A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy.

The transcriptional co-regulators YAP and TAZ pair primarily with the TEAD family of transcription factors to elicit a gene expression signature that plays a prominent role in cancer development, progression and metastasis. YAP and TAZ endow cells with various oncogenic traits such that they sustain proliferation, inhibit apoptosis, maintain stemness, respond to mechanical stimuli, engineer metabolism, promote angiogenesis, suppress immune response and develop resistance to therapies. Therefore, inhibiting YAP/TAZ- TEAD is an attractive and viable option for novel cancer therapy. It is exciting to know that many drugs already in the clinic restrict YAP/TAZ activities and several novel YAP/TAZ inhibitors are currently under development. We have classified YAP/TAZ-inhibiting drugs into three groups. Group I drugs act on the upstream regulators that are stimulators of YAP/TAZ activities. Many of the Group I drugs have the potential to be repurposed as YAP/TAZ indirect inhibitors to treat various solid cancers. Group II modalities act directly on YAP/TAZ or TEADs and disrupt their interaction; targeting TEADs has emerged as a novel option to inhibit YAP/TAZ, as TEADs are major mediators of their oncogenic programs. TEADs can also be leveraged on using small molecules to activate YAP/TAZ-dependent gene expression for use in regenerative medicine. Group III drugs focus on targeting one of the oncogenic downstream YAP/TAZ transcriptional target genes. With the right strategy and impetus, it is not far-fetched to expect a repurposed group I drug or a novel group II drug to combat YAP and TAZ in cancers in the near future.

Identification of the natural product paeonol derived from peony bark as an inhibitor of the Salmonella enterica serovar Typhimurium type III secretion system.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic pathogen in public health and food safety. The type III secretion system (T3SS) encoded by Salmonella pathogenicity island (SPI) is a sophisticated molecular machine that facilitates active invasion, intracellular replication, and host inflammation. Due to increasing antibiotic resistance, new therapeutic strategies that target the Salmonella T3SS have received considerable attention. In this study, paeonol was identified as an inhibitor of the S. Typhimurium T3SS. Paeonol significantly blocked the translocation of SipA into host cells and suppressed the expression of effector proteins without affecting bacterial growth in the effective concentration range. Additionally, S. Typhimurium-mediated cell injury and invasion levels were significantly reduced after treatment with paeonol, without cytotoxicity. Most importantly, the comprehensive protective effect of paeonol was confirmed in an S. Typhimurium mouse infection model. Preliminary mechanistic studies suggest that paeonol inhibits the expression of effector proteins by reducing the transcription level of the SPI-1 regulatory pathway gene hilA. This work provides proof that paeonol could be used as a potential drug to treat infections caused by Salmonella.

A high-throughput system to identify inhibitors of Candidatus Liberibacter asiaticus transcription regulators.

Citrus greening disease, also known as huanglongbing (HLB), is the most devastating disease of Citrus worldwide. This incurable disease is caused primarily by the bacterium Candidatus Liberibacter asiaticus and spread by feeding of the Asian Citrus Psyllid, Diaphorina citriCa L. asiaticus cannot be cultured; its growth is restricted to citrus phloem and the psyllid insect. Management of infected trees includes use of broad-spectrum antibiotics, which have disadvantages. Recent work has sought to identify small molecules that inhibit Ca L. asiaticus transcription regulators, based on a premise that at least some regulators control expression of genes necessary for virulence. We describe a synthetic, high-throughput screening system to identify compounds that inhibit activity of Ca L. asiaticus transcription activators LdtR, RpoH, and VisNR. Our system uses the closely related model bacterium, Sinorhizobium meliloti, as a heterologous host for expression of a Ca L. asiaticus transcription activator, the activity of which is detected through expression of an enhanced green fluorescent protein (EGFP) gene fused to a target promoter. We used this system to screen more than 120,000 compounds for compounds that inhibited regulator activity, but not growth. Our screen identified several dozen compounds that inhibit regulator activity in our assay. This work shows that, in addition to providing a means of characterizing Ca L. asiaticus regulators, an S. meliloti host can be used for preliminary identification of candidate inhibitory molecules.

Honeysuckle-derived microRNA2911 directly inhibits varicella-zoster virus replication by targeting IE62 gene.

Varicella-zoster virus (VZV) leads to chicken pox on primary infection and herpes zoster on reactivation. Recent studies suggest that microRNA2911 (MIR2911), honeysuckle (HS)-encoded atypical microRNA, has potential as a therapeutic agent against influenza and EV71 virus infections. Here, we report that MIR2911 directly inhibits VZV replication by targeting the IE62 gene. The luciferase reporter assay and bioinformatics prediction revealed that MIR2911 could target the IE62 gene of VZV. The VZV-encoded IE62 protein expression was inhibited significantly by synthetic MIR2911, while the expression of the mutants, whose MIR2911-binding sites were modified, was not inhibited. The RNA extracted from HS decoction and synthetic MIR2911 considerably suppressed VZV infection. However, it did not influence viral replication of a mutant virus with alterations in the nucleotide sequences of IE62. At the same time, the RNA extracted from HS decoction treated with the anti-MIR2911 antagomir could not inhibit the VZV replication, demonstrating that VZV replication was specifically and sufficiently inhibited by MIR2911. These results indicated that, by targeting the IE62 gene, MIR2911 may effectively inhibit VZV replication. Our results also suggest a potential novel strategy for the treatment and prevention of diseases caused by VZV infection.

Discovery of a New Pterocarpan-Type Antineuroinflammatory Compound from Sophora tonkinensis through Suppression of the TLR4/NFκB/MAPK Signaling Pathway with PU.1 as a Potential Target.

Neuroinflammation underlies many neuro-degenerative diseases. In this paper, we report the identification of a new pterocarpan-type anti-inflammatory compound named sophotokin isolated from Sophora tonkinensis. S. tonkinensis has been used traditionally for treatment of conditions related to inflammation. Our initial screening showed that sophotokin dose-dependently inhibits lipopolysaccharide (LPS)-stimulated production of NO, TNF-α, PGE2, and IL-1ß in microglial cells. This antineuroinflammatory effect was associated with sophotokin's blockade of LPS-induced production of the inflammatory mediators iNOS and COX-2. Western blot and qPCR analysis demonstrated that sophotokin inhibits both the p38-MAPK and NF-κB signal pathways. Further studies revealed that sophotokin also suppresses the expression of cluster differentiation 14 (CD14) in the toll-like receptor 4 (TLR4) signaling pathway. Following down-regulation of MyD88 and TRAF6, sophotokin inhibits the activation of the NF-κB and MAPK signal pathways in LPS-induced BV-2 cells. In silico studies suggested that sophotokin could interact with PU.1-DNA complex through hydrogen binding at sites 1 and 2 of the complex, blocking the DNA binding. This suggests that PU.1 may be a potential target of sophotokin. Taken together, these results suggest that sophotokin may have therapeutic potential for diseases related to neuroinflammation. The mechanism of antineuroinflammatory effects involves inhibition of the TLR4 signal pathway at the sites of NF-κB and MAPK with PU.1 as a likely upstream target.

Combination Therapy Strategy of Quorum Quenching Enzyme and Quorum Sensing Inhibitor in Suppressing Multiple Quorum Sensing Pathways of P. aeruginosa.

The threat of antibiotic resistant bacteria has called for alternative antimicrobial strategies that would mitigate the increase of classical resistance mechanism. Many bacteria employ quorum sensing (QS) to govern the production of virulence factors and formation of drug-resistant biofilms. Targeting the mechanism of QS has proven to be a functional alternative to conventional antibiotic control of infections. However, the presence of multiple QS systems in individual bacterial species poses a challenge to this approach. Quorum sensing inhibitors (QSI) and quorum quenching enzymes (QQE) have been both investigated for their QS interfering capabilities. Here, we first simulated the combination effect of QQE and QSI in blocking bacterial QS. The effect was next validated by experiments using AiiA as QQE and G1 as QSI on Pseudomonas aeruginosa LasR/I and RhlR/I QS circuits. Combination of QQE and QSI almost completely blocked the P. aeruginosa las and rhl QS systems. Our findings provide a potential chemical biology application strategy for bacterial QS disruption.

Virtual screening and biological evaluation of biofilm inhibitors on dual targets in quorum sensing system.

AIM: Resistance to conventional antibiotics has spurred interest in exploring new antimicrobial strategies. Suppressing quorum sensing within biofilm is a promising antimicrobial strategy. LasR in quorum sensing system of the Gram-negative bacteria, Pseudomonas aeruginosa, directly enhances virulence and antibiotic resistance, with QscR as its indirect suppressor, so targeting both of them can synergistically take the effect. METHODOLOGY/RESULTS: An in silico protocol combining pharmacophores with molecular docking was applied. Pharmacophores of QscR agonists and LasR antagonists were prepared for preliminary screening, followed by counter-screen using a pharmacophore model of LasR agonists and molecular docking of LasR. Four compounds with novel scaffolds were confirmed as potential biofilm inhibitors with preliminary experimental data. CONCLUSION: Novel biofilm inhibitors can be found with the method.

Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis.

The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway represents a promising therapeutic target to prevent fibrosis. We have tested the effects of new pharmacological inhibitors of MRTF/SRF signalling in a preclinical model of fibrosis. CCG-222740, a novel MRTF/SRF inhibitor, markedly decreased SRF reporter gene activity and showed a greater inhibitory effect on MRTF/SRF target genes than the previously described MRTF-A inhibitor CCG-203971. CCG-222740 was also five times more potent, with an IC50 of 5 µM, in a fibroblast-mediated collagen contraction assay, was less cytotoxic, and a more potent inhibitor of alpha-smooth muscle actin protein expression than CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery increased the long-term success of the surgery by 67% (P < 0.0005) and 33% (P < 0.01), respectively, and significantly decreased fibrosis and scarring histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG-203971 caused any detectable epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous, and serum. We conclude that inhibitors of MRTF/SRF-regulated gene transcription such as CCG-222740, potentially represent a new therapeutic strategy to prevent scar tissue formation in the eye and other tissues.

The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σ(E) -dependent sRNA RybB.

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self-produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ(E) cell envelope stress response and thereby reducing the expression of CsgD - a crucial activator of curli and cellulose biosynthesis - due to csgD mRNA targeting by the σ(E) -dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm-associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.

The Chinese medicine Sini-San inhibits HBx-induced migration and invasiveness of human hepatocellular carcinoma cells.

BACKGROUND: Sini-San (SNS) is a formulation of four Traditional Chinese Drugs that exhibits beneficial therapeutic effects in liver injury and hepatitis. However, there are no reports describing its effects on the hepatitis B X-protein (HBx)-induced invasion and metastasis in hepatoma cells, and the detailed molecular mechanisms of its actions are still unclear. METHODS: In this study, we investigated the mechanisms underlying SNS-mediated inhibition of HBx-induced cell invasion and the inhibition of secreted and cytosolic MMP-9 production, using gelatin zymography and Western blot analysis in a human hepatoma cell line (HepG2). Relative luciferase activity was assessed for MMP-9, NF-κB, or AP-1 reporter plasmid-transfected cells. RESULTS: SNS suppressed MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-κ B (NF-κB) activity. SNS suppressed HBx-induced AP-1 activity through inhibition of phosphorylation in the extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. SNS also suppressed HBx-induced inhibition of NF-κB nuclear translocation through IκB and suppressed HBx-induced activation of ERK/phosphatidylinositol 3-kinase/Akt upstream of NF-κB and AP-1. CONCLUSIONS: SNS suppresses the invasiveness and metastatic potential of hepatocellular carcinoma cells by inhibiting multiple signal transduction pathways.

Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance.

The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56-25 µg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract's lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies.

AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

Predicted drug-induced bradycardia related cardio toxicity using a zebrafish in vivo model is highly correlated with results from in vitro tests.

Several in vivo and in vitro studies have assessed methods of evaluating the cardio toxicity of compounds during drug development due to its importance for predicting human toxicity. However, in vivo/in vitro relationships have not yet been reported using a zebrafish model. This study determined the bradycardia of 15 compounds by evaluating the change in heart beat rate (HBR) in zebrafish, hERG fluorescence polarization (hERG-FP), and ionic current change using a patch clamp (hERG-PC). In addition, a model for prediction of drug-induced bradycardia was established using in vivo and in vitro assays designed for high-throughput toxicological screening. The IC(50) values correlated well in two in vitro studies (R(2)=0.9). The change in HBR in zebrafish caused by the compounds could be estimated using the IC(50) from the hERG-FP assay [(i.e., % of HBR=19.5×log(IC(50), hERG-FP)] or hERG-PC assay [(i.e., % of HBR=19.6×log(IC(50), hERG-FP)]. To validate the predictive model, 10 unknown compounds were used and the percentages of the HBR were estimated using the model. The observed and predicted HBR% for the compounds in zebrafish were well-correlated (R(2)=0.948). Therefore, the proposed models were useful for prediction of drug-induced bradycardia related cardio toxicity.

Screening of SdiA inhibitors from Melia dubia seeds extracts towards the hold back of uropathogenic E.coli quorum sensing-regulated factors.

Plants have always been a supreme source of drugs and India is endowed with a wide variety of them with high medicinal values. The Quorum Sensing (QS) quenching efficiency of various solvent extracts of Melia dubia seeds was investigated against uropathogenic Escherichia coli (UPEC) to screen the competitive inhibitor of SdiA, a transcriptional activator of quorum sensing in E. coli. In this study, potentiality of five different extracts of Melia dubia seeds for quorum sensing inhibitory activity was investigated against uropathogenic Escherichia coli (UPEC). Assays such as cell density, swarming motility, protein, protease, hemolysis, hemagglutination, hydrophobicity and biofilm inhibition were performed. Biofilm, hemolysis and swarming motility were found to be inhibited by 92.1%, 20.9 % and 48.52% respectively, when the medium was supplemented with 30 mg/ml of the ethanolic extract. GC-MS spectrum of the ethanolic extract showed an array of 27 structurally unlinked compounds with natural ligand C8HSL. The docking against QS transcriptional regulator SdiA was predicted by in silico studies and the ligand C6 showed significant activity with -10.8 GScore. In vitro and in silico docking analysis showed fairly a good correlation, suggesting that the ethanolic extract showed potency to attenuate quorum sensing of uropathogenic E. coli. Further studies by in vitro and in vivo strategies are necessary to foresee the quorum quenching effect of the ligands.

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines.

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.

Inhibition of the mTORC2 and chaperone pathways to treat leukemia.

Constitutive activation of the kinases Akt or protein kinase C (PKC) in blood cancers promotes tumor-cell proliferation and survival and is associated with poor patient survival. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) regulates the stability of Akt and conventional PKC (cPKC; PKCα and PKCß) proteins by phosphorylating the highly conserved turn motif of these proteins. In cells that lack mTORC2 function, the turn motif phosphorylation of Akt and cPKC is abolished and therefore Akt and cPKC protein stability is impaired. However, the chaperone protein HSP90 can stabilize Akt and cPKC, partially rescuing the expression of these proteins. In the present study, we investigated the antitumor effects of inhibiting mTORC2 plus HSP90 in mouse and human leukemia cell models and show that the HSP90 inhibitor 17-allylaminogeldanamycin (17-AAG) preferentially inhibits Akt and cPKC expression and promotes cell death in mTORC2 deficient pre-B leukemia cells. Furthermore, we show that 17-AAG selectively inhibits mTORC2 deficient leukemia cell growth in vivo. Finally, we show that the mTOR inhibitors rapamycin and pp242 work together with 17-AAG to inhibit leukemia cell growth to a greater extent than either drug alone. These studies provide a mechanistic and clinical rationale to combine mTOR inhibitors with chaperone protein inhibitors to treat human blood cancers.

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.

Structure-based virtual screening for plant-derived SdiA-selective ligands as potential antivirulent agents against uropathogenic Escherichia coli.

The uropathogenic Escherichia coli pathogenecity is affected by quorum sensing transcriptional regulator SdiA. In this study, in vitro characterization of the active principles that could potentially antagonize with SdiA from the Melia dubia bark extracts has been described. After in vitro assays carried out to evaluate the inhibitory activities against the uropathogenic E. coli, the ethanolic extract (30 mg/ml) which showed the strongest suppression of haemolysis, swarming motility, hydrophobicity and biofilm formation, was subjected to GC-MS analysis and an array of 40 unrelated compounds was identified. Docking studies was conducted to screen for plant-based SdiA inhibitors. Five hits were assessed for their binding profiles and 7-(1-bromoethyl)-3, 3-dimethyl-bicyclo [4.1.0]heptan-2-one showed 66.95% binding ability with respect to C(8)HSL.

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.