traced back to Australian red marine algae Delisea pulchra. It produces secondary metabolites, a number of halogenated furanones that have structural similarities to AHL molecules. These metabolites can interfere with the bacterial processes that involve AHL-driven quorum-sensing systems [6]. To date, many compounds from various natural sources, molecules with structural similarities to quorum-sensing signals and synthetic chemicals have been reported to act as quorum-sensing inhibitors. In recent Figure 1. Three main QS systems in P. aeruginosa, las system, rhl system, and pqs system and their signal molecules. Las is indicated in green, rhl is indicated in orange, and pqs is indicated in blue. Las is at the top of the QS hierarchy and influences rhl and pqs. On the other hand, rhl is under the control of both las and pqs.
There are many reports on the inhibitors [13][14][15][16][17][18] of P. aeruginosa; flavonoids, allicin analogs from plants, equisetin isolated from marine fungus, and so on can show potent inhibitory activity. Existing drugs also deserve further study; for example, studies showed that piroxicam had potential QS inhibitory activity. Thus, reusing old drugs is an important method of developing new QSIs. Regarding the working mechanisms, some of them have been studied clearly, but there are still many compounds that can act as inhibitors with unclear action mechanisms. This paper reviews the research progress of quorum-sensing inhibitors against P. aeruginosa from the aspects of screening from various sources and working mechanisms.

The Introduction of las
Las plays a fundamental role in the regulation of the QS network of P. aeruginosa; the signaling molecule N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL, OdDHL) is synthesized by LasI. When the signal concentration reaches the threshold, it binds to the receptor protein LasR to form a complex and initiates the expression of downstream target genes [19]. The las signaling system regulates virulence factors such as exotoxin A and the expression of LasA staphylolytic protease, LasB elastase, and Apr alkaline protease as well as biofilm formation [20]. LasA and LasB play a vital role in regulating protease activity; they are in charge of the degradation of elastin, fibrin, complement, interferon, and collagen during infection. They can also influence toxin levels [21]. Therefore, suppressing las is an effective way to inhibit the QS system. There have been many reports about las inhibitors including molecules from natural plant extracts, similar structures of AHL signals, and artificial compounds. Las pathways involved with the QS controls of virulence and other phenotypes are shown in Figure 2.

Inhibitors Acting on las
Currently, inhibitors of the las system are the most studied. Specific las inhibition is measured based on expression levels of the respective genes through various approaches, such as RNA extraction and microarray analysis, high-throughput RNA sequencing, molecular docking, gene expression assay, and quantitative real-time PCR. Specific rhl and pqs inhibition are measured through similar approaches to those for las. Many researchers found that some natural materials from different sources had QS inhibitory activities by targeting las and most are from plants. The results showed that ethyl acetate extract of TemuIreng rhizomes (Curcuma aeruginosa) can decrease P. aeruginosa virulence responses such as protease LasA, LasB, and biofilm formation, which is controlled by quorum sensing [21]. As we all know, many organosulfur compounds have bio-inhibitory activities, so many researchers have devoted effort to studying the related compounds. It was reported that garlic-derived natural vinyl dithiins 1 and 2 ( Figure 3a) inhibited LasI/LasR-based QS but exclusively in LuxR monitoring system [13]. Sulforaphane and erucin (Figure 3a) are natural isothiocyanates from broccoli. They can effectively combine with LasR to inhibit the QS system [22]. Many essential oils also can serve as inhibitors such as the oil of Murraya koenigii, of which the components can target las and thereby inhibit the production of pigment and siderophore in P. aeruginosa [23]. Bettadaiah et al. [14] found that the key phenolic compounds of ginger; 6-gingerol; 6-shogaol; zingerone; a derivative of 6-shogaol, 6-azashogaol; and an isoxazoline derivative of 6-gingerol ( Figure 3b) showed good QSI activities against P. aeruginosa. These compounds, consisting of long alkyl chains and moieties of N-acyl-homoserine lactone, can inhibit LasR-dependent gene expression. Among these compounds, 6-gingerol showed the best QSI activity against P. aeruginosa. Many flavonoids were also proved to have QSI activities and can be inhibitors. Quercetin (Figure 3c) is a flavonoid that interacts with transcriptional regulator LasR as well as suppressing QS circuitry, biofilm formation, and virulence gene expression [15]. The structure of naringenin is similar to quercetin, so it plays a similar role to the latter. It showed its effect by directly binding the QS regulator LasR and competing with 3-oxo-C12-HSL. The compound reduced the production of AHLs in P. aeruginosa and therefore inhibited the production of QS-regulated virulence factors [16]. Studies have shown that other flavonoids ( Figure 3c) such as catechin-7-xyloside (C7X, a flavan-3-ols from Spiraea hypericifolia), sappanol (a 3,4-dihydroxyhomoisoflavan from Caesalpinia sappan), and butein (a chalcone from Toxicodendron vernicifluum) were capable of interacting with LasR of P. aeruginosa (a LuxRtype quorum-sensing regulator) without killing the bacteria [24]. Phyllanthus emblica is a traditional medicinal plant that has various biological activities. Docking studies showed that its component cardamonin belonged to the chalcone compounds and might have good inhibitory activity against QS by binding with LasR receptor [25]. Other natural products have also been reported to have inhibitory activities ( Figure 4). Furvina is a nitro-vinylfuran antibiotic that was first isolated from bagasse in Cuba. It was reported that it can interact with las as well as interfere with 3-oxo-C12-HSL signal molecule and bioluminescence emission [26]. Therefore, it can be a promising complementary strategy that furvina might be applied to combine with less effective antibiotics to cure biofilmrelated infections. Trans-anethole is the major component of anise (Pimpinella anisum) oil, having similar structure to that of natural ligand OdDHL, and it shows antimicrobial and antifungal activities [27]. Moreover, it was demonstrated that trans-anethole reduced the production of virulence factor by binding to LasR protein [27]. 1,8-Cineole is the main component of niaouli essential oil, and it shows an inhibitory effect on biofilm formation in P. aeruginosa. A molecular docking study confirmed that it interfered the binding with LasR and inhibited las [28]. Some catabolites were reported to inhibit quorum sensing by inhibiting las. For example, 4-methylenebut-2-en-4-olide, a protoanemonin, is produced by the pseudomonads Pseudomonas sp. B13 and Pseudomonas reinekei MT1. It inhibits a lasB-based QS monitor and the production of elastase LasB and phenazine [29]. Because many natural products can be used as QSIs, researchers tried to synthesize new compounds by changing the structure of natural products. (R)-bgugaine has antibacterial activity, while synthesized norbgugaine is a demethylated form of natural bgugaine (R)-norbgugaine showed inhibition to various motilities, pyocyanin pigmentation, the production of LasA protease, and biofilm formation in P. aeruginosa [30]. (4-((E)-(4-hydroxy-2-methylphenylimino)methyl)-2methoxyphenol (MMP) is a vanillin derivative. After in-depth analysis, it was found to have a strong binding affinity to the las system [31]. To develop more QSI against P. aeruginosa, some researchers attempted to design inhibitors based on the AHL signaling molecules. The structures of N-(4-{4-fluoroanilno}butanoyl)-Lhomoserine lactone (FABHL) and N-(4-{4-chlororoanilno}butanoyl)-L-homoserine lactone (CABHL) are similar to HSLs (Figure 5a). These two compounds can influence las via interacting with LasR protein [32]. Based on the fact that chalcones have widespread biological activity, Wu et al. focused on central amide moieties and synthesized a series of chalcone-based L-homoserine lactones; among these compounds, (S)-2-((4-(3-(4bromo-2-fluorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofur-an-3-yl) acetamide 3 ( Figure 5a) was found to inhibit the LasR-dependent QS system of P. aeruginosa [33]. Over the past years, designing new QSIs by modifying the structure of signaling molecules has always been a widely used approach. In 2011, N 1 -(5-chloro-2-hydroxyphenyl)-N 3octylmalonamide 4 ( Figure 5b) was developed structurally in the L-HSL ring system. It was modified by replacing the methylene group to β-ketoamide in the acyl chain of OdDHL with an NH group, and at the same time, the replacement of the homoserine lactone system with stable cyclic groups can be conducted [34]. This compound displayed LasR-induction antagonist activity and influenced the host immune system. In 2013, an OdDHL analogue 5 ( Figure 5b) containing a nonnative head group was found to be a potent inhibitor of LasR-based quorum sensing, reducing the production of pyocyanin by disrupting LasR [35]. In 2015, Byun et al. [36] designed several novel QS compounds by replacing the lactone ring of OdDHL with the pyrone ring. By varying alkyl chain lengths to inhibit the binding between OdDHL and LasR of P. aeruginosa, 6 ( Figure 5b) was the most potent compound that displayed strong inhibitory activities against biofilm formation. In the same year, 7 (V-06-018, Figure 5b) was designed to modulate LasR strongly and influence QS-dependent phenotypes in P. aeruginosa [37]. In 2017, it was investigated that (S,E)-2-hydroxy-N-(2-hydroxy-5-nitrobenzylidene)propanehydrazide(lactohydrazone) 8 ( Figure 5b) displayed interactions as a natural auto-inducer with LasR and then inhibited virulence factors [38]. The indole-based AHL analogue 9 ( Figure 5c) and salicylic acid (SA) (Figure 5c) can effectively inhibit the P. aeruginosa LasR. Accordingly, in 2018, Sekhar et al. designed and synthesized some derivatives of 2-phenylindole-amide-triazole and salicylic acid-triazole; among the compounds, four (10a-c, 11) were found to have inhibitory activity (Figure 5c) [39]. Later, the same group developed 12a-d (Figure 5c) as novel inhibitors of LasR-dependent quorum sensing; these compounds exhibited quorum-sensing inhibitory activity without showing toxic effects on normal cells [40]. Some other researchers tried to focus on some different scaffolds and functional groups. Lactams were designed and synthesized through the reductive amination of mucochloric acid and mucobromic acid. 13a-d ( Figure 6a) were found to be the most active compounds and can dock to the LasR receptor protein and serve as new lead compounds for developing potent QSI compounds [41]. Triphenyl 14a and a nonnatural triaryl analogue 14b (Figure 6a) containing chlorine atom show high LasR antagonist activity [42]. Nonnatural irreversible antagonists 15a and 15b ( Figure 6a) were identified to exhibit effective LasR antagonist activity and inhibit the expression of virulence factors such as pyocyanin and the formation of biofilm [43]. Based on triphenyl scaffold, some researchers designed various new inhibitors to strongly agonize LasR, and then they found that 16 (Figure 6a) was the most potent LasR antagonist [44]. Computer-aided approaches were also a good way to develop novel compounds; some researchers designed five new compounds (17a-e) ( Figure 6b); these can act as inhibitors of the LasR QS system of P. aeruginosa in a dose-dependent manner [45]. Novel compounds containing benzothiazole moiety were synthesized. Among them, 18a-c ( Figure 6b) have more potent inhibitory activity against LasR in P. aeruginosa [46]. A series of unsymmetrical azines were designed and evaluated, and docking analysis showed that 19a and 19b (Figure 6b) can bind to LasR protein [10]. Celecoxib derivatives SGK 324, 327 and 330 ( Figure 6c) have favorable interactions with LasR binding sites. Among these compounds, SGK 330 is the most active because at the place of the lactone ring of the natural ligand HSL, the interaction site was occupied by the thiazolidinone ring [47]. A range of dihydropyrrolone analogues were synthesized and can dock to LasR, and 20 ( Figure 6c) was the most potent inhibitor of biofilm formation [48]. Two novel Mannich bases, 1-(phenyl (o-tolylamino) methyl) urea 21 and 3-((1H-benzo[d]imidazol-1-yl)methyl)naphthalen-2-ol 22 (Figure 6c), were synthesized by Mannich reaction, showing LasR antagonistic activities and inhibiting biofilm formation and pyocyanin production in a dose-dependent manner [49]. Chrysin derivative 23 (Figure 6c) can bind to regulator LasR and shows anti-virulence activity [50]. Employing a novel specialized multilevel in silico approach, plenty of compounds were assessed, and eight compounds (24a-h) ( Figure 6d) were identified as promising LasR inhibitors [51].

The Introduction of rhl
Another system belonging to the LuxR-type receptor family in the QS system of P. aeruginosa is rhl, which is similar to las. The difference is that the AHL-signaling molecule of rhl is N-butanoyl-L-homoserine lactone (BHL or C4-HSL), which is produced by RhlI, and the signal molecule binds to its receptor and activates transcriptional regulator RhlR [52]. Rhl is related to the production of some virulence factors such as rhamnolipids and the toxic exo-factors hydrogen cyanide, pyocyanin, and so on [53]. Among these, rhamnolipids are biodegradable surfactants that contain two fatty acid molecules and rhamnose residues [54]. They play a vital role in protecting cells from oxidative stress and participating in biofilm growth and maturation [55]. Pyocyanin, a greenish pigment secreted by P. aeruginosa, can cause severe toxic effects on the host [56]. Therefore, targeting rhl is a good strategy for controlling quorum sensing infections. Rhl pathways involved with the QS controls of virulence and other phenotypes are shown in Figure 7.

Inhibitors Acting on rhl
Inhibitors acting on rhl systems can be divided into three sources: natural products, analogues of signaling molecules, and synthetic compounds. Some inhibitors are from natural products. Three ellagic acid derivatives (25a-c, Figure 8a) isolated from Anogeissus leiocarpus (DC) have been reported to inhibit the expression of QS-regulated virulence factors and down-regulate the QS regulator gene rhlR [1]. Tannic acid (TA) (Figure 8a) was tested to suppress the synthesis of pyocyanin, which is controlled by rhl, including the transcriptional activator RhlR and the auto-inducer synthase RhlI [57]. The plant compound rosmarinic acid (RA) (Figure 8a) was shown to specifically bind to the RhlR QS receptor of P. aeruginosa and induce some QS-mediated behaviors [58]. Some are analogues of signaling molecules. It was identified that mBTL (Figure 8b), an analogue of the native P. aeruginosa autoinducers, can inhibit the quorum-sensing receptor RhlR and prevent biofilm formation [59]. A series of hybrid AHL analogs was designed, and among those evaluated compounds, N-acyl-L-homocysteine thiolactones 26a-c (Figure 8b), which contained homocysteine thiolactone head groups, were identified to be promising compounds that showed high selectivity for RhlR [60]. On the other side, synthetic compounds such as 2, 8-bit derivatives of quinoline synthesized by Suzuki coupling reaction were found to be potent inhibitors against biofilm formation. After optimization by Sun and co-workers, 27a and 27b (Figure 8c) showed the most potential candidates mainly inhibiting the expression of rhl [61]. A set of 4-gingerol analogues were synthesized, and structure-activity relationship studies identified that the alkynyl ketone 28 (Figure 8c) can act as a strong RhlR antagonist and shows inhibitory activities on biofilm formation and rhamnolipid production in P. aeruginosa [62].

The Introduction of pqs
The third QS system in P. aeruginosa is called pqs, and it is closely related to two AHLbased systems. It puts 2-heptyl-3-hydroxy-4-quinolone (PQS) as its signal molecules, and the precursor of PQS, 2-heptyl-4-hydroxyquinoline (HHQ), is commonly associated with QS along with pqs [63]. The biosynthesis of these two signal molecules requires a transcriptional regulator PqsR (MvfR) and pqsABCDE operon, whose expression is controlled by the former [64]. PqsR plays a key role in pqs and regulates the expression of rhlI as well as some virulence functions such as hydrogen cyanide, pyocyanin, bacterial motility, and biofilm formation, of which the expression is activated by LasR but inhibited by RhlR [65]. Hence, PqsR is a potential target for the study of novel QS inhibitors. Pqs pathways involved with the QS controls of virulence and other phenotypes are shown in Figure 9.

Inhibitors Acting on pqs
In recent years, increasing numbers of researchers have paid attention to QS inhibitors targeting pqs. There are some inhibitors from natural sources such as stigmatellin Y and wogonin ( Figure 10), with similar structural skeletons. Stigmatellin Y was from Bacillus subtilis BR4, and it showed strong affinity to PqsR as well as competing with pqs to distract PQS-PqsR mediated communication [66]. Wogonin is the active ingredient from Agrimonia pilosa. One study showed that it can target the PqsR of pqs and be a potent QS inhibitor [67]. Farnesol ( Figure 10) is a natural sesquiterpene alcohol, and it can suppress the transcription and protein expression of the pqsABCDE and pqsH genes and interfere with the synthesis of pyocyanin [68]. Perillaldehyde ( Figure 10) is a natural, widely used, and nontoxic food additive from Perilla frutescens. It showed anti-virulence activity and repressed biofilm formation. Molecular mechanism studies showed that it can interact with PqsR protein [69].  However, many QS inhibitors still rely on synthesis. Hartmann et al. have reported that antagonists of PqsR (29a and 29b, Figure 11a) can inhibit the production of pyocyanin and may have potential to become QS inhibitors [70]. The thiazolyl-containing quinazolinones 30a and 30b (Figure 11a) play similar roles as 29a and 29b, and they were demonstrated to attenuate the production of pyocyanin and to reduce many other virulence responses via targeting PqsR [71]. Quinazolinone 31 (Figure 11a) can also act as a PqsR inhibitor of biofilm formation in P. aeruginosa [72]. Two series of chromones-based compounds were synthesized with the help of molecular docking. Based on the bioactive results, 32 (Figure 11a) was the most potent inhibitor of biofilm; it showed good predicted affinities for PqsR and may be a promising QS inhibitor targeting pqs [73]. Many compounds containing amide bonds or ureido motifs can act as inhibitors. It was identified that ureidothiophene-2-carboxylic acids 33 (Figure 11b) can be inhibitors of PqsD. Through combining in silico analysis and biophysical methods, 34 was synthesized based on 33 (Figure 11b), which carried a phenylalanine substituent at the ureido motif as the most active inhibitor [64,74]. 3-Cl substituted benzamidobenzoic acid 35 (Figure 11b) can also be used as an inhibitor of PqsD, playing a key role in the QS system and mediating the formation of HHQ [75]. Through a scaffold-based approach, Hartmann et al. developed a novel inhibitor of PqsD 36 (Figure 11c). It reduced the HHQ and PQS levels and significantly inhibited the formation of biofilm with no antibiotic effects [76]. PqsD shared some features with chalcone synthase, and thereby, a novel class of inhibitors composed of catechol moieties was discovered. Among those compounds, 37a, 37b, and 37c (Figure 11c) showed inhibitory activities [77]. A series of N-1, C-2, C-6-substituted 3-hydroxy-pyridin-4(1H)-one derivatives were synthesized and evaluated, 38 (Figure 11c) was found to be the most active compound, it strongly suppressed biofilm formation at low concentrations through inhibiting the expression of PqsA and reducing the production of virulence factors. It was considered a potentially novel QS inhibitor [78]. Compounds containing aminoquinolines were reported to have inhibitory activities. Quinolone-derived molecule 39 (Figure 11d) was identified to show inhibitory activity in the production of alkyl quinolone (AQ), a pqs signal molecule [79]. It was found that the 7-Cl and 7-CF 3 substituted long-chain Ndodecylamino-4-aminoquinolines 40a and 40b (Figure 11d) can act as PQS antagonists [3]. A series of N-octaneamino-4-amino-quinoline derivatives were also synthesized, such as 41a and 41b (Figure 11d) with benzofuran and benzothiophene substituents respectively; they showed the effective inhibition of the production of pyocyanin and can reduce the motility of P. aeruginosa via interfering with the pqs signaling pathway [65]. M64 (Figure 11d), one of the benzamide-benzimidazole (BB) series compounds, was the first PqsR inhibitor to show in vivo activity; it targeted PqsR and interfered with the early steps of biofilm formation [80]. Through further optimization, 42 (Figure 11d) was designed and reported to act as a potent PqsR antagonist, inhibiting the production of HHQ and PQS at sub-micromolar concentrations [81]. 43 (Figure 11e) is a novel lead QSI and shows high potency, targeting the PqsR of pqs and inhibits the production of pyocyanin [82]. Recently a study showed that vitamin E and vitamin K 1 (Figure 11e) significantly inhibited the formation of biofilm and the production of virulence factors, which had potential as QS inhibitors through inhibiting PqsR protein [83]. In recent years, many antibacterial drugs have been developed, some of which can be used as QSIs. The FDA-approved antivirulence drugs nitrofurazone and erythromycin estolate (Figure 12), which belong to the nitrofuran and macrolide structural classes, respectively, are able to inhibit the main effector protein PqsE of pqs and reduce the production of the PqsE-controlled virulence factors such as pyocyanin and rhamnolipids [17]. Some researchers have applied a combined multilevel computational approach to find possible QS inhibitors that target the PqsR of P. aeruginosa in FDA-approved drugs. They found that nilotinib, indocyanine green, cabozantinib, venetoclax, and montelukast ( Figure 12) can act as QS inhibitors [84]. In short, present drugs may have new uses and are promising candidates for QS inhibitors.

Inhibitors Acting on Multiple QS Systems
Las is closely connected with rhl and pqs. It is known that many inhibitors can show inhibition of both las and rhl. Some inhibitors belong to natural products or their derivatives. The human sex hormones hordenine and estrone (Figure 13a) as well as their structural relatives estriol and estradiol (Figure 13a) were found to decrease AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in P. aeruginosa [85]. Researchers have reported that many natural flavonoids had inhibitory activities. For instance, 3,5,7-trihydroxyflavone (TF, Figure 13b) from Alstoniascholaris leaf extract and luteolin from traditional Chinese herbal medicine were found to inhibit the QS signaling molecules OdDHL in las and BHL in rhl through molecular docking study; they had inhibitory effect on the formation of biofilm and the production of QS-dependent virulence factors [86,87]. Baicalein (BCL) and naringin (Figure 13b) also belong to natural flavonoids and play similar roles to those of TF and luteolin in P. aeruginosa [8,88]. 6-methylcoumarin (6-MC, Figure 13b) is one of the coumarin derivatives. Molecular docking studies showed that it inhibited LasI in las and RhlI/RhlR in rhl [89]. Natural products of other structures also exhibit some inhibitory abilities. The isoquinoline alkaloid berberine and polyphenol chlorogenic acid (Figure 13c) were extracted from plants, and they displayed significant inhibition of quorum-sensing-regulated phenotypes. Molecular docking analysis showed that these two compounds appeared to be potent inhibitors of LasR and RhlR [8,90]. The natural pentacyclic triterpenes betulin (BT, Figure 13c) and betulinic acid (BA, Figure 13c) can exhibit significant attenuation of the QS receptors, LasR, and RhlR [88]. A triterpenoid coumarate ester isolated from Dalbergia trichocarpa has been identified as oleanolic aldehyde coumarate (OALC, Figure 13c). Evernic acid (Figure 13c) was a lichen secondary metabolite that can be isolated from Evernia species. These two compounds can inhibit the expression of QS-regulated lasB genes and rhlA genes and reduce virulence factors [91,92]. 1-(4amino-2-hydroxyphenyl)ethanone (AHE, Figure 13c) was identified from the crude extract of Phomopsis liquidambariS47, and it had strong binding affinity with LasR and RhlR and repressed the transcriptional levels of LasI and RhlI [93]. Many natural plant extracts, for example, the extract of Dalbergia trichocarpa bark (DTB) and aqueous extract of Yunnan Baiyao, showed significant interference with las and rhl of QS signaling circuit [94,95]. There are four phytoconstituents from Cassia fistula influence AHL activity, rhein, 3-aminodibenzofuran, 5-(hydroxymethyl)-2-(dimethoxymethyl)furan and dihydrorhodamine (Figure 13d). They target las and rhl, down-regulate the two system-related genes, and have effects on biofilm formation [96]. Gingerol analogues 44a and 44b (Figure 14a) can reduce biofilm formation effectively and have potent binding affinity to LasR by increasing acyl chain length and modifying absolute configuration [97]. Meanwhile, it can bind to another QS receptor RhlR. Potassium 2-methoxy-4-vinylphenolate 45 (Figure 14a) has a partially similar structure to that of 6-gingerol, and it was found to target P. aeruginosa las and rhl circuitry and to inhibit biofilm formation along with various virulence factors such as LasA protease, LasB elastase and pyocyanin [98]. The potential active compound 46 (Figure 14a) isolated from the extract of Delftia tsuruhatensis SJ01 was identified as 1,2-benzenedicarboxylic acid diisooctylester, having a similar structure with AHL. It competes with LasR and then down-regulates the protease and elastase activity along with the rhl system expression [99]. In 2012, a novel QS inhibitor C2 (N-decanoyl-L-homoserine benzyl ester) (Figure 14b), which belongs to the structural analogues of AHLs, was found to repress the expression of lasR, lasI, rhlR, and rhlI to varying degrees and to inhibit las and rhl [100]. Some compounds containing five-membered rings also show inhibitory activities. Terrein was first isolated from Aspergillus terreus as a secondary bioactive metabolite, 5-hydroxymethylfurfural (5-HMF) (Figure 14b); it can be found in honey, dried fruits, wine, coffee, and so on. These two compounds antagonize both regulatory proteins LasR and RhlR, inhibiting the production of virulence factors and the formation of biofilm [101,102]. Moreover, the extracts of Quercus infectoria gall, Hypericum perforatum L., and olive leaf were also revealed to reduce the expression of las and rhl related genes levels [56,103]. Other extracts, for instance Senna alexandriana mill extracted by acetone and hexane; 3-isobutylhexahydropyrrolo[1,2-a] pyrazine-1,4-dione (47, Figure 14b), a component of Phomopsis tersa, a kind of cranberry extract rich in proanthocyanidins (cerPAC); and chitosan extracted from Aspergillus flavus were demonstrated to interfere with both the rhl pathway and the las pathway [19,104,105]. Several reported drugs exhibit QS inhibitory activities. Ibuprofen belongs to the NSAID compounds, and it is one of the most popular nonprescription drugs; it was discovered that ibuprofen can bind with LuxR, LasR, LasI, and RhlR at high binding scores and inhibit both las and rhl as well as biofilm formation in P. aeruginosa [106]. Aspirin can cause significant reductions of elastase, protease, and so on at sub-MICs, and the antagonist activity of aspirin can be attributed to las and rhl [107]. More recently, there was a new discovery that the FDA-approved drug allopurinol can act as a QS inhibitor against P. aeruginosa, and through molecular docking study, it exerted inhibitory effects by binding LasR and RhlR receptors [108]. Amorfrutins B and synthesized 48a-c (Figure 15a) were analogues of the natural products cajaninstilbene acid (CSA). They can inhibit the las system and las system-related virulence factors by competitively binding with the receptor OdDHL; they can also interrupt pqs and show biofilm inhibitory activity [109,110]. It was found that a caffeic acid derivative with phenolic hydroxyl group 49 (Figure 15a) played the same role as amorfrutins B. It inhibits las and pqs as well and has favorable inhibition of the expression of virulence factors [111]. Through the research and exploration of Chinese herbal medicine, falcarindiol (Figure 15b), the main ingredient of Notopterygiumincisum, was found to have inhibitory effects on both las and pqs. Molecular docking analysis revealed that it competed with natural ligands and combined with LasR [112]. Synthetic furanone C-56 and furanone C-30 ( Figure 15b) are analogues of AHL signaling molecule. They were reported to interfere with N-acylhomoserine lactone and had potent inhibitory ability against QS in P. aeruginosa [113]. According to the previous reports, dihydropyrrol-2-ones (DHP) molecules can be potential QS inhibitors, so both halogenated 50a-c and non-halogenated 50d (Figure 15b) compounds were evaluated. They showed good inhibition of LasR and PqsR receptors; hence, it inhibited las and pqs [114]. Cefoperazone (CFP, Figure 15b) is one of the most widely used β-lactam antibiotics. Some researchers tried to explore some other activities of CFP, and its metallic derivatives cefoperazone iron complex (CFPF) and cefoperazone cobalt complex (CFPC) were found to interact more strongly with LasI, LasR, and PqsR receptors than CFP, CFPF, and CFPC and to eliminated QS-associated virulence factors [115]. Hadizadeh et al. found that oxicams (piroxicam and meloxicam, Figure 15b) can serve as the best inhibitors of LasR from the studied nonsteroidal anti-inflammatory drug (NSAID) compounds and also have potential antagonist activity against PqsE in the pqs system [116]. There were also some other compounds that inhibited both rhl and pqs. 3-phenyllactic acid (PLA, Figure 15c) is produced by Lactobacillus species and strongly binds to receptors RhlR and PqsR. S4 (Figure 15c) was designed to disrupt rhl-pqs crosstalk [117,118]. In addition, sulfur-containing compounds tend to have inhibitory activity. Allicin (diallyl thiosulfinate) (Figure 15c), which was extracted from the edible plant garlic, was found to inhibit C4-HSLs synthesis-related gene rhlI. It also suppresses the expression levels of genes like rhlA, rhlB, and rhlC, which is related to rhamnolipid synthesis and inhibits PQS molecule synthesis [119].
In many cases, the three systems have mutual influence, and partial inhibitors can work on the three systems at the same time. Some are from natural sources. Baicalin (an active natural flavonoid extracted from the traditional Chinese medicinal Scutellaria baicalensis, Figure 16a), soy isoflavones (a group of phenolic compounds rich in soybeans and other legumes, Figure 16a), epigallocatechin-3-gallate (EGCG, a bioactive component of green tea, Figure 16a), and 1H-pyrrole-2-carboxylic acid 51 (a compound isolated from Streptomyces coelicoflavus, Figure 16a) showed inhibitory activities to las, rhl, and pqs and attenuated various virulence phenotypes (Figure 16a) [120][121][122][123]. The bacterial extract rhodamine isothiocyanate (Figure 16a) is a potential bioactive compound that can downregulate QS regulatory genes and virulence-related genes [124]. As we all know, ajoene from garlic can inhibit quorum sensing. On the basis of mimicking the active skeleton of ajoene, Givskov et al. synthesized a series of sulfur-containing ajoene-derived compounds by simply changing substituents. Among those compounds, 52a and 52b (Figure 16b) display efficient inhibitory activity to the las-rhl-pqs system without affecting bacterial activity [125]. Diallyl sulfide (DAS) and diallyl disulfide (DADS) (Figure 16b) from garlic oil work in the same way and inhibit the essential gene expression of the three QS systems and the production of virulence factors [126]. Other compounds such as L-homoserine lactone analogues and phenylurea-containing N-dithiocarbamated homoserine lactones, especially 53 (Figure 16c), can selectively attenuate the expression of virulence factors, swarming motility and biofilm formation. 53 can competed with OdDHL in binding to LasR by molecular docking analysis and also inhibited the expression of the las system genes, rhl system genes, and pqs system genes [127]. The synthesized itaconimides 54a and 54b and EDTA ( Figure 16c) were used as antivirulence compounds that suppress the las, rhl, and pqs systems of P. aeruginosa [128]. Sodium ascorbate is a vitamin that may act as an AHL analogue; it shows strong inhibition of the expression of LasR in the las system, reduces related virulence factors, and then inhibits rhl and pqs [129]. D-cycloserine has a similar parent nucleus structure with the AHL signal molecule, so it effectively suppresses las, rhl, and pqs [130].

Inhibition Mechanism Undetermined
There are also many other compounds that can inhibit QS, but their working mechanisms are not yet clear. For instance, the flavone compound chrysin (Parkia javanica fruit extract) and the anthraquinone derivative emodin (extracted from rhubarb) were found to have significant antibiofilm activities against P. aeruginosa (Figure 17a) [7,131]. Studies showed that terpinen-4-ol (Figure 17b), the main constituent of Melaleuca alternifolia essential oil, displayed inhibitory activity to QS-mediated virulence factors and biofilm formation [132]. Some phenolic compounds such as carvacrol from the oregano essential oil and active compounds tyramine and N-acetyltyramine from marine bacteria (Figure 17b) can be potential quorum-sensing inhibitors by reducing both pyocyanin production and biofilm formation [133]. The plant-sourced unsaturated fatty acid linoleic acid (LA, Figure 17c) was found to inhibit the formation of biofilm without inhibiting the growth of P. aeruginosa [134]. Three sesquiterpene lactones (SLs), 55a-c (Figure 17c) isolated from Argentine herb Centratherum punctatum were reported to show the QS inhibition of P. aeruginosa [135]. The main pyranoanthocyanin 56a (carboxypyranoanthocyanin, red wine extract) and the pure compound carboxypyranocyanidin-3-O-glucoside 56b (Figure 17d) significantly interfered with the expression of several QS-related genes in P. aeruginosa and have potential as QS inhibitors [136]. Another possibility being investigated is synthetic compounds. Compounds with a benzene ring such as three hydrazine-carboxamide hybrid derivatives 57a-c and the synthetic molecule isoeugenol (Figure 18a) show anti-biofilm activity against P. aeruginosa [137,138]. Additionally, a benzalacetone analog 4-methoxybenzalacetone shows the suppression of some virulence phenotypes of P. aeruginosa [139]. Studies reported that the indole derivatives 7-fluoroindole, 7-hydroxyindole, and 3-indolylacetonitrile ( Figure 18a) were potent antivirulence compounds. They reduced the production of QS-regulated virulence factors in P. aeruginosa [39]. Benzimidazole derivatives 5,6-dimethyl 2-aminobenzimidazole 58 and two benzimidazolium salts 59a, 59b (Figure 18b) were found to have inhibition effects on the production of QS-regulated virulence factors and biofilm formation in P. aeruginosa [140,141]. Glyoxamide derivatives 60a-e and 61 (Figure 18c) display great quorum-sensing inhibition activity and exhibit the inhibition of P. aeruginosa biofilms [142]. Some compounds containing a benzene ring and a five-membered heterocycle can possess QS inhibitory activities. For example, 4-(o-methoxyphenyl)-2-aminothiazole (MPAT), 3-amino-2-oxazolidinone derivative YXL-13, and nitrofuran antibiotic furazolidone were identified as showing potent biofilm inhibition (Figure 18d) [143][144][145]. A set of thymi-dine derivatives bearing isoxazole and 1,2,3-triazole rings (TITL) were synthesized, and TITL-8f (Figure 18d) showed the best inhibitory activity against biofilm formation and virulence factors as a promising inhibitor [146]. A novel synthesized molecule, N-(2pyrimidyl)butanamide (Figure 18d), was confirmed to reduce the expression of QS genes and was related to virulence factors in an animal embryo model [147]. A range of novel acetylene analogues of dihydropyrrolones from brominated dihydropyrrolones and acetylene analogues of fimbrolides brominated furanones were synthesized via Sonogashira coupling reactions. Biological test revealed that 62a-c, 63a and 63b (Figure 18d) exhibited higher QS inhibitory activity against P. aeruginosa [4,148]. The flavone acacetin was investigated and showed strong inhibition capacity against the production of AHL-intermediated virulence factors and downregulated QS-related genes, but the exact action mechanism of it is still unknown [149]. Tenoxicam (Figure 18e) plays a role in the reduction of quorumsensing-dependent antivirulence factor production, and therefore affects its pathogenesis in the host [150]. Diethylene triamine pentaacetic acid (DTPA, Figure 18e) is an FDA-approved drug that can also repress the production of elastase [151]. There are also many structurally uncertain substances with QS inhibitory abilities, and most substances are plant extracts. Extracts from four different Chinese herbal plants (Poria cum Radix pini, Angelica dahurica, Rhizomacibotii, and Schizonepeta tenuifolia) and polyphenolic extract from Rosa rugosa tea showede antibiofilm and quorum-sensing inhibitory potential [152,153]. The extracts of Citrus sinensis, Laurus nobilis, Allium cepa, Elettaria cardamomum, and Coriandrum sativum also show potent quorum-quenching effects on P. aeruginosa and eliminate pyocyanin formation [154]. In addition, Acacia seyal Del Bark extracts and plantain herb extracts affect QS-controlled extracellular virulence factors production [54,155]. Piper nigrum containing phenolic and flavonoid components, Punica granatum containing tannins and alkaloids, and Pisum sativum containing tannins and phenolic compounds have quorum-sensing inhibitory activity and can control the formation of biofilm [156]. Ferula oil and Dorema oil, belonging to the Apiaceae family, and grapefruit (Citrus paradisi) essential oils can produce flavonoids, which were found to reduce virulence factor production and interfere with the communication between bacteria [157,158]. Extracts from other natural sources such as the methanol extract of T. bellerica and extracts of Oscillatoria subuliformis, Moringa oleifera, Cuphea carthagenensis, Coccinia indica, and Muntingia calabura L. leaves, have been reported to interfere with the biofilm development and QS of P. aeruginosa [159][160][161][162][163][164]. The extracted metabolites of the endolichenic fungi (ELF) Aspergillus quandricinctus of Usnea longissima affect the activities of elastase and protease and prevent the biofilm formation of P. aeruginosa [165]. Many kinds of oils also can act as inhibitors. Rose, clove, and chamomile essential oils, lemon oil, and pine turpentine were found to work as QSIs [166,167]. Other compounds such as the aromadendrane-type sesquiterpenoid viridiflorol isolated from an Argentinian collection of the liverwort Lepidoziachordulifera showed potent biofilm formation inhibition against P. aeruginosa [167]. Polyphenols from Salix tetrasperma were reported to suppress virulence responses and inhibit the QS of P. aeruginosa [168].

QS Inhibitors from Marine Resources
The ocean is rich in resources, and researchers have reported that many compounds derived from marine organisms are biologically active. Marine sponges make up a large part of marine organisms, which makes them always the subject of research. Three secondary metabolites from marine sponges, manoalide, manoalide monoacetate, and secomanoalide (Figure 19a), were shown to be strong inhibitors of the las QS system [8]. Psammaplin A and bisaprasin (Figure 19b) are psammaplin-type compounds that derive from the marine sponge Aplysinellarhax; they can as QS inhibitors and show inhibitory effects on las and rhl, specifically on LasR and RhlR [169]. Marine plants are also good sources of QS inhibitors. The low molecular weight alginate oligomer (OligoG CF-5/20), which is produced by brown seaweed Laminaria hyperborea, can act as a QS antagonist to las and rhl QS pathway expression in P. aeruginosa and to inhibit biofilm formation [170]. Studies showed that an active constituent of seagrass, Halodulepinifolia, 4-methoxybenzioic acid (Figure 19b), displayed inhibitory activity to QS-mediated virulence factors and biofilm formation [171]. Marine fungi also cannot be ignored as a main source. Equisetin (Figure 19c) was a tetramate-containing secondary metabolite of the marine fungus Fusarium sp. Z10 [18]. 64a-c ( Figure 19c) were isolated from the marine bacteria Oceanobacillus sp. XC22919 [172]. The active compounds tyramine and N-acetyltyramine were from marine bacteria [173]. These compounds were reported to show QS inhibition to P. aeruginosa. Therefore, compounds derived from marine organisms can be promising QS inhibitors and should be a mainstream subject in further research.

Conclusions
Quorum sensing is becoming a global concern; there have been many studies about QSIs. Among the existing reports, QS inhibitors targeting las account for the majority, and QS inhibitors targeting rhl are the least studied. There are many inhibitors that act on multiple systems. Many of the reported QSIs are from natural origins including plants and marine organisms. Apparently, marine biological resources are so rich that there are still many that we have not yet developed, and there are still many unrevealed QSIs from natural sources; therefore, it is worth continuing to explore more novel candidates. Another way to develop new QSIs is to modify the structures of natural products or to modify the structures of signal molecules, and long alkyl chains are important in transforming signal molecules. Moreover, designing compounds with the help of computational technology effectively provides ideas for the synthesis of new inhibitors. Meanwhile, it must be acknowledged that many effective inhibitors have unclear working models. Thus, studying the mechanisms of their bioactivities can be a future research direction. Some existing FDAapproved drugs were found to have inhibitory activity after restudying them. However, there are still many limitations to clinical use because there may be side effects. In general, it is meaningful to study QSIs, as it gives new hope for treating bacterial infections.