Amide bioisosteric replacement in the design and synthesis of quorum sensing modulators.

The prevention or control of bacterial infections requires continuous search for novel approaches among which bacterial quorum sensing inhibition is considered as a complementary antibacterial strategy. Quorum sensing, used by many different bacteria, functions through a cell-to-cell communication mechanism relying on chemical signals, referred to as autoinducers, such as N-acyl homoserine lactones (AHLs) which are the most common chemical signals in this system. Designing analogs of these autoinducers is one of the possible ways to interfere with quorum sensing. Since bioisosteres are powerful tools in medicinal chemistry, targeting analogs of AHLs or other signal molecules and mimics of known QS modulators built on amide bond bioisosteres is a relevant strategy in molecular design and synthetic routes. This review highlights the application of amide bond bioisosteric replacement in the design and synthesis of novel quorum sensing inhibitors.

ß-Cyclodextrin Encapsulation of Synthetic AHLs: Drug Delivery Implications and Quorum-Quenching Exploits.

Many bacteria, such as Pseudomonas aeruginosa, regulate phenotypic switching in a population density-dependent manner through a phenomenon known as quorum sensing (QS). For Gram-negative bacteria, QS relies on the synthesis, transmission, and perception of low-molecular-weight signal molecules that are predominantly N-acyl-l-homoserine lactones (AHLs). Efforts to disrupt AHL-mediated QS have largely focused on the development of synthetic AHL analogues (SAHLAs) that are structurally similar to native AHLs. However, like AHLs, these molecules tend to be hydrophobic and are poorly soluble under aqueous conditions. Water-soluble macrocycles, such as cyclodextrins (CDs), that encapsulate hydrophobic guests have long been used by both the agricultural and pharmaceutical industries to overcome the solubility issues associated with hydrophobic compounds of interest. Conveniently, CDs have also demonstrated anti-AHL-mediated QS effects. Here, using fluorescence spectroscopy, NMR spectrometry, and mass spectrometry, we evaluate the affinity of SAHLAs, as well as their hydrolysis products, for ß-CD inclusion. We also evaluated the ability of these complexes to inhibit wild-type P. aeruginosa virulence in a Caenorhabditis elegans host infection study, for the first time. Our efforts confirm the potential of ß-CDs for the improved delivery of SAHLAs at the host/microbial interface, expanding the utility of this approach as a strategy for probing and controlling QS.

Quorum sensing N-Acyl homoserine lactones are a new class of anti-schistosomal.

BACKGROUND: Schistosomiasis is a prevalent neglected tropical disease that affects approximately 300 million people worldwide. Its treatment is through a single class chemotherapy, praziquantel. Concerns surrounding the emergence of praziquantel insensitivity have led to a need for developing novel anthelmintics. METHODOLOGY/PRINCIPLE FINDINGS: Through evaluating and screening fourteen compounds (initially developed for anti-cancer and anti-viral projects) against Schistosoma mansoni, one of three species responsible for most cases of human schistosomiasis, a racemic N-acyl homoserine (1) demonstrated good efficacy against all intra mammalian lifecycle stages including schistosomula (EC50 = 4.7 µM), juvenile worms (EC50 = 4.3 µM) and adult worms (EC50 = 8.3 µM). To begin exploring structural activity relationships, a further 8 analogues of this compound were generated, including individual (R)- and (S)- enantiomers. Upon anti-schistosomal screening of these analogues, the (R)- enantiomer retained activity, whereas the (S)- lost activity. Furthermore, modification of the lactone ring to a thiolactone ring (3) improved potency against schistosomula (EC50 = 2.1 µM), juvenile worms (EC50 = 0.5 µM) and adult worms (EC50 = 4.8 µM). As the effective racemic parent compound is structurally similar to quorum sensing signaling peptides used by bacteria, further evaluation of its effect (along with its stereoisomers and the thiolactone analogues) against Gram+ (Staphylococcus aureus) and Gram- (Escherichia coli) species was conducted. While some activity was observed against both Gram+ and Gram- bacteria species for the racemic compound 1 (MIC 125 mg/L), the (R) stereoisomer had better activity (125 mg/L) than the (S) (>125mg/L). However, the greatest antimicrobial activity (MIC 31.25 mg/L against S. aureus) was observed for the thiolactone containing analogue (3). CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this is the first demonstration that N-Acyl homoserines exhibit anthelmintic activities. Furthermore, their additional action on Gram+ bacteria opens a new avenue for exploring these molecules more broadly as part of future anti-infective initiatives.

Identification and Quantification of N-Acyl Homoserine Lactones Involved in Bacterial Communication by Small-Scale Synthesis of Internal Standards and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. Graphical abstract ᅟ.

Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume-Rhizobia Symbiosis.

Density-dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram-negative bacteria, QS is regulated by N-acylated-l-homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host-symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume-rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS-reporter line of S. meliloti and nodulation assays with wild-type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host.

Solid-phase synthesis and biological evaluation of N-dipeptido L-homoserine lactones as quorum sensing activators.

Bacteria use small signaling molecules to communicate in a process termed "quorum sensing" (QS), which enables the coordination of survival strategies, such as production of virulence factors and biofilm formation. In Gram-negative bacteria, these signaling molecules are a series of N-acylated L-homoserine lactones. With the goal of identifying non-native compounds capable of modulating bacterial QS, a virtual library of N-dipeptido L-homoserine lactones was screened in silico with two different crystal structures of LasR. The 30 most promising hits were synthesized on HMBA-functionalized PEGA resin and released through an efficient acid-mediated cyclative release mechanism. Subsequent screening for modulation of QS in Pseudomonas aeruginosa and E. coli identified six moderately strong activators. A follow-up library designed from the preliminary derived structure-activity relationships was synthesized and evaluated for their ability to activate the QS system in this bacterium. This resulted in the identification of another six QS activators (two with low micromolar activity) thus illuminating structural features required for QS modulation.

Selective imaging of quorum sensing receptors in bacteria using fluorescent Au nanocluster probes surface functionalized with signal molecules.

Fluorescent ultrasmall gold clusters decorated with bacterial quorum sensing signal molecules, acyl homoserine lactone, are synthesized. These fluorescent probes are found to have emission in the near-infrared spectral region advantageous for bioimaging. Imaging studies using different strains of bacteria with and without acyl homoserine lactone receptors with the aid of confocal microscopy have shown that the probe interacts preferentially with cells possessing these receptors. This indicates that, with appropriate surface functionalization, the Au clusters can be used for receptor specific detection with enhanced selectivity.

N-acylated alanine methyl esters (NAMEs) from Roseovarius tolerans, structural analogs of quorum-sensing autoinducers, N-acylhomoserine lactones.

The Roseobacter clade is one of the most important bacteria group living in the ocean. Liquid cultures of Roseovarius tolerans EL 164 were investigated for the production of autoinducers such as N-acylhomoserine lactones (AHLs) and other secondary metabolites. The XAD extracts were analyzed by GC/MS. Two AHLs, Z7-C14 : 1-homoserine lactone (HSL) and C15 : 1-HSL, were identified. Additionally, the extract contained five compounds with molecular-ion peaks at m/z 104, 145, and 158, thus exhibiting mass spectra similar to those of AHLs with corresponding peaks at m/z 102, 143, and 156. Isolation of the main compound by column chromatography, NMR analysis, dimethyl disulfide derivatization for the determination of the location of the CC bond and finally synthesis of the compound with the proposed structure confirmed the compound to be (Z)-N-(hexadec-9-enoyl)alanine methyl ester. Four additional minor compounds were identified as C14 : 0-, C15 : 0-, C16 : 0-, and C17 : 1-N-acylated alanine methyl esters (NAMEs). All NAMEs have not been described from natural sources before. A BLASTp search showed the presence of AHL-producing luxI genes, but no homologous genes potentially responsible for the structurally closely related NAMEs were found. The involvement of the NAMEs in chemical communication processes of the bacteria is discussed.

Modified N-acyl-homoserine lactones as chemical probes for the elucidation of plant-microbe interactions.

Gram-negative bacteria often use N-acyl-homoserine lactones (AHLs) as signal molecules to monitor their local population densities and to regulate gene-expression in a process called "Quorum Sensing" (QS). This cell-to-cell communication allows bacteria to adapt to environmental changes and to behave as multicellular communities. QS plays a key role in both bacterial virulence towards the host and symbiotic interactions with other organisms. Plants also perceive AHLs and respond to them with changes in gene expression or modifications in development. Herein, we report the synthesis of new AHL-derivatives for the investigation and identification of AHL-interacting proteins. We show that our new compounds are still recognised by different bacteria and that a novel biotin-tagged-AHL derivative interacts with a bacterial AHL receptor.

Fluorescent labeling agents for quorum-sensing receptors (FLAQS) in live cells.

Lighten up: A selective fluorescent-labeling agent for quorum sensing (FLAQS) can be used for the visualization of the communication pathway of bacteria in live cells (see figure). This represents a new, operationally simple, fast, and inexpensive tool for the imaging of quorum-sensing receptors by using fluorescently labeled signaling-molecule analogues.

Synthesis and biological evaluation of triazole-containing N-acyl homoserine lactones as quorum sensing modulators.

Many bacterial species are capable of assessing their local population densities through a cell-cell signaling mechanism termed quorum sensing (QS). This intercellular communication process is mediated by small molecule or peptide ligands and their cognate protein receptors. Numerous pathogens use QS to initiate virulence once they achieve a threshold cell number on a host. Consequently, approaches to intercept QS have attracted considerable attention as potential anti-infective therapies. Our interest in the development of small molecule tools to modulate QS pathways motivated us to evaluate triazole-containing analogs of natural N-acyl L-homoserine lactone (AHL) signals as non-native QS agonists and antagonists in Gram-negative bacteria. We synthesized 72 triazole derivatives of five broad structure types in high yields and purities using efficient Cu(I)-catalyzed azide-alkyne couplings. These compounds were evaluated for their ability to activate or inhibit two QS receptors from two prevalent pathogens - LasR from Pseudomonas aeruginosa and AbaR from Acinetobacter baumannii- using bacterial reporter strains. Several triazole derivatives were identified that were capable of strongly modulating the activity of LasR and AbaR. These compounds represent a new and synthetically accessible class of AHL analogs, and could find utility as chemical tools to study QS and its role in bacterial virulence.

Imidazolines as non-classical bioisosteres of N-acyl homoserine lactones and quorum sensing inhibitors.

A series of selected 2-substituted imidazolines were synthesized in moderate to excellent yields by a modification of protocols reported in the literature. They were evaluated as potential non-classical bioisosteres of AHL with the aim of counteracting bacterial pathogenicity. Imidazolines 18a, 18e and 18f at various concentrations reduced the violacein production by Chromobacterium violaceum, suggesting an anti-quorum sensing profile against Gram-negative bacteria. Imidazoline 18b did not affect the production of violacein, but had a bacteriostatic effect at 100 µM and a bactericidal effect at 1 mM. Imidazoline 18a bearing a hexyl phenoxy moiety was the most active compound of the series, rendering a 72% inhibitory effect of quorum sensing at 100 µM. Imidazoline 18f bearing a phenyl nonamide substituent presented an inhibitory effect on quorum sensing at a very low concentration (1 nM), with a reduction percentage of 28%. This compound showed an irregular performance, decreasing inhibition at concentrations higher than 10 µM, until reaching 100 µM, at which concentration it increased the inhibitory effect with a 49% reduction percentage. When evaluated on Serratia marcescens, compound 18f inhibited the production of prodigiosin by 40% at 100 µM.

Deuterium-labelled N-acyl-L-homoserine lactones (AHLs)--inter-kingdom signalling molecules--synthesis, structural studies, and interactions with model lipid membranes.

N-Acyl-L-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-L-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum's acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein.

Functional properties of synthetic N-acyl-L-homoserine lactone analogs of quorum-sensing gram-negative bacteria on the growth of human oral squamous carcinoma cells.

Quorum sensing is defined as the ability of microorganisms to sense their population density via the release of signaling molecules composed of acyl-homoserine lactone (AHL), which is a type of autoinducer (AI). Previous structure-activity relationship (SAR) studies demonstrated that the 3-oxo group, homoserine lactone of L-form, and long acyl side chain have crucial roles in apoptosis induction. Various types of synthetic AI analogs of Pseudomonas aeruginosa were prepared, and SAR study was conducted to determine their effects against human oral squamous carcinoma cells derived from gingival carcinoma Ca9-22 cells and tongue cancer SAS cells. Not only the antiproliferative potential but also the radiation-sensitizing effects against these cells were examined. It was found that antiproliferative activity partly depended on HSL structure and acyl side chain length. Moreover, a few compounds, compound 5 and 87, showed antiproliferative effects against both Ca9-22 and SAS cells, and also induced radiation-sensitizing effects against Ca9-22 cells. Compound 5 alone induced apoptotic cell death accompanied by sub-G1 phase accumulation in cell cycle and caspase-3 activation, and radiation-sensitizing effects of compound 5 could be attributed to enhanced apoptosis induction. In contrast, there were no remarkable alterations in cell cycle distribution in Ca9-22 treated with compound 87 alone or in combination. However, both compounds lack 3-oxo and their acyl side chain lengths are not necessarily long. This SAR study demonstrated that HSL analogs, which lacked the recommended characteristics for apoptosis induction clearly showed antiproliferative and radiation-sensitizing activity in Ca9-22 cells.

Thiolactone modulators of quorum sensing revealed through library design and screening.

Quorum sensing (QS) is a process by which bacteria use small molecules or peptidic signals to assess their local population densities. At sufficiently high density, bacteria can alter gene expression levels to regulate group behaviors involved in a range of important and diverse phenotypes, including virulence factor production, biofilm formation, root nodulation, and bioluminescence. Gram-negative bacteria most commonly use N-acylated l-homoserine lactones (AHLs) as their QS signals. The AHL lactone ring is hydrolyzed relatively rapidly at biological pH, and the ring-opened product is QS inactive. We seek to identify AHL analogues with heightened hydrolytic stability, and thereby potentially heightened activity, for use as non-native modulators of bacterial QS. As part of this effort, we probed the utility of thiolactone analogues in the current study as QS agonists and antagonists in Gram-negative bacteria. A focused library of thiolactone analogs was designed and rapidly synthesized in solution. We examined the activity of the library as agonists and antagonists of LuxR-type QS receptors in Pseudomonas aeruginosa (LasR), Vibrio fischeri (LuxR), and Agrobacterium tumefaciens (TraR) using bacterial reporter strains. The thiolactone library contained several highly active compounds, including some of the most active LuxR inhibitors and the most active synthetic TraR agonist reported to date. Analysis of a representative thiolactone analog revealed that its hydrolysis half-life was almost double that of its parent AHL in bacterial growth medium.

Design, synthesis, and biological evaluation of abiotic, non-lactone modulators of LuxR-type quorum sensing.

Quorum sensing (QS) is a cell-cell signaling mechanism that allows bacteria to monitor their population size and alter their behavior at high cell densities. Gram-negative bacteria use N-acylated L-homoserine lactones (AHLs) as their primary signals for QS. These signals are susceptible to lactone hydrolysis in biologically relevant media, and the ring-opened products are inactive QS signals. We have previously identified a range of non-native AHLs capable of strongly agonizing and antagonizing QS in Gram-negative bacteria. However, these abiotic AHLs are also prone to hydrolysis and inactivation and thereby have a relatively short time window for use (∼12-48 h). Non-native QS modulators with reduced or no hydrolytic instability could have enhanced potencies and would be valuable as tools to study the mechanisms of QS in a range of environments (for example, on eukaryotic hosts). This study reports the design and synthesis of two libraries of new, non-hydrolyzable AHL mimics. The libraries were screened for QS modulatory activity using LasR, LuxR, and TraR bacterial reporter strains, and several new, abiotic agonists and antagonists of these receptors were identified.

Potent and selective synthetic modulators of a quorum sensing repressor in Pseudomonas aeruginosa identified from second-generation libraries of N-acylated L-homoserine lactones.

Bacteria can coordinate group behavior using chemical signals in a process called quorum sensing (QS). The QS system in the opportunistic pathogen Pseudomonas aeruginosa is largely governed by the LasR receptor and its cognate chemical signal, N-(3-oxo)-dodecanoyl L-homoserine lactone (OdDHL). LasR also appears to share this signal with an orphan LuxR-type receptor in P. aeruginosa, termed QscR, which represses LasR activity. Non-native molecules that modulate QscR would represent valuable tools to study the role of this novel QS repressor protein in P. aeruginosa. We performed a critical analysis of previously identified, non-native N-acylated L-homoserine lactone (AHL) activators and inhibitors of QscR to determine a set of structure-activity relationships (SARs). Based on these SAR data, we designed, synthesized, and screened several second-generation libraries of AHLs for new ligands that could target QscR. These studies revealed the most active AHL agonists and antagonists of QscR reported to date, with activities ranging from nanomolar to low micromolar in a QscR bacterial reporter strain. Several of these AHLs were highly selective for QscR over LasR and other LuxR-type receptors. A small subset of the new QscR activators, however, were also found to inhibit LasR; this demonstrates the exciting potential for the synergistic modulation of these integral P. aeruginosa QS receptors by using a single synthetic compound.

Custom synthesis of autoinducers and their analogues.

Bacterial quorum sensing (QS) system is a unique target for the development of a new class of drugs that potentially control pathogenicity and attenuate virulence. Thus, it has been of significant interest to discover small organic molecules that modulate QS circuits by competing with the signaling molecules, or so-called autoinducers (AIs), for binding to QS proteins. In this chapter, we summarize synthetic methodology for custom QS agonists and antagonists against the Lux system in Gram-negative bacteria.

Facile preparation of deuterium-labeled N-acylhomoserine lactones as internal standards for isotope dilution mass spectrometry.

N-Acylhomoserine lactones (AHLs) are widely conserved signal molecules that mediate quorum sensing in Gram-negative bacteria. In this study, deuterium-labeled AHLs were prepared for use as internal standards for isotope dilution mass spectrometry. Their utility in the sensitive and precise quantification of AHLs in culture supernatants of bacteria by GC/MS was demonstrated.

Long-chain acyl-homoserine lactones from Methylobacterium mesophilicum: synthesis and absolute configuration.

The acyl-homoserine lactones (acyl-HSLs) produced by Methylobacterium mesophilicum isolated from orange trees infected with the citrus variegated chlorosis (CVC) disease have been studied, revealing the occurrence of six long-chain acyl-HSLs, i.e., the saturated homologues (S)-N-dodecanoyl (1) and (S)-N-tetradecanoyl-HSL (5), the uncommon odd-chain N-tridecanoyl-HSL (3), the new natural product (S)-N-(2E)-dodecenoyl-HSL (2), and the rare unsaturated homologues (S)-N-(7Z)-tetradecenoyl (4) and (S)-N-(2E,7Z)-tetradecadienyl-HSL (6). The absolute configurations of all HSLs were determined as 3S. Compounds 2 and 6 were synthesized for the first time. Antimicrobial assays with synthetic acyl-HSLs against Gram-positive bacterial endophytes co-isolated with M. mesophilicum from CVC-infected trees revealed low or no antibacterial activity.