AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

Synthesis and biological evaluation of new N-acyl-homoserine-lactone analogues, based on triazole and tetrazole scaffolds, acting as LuxR-dependent quorum sensing modulators.

New analogues of N-acyl-homoserine-lactone (AHL), in which the amide was replaced by a triazole or tetrazole ring, were prepared and tested for their activity as LuxR-dependent QS modulators. Several compounds showed a level of antagonistic or agonistic activity, notably some 1,4-triazolic and 1,5-tetrazolic derivatives, whereas the 2,5-tetrazolic compounds were inactive. In 1,5-tetrazoles, substituted with butyrolactone and an alkyl chain, the activity was reversed, depending on the connection between the lactone and the tetrazole. The C-N connected compounds were agonists whereas the C-C connected ones were antagonists.

AHL-dependent quorum sensing inhibition: synthesis and biological evaluation of α-(N-alkyl-carboxamide)-γ-butyrolactones and α-(N-alkyl-sulfonamide)-γ-butyrolactones.

New N-acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a reverse-amide one have been studied as AHL QS modulators. The series of compounds consists of α-(N-alkyl-carboxamide)-γ-butyrolactones, α-(N-alkyl-sulfonamide)-γ-butyrolactones, and 2-(N-alkyl-carboxamide)-cyclopentanones and cyclopentanols. Most active compounds exhibited antagonist activities against LuxR reaching the 30 µM range.

LuxR dependent quorum sensing inhibition by N,N'-disubstituted imidazolium salts.

Thirty N,N'-disubstituted imidazolium salts have been synthesized and evaluated as LuxR antagonists. Substitution on one of the imidazolium nitrogen atoms includes benzhydryl, fluorenyl or cyclopentyl substituent, and alkyl chains of various lengths on the second one. Most of these compounds displayed antagonist activity, with IC(50) reaching the micromolar range for the most active ones. The disubstituted imidazolium scaffold is thus shown to be a new pertinent pharmacophore in the field of AHL dependent QS inhibition.

Quantitative structure-activity relationship for 4-hydroxy-2-alkenal induced cytotoxicity in L6 muscle cells.

Lipid peroxidation is one of the most important sources of endogenous toxic metabolites. 4-Hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexenal (HHE) are produced in several oxidative stress associated diseases from peroxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Both are able to form covalent adducts with many biomolecules. Particularly, proteins adduction can induce structural and conformational changes and impair biological function, which may be involved in the toxicity of hydroxy-alkenals. The aim of this study was to compare the effect of 4-hydroxy-2-alkenals to several chemically related derivatives in order to clarify the physico-chemical requirement of their toxicity. L6 muscle cells were treated with HHE, HNE and parent derivatives (acetal derivative, trans-alkenals and alkanals). Viability and necrosis were estimated using MTT, LDH and caspase-3 tests. LogLC50 (Lethal Concentration 50) was then tested for correlation with adducts formation (estimated using dinitrophenylhydrazine) and several molecular descriptors in order to establish quantitative structure-toxicity relationship (QSTR) models. The rank of derivatives toxicity, based on LC50 was: hydroxy-alkenals>acetal derivatives approximately 2-alkenals>alkanals and a high correlation was found between logLC50 and protein carbonylation. Moreover, logLC50 was correlated to the electrophilic descriptor LUMO (lowest unoccupied molecular orbital) as well as with electronegativity-related molecular descriptors such as number of oxygen atoms, partial negative surface area (PNSA3) and partial positive surface area (PPSA3). Together, these results point out the important role of the electrophilic structure and adduct formation in hydroxy-alkenals toxicity. Our present study demonstrates that 4-hydroxy-2-alkenals dramatic effects on cell viability are due to covalent adducts formation, particularly Michael adducts. This capacity is related to the electrophilic structure and reactive CC double bond, making it highly accessible for nucleophilic addition. The present study suggests that nucleophilic scavengers might protect cells against electrophile compounds and might be of possible therapeutic value in oxidative stress associated diseases.

LuxR-dependent quorum sensing: computer aided discovery of new inhibitors structurally unrelated to N-acylhomoserine lactones.

A virtual screening, involving flexible docking sequences within the LuxR, TraR and LasR binding sites, was used as a structural binding sites similarity filter to specifically target conserved residues in the proteins of the LuxR family (namely Tyr62, Trp66, Tyr70, Asp79, Trp94 for LuxR). This docking-based screening, employing a genetic algorithm, was performed on a 2344 chemical compounds library, together with empirical binding free energy (DeltaG(bind)) calculations. Docking results were analysed, and the compounds detected with reproducible low DeltaG(bind) values or identified as being in the top 120 for most of the docking sequences, were selected as hits candidates which interact with conserved residues. Biological evaluation with LuxR-dependent quorum sensing led to the discovery of some new inhibitors, namely tamoxifen, sertraline, pimethixene, terfenadine, fendiline and calmidazolium. Notably, calmidazolium was identified as one of the most potent AHL-structurally unrelated inhibitors of LuxR-dependent quorum sensing, with an IC(50) value of 7.0+/-0.2 microM.

N-Acyl-3-amino-5H-furanone derivatives as new inhibitors of LuxR-dependent quorum sensing: Synthesis, biological evaluation and binding mode study.

New N-acyl homoserine lactone analogues, N-acyl-3-amino-5H-furanone derivatives and some 4-halogeno counterparts, were synthesised and tested for their ability to modulate LuxR-dependent bacterial quorum sensing. Both types of analogues proved to be inhibitors, the halogenated compounds being significantly more active. Molecular modelling suggested that the conjugated enamide group induces two preferential conformations leading to specific binding modes. In addition, the presence of the halogen atom could enhance the fitting of the lactone ring through specific interactions with strictly conserved or conservatively replaceable residues in the LuxR protein family, namely Asp79, Trp94 and Ile81.

Molecular insights into quorum sensing in Acidithiobacillus ferrooxidans bacteria via molecular modelling of the transcriptional regulator AfeR and of the binding mode of long-chain acyl homoserine lactones.

Amino acid sequence alignments of the transcriptional regulator AfeR, which is involved in type 1 quorum sensing (QS) in Acidithiobacillus ferrooxidans bacteria, with other acyl homoserine lactone (AHL)-dependent QS regulators, revealed the presence of strictly or highly conserved residues located in the active site of these proteins. As a consequence, a model of AfeR was constructed to study the binding mode of long-chain AHLs using molecular dynamics and subsequent rigid ligand docking. This study, performed on the tetradecanoyl homoserine lactone C14-AHL, showed that the binding mode involved a curved conformation. Based on these results, the binding mode of tetradec-7-Z enoyl homoserine lactone, an AHL that is conformationally constrained due to the presence of the cis double bond, was investigated. This mono-unsaturated AHL with its preferential curved shape conformation was found to be particularly well adapted to the active site of AfeR. These results should be helpful in the rational design of QS modulators with potential biotechnological applications and especially in the improvement of industrial bioleaching from ores.

Synthetic homoserine lactone-derived sulfonylureas as inhibitors of Vibrio fischeri quorum sensing regulator.

A series of 9 homoserine lactone-derived sulfonylureas substituted by an alkyl chain, some of them bearing a phenyl group at the extremity, have been prepared. All compounds were found to inhibit the action of 3-oxo-hexanoyl-L-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri, the aliphatic compounds being more active than their phenyl-substituted counterparts. Molecular modelling studies performed on the most active compound in each series suggest that the antagonist activity could be related to the perturbation of the hydrogen-bond network in the ligand-protein complexes.

An expeditious synthesis of 4-hydroxy-2E-nonenal (4-HNE), its dimethyl acetal and of related compounds.

The facile one step synthesis of 4-hydroxy-2E-nonenal and its dimethyl acetal via a cross-metathesis reaction between commercially available octen-3-ol and acrolein or its dimethyl acetal is reported. The method was extended to the synthesis of C6 and C12 4-hydroxy-2E-enals, their dimethyl acetal and of the 4-hydroxy-2E-nonenoic acid (4-HNA).

Exploring the active site of acyl homoserine lactones-dependent transcriptional regulators with bacterial quorum sensing modulators using molecular mechanics and docking studies.

A comparative molecular modelling study of acyl homoserine lactones-dependent transcriptional regulators (TraR, SdiA, LuxR and LasR) involved in bacterial quorum sensing (QS) revealed a high structural homology of their active site. Docking studies within the active site of TraR of fixed conformations obtained using molecular mechanics calculations showed that TraR, for which the crystalline structure is known, is a relevant model for the study of other protein-ligand interactions in the same protein family. Structure-activity relationships of AHLs derived QS modulators including carboxamides, sulfonamides and ureas were thus investigated. The results show that Tyr61, a residue conserved in the LuxR-proteins family, is involved in attractive interactions with aromatic carboxamide antagonists. Tyr53, Tyr61 and Asp70, conserved residues, are implicated in both the development of additional hydrogen bonds and attractive interactions with the N-sulfonyl homoserine lactones and AHLs derived ureas antagonists.

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals.

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.

Ac2-DPD, the bis-(O)-acetylated derivative of 4,5-dihydroxy-2,3-pentanedione (DPD) is a convenient stable precursor of bacterial quorum sensing autoinducer AI-2.

Ac2-DPD, the bis-(O)-acetylated derivative of 4,5-dihydroxy-2,3-pentanedione (DPD), was prepared both as a racemic mixture and in the optically active form found in naturally occurring DPD. It was shown to exhibit the same ability as DPD to induce bioluminescence in Vibrio harveyi and beta-galactosidase activity in Salmonella enterica Typhimurium, both gram-negative bacteria. Likewise, it was also shown to inhibit biofilm formation in gram-positive Bacillus cereus. The most likely hypothesis is that Ac2-DPD activity is due to the release of DPD by in situ hydrolysis of the ester groups. Importantly, by contrast with DPD, Ac2-DPD proved to be a stable compound which can be purified and stored.

Synthetic studies on the MARDi cascade: stereoselective synthesis of heterocyclic seven-membered rings.

[reaction: see text] A versatile stereoselective synthesis of substituted and functionalized heterocyclic seven-membered rings is described. The approach involves a formal two-carbon ring expansion of heterocyclic cyclopentanones through a base-induced anionic domino three-component transformation named the MARDi cascade leading either to oxa-, aza-, or thiacycloheptanes bearing up to five contiguous stereogenic centers.

Differential membrane packing of stereoisomers of bis(monoacylglycero)phosphate.

Bis(monoacylglycero)phosphate (BMP) reveals an unusual sn-1,sn-1' stereoconfiguration of glycerophosphate. We synthesized sn-(3-myristoyl-2-hydroxy)glycerol-1-phospho-sn-1'-(3'-myristoyl-2'-hydroxy)glycerol (1,1'-DMBMP) and characterized the thermotropic phase behavior and membrane structure, in comparison with those of the corresponding sn-3:sn-1' stereoisomer (3,1'-DMBMP), by means of differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SAXS and WAXS, respectively), pressure-area (pi-A) isotherms, epifluorescence microscopy of monolayers, and molecular dynamics (MD) simulations. In DSC, these lipids exhibited weakly energetic broad peaks with an onset temperature of 9 degrees C for 1,1'-DMBMP and 18 degrees C for 3,1'-DMBMP. In addition, a highly cooperative, strongly energetic transition peak was observed at approximately 40 degrees C for 1,1'-DMBMP and approximately 42 degrees C for 3,1'-DMBMP. These results are supported by the observation that 1,1'-DMBMP exhibited a larger phase transition pressure (pi(c)) than 3,1'-DMBMP. Small- and wide-angle X-ray scattering measurements identified these small and large energetic transitions as a quasi-crystalline (L(c1))-quasi-crystalline with different tilt angle (L(c2)) phase transition and an L(c2)-L(alpha) main phase transition, respectively. X-ray measurements also revealed that these DMBMPs undergo an unbinding at the main phase transition temperature. The MD simulations estimated stronger hydrogen bonding formation in the 3,1'-DMBMP membrane than in 1,1'-DMBMP, supporting the experimental data.

Synthesis and biological evaluation of homoserine lactone derived ureas as antagonists of bacterial quorum sensing.

A series of 15 racemic alkyl- and aryl-N-substituted ureas, derived from homoserine lactone, were synthesized and tested for their ability to competitively inhibit the action of 3-oxohexanoyl-l-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri. N-alkyl ureas with an alkyl chain of at least 4 carbon atoms, as well as certain ureas bearing a phenyl group at the extremity of the alkyl chain, were found to be significant antagonists. In the case of N-butyl urea, it has been shown that the antagonist activity was related to the inhibition of the dimerisation of the N-terminal domain of ExpR, a protein of the receptor LuxR family. Molecular modelling suggested that this would result from the formation of an additional hydrogen bond in the protein acylhomoserine lactone binding cavity.

N-Sulfonyl homoserine lactones as antagonists of bacterial quorum sensing.

A series of 11 new analogues of N-acylhomoserine lactones in which the carboxamide bond was replaced by a sulfonamide one, has been synthesised. These compounds were evaluated for their ability to competitively inhibit the action of 3-oxohexanoyl-L-homoserine lactone, the natural ligand of the quorum sensing transcriptional regulator LuxR, which in turn activates expression of bioluminescence in the model bacterium Vibrio fischeri. Several compounds were found to display antagonist activity. Molecular modeling suggests that the latter prevent a cascade of structural rearrangements necessary for the formation of the active LuxR dimer.

Plasmalogens protect unsaturated lipids against UV-induced oxidation in monolayer.

Oxidative stress results from the attack by free radicals of several cellular targets (proteins, DNA and lipids). The cell equilibrium is a direct consequence of the pro-/antioxidant balance. In order to understand the physiological processes involved in oxidative stress, we followed oxidation of unsaturated lipids using a biomimetic system: Langmuir monolayers. The oxidation mode chosen was UV-irradiation and the lipid model was a polyunsaturated phospholipid: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). The monomolecular film technique was used to measure membrane rheology before and after UV-irradiation. We showed that the UV-irradiation of a DLPC monomolecular film led to a molecular area and surface elasticity modulus decrease that attests to the apparition of new molecular species at the air-water interface. The antioxidant effect of a synthetic plasmalogen (1-O-(1'-(Z)-hexadecenyl)-2-O-oleoyl-sn-glycero-3-phosphocholine or P(PLM)OPE) was tested on the oxidation of DLPC. Indeed, for about 25% mol P(PLM)OPE in mixed DLPC/P(PLM)OPE monolayers, a complete inhibition of the molecular area and the surface elasticity modulus decreases was observed in our experimental conditions. Lower P(PLM)OPE quantities delayed but did not prevent the DLPC oxidation in mixed monolayers.

Covalent binding of hydroxy-alkenals 4-HDDE, 4-HHE, and 4-HNE to ethanolamine phospholipid subclasses.

Lipid oxidation is implicated in a wide range of pathophysiogical disorders, and leads to reactive compounds such as fatty aldehydes, of which the most well known is 4-hydroxy-2E-nonenal (4-HNE) issued from 15-hydroperoxyeicosatetraenoic acid (15-HpETE), an arachidonic acid (AA) product. In addition to 15-HpETE, 12(S)-HpETE is synthesized by 12-lipoxygenation of platelet AA. We first show that 12-HpETE can be degraded in vitro into 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), a specific aldehyde homologous to 4-HNE. Moreover, 4-HDDE can be detected in human plasma. Second, we compare the ability of 4-HNE, 4-HDDE, and 4-hydroxy-2E-hexenal (4-HHE) from n-3 fatty acids to covalently modify different ethanolamine phospholipids (PEs) chosen for their biological relevance, namely AA- (20: 4n-6) or docosahexaenoic acid- (22:6n-3) containing diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) molecular species. The most hydrophobic aldehyde used, 4-HDDE, generates more adducts with the PE subclasses than does 4-HNE, which itself appears more reactive than 4-HHE. Moreover, the aldehydes show higher reactivity toward alkenylacyl-GPE compared with diacyl-GPE, because the docosahexaenoyl-containing species are more reactive than those containing arachidonoyl. We conclude that the different PE species are differently targeted by fatty aldehydes: the higher their hydrophobicity, the higher the amount of adducts made. In addition to their antioxidant potential, alkenylacyl-GPEs may efficiently scavenge fatty aldehydes.