Bacterial Alkyl-4-quinolones: Discovery, Structural Diversity and Biological Properties

The alkyl-4-quinolones (AQs) are a class of metabolites produced primarily by members of the Pseudomonas and Burkholderia genera, consisting of a 4-quinolone core substituted by a range of pendant groups, most commonly at the C-2 position. The history of this class of compounds dates back to the 1940s, when a range of alkylquinolones with notable antibiotic properties were first isolated from Pseudomonas aeruginosa. More recently, it was discovered that an alkylquinolone derivative, the Pseudomonas Quinolone Signal (PQS) plays a key role in bacterial communication and quorum sensing in Pseudomonas aeruginosa. Many of the best-studied examples contain simple hydrocarbon side-chains, but more recent studies have revealed a wide range of structurally diverse examples from multiple bacterial genera, including those with aromatic, isoprenoid, or sulfur-containing side-chains. In addition to their well-known antimicrobial properties, alkylquinolones have been reported with antimalarial, antifungal, antialgal, and antioxidant properties. Here we review the structural diversity and biological activity of these intriguing metabolites.


Introduction
The bacterial alkylquinolones are a class of microbial metabolites consisting of a 4-quinolone core, typically substituted with alkyl groups, most often at the 2 position [1]. The best-known of these are the Pseudomonas Quinolone Signal (PQS, O7), a potent modulator of quorum sensing behaviour in the Pseudomonas genus, and its biosynthetic precursor, 4-hydroxy-2-heptylquinoline (HHQ, H7). The discovery of these alkylquinolones begin with the use of a Pseudomonas aeruginosa (Bacillus pyocyaneus) extract by Bouchard for the prevention of anthrax in rabbits [2]. Emmerich and Low later demonstrated the antibiotic activity of the cell-free extracts of P. aeruginosa and named the antibiotic extract 'pyocynase', because at that time the antibiotic activity was attributed to the presence of enzymes [3]. Several years later Hays et al. isolated the actual antibiotic substances from P. aeruginosa and confirmed that they were small molecules. The names Pyo Ib, Ic, II, III and IV were proposed, and their antibiotic activity was demonstrated [4], though it took several years for the exact structures of these compounds to be determined. After studies involving chemical degradation, UV spectrophotometry, and total synthesis, the Pyo compounds were identified as alkylquinolones [5,6].
Initially, only the antimicrobial activity of these molecules was the focus of attention, but in 1999 Pesci et al. made a breakthrough when they found that cell-cell communication in P. aeruginosa was not solely a function of the homoserine lactones. They isolated 3-hydroxy-2-heptylquinolone (O7) and named it the Pseudomonas Quinolone Signal (PQS), describing its activity as an auto-inducer, acting as a pivotal element in the quorum sensing system [12]. Later, other alkylquinolones responsible for quorum sensing were also identified [13]. It was also discovered that P. aeruginosa was not the only microbial species capable of producing alkylquinolones: bacteria belonging to Alteromonas, Pseudoaltermonas and Burkholderia genera have been shown to produce compounds from this class: so far, more than 57 alkylquinolones have been isolated from different sources. The Pseudomonas Quinolone Signal itself has been recently reviewed in detail [14], however, the properties of the wider alkylquinolone class are less well-described. Thus, in this review, we take a look at the microbial alkylquinolones in general, their structural diversity, bioactivity and their role in quorum sensing.

Structural Diversity and Distribution
The central structural motif in the alkylquinolones is the 4-quinoline core, an aromatic nitrogencontaining heterocyclic compound that can participate in both electrophilic and nucleophilic substitution reactions (Figure 1). The quinoline scaffold commonly exists in various natural products, exhibiting a broad range of biological activities [15]. In the context of the alkylquinolones, the quinolone nucleus forms the core around which the diverse substituents are arranged. Most of these molecules have alkyl (both saturated and unsaturated) substitution at position 2, though substitution at the quinolone nitrogen atom and C-3 positions are also relatively common [1]. Many of the quinolones exist in equilibrium between the 4-quinolone and 4-hydroxy-quinoline tautomeric forms [16]; the predominance of one over the other is determined largely by the pH [17,18] (Figure 1). Heeb et al. described a nomenclature based on structural predominance at physiological pH [19]. For the purposes of this review, we have represented the structures in the quinolone form wherever possible. The bacterial quinolones discussed in this review can be divided into six major categories, some of which are widely distributed, whilst others are found in only a few rare microbial strains ( Figure 2).   The first of these are the classical alkyl-4-quinolones, also known as the hydroxyalkylquinolones (HAQs) characterized by HHQ and its derivatives, which are substituted solely at the 2-position. The side-chains are most often linear saturated or monounsaturated alkyl chains. While these are most strongly associated with the Pseudomonas genus, examples have also been reported from the Alteromonas, Pseudoalteromonas, and Burkholderia genera (Table A1). By far the most widely distributed of these compounds is HHQ itself, but numerous other examples have been elucidated over the years. Alkylquinolones with 1, and 4-11-carbon linear chains have been isolated and fully characterized, though MS-based studies have detected the presence of quinolone derivatives with 1-13 carbon side-chains [20][21][22]. Alkylquinolones with odd-numbered numbers of carbons are more commonly found than those with even numbers of carbon atoms, with sevencarbon and nine-carbon examples being particularly prominent; this may reflect their biosynthesis [23]. More recently, several branched chain examples have been described from Pseudomonas (H7a, H8b) [24] and Pseudoalteromonas (H5a, H6a) strains, respectively [22]. Numerous alkylquinolones with unsaturated alkyl chains have also been described (Figure 2), the best known and most widely distributed of which is 2-(Δ1′-nonenyl-)-4-quinolone (H9Δ1), one of the originally reported Pyo series of compounds [6]. The position of the double bond can vary, with quinolones being reported with double bonds at the 1, 2, 3, and 4 positions (Table A1). While the majority of alkylquinolones reported have simple saturated or unsaturated hydrocarbon side chains, a handful of quinolones have been reported with more complex substituents. These include one example containing a cyclopropyl ring in the side chain (H12a), first reported from a P. aeruginosa strain [25], and sulfide-and benzylsubstituted quinolones (H3a, H7b), from a Chinese P. aeruginosa isolate [24]. The first of these are the classical alkyl-4-quinolones, also known as the hydroxy-alkylquinolones (HAQs) characterized by HHQ and its derivatives, which are substituted solely at the 2-position. The side-chains are most often linear saturated or monounsaturated alkyl chains. While these are most strongly associated with the Pseudomonas genus, examples have also been reported from the Alteromonas, Pseudoalteromonas, and Burkholderia genera (Table A1). By far the most widely distributed of these compounds is HHQ itself, but numerous other examples have been elucidated over the years. Alkylquinolones with 1, and 4-11-carbon linear chains have been isolated and fully characterized, though MS-based studies have detected the presence of quinolone derivatives with 1-13 carbon side-chains [20][21][22]. Alkylquinolones with odd-numbered numbers of carbons are more commonly found than those with even numbers of carbon atoms, with seven-carbon and nine-carbon examples being particularly prominent; this may reflect their biosynthesis [23]. More recently, several branched chain examples have been described from Pseudomonas (H7a, H8b) [24] and Pseudoalteromonas (H5a, H6a) strains, respectively [22]. Numerous alkylquinolones with unsaturated alkyl chains have also been described (Figure 2), the best known and most widely distributed of which is 2-(∆1 -nonenyl-)-4-quinolone (H9∆1), one of the originally reported Pyo series of compounds [6]. The position of the double bond can vary, with quinolones being reported with double bonds at the 1, 2, 3, and 4 positions (Table A1). While the majority of alkylquinolones reported have simple saturated or unsaturated hydrocarbon side chains, a handful of quinolones have been reported with more complex substituents. These include one example containing a cyclopropyl ring in the side chain (H12a), first reported from a P. aeruginosa strain [25], and sulfide-and benzyl-substituted quinolones (H3a, H7b), from a Chinese P. aeruginosa isolate [24].
The second class are the Pseudomonas Quinolone Signal (PQS) and its derivatives, which are hydroxylated at the 3 position. PQS itself (O7), which possesses a seven-carbon alkyl chain at the 2-position, was initially reported from a P. aeruginosa strain in 1959, long before its importance as a quorum sensing agent was known [26]. Since then, only a single additional analogue has been isolated, Molecules 2020, 25, 5689 4 of 26 the nonyl-substituted O9 from a Streptomyces species [27], however, other hydroxylated derivatives have been detected using mass spectrometry in several Pseudomonas strains [21,28].
Next are the alkyl-4-quinolone N-oxides (AQNOs), which attracted much of the early attention on the quinolones due to their significant antibacterial activity. The N-oxides also exist as tautomers (hydroxylamine and N-oxide forms). The most commonly encountered examples are the heptyl and nonyl-substituted derivatives, though eight and eleven-carbon analogues have also been reported, along with small suite of unsaturated derivatives (Table A1). To date, bacterial quinolone N-oxides have only been isolated from members of the Pseudomonas genus, with only one exception: a C-3 methylated N-oxide (CN9∆2) produced by an Arthrobacter species [29]. However, a range of unsaturated derivatives have been detected by MS in Burkholderia strains [30].
While the majority of quinolones are alkylated solely at the 2-position, a small number are also alkylated at C-3. The most common of these are a group of quinolones bearing a methyl group at the 3 position, known as hydroxy-methyl-alkylquinolines (HMAQs) or methylalkylquinolones (MAQs), which are widely distributed in Burkholderia species [31]. The first example, 2-(2-heptenyl)-3-methyl-4-quinolone (C7∆2), was reported from Burkholderia pyrrocinia (originally described as Pseudomonas pyrrocinia) in 1967 [32]. Several derivatives have since been discovered: the range of sizes of the C-2 substituents are similar to those for other alkylquinolones, with isolated examples incorporating hydrocarbon chains between 5 and 9 carbon atoms long (Table A1). In contrast to the HAQs, all unsaturated HMAQs reported thus far from the Burkholderia genus possess unsaturation exclusively at the 2-position of the side-chain. A number of C-3 methylated N-oxides have also been detected by metabolic profiling methods (see Section 2), though only one has been isolated and fully characterized [29]. Overall, across all of the quinolone sub-classes substituted at the 2-position, it can be seen that seven and nine-carbon side-chains are the most common ( Figure 3). Special note should be made of an intriguing subclass of the alkylquinolones: those containing prenylated side-chains. In a report by Dekker et al. a suite of quinolones (HG-HGc and CG-CGc) incorporating geranyl-derived side chains were described from a marine Pseudonocardia species, half of which were also methylated at the C-3 position [33]. Some of these compounds were also N-alkylated, a feature common in the plant quinolones but very rare in bacterial examples.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 29 quorum sensing agent was known [26]. Since then, only a single additional analogue has been isolated, the nonyl-substituted O9 from a Streptomyces species [27], however, other hydroxylated derivatives have been detected using mass spectrometry in several Pseudomonas strains [21,28].
Next are the alkyl-4-quinolone N-oxides (AQNOs), which attracted much of the early attention on the quinolones due to their significant antibacterial activity. The N-oxides also exist as tautomers (hydroxylamine and N-oxide forms). The most commonly encountered examples are the heptyl and nonyl-substituted derivatives, though eight and eleven-carbon analogues have also been reported, along with small suite of unsaturated derivatives (Table A1). To date, bacterial quinolone N-oxides have only been isolated from members of the Pseudomonas genus, with only one exception: a C-3 methylated N-oxide (CN9Δ2) produced by an Arthrobacter species [29]. However, a range of unsaturated derivatives have been detected by MS in Burkholderia strains [30].
While the majority of quinolones are alkylated solely at the 2-position, a small number are also alkylated at C-3. The most common of these are a group of quinolones bearing a methyl group at the 3 position, known as hydroxy-methyl-alkylquinolines (HMAQs) or methylalkylquinolones (MAQs), which are widely distributed in Burkholderia species [31]. The first example, 2-(2-heptenyl)-3-methyl-4-quinolone (C7Δ2), was reported from Burkholderia pyrrocinia (originally described as Pseudomonas pyrrocinia) in 1967 [32]. Several derivatives have since been discovered: the range of sizes of the C-2 substituents are similar to those for other alkylquinolones, with isolated examples incorporating hydrocarbon chains between 5 and 9 carbon atoms long (Table A1). In contrast to the HAQs, all unsaturated HMAQs reported thus far from the Burkholderia genus possess unsaturation exclusively at the 2-position of the side-chain. A number of C-3 methylated N-oxides have also been detected by metabolic profiling methods (see Section 2), though only one has been isolated and fully characterized [29]. Overall, across all of the quinolone sub-classes substituted at the 2-position, it can be seen that seven and nine-carbon side-chains are the most common ( Figure 3). Special note should be made of an intriguing subclass of the alkylquinolones: those containing prenylated side-chains. In a report by Dekker et al. a suite of quinolones (HG-HGc and CG-CGc) incorporating geranyl-derived side chains were described from a marine Pseudonocardia species, half of which were also methylated at the C-3 position [33]. Some of these compounds were also N-alkylated, a feature common in the plant quinolones but very rare in bacterial examples. Another class of structurally distinct prenylated quinolones are the quinolone-type aurachins, which are substituted by isoprenoid chains at the C-3 position (Table A2), and have been reported from Stigmatella, Rhodococcus, and Streptomyces species [34][35][36][37]. These are unique in that they are the Another class of structurally distinct prenylated quinolones are the quinolone-type aurachins, which are substituted by isoprenoid chains at the C-3 position (Table A2), and have been reported from Stigmatella, Rhodococcus, and Streptomyces species [34][35][36][37]. These are unique in that they are the only reported bacterial 4-quinolones to possess alkyl chains larger than a methyl group at the C-3 position. The last class, the tetrahydroquinolines, are included here as they commonly co-occur with the alkylquinolones and share a biosynthetic origin (Table A2). To date, they have only been reported from Pseudomonas species [4,8,9,24,25]. The first isolation of 3-n-heptyl-3-hydroxy-1,2,3,4-tetra-hydro quinoline-2,4-dione (T7) and 3-n-nonyl-3-hydroxy-1,2,3,4-tetra-hydroquinoline-2,4-dione (T9) from P. aeruginosa was reported by Hays in 1945 as part of the original Pyo series of quinolones [4]. Subsequently, Kitamura isolated T7 from P. methanica [38]. Budzikiewicz reported that T7 and T9 were formed when P. aeruginosa is grown under iron deficiency [9]. MS profiling studies have revealed the presence of additional analogues with alternative side chains [23].
A handful of bacterial quinolones do not fit into any of these classes. The siderophores quinolobactin and thioquinolobactin are produced by several Pseudomonas fluorescens strains, especially under conditions of iron limitation [39,40]. The metabolite 2,4-dihydroxyquinoline (DHQ) has been reported from both Pseudomonas aeruginosa and Streptomyces sindenensis [20,27].

Metabolic Profiling
In addition to those quinolones that have actually been isolated and characterized, numerous additional derivatives have been detected by MS-based metabolic profiling. One of the first of these studies was conducted by Taylor et al. in 1995, where GCMS profiling of a clinical isolate of Pseudomonas aeruginosa revealed the presence of a large number of alkylquinolones (Table A3) [20]. The major components were the saturated quinolones H7 and H9, in addition to a number of unsaturated analogues with 7, 9, and 11 carbon-chains. GC-MS profiling was also used during the course of a biosynthetic study by Brendenbruch et al., which revealed the presence both PQS and AQ derivatives [28]. Lepine et al. used an LCMS-based method for the analysis of a P. aeruginosa strain, which revealed several series of quinolone derivatives: including saturated and unsaturated HAQ derivatives, N-oxides, and tetrahydroquinolines. Perhaps most significant were the first detections of C-3 hydroxylated quinolones that were not PQS itself. In a 2017 study, Depke et al. used a new MS-based unsupervised clustering method to analyse an extract from P. aeruginosa PA14, identifying a wide range of HAQ, AQNOs, and PQS derivatives. Most notable was the first detection of quinolones containing polyunsaturated side-chains, though only for the longer-chain derivatives (C9 and higher) [41]. In a recent study, a rapid LC-MS method was developed for the analysis of both microbial strains and clinical samples: a total of 28 quinolone derivatives were detected [42]. Other genera also produce quinolones: LC-MS analysis of a marine Pseudoalteromonas isolate revealed a suite of saturated alkylquinolone derivatives, including two branched-chain derivatives [22].
Burkholderia strains have also been studied-in a 2008 report eleven different Burkholderia strains were profiled using LCMS, three of which produced a range of medium-chain HMAQs, HAQs, and methyl-alkylquinoline N-oxides (MAQNOs), with dramatically different distributions between the three species [31]. In a biosynthetic study on the effects of KynB on quinolone biosynthesis in Burkholderia pseudomalleii, LC-MS profiling was carried out, revealing a similar chemical profile to the prior study [43]. A molecular networking study on the effects of the antibiotic trimethoprim on the secondary metabolites of a Burkholderia thailandensis isolate revealed a small suite of C-3 methylated and non-methylated alkylquinolones [44]. A 2020 report described the LC-MS analysis of quinolone derivatives in three microbial strains by multiple reaction monitoring (MRM). With the aid of synthetic standards the authors were able to confirm the location of the double bonds in the unsaturated derivatives: all quinolones with an unsaturated side chain produced by the Burkholderia strains possessed a double bond at the 2 -position, while those produced by Pseudomonas species had double bonds at either the 1 or 2 positions [30].

Biosynthesis
Over the past few decades, the biosynthesis of the alkylquinolones has been elucidated [21,45]. The synthesis of HHQ and PQS ( Figure 4) is initiated by the coenzyme (CoA) ligase PqsA, which catalyzes the activation of anthranilic acid with ATP/Mg 2+ to produce the intermediate anthranilyl-AMP Although 2-ABA-CoA is highly susceptible to spontaneous cyclization to form 2,4-dihydroxyquinoline (DHQ), which has been shown to be fundamental in P. aeruginosa pathogenicity [50], in vivo this is counterbalanced by the activity of PqsE, which acts as a 2-ABA-CoA thioesterase to release 2-aminobenzoylacetate (2-ABA) [45]. 2-ABA is another branching point in the pathway: it can undergo decarboxylation to 2-aminoacetophenone (2-AA), a secondary metabolite reported to promote chronic infection phenotypes of P. aeruginosa and to modulate the host innate immune response. 2-ABA is transformed into HHQ by the heterodimeric PqsBC bearing an octanoyl chain. Finally, the flavin monooxygenase PqsH oxidizes HHQ into PQS [51]. In addition, 2-ABA could be converted into its hydroxylamine form by the oxidase PqsL and subsequently transformed into 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) by the octanoyl-PqsBC complex [52].

Biological Activity
With their structural diversity, the alkylquinolones also bring a significant number of biological effects. Although most recently the focus has centred on their quorum sensing properties of alkylquinolones, these molecules were initially discovered as antimicrobial agents. Since then, many of these compounds have been isolated for their antibiotic, antifungal and anti-algal activity against human, plant and animal pathogens.

Earlier Discoveries
Based on the works of Bouchard, Emmerich and Löw, Hays et al. described their antibiotic effect and partially characterized the alkylquinolone antibiotics produced by P. aeruginosa [4]. They employed serial-dilution assays to test the antibiotic effect of crude extracts, which were later identified as mixtures of 2-alkyl-4-quinolones and their N-oxides [4]. These compounds were found to be highly active against Gram-positive bacteria but showed only slight activity against Gram-negative bacteria [4]. It was also shown that the N-oxides N7-N9, N11 (designated as Pyo-II by Hays et al.) were ten times more potent than the reduced compounds, displaying bactericidal activity at high concentrations and bacteriostatic effects at lower concentrations [4,6,7]. Lightbrown and Jackson later reported that the N-oxides are also potent antagonists of streptomycin and dihydrostreptomycin, and inhibit the cytochrome systems of heart muscle and certain bacteria by interfering with the respiratory chain [53,54].

Antibacterial Activity
After the earlier discoveries and introduction of quinolones as front-line antibiotics, a hunt for new natural antimicrobial molecules based on the quinolone scaffold started [16]. The naturally occurring alkylquinolones have proven to be interesting starting point for synthesis of molecules with broad spectrum of activity and less toxicity [16,19]. Wratten et al. reported the isolation of HHQ (H7) and its shorter congener, 2-n-pentyl-4-quinolone (H5/PQ) from a marine Pseudomonas bromoutalis isolate. These molecules showed antibiotic activity against both Gram-positive and Gram-negative bacteria including Staphylococcus aureus, and the common marine pathogens, Vibrio harveyi and Vibrio anguillarum [55]. While investigating the ecological and biogeochemical role of a previously-reported alkylquinolone, 2-n-pentyl-4-quinolone (H5/PQ) from a marine Alteromonas sp. SWAT 5, it was determined that H5 inhibits the growth and motility of several particle-associated bacteria from different phyla including α-Proteobacteria, Bacteroidetes and γ-Proteobacteria. It was also found that H5 targets DNA synthesis and motility at concentrations as low as 10 µM [56]. In a 1998 report, Debitus and co-workers isolated several quinolone derivatives (H7, H9, H9∆1, T7, N7) from a sponge-derived Pseudomonad, with H7 displaying strong antimicrobial activity [57].
Homma et al. discovered that Pseudomonas cepacia RFM25 (subsequently reclassified as Burkholderia cepacia) was inhibitory to several soil borne plant pathogens [58]. Later they managed to isolate 2-(2-heptenyl)-3-methyl-4-quinolone (C7∆2), previously isolated by Hashimoto and Hattori, and 2-(2-nonenyl)-3-methyl-4-quinolone (C9∆2) from the same strain [32]. Both molecules displayed similar activity against Corynebacterium michiganense [58]. Helicobacter pylori infection is one of the most common causes of gastritis and peptic ulcers. While exploring the effects of extracellular substances produced by different bacteria on H. pylori it was found that a clinical strain of P. aeruginosa inhibited the growth of H. pylori. The active fractions were found to contain 2-heptyl-quinolone (H7), 2-nonyl-quinolone (H9) and their corresponding N-oxides. H7 and N7 were found to be active against metronidazole and metronidazole resistant strains of H. pylori with MIC values of 0.1-0.5 mg/mL in an agar-well plate assay [59]. Another group of bacterial quinolones with antimicrobial activity against H. pylori are the geranylated quinolones (CG-CGc; HG-HGc) isolated from Pseudonocardia spp. CL38489, several of which possessed nanomolar activity. The epoxide derivative 3-dimethyl-2-(6,7-epoxygeranyl)-4-hydroxy-quinolone (CGb) was found to be the most potent of all isolates, with a MIC of 0.1 ng/mL [33]. Because of their inactivity against other microbes, and potential of posing no potential harm to normal gut flora, these molecules hold great promise in clinical settings to be used as anti-ulcer agents. One possible explanation for their selective action was postulated to be the selective inhibition of a part of microaerophilic respiratory chain of H. pylori.
Later, 4-hydroxy-3-methyl-2(1H)-quinolone (CX1) was isolated from Burkholderia sp. 3Y-MMP [66]. This compound was previously isolated as a plant metabolite from Isatis tinctoria (Brassicaceae) and displayed anti-tuberculosis activity with an IC 90 of 6.8 µM [66][67][68]. An extensive isolation report in 2020 described the isolation of six new HAQs, including the highly unusual sulfide and benzyl derivatives H3a and H7b, in addition to fifteen known AQs, three AQNOs, and the tetrhydroquinoline T7 [24]. While no significant biological studies were reported in the initial isolation report, a subsequent synthetic investigation revealed that the natural products H3a, H7b and H7∆2 inhibited the growth of S. aureus, with the latter reducing growth by over 80% at 1.1 µM. The latter two compounds also reduced the swarming motility of B. subtilis [69].
Kunze et al. described the antimicrobial effect of the aurachins, several of which of are isoprenoid quinolone alkaloids characterized by a farnesyl residue, from the myxobacterium Stigmatella aurantiaca [34]. Aurachins C (AC15) and D (AD15) were shown to block NADH oxidation and inhibit the growth of several gram-positive bacteria including B. subtilis, S. aureus, Arthrobacter aurescens, Brevibacterium ammoniagenes, and Corynebacterium fascians; potentially by interfering with bacterial respiration [34]. Additional antimicrobial aurachins have been reported: Kitagawa et al. isolated aurachin RE (ARE15) from Rhodococcus erythropolis JCM 6824 which exhibited a broader and more potent activity than aurachin C [34,35]. It was found to inhibit both Gram positive (B. subtilis, Nocardia pseudosporangifera, Streptomyces griseus) and Gram-negative bacteria (Sinorhizobium meliloti and Deinococcus grandis). Aurachins Q (AQ15) and R (AR15) from Rhodococcus sp. Acta 2259 displayed low to moderate activity against S. epidermidis, B. subtilis and P. acnes, respectively [34][35][36]. The O-methylated aurachin SS (ASS10), isolated from a Streptomyces strain, possessed reduced antimicrobial activity compared to aurachins C and D, though it is unclear whether this is due to the presence of the methoxy group, or the shortened side-chain [37].
Many researchers have studied the structure-activity relationships of the synthetic quinolone antibiotics [70], but the relationships of the bacterial alkylquinolones have not been as well-established. While it has been commonly observed that the alkylquinolone N-oxides (AQNOs) have a broader spectrum of activity than their reduced counterparts it is clear that the length, degree of unsaturation and branching of the alkyl chain also play a role. For example, Szamosvári (2017) reported that the unsaturated compound trans-(non-1-enyl)-4-quinolone N-oxide exhibited up to 20-fold higher bacterio-static activity against Staphylococcus aureus strains than the most potent saturated AQNO [71]. Due to the diverse test organisms and methods used in the studies overviewed in this section, it is difficult to establish firm rules for antibacterial activity. Nonetheless, alkylquinolones possessing an unsaturated chain and either N-methylation or N-oxidation tend to have improved antimicrobial properties. Alkylquinolones with unsaturated carbon chains and methylation at R-3 do tend to possess good anti-bacterial activity, but such structural modifications are not essential for good anti-bacterial activity [32,64]. An unusual example seems to be the geranylated quinolones, which possess potent and selective activity against H. pylori, with epoxidation of the geranyl residue (CGb) significantly increased the potency of the molecule [33]. For the aurachins, the addition of a double bond at position 4 of the prenylated chain (AQ15, AR15) results in loss of activity, whereas addition of hydroxy group in the side-chain (ARE15) increased the spectrum of activity to a great extent [34,35].

Antifungal and Anti-Oomycete Activity
Fungal and oomycete pathogens cause massive economic losses by effecting crops in both temperate and tropical areas.
During a co-culture experiment it was found that P. aeruginosa exhibited a fungicidal effect on Cryptococcus neoformans. On further investigation it was discovered that the anti-fungal action was due to pyocyanin and the alkylquinolones PQS (O7) and HHQ (H7) produced in the co-culture [77].The quinolones H1, H7, H9, H9∆1, H11, O7 and O9 from an alkali-tolerant Streptomyces sindenensis were found to inhibit the growth of C. albicans one of the most prevalent causes of opportunistic fungal infections in humans [27]. Aurachins C and D were also shown to inhibit the growth of the yeasts Saccharomyces cerevisiae and Debaryomyces hansenii [34]. A small suite of quinolones (C7, C7∆2, H9∆2, C9∆2), isolated from a Burkholderia species, displayed antifungal activity against the fungal pathogens Rhizopus oryzae and Trichophyton rubrum [64]. Mossialos et al. discovered the iron-chelating properties of quinolobactin, isolated from a Pseudomonas fluorescens ATCC 17400 strain [39]. Although quinolobactin (QB), originally reported along with thioquinolobactin in 1980 [78], did not show any significant activity besides iron chelation it was later demonstrated that quinolobactin is a produced by the hydrolysis of the unstable but highly active 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Thioquinolobactin (TQB) not only possesses iron chelation properties but also significantly inhibits the growth of Pythium debaryanum, Rhizoctonia solani and Sclerotinia sepivorium [40]. Later Kilani-Feki et al. also isolated C7 and C7∆2 from B. cepacia and demonstrated their activity against common food pathogen Aspergillus niger [79,80]. C9∆2 from outer membrane vesicles (OMVs) of B. thailandensis displayed anti-fungal activity at 100 µM against C. albicans and Cryptococcus neoformans [63]. Microbial alkylquinolones C7∆2, C8∆2, C9∆2, obtained by employing Conrad−Limpach approach and Suzuki−Miyaura coupling reactions, showed moderate activity against C. neoformans [65]. Overall, for anti-fungal and anti-oocyte activity of the alkylquinolones, methylation of position-3 and the presence of an unsaturated alkyl chain at C-2 are strong predictors of antifungal activity.

Quorum Sensing/Biofilm Formation
The ability of bacteria to communicate and act as a community for collective tasks was underestimated for many years. It was believed that these organisms act on the principle of 'every cell for itself' but later it was realized that the bacterial cells communicate by a phenomenon called quorum sensing, or auto-induction [81]. The concentrations of auto-inducer molecules released by a single bacterium are insufficient to bring about behavioural and metabolic changes. Thus, this phenomenon occurs in a cell density-dependent manner, where the bacteria must reach a critical mass necessary for collective action, to activate or supress target genes by releasing auto-inducers [82]. The collective action of such molecules and the regulation of an array of genes help these bacteria move to a more friendly environment (with better nutrients), adapt a new growth strategy, or aid in protection from harsh/deleterious environments by biofilm formation [82].
At a transcriptional level, the biosynthesis of alkylquinolones is controlled by PqsR, which activates the expression of pqsABCDE and phnAB operons in P. aeruginosa [85]. A homologue of the pqsABCDE operon named hmqABCDEFG has been found in Burkholderia spp. (Figure 4) [31,86]. Some of these autoinducers i.e., PQS and HHQ, regulate their own expression by binding to PqsR itself [87]. It has been revealed that HHQ induces a conformational change in PqsR, as binding of PqsR to the pqsA promoter in vitro is enhanced by HHQ, although not as much as with PQS [88]. Other AQs such as 2-nonyl-4-quinolone (H9) can also activate PqsR and as such could potentially be considered as autoinducers, although not as potent as PQS [84]. Alkylquinolones are highly lipophilic molecules, which might hinder their ability to act as effective signaling molecules, however, several of the biochemical changes they induce work to minimize this problem. PQS increases the production of rhamnolipids, which increases the solubility of this lipophilic molecule within aqueous solutions [89]. Another induced change that may help to overcome solubility issues is via promoting the biogenesis of outer membrane vesicles (OMVs) that package the highly lipophilic PQS for transport within the population [90]. It has been found that the third hydroxy position is absolutely critical for the formations of OMVs and is the reason why PQS and not HHQ can stimulate OMV formation [91]. The kinetics of both HAQs and HMAQs show that these molecules start to accumulate near the end of the log phase of growth of the respective bacteria [31].
Besides regulating adaptation responses and signal integration, these alkylquinolone derivatives are primarily involved in the regulation of various genes responsible for virulence. The virulence factors mainly regulated by these auto-inducers include elastase, pyocyanin, hydrogen cyanide, rhamnolipids and LecA lectin [86,92]. The production of these molecules gives a competitive advantage to the bacterium producing them [93]. This advantage is demonstrated by modulating the swarming motility, growth repression by depriving the rival bacteria of iron i.e., iron chelation, cytotoxicity via production of reactive oxygen species, and regulating the production of antimicrobial AQs like 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) (N7) [91,[94][95][96][97][98]. Although not an actual quorum sensing molecule itself, HQNO has been found to induce antibiotic tolerance by triggering programmed cell death. The DNA released due to cell death can induce biofilm formation making the bacteria resistant to antibiotics. This antibiotic tolerance can be triggered not only in the species producing the molecule i.e., P. aeruginosa, but also in the bacteria in the surrounding environment e.g., S. aureus [99,100]. It should be noted that the growth repression function is distinct from bacteriostatic or bactericidal effect of antibiotics [94]. Biofilm formation is a complex phenomenon, the mechanism of which is not fully understood, but it has been inferred that alkylquinolone quorum sensing molecules promote biofilm formation via LecA activation and/or formation of extracellular DNA and may also promote or inhibit the biofilm formation in other bacteria [97,101,102].
Reen et al. discovered that the C-3 position is crucial for the wide range of activities including production of virulence factors and biofilm formation exhibited by PQS and HHQ. Absence or substitution of this position with halogens or an NH group results in loss of activity in modulating inter-species and intra-species behaviour [103]. It has also been observed that PQS not only acts by transcriptional regulation within the cells but may also interact directly with the proteins e.g PQS binds with MexG (RND-type efflux pump) and MgtA (Mg 2+ transporter) [104]. It has also been demonstrated that the two quorum sensing systems in Pseudomonas and Burkholderia spp. work interdependently. The acyl-homoserine lactones (AHLs) regulate the expression of HAQs via LasR and RhlR which bind to PqsR [105]. On the other hand, hydroxymethyl-alkylquinolines (HMAQs) found in Burkholderia spp. regulate the expression of AHLs and it has been demonstrated that the methyl group of HMAQs is essential for this function [31]. The role of quinolones in quorum sensing, virulence, and interspecies interactions is summarized in Figure 5.
Molecules 2020, 25, x FOR PEER REVIEW 12 of 29 found in Burkholderia spp. regulate the expression of AHLs and it has been demonstrated that the methyl group of HMAQs is essential for this function [31]. The role of quinolones in quorum sensing, virulence, and interspecies interactions is summarized in Figure 5.

Miscellaneous Activities
Alkylquinolones have mostly been analyzed for their antimicrobial and quorum sensing abilities. But these unique quinolones have proven to be very diverse compounds in terms of their activity despite of their limited structural diversity. 2-nonyl-4-quinolone (H9), one of the oldest alkylquinolones known from P. aeruginosa was isolated from Brazilian shrub Raulinoa echinata by

Miscellaneous Activities
Alkylquinolones have mostly been analyzed for their antimicrobial and quorum sensing abilities. But these unique quinolones have proven to be very diverse compounds in terms of their activity despite of their limited structural diversity. 2-nonyl-4-quinolone (H9), one of the oldest alkylquinolones known from P. aeruginosa was isolated from Brazilian shrub Raulinoa echinata by Biavatti et al. in 2002 and showed moderate antitrypanosomal activity against T. cruzi with an IC 50 100.9 µg/mL [6,107]. 2-undecyl-4-quinolone isolated from Pseudomonas sp. 1531 E7 displayed antiviral against HIV at an ID 50 of 10 −3 µg/mL.
2-n-Heptyl-4-hydroxy-quinoline-N-oxide (N7), 2-n-heptyl-4-quinolone (H7), 3-n-heptyl-3hydroxy-1,2,3,4-tetrahydroquinoline-2,4-dione (T7) were analyzed for their anti-asthma activity via 5-lipooxygenase. Although all these compounds showed 5-lipooxygenase inhibition activity, N7 proved to be the most potent and selective inhibitor (IC 50 = 1.5 × 10 −7 M) [38]. Later N7 was also shown to significantly suppress the antigen-induced bronchoconstriction in guinea pigs and inhibition of histamine release [108]. 2-n-heptyl-4-quinolone (H7) from marine Pseudoalteromonas sp. M2 was investigated for its anti-inflammatory activity and proved to be a very promising candidate for neuro-inflammatory disorders, owing to its ability to inhibit NO, ROS production and the expression of iNOS and COX-2 [109,110]. The same compound (H7) proved to be the most potent inhibitor of melanin synthesis when tested for anti-melanogenic activity. A range of AQs also showed strong activity against Hep C virus with an IC 50 of 1.4 ± 0.2 µg/mL and no cytotoxicity. This activity was stronger than that of ribavirin and an investigation into the mechanism of action revealed that H9 acts against HCV by inhibiting the viral entry and replication [111]. H9 has also been found to possess iron-chelation properties [112]. Aurachins, including AC12 and AD12 from Rhodococcus sp. Acta 2259 showed weak inhibition of glycogen-synthase-kinase 3β (GSK-3β) which could lead to exploration of these molecules for treatment of neurodegenerative and other disorders caused by perturbation of GSK-3β [36].

Conclusions
Overall, the alkylquinolones are a chemically and biologically diverse class of compounds. Initially discovered from the Pseudomonas genus, recent studies have shown that these compounds are distributed amongst several genera, including not only Burkholderia, but also more distantly related genera such as Streptomyces and Rhodococcus. Likewise, while many of the earlier-discovered alkylquinolones incorporated only linear C-2 side chains, more recent isolation studies have greatly diversified the number of structural motifs encountered in this structure class. While the majority of studies have focused on their antimicrobial properties (Table A1 in Appendix A, Figure 6), it is clear that these compounds have potential as anti-fungal, anti-malarial, and anti-inflammatory agents as well, and that future investigators would do well to broaden the scope of biological activities under investigation.

Conflicts of Interest:
The authors declare no conflict of interest.