Divergent synthesis and biological evaluation of 2-(trifluoromethyl)pyridines as virulence-attenuating inverse agonists targeting PqsR.

A short and divergent route towards new derivatives of 2-(trifluoromethyl)pyridines as potent inverse agonists of the bacterial target PqsR against Pseudomonas aeruginosa (PA) infections is described. This Gram-negative pathogen causes severe nosocomial infections and common antibiotic treatment options are rendered ineffective due to resistance issues. Based on an earlier identified optimized hit, we conducted derivatization and rigidification attempts employing two central building blocks. The western part of the molecule is built up via a 2-(trifluoromethyl)pyridine head group equipped with a terminal alkyne. The eastern section is then introduced through aryliode motifs exploiting Sonogashira as well as Suzuki-type chemistry. Subsequent modification provided quick access to an array of compounds, allowed for deep SAR insights, and enabled to optimize the hit scaffold into a lead structure of nanomolar potency combined with favorable in vitro ADME/T features.

SIX4 acts as a master regulator of oncogenes that promotes tumorigenesis in non-small-cell lung cancer cells.

A number of homeobox genes are implicated in the malignancy of various cancers. Here, we investigated the role of the homeobox gene SIX4 in non-small-cell lung cancer (NSCLC). The sine oculis homeobox (SIX4) gene was found to be highly expressed at both mRNA and protein levels in NSCLC tumor tissues as compared with matching normal counterparts. In this study, the SIX4 gene of two human NSCLC cell lines (A549 and PC9) was overexpressed or silenced using the lentiviral system. We evaluated the malignancy-associated phenotype of transfected cells, which demonstrated that exogenous expression of the SIX4 gene greatly enhanced the proliferation, migration, and invasion of NSCLC cells. The opposite was true in the SIX4-silenced cells. Transcriptomic profiling analysis revealed that the SIX4 gene modulated the expression of hundreds of downstream target genes in a cell context-dependent manner. Most notably, the SIX4 gene controls the expression of crucial genes with evidently oncogenic function. We conclude that SIX4 plays an oncogenic role and may be potentially utilized as a diagnostic and therapeutic marker for NSCLC.

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop "Out" Conformation for LasR.

Chemical strategies to block quorum sensing (QS) could provide a route to attenuate virulence in bacterial pathogens. Considerable research has focused on this approach in Pseudomonas aeruginosa, which uses the LuxR-type receptor LasR to regulate much of its QS network. Non-native ligands that antagonize LasR have been developed, yet we have little understanding of the mode by which these compounds interact with LasR and alter its function, as the receptor is unstable in their presence. Herein, we report an approach to circumvent this challenge through the study of a series of synthetic LasR agonists with varying levels of potency. Structural investigations of these ligands with the LasR ligand-binding domain reveal that certain agonists can enforce a conformation that deviates from that observed for other, often more potent agonists. These results, when combined with cell-based and biophysical analyses, suggest a functional model for LasR that could guide future ligand design.

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence.

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.

Hepatitis B virus X protein and proinflammatory cytokines synergize to enhance TRAIL-induced apoptosis of renal tubular cells by upregulation of DR4.

Persistent infection with hepatitis B virus (HBV) may lead to HBV-associated glomerulonephritis (HBV-GN). Presence of HBV-DNA and -RNA in renal tubular epithelial cells (RTECs) suggests direct virus-induced injury. Increase in proinflammatory cytokines is also observed under these conditions. Apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays a significant role in the pathogenesis of HBV-infections. However, the effects of HBV X protein (HBx) on TRAIL-induced apoptosis of RTECs especially under certain inflammatory conditions remain obscure. Here, we show that HBx synergizes with proinflammatory cytokines to significantly increase TRAIL-induced apoptosis of RTECs. HBx markedly up-regulates death receptor-4 (DR4) expression by enhancing the activation of nuclear factor-kappa B (NF-κB) in the presence of proinflammatory cytokines. Dramatic increase in DR4 expression leads to the sensitization of RTECs to TRAIL-induced apoptosis. Furthermore, in patients with HBV-GN, DR4 expression in the kidneys is significantly elevated and is positively correlated with the HBx and proinflammatory cytokines expression. These findings provide a novel insight into the underlying mechanisms of renal tubule lesions induced by HBx in HBV-GN.

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans.

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH-nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

Development of a novel series of non-natural triaryl agonists and antagonists of the Pseudomonas aeruginosa LasR quorum sensing receptor.

Bacterial chemical communication, through a process called quorum sensing (QS), plays a central role in infection in numerous bacterial pathogens. Quorum sensing in Pseudomonas aeruginosa employs a series of small molecule receptors including the master QS regulator, LasR. In this study we investigate a non-natural triaryl series of LasR ligands using a combination of structure activity relationship studies and computational modeling. These studies have enabled the identification of key structural requirements for ligand binding and have revealed a new strategy for inducing the therapeutically relevant antagonism of LasR.

Fine-Tuning Covalent Inhibition of Bacterial Quorum Sensing.

Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population-wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro-substituted ITC-12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P. aeruginosa.

Dickkopf Homolog 3 Induces Stem Cell Differentiation into Smooth Muscle Lineage via ATF6 Signalling.

Smooth muscle cells (SMCs) are a key component of healthy and tissue engineered vessels and play a crucial role in vascular development and the pathogenic events of vascular remodeling i.e. restenosis. However, the cell source from which they can be isolated is limited. Embryonic stem (ES) cells that have the remarkable capability to differentiate into vascular SMCs in response to specific stimuli provide a useful model for studying SMC differentiation. Previous studies suggested that dickkopf homolog 3 (DKK3) has a role in human partially induced pluripotent stem cell to SMC differentiation. Here, we demonstrate that the expression of DKK3 is essential for the expression of SMC markers and myocardin at both the mRNA and protein levels during mouse ES cell differentiation into SMCs (ESC-SMC differentiation). Overexpression of DKK3 leads to further up-regulation of the aforementioned markers. Further investigation indicates that DKK3 added as a cytokine activates activating transcription factor 6 (ATF6), leading to the increased binding of ATF6 on the myocardin promoter and increased its expression. In addition, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) promotes the expression of ATF6 and leads to further increase of myocardin transcription. Our findings offer a novel mechanism by which DKK3 regulates ESC-SMC differentiation by activating ATF6 and promoting myocardin expression.

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.

Non-native N-aroyl L-homoserine lactones are potent modulators of the quorum sensing receptor RpaR in Rhodopseudomonas palustris.

Quorum sensing (QS) is a process by which bacteria use low-molecular-weight signaling molecules (or autoinducers) to assess their local population densities and alter gene expression levels at high cell numbers. Many Gram-negative bacteria use N-acyl L-homoserine lactones (AHLs) with aliphatic acyl groups as signaling molecules for QS. However, bacteria that utilize AHLs with aroyl acyl groups have been recently discovered; they include the metabolically versatile soil bacterium Rhodopseudomonas palustris, which uses p-coumaroyl HL (p-cAHL) as its QS signal. This autoinducer is especially unusual because its acyl group is believed to originate from a monolignol (i.e., p-coumarate) produced exogenously by plants in the R. palustris environment, rather than through the endogenous fatty acid biosynthesis pathway like other native AHLs. As such, p-cAHL could signal not only bacterial density, but also the availability of an exogenous plant-derived substrate and might even constitute an interkingdom signal. Like other Gram-negative bacteria, QS in R. palustris is controlled by the p-cAHL signal binding its cognate LuxR-type receptor, RpaR. We sought to determine if non-native aroyl HLs (ArHLs) could potentially activate or inhibit RpaR in R. palustris, and thereby modulate QS in this bacterium. Herein, we report the testing of a set of synthetic ArHLs for RpaR agonism and antagonism by using a R. palustris reporter strain. Several potent non-native RpaR agonists and antagonists were identified. Additionally, the screening data revealed that lower concentrations of ArHL are required to strongly agonize RpaR than to antagonize it. Structure-activity relationship analyses of the active ArHLs indicated that potent RpaR agonists tend to have sterically small substituents on their aryl groups, most notably in the ortho position. In turn, the most potent RpaR antagonists were based on either the phenylpropionyl HL (PPHL) or the phenoxyacetyl HL (POHL) scaffold, and many contained an electron-withdrawing group at either the meta or para positions of the aryl ring. To our knowledge, the compounds reported herein represent the first abiotic chemical modulators of RpaR, and more generally, the first abiotic ligands capable of intercepting QS in bacteria that utilize native ArHL signals. In view of the origins of the p-cAHL signal in R. palustris, the largely unknown role of QS in this bacterium, and R. palustris' unique environmental lifestyles, we anticipate that these compounds could be valuable as chemical probes to study QS in R. palustris in a range of fundamental and applied contexts.

Tailor-made LasR agonists modulate quorum sensing in Pseudomonas aeruginosa.

The primary quorum sensing system in the opportunistic pathogen Pseudomonas aeruginosa is regulated through the synthesis and secretion of N-3-oxo-dodecanoyl-L-homoserine lactone (C12) which binds the transcriptional activator LasR. In this study we report the design, synthesis and biological evaluation of new analogs of C12. Analysis of the autoinducer binding site cavity of LasR revealed a positively charged cavity near the center of bound C12. Accordingly, we synthesized two piperidine-C12 diastereoisomers and tested their biological activity. Both analogs proved to be strong LasR agonists that showed a synergistic effect when presented together with the natural ligand. Moreover, binding of the analogs resulted in phenotypic changes characteristic of QS controlled receptor activation.

Interactions among quorum sensing inhibitors.

Many pathogenic bacteria use quorum sensing (QS) systems to regulate the expression of virulence genes in a density-dependent manner. In one widespread QS paradigm the enzyme LuxI generates a small diffusible molecule of the acyl-homoserine lactone (AHL) family; high cell densities lead to high AHL levels; AHL binds the transcription factor LuxR, triggering it to activate gene expression at a virulence promoter. The emergence of antibiotic resistance has generated interest in alternative anti-microbial therapies that target QS. Inhibitors of LuxI and LuxR have been developed and tested in vivo, and can act at various levels: inhibiting the synthesis of AHL by LuxI, competitively or non-competitively inhibiting LuxR, or increasing the turnover of LuxI, LuxR, or AHL. Here use an experimentally validated computational model of LuxI/LuxR QS to study the effects of using inhibitors individually and in combination. The model includes the effect of transcriptional feedback, which generates highly non-linear responses as inhibitor levels are increased. For the ubiquitous LuxI-feedback virulence systems, inhibitors of LuxI are more effective than those of LuxR when used individually. Paradoxically, we find that LuxR competitive inhibitors, either individually or in combination with other inhibitors, can sometimes increase virulence by weakly activating LuxR. For both LuxI-feedback and LuxR-feedback systems, a combination of LuxR non-competitive inhibitors and LuxI inhibitors act multiplicatively over a broad parameter range. In our analysis, this final strategy emerges as the only robust therapeutic option.

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.

Identification of LasR ligands through a virtual screening approach.

With the widespread occurrence of bacterial resistance to antibiotics, the development of new strategies beyond conventional treatments is a pursuit taken by public health institutions worldwide. LasR, a transcription factor that controls quorum sensing in Pseudomonas aeruginosa, has emerged as an attractive therapeutic target for the next generation of antimicrobial agents. In the present study, a virtual screening workflow combining pharmacophore- and structure-based approaches was used to identify new LasR ligands. Five novel inducers and three inhibitors of LasR activity were validated experimentally by use of a cell-based assay. Interestingly, these compounds are molecularly distinct from the native signal molecule, N-3-oxododecanoyl-L-homoserine lactone (OHN), and may serve as lead structures for the design of new drugs. The binding modes of these compounds to the OHN binding site in LasR were predicted and used to identify the key interactions that contribute to the induction and inhibition of LasR activity.

Activating mitochondrial regulator PGC-1α expression by astrocytic NGF is a therapeutic strategy for Huntington's disease.

Mitochondrial dysfunction plays an important role in Huntington's disease (HD). NGF gene delivery in AD patients showed an increase in brain energy metabolism and NGF has been shown neuroprotective effects against mitochondrial toxins. However, the role of NGF in regulating mitochondrial function is unclear. Here, we found that NGF-stimulated mitochondrial biogenesis in PC12 and primary neuron cells. Our results demonstrated that peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is a downstream key target of the NGF signalling pathway. In a 3-nitropropionic acid (3-NP) cell model, NGF treatment rescued the defects in mitochondrial activity and mitochondrial membrane potential. Since NGF cannot freely cross blood-brain barrier, we found an astrocytic NGF inducer, Ganoderma lucidum (GaLu) extract. Its active constituents had potent effects on the induction of NGF in primary astrocytes. Among the identified ingredients, ganoderic acid C2 was most effective. We further found that GaLu-conditioned media can enhance mitochondrial biogenesis in PC12 cells and preventing NGF signalling using NGF antibody or PGC-1α siRNA blocked these effects. Moreover, GaLu and ganoderic acid C2-conditioned media treatment attenuated mitochondrial defects in 3-NP cell model. After 3-NP-induced behavioural impairment and striatal degeneration in mice, GaLu treatment therapeutically restored the behaviour score, sensorimotor ability and neuronal loss. We found that striatal NGF, PGC-1α expression level and succinate dehydrogenase activity were recovered in GaLu-fed mice. These results suggest that the NGF-signalling pathway connected to the mitochondrial regulator, PGC-1α, expression. This signalling triggered by astrocytic NGF with small molecule inducers may offer a therapeutic strategy for HD.

Design, synthesis and biological evaluation of non-natural modulators of quorum sensing in Pseudomonas aeruginosa.

Many species of bacteria employ a mechanism of intercellular communication known as quorum sensing which is mediated by small diffusible signalling molecules termed autoinducers. The most common class of autoinducer used by Gram-negative bacteria are N-acylated-L-homoserine lactones (AHLs). Pseudomonas aeruginosa is a clinically important bacterium which is known to use AHL-mediated quorum sensing systems to regulate a variety of processes associated with virulence. Thus the selective disruption of AHL-based quorum sensing represents a strategy to attenuate the pathogenicity of this bacterium. Herein we describe the design, synthesis and biological evaluation of a collection of structurally novel AHL mimics. A number of new compounds capable of modulating the LasR-dependent quorum sensing system of P. aeruginosa were identified, which could have value as molecular tools to study and manipulate this signalling pathway. Worthy of particular note, this research has delivered novel potent quorum sensing antagonists, which strongly inhibit the production of virulence factors in a wild type strain of this pathogenic bacterium.

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.

Quorum sensing in bacterial species that use degenerate autoinducers can be tuned by using structurally identical non-native ligands.

Many bacteria use quorum sensing (QS) to regulate cell-density dependent phenotypes that play critical roles in the maintenance of their associations with eukaryotic hosts. In Gram-negative bacteria, QS is primarily controlled by N-acylated L-homoserine lactone (AHL) signals and their cognate LuxR-type receptors. AHL-LuxR-type receptor binding regulates the expression of target genes necessary for QS phenotypes. We recently identified a series of non-native AHLs capable of intercepting AHL-LuxR binding in the marine symbiont Vibrio fischeri, and thereby strongly promoting or inhibiting QS in this organism. V. fischeri utilizes N-(3-oxo)-hexanoyl L-HL (OHHL) as its primary QS signal, and OHHL is also used by several other bacterial species for QS. Such signal degeneracy is common among bacteria, and we sought to determine if our non-native LuxR agonists and antagonists, which are active in V. fischeri, would also modulate QS phenotypes in other bacteria that use OHHL. Herein, we report investigations into the activity of a set of synthetic LuxR modulators in the plant pathogen Pectobacterium carotovora subsp. carotovora Ecc71. This pathogen uses OHHL and two closely related LuxR-type receptors, ExpR1 and ExpR2, to control virulence, and we evaluated their responses to synthetic ligands by quantifying virulence factor production. Our results suggest an overall conservation in the activity trends of the ligands between the ExpR receptors in P. carotovora Ecc71 and LuxR in V. fischeri, and indicate that these compounds could be used as tools to study QS in an expanded set of bacteria. Notable differences in activity were apparent for certain compounds, however, and suggest that it might be possible to selectively regulate QS in bacteria that utilize degenerate AHLs.