Fungal-derived xenobiotic exhibits antibacterial and antibiofilm activity against Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen, responsible for superficial and invasive infections both in nosocomial and community-acquired settings. The incidences of infection have become more problematic attributable to emerging drug resistance and biofilm formation. These challenges suggest the need for new antimicrobial agents against S. aureus. In present work, we purified a fungal xenobiotic (FI3) which elicits a potent antimicrobial activity against a list of tested microbes including methicillin sensitive (MSSA) and methicillin resistance (MRSA) S. aureus. The cell growth of MSSA and MRSA were completely ceased with the 1× minimum inhibitory concentration (MIC); 32 µg/mL and 128 µg/mL, respectively. The cell viability severely decreased within 90 min, due to disturbance of membrane homeostasis. This bactericidal effect was enhanced at lower pH (pH 4) with a speculation to retain positive charge. The FI3 potently disrupts biofilm adherence at 64 µg/mL and found to be a safe with no toxic effect on mammalian tissue. FI3 also leads to increase the potency of tested antibiotics. Taken together, we established that FI3 has a potent antimicrobial activity against tested microbes and safer to human tissue. It may be proven a leading molecule for the treatment of bacterial infections.

Identification of a xenobiotic as a potential environmental trigger in primary biliary cholangitis.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is an autoimmune-associated chronic liver disease triggered by environmental factors, such as exposure to xenobiotics, which leads to a loss of tolerance to the lipoic acid-conjugated regions of the mitochondrial pyruvate dehydrogenase complex, typically to the E2 component. We aimed to identify xenobiotics that might be involved in the environmental triggering of PBC. METHODS: Urban landfill and control soil samples from a region with high PBC incidence were screened for xenobiotic activities using analytical, cell-based xenobiotic receptor activation assays and toxicity screens. RESULTS: A variety of potential xenobiotic classes were ubiquitously present, as identified by their interaction with xenobiotic receptors - aryl hydrocarbon receptor, androgen receptor and peroxisome proliferator activated receptor alpha - in cell-based screens. In contrast, xenoestrogens were present at higher levels in soil extracts from around an urban landfill. Furthermore, two landfill sampling sites contained a chemical(s) that inhibited mitochondrial oxidative phosphorylation and induced the apoptosis of a hepatic progenitor cell. The mitochondrial effect was also demonstrated in human liver cholangiocytes from three separate donors. The chemical was identified as the ionic liquid [3-methyl-1-octyl-1H-imidazol-3-ium]+ (M8OI) and the toxic effects were recapitulated using authentic pure chemical. A carboxylate-containing human hepatocyte metabolite of M8OI, bearing structural similarity to lipoic acid, was also enzymatically incorporated into the E2 component of the pyruvate dehydrogenase complex via the exogenous lipoylation pathway in vitro. CONCLUSIONS: These results identify, for the first time, a xenobiotic in the environment that may be related to and/or be a component of an environmental trigger for PBC. Therefore, further study in experimental animal models is warranted, to determine the risk of exposure to these ionic liquids. LAY SUMMARY: Primary biliary cholangitis is a liver disease in which most patients have antibodies to mitochondrial proteins containing lipoic acid binding site(s). This paper identified a man-made chemical present in soils around a waste site. It was then shown that this chemical was metabolized into a product with structural similarity to lipoic acid, which was capable of replacing lipoic acid in mitochondrial proteins.

A review of nanoscale LC-ESI for metabolomics and its potential to enhance the metabolome coverage.

Liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) platforms are widely used to perform high throughput untargeted profiling of biological samples for metabolomics-based approaches. However, these LC-ESI platforms usually favour the detection of metabolites present at relatively high concentrations because of analytical limitations such as ion suppression, thus reducing overall sensitivity. To counter this issue of sensitivity, the latest in terms of analytical platforms can be adopted to enable a greater portion of the metabolome to be analysed in a single analytical run. Here, nanoflow liquid chromatography-nanoelectrospray ionisation (nLC-nESI), which has previously been utilised successfully in proteomics, is explored for use in metabolomic and exposomic research. As a discovery based field, the markedly increased sensitivity of these nLC-nESI platforms offer the potential to uncover the roles played by low abundant signalling metabolites (e.g. steroids, eicosanoids) in health and disease studies, and would also enable an improvement in the detection of xenobiotics present at trace levels in biological matrices to better characterise the chemical exposome. This review aims to give an insight into the advantages associated with nLC-nESI for metabolomics-based approaches. Initially we detail the source of improved sensitivity prior to reviewing the available approaches to achieving nanoflow rates and nanospray ionisation for metabolomics. The robustness of nLC-nESI platforms was then assessed using the literature available from a metabolomic viewpoint. We also discuss the challenging point of sample preparation which needs to be addressed to fully enjoy the benefits of these nLC-nESI platforms. Finally, we assess metabolomic analysis utilising nano scale platforms and look ahead to the future of metabolomics using these new highly sensitive platforms.

Organ-on-a-chip for assessing environmental toxicants.

Man-made xenobiotics, whose potential toxicological effects are not fully understood, are oversaturating the already-contaminated environment. Due to the rate of toxicant accumulation, unmanaged disposal, and unknown adverse effects to the environment and the human population, there is a crucial need to screen for environmental toxicants. Animal models and in vitro models are ineffective models in predicting in vivo responses due to inter-species difference and/or lack of physiologically-relevant 3D tissue environment. Such conventional screening assays possess limitations that prevent dynamic understanding of toxicants and their metabolites produced in the human body. Organ-on-a-chip systems can recapitulate in vivo like environment and subsequently in vivo like responses generating a realistic mock-up of human organs of interest, which can potentially provide human physiology-relevant models for studying environmental toxicology. Feasibility, tunability, and low-maintenance features of organ-on-chips can also make possible to construct an interconnected network of multiple-organs-on-chip toward a realistic human-on-a-chip system. Such interconnected organ-on-a-chip network can be efficiently utilized for toxicological studies by enabling the study of metabolism, collective response, and fate of toxicants through its journey in the human body. Further advancements can address the challenges of this technology, which potentiates high predictive power for environmental toxicology studies.

Nutrition effects on the biofilm immobilization and 3,5-DNBA degradation of Comamonas testosteroni A3 during bioaugmentation treatment.

The survival of inoculated microbes is critical for successful bioaugmentation in wastewater treatment. The influence of readily available nutrients (RANs) on the colonization of two functional bacteria, Pseudomonas putida M9, a strong biofilm-forming strain, and Comamonas testosteroni A3, a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading strain, in biofilms was studied with 3,5-dinitrobenoic acid synthetic wastewater (DCMM) complemented with various ratios of Luria-Bertani broth (LB). With the increase in LB rate, the biofilm biomass was increased, the percentage of gfp-labeled M9 measured in the mixed culture enhanced, and also M9 became dominant. In laboratory-scale sequencing batch biofilm reactors, with the increase in 3,5-DNBA concentration and extension of the running time, the 3,5-DNBA removal in DCMM wastewater complemented with RANs tended to be more efficient and its removal rates increased gradually over the experimental period. Our study demonstrated that supplementing RANs could be a useful strategy for enhancing colonization of degrading bacteria in wastewater treatment systems.

The use of used automobile tyres in a partitioning bioreactor for the biodegradation of xenobiotic mixtures.

Waste tyres were utilized as the sorption phase in a two-phase partitioning bioreactor (TPPB) for the biodegradation of a binary mixture of 2,4-dichlorophenol (DCP) and 4-nitrophenol (4NP). These compounds are extensively used in the chemical industry and are found in many industrial effluents. Although both compounds are toxic and are on the EPA list of priority pollutants, a higher inhibitory effect on microorganisms is exerted by DCP, and our experimental tests were focused on strategies to reduce its negative impact on microbial activity. Sorption/desorption tests for the DCP-4NP mixture were first performed to verify the related uptake/release rates by the tyres, which showed that the tyres had a higher capacity for DCP uptake and practically no affinity for 4NP. An acclimatized mixed culture was then utilized in a sequencing batch reactor (SBR) operated in conventional and two-phase mode. For the binary DCP-4NP mixture a significant reduction in DCP toxicity, and a concomitant enhancement in substrate removal efficiency (up to 83%for DCP and approximate 100% for 4NP) were clearly seen for the TPPB operated with 10% and 15% v/v tyres, for influent concentrations up to 180 mg/L, with practically negligible biodegradation in the conventional single phase reactor. The long-term utilization of tyres was confirmed at an influent loading of 180 mg/L with a test performed over 20 work cycles showing an improvement of the removal performance for both compounds.

Use of on-site bioreactors to estimate the biotransformation rate of N-ethyl perfluorooctane sulfonamidoethanol (N-EtFOSE) during activated sludge treatment.

Accurate rates are needed for models that predict the fate of xenobiotic chemicals and impact of inhibitors at full-scale wastewater treatment plants. On-site rates for aerobic biotransformation of N-ethyl perfluorooctane sulfonamidoethanol (N-EtFOSE), a fluorinated repellent, were determined by continuously pumping mixed liquor from an aeration basin into two well-mixed acrylic bioreactors (4-L) operated in parallel. Known masses of N-EtFOSE and bromide were continuously added to the reactors. Reactor effluents were then monitored for bromide, N-EtFOSE, and metabolites of N-EtFOSE. Of the six transformation products reported in batch studies, only N-ethyl perfluorooctane sulfonamido acetate (N-EtFOSAA) was detected in the effluents. Bromide addition to the reactors enabled rate estimates despite variations in flow rate. Pseudo-second order rate coefficients for the N-EtFOSE biotransformation to N-EtFOSAA, predicted using a dynamic model of the reactor system, were k=2.0 and 2.4Lg(-1)VSSd(-1) for the two reactors, which are slower than the rates previously obtained using batch reactors. Given the relatively slow rate of N-EtFOSE transformation, its sorption and volatilization may be important in wastewater processes. The methodology used in this study should be suitable for similar on-site rate assessments with other contaminants or inhibitors.

Validated liquid chromatography-tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDP-glucuronosyltransferases.

A simplified, rapid, and selective liquid chromatography-tandem mass spectrometry method for the determination of the activities of cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in two separate settings was developed and successfully applied to 8 CYP isoenzymes and UGT2B7 enzyme activities in rat liver microsomes. The triple-quadrupole mass spectrometric detection was operated in positive mode for the probe metabolites: CYP1A2 (resorufin), CYP2B6 (hydroxybupropion), CYP2C19 (5-hydroxyomeprazole), CYP2D6 (dextrophan), CYP3A4 (6ß-hydroxytestosterone), and UGT2B7 (morphine-3-glucuronide); also in negative mode for CYP2C9 (4-hydroxytolbutamide), CYP2E1 (6-hydroxychloroxazone), and CYP4A (hydroxylauric acid). The metabolic reactions were terminated with acetonitrile, containing metoprolol and acetaminophen as the internal standard for positive and negative ion electrospray ionization, respectively. The method was validated over the concentration range of 25-2500 ng/mL for 5-hydroxyomeprazole, dextrophan, hydroxylauric acid, and morphine-3-glucuronide; 5-500 ng/mL for resorufin; 3-300 ng/mL for hydroxybupropion; 10-1000 ng/mL for 4-hydroxytolbutamide; 40-4000 ng/mL for 6-hydroxychloroxazone; and 63-6300 ng/mL for 6ß-hydroxytestosterone. All of the extraction recoveries of these analytes were greater than 85%, except for hydroxylauric acid at mid-concentration with a recovery of 83.2% ± 3.2%. The matrix effects were between 85.8% and 119.9%; the respective within- and between-run precisions were 0.9-12.0% and 2.0-13.9%; and the within- and between-run accuracy levels were 0.6-17.2% and 0.1-15.1%, respectively, for all these analytes. All of the analytes were stable during the assay and storage in the liver microsomes of Sprague-Dawley rats. The measurement activity of multiple enzymes was feasible using a cocktail approach. This method proved to be a robust, fast, accurate, specific and sensitive assay, and was successfully used to investigate in vivo enzyme activities of 8 major CYP isoenzymes and UGT2B7 in Sprague-Dawley rats with fatty livers. By the end of the eighth week, the CD-fed induced fatty liver rats showed a significant decrease in the activities of CYP1A2 and UGT2B7 as compared to the standard diet group.

A case-study on the accuracy of mass balances for xenobiotics in full-scale wastewater treatment plants.

Removal efficiencies of micropollutants in wastewater treatment plants (WWTPs) are usually evaluated from mass balance calculations using a small number of observations drawn from short sampling campaigns. Since micropollutant loads can vary greatly in both influent and effluent and reactor tanks exhibit specific hydraulic residence times, these short-term approaches are particularly prone to yield erroneous removal values. A detailed investigation of micropollutant transit times at full-scale and on how this affects mass balancing results was still lacking. The present study used hydraulic residence time distributions to scrutinize the match of influent loads to effluent loads of 10 polar micropollutants with different influent dynamics in a full-scale WWTP. Prior hydraulic modeling indicated that a load sampled over one day in the effluent is composed of influent load fractions of five preceding days. Results showed that the error of the mass balance can be reduced with increasing influent sampling duration. The approach presented leads to a more reliable estimation of the removal efficiencies of those micropollutants which can be constantly detected in influents, such as pharmaceuticals, but provides no advantage for pesticides due to their sporadic occurrence. The mismatch between sampled influent and effluent loads was identified as a major error source and an explanation was provided for the occurrence of negative mass balances regularly reported. This study indicates that the accurate determination of global removal values is only feasible in full-scale investigations with sampling durations much longer than 1 day. In any case, the uncertainty of these values needs to be reported when used in removal assessment, model selection or validation.

[Role of ancestral genotypes of the genes of xenobiotics biotransformation in susceptibility to lung cancer among Mayak workers].

An association between polymorphous (allelic) variants of genes of the 1st Phase of enzymatic xenobiotic biotransformation (EXB) in the superfamily of cytochromes P450 gene CYP1A1 * 2A (ml polymorphism), CYP1A1 * 2C (m2 polymorphism), gene CYP2E1 * 6 (C polymorphism), and between polymorphous vari- ants of gene of the 2nd EXB Phase of mircosomal epoxide hydrolase mEPOX Tyr113His (EH3 polymor- phism), and mEPOXHis139Arg (EH4 polymorphism) and lung cancer in Mayak workers with occupational prolonged exposure was studied. Analysis of population genotype frequency and alleles of genes CYP1A1 * 2A, CYP1A1 * 2C, CYP2E1 * 6, mEPOX Tyr113His, and mEPOXHis139Arg was conducted in the group of Mayak workers (289 individuals). The study has shown their compliance with the Hardy-Weinberg equilibrium, and correspondence of the genotype frequency to their frequency in European population. The study was per- formed using case-control method. Case-group consisted of 49 Mayak workers with verified diagnosis of lung cancer. Control-group consisted of 172 Mayak workers without lung cancer registered at the moment of the research. There was an increasing trend in odds ratio (OR) of lung cancer among homozygous carriers of the mutation in pp gene CYP1A1 * 2A, heterozygous carriers of the mutation in wp gene mEPOX Tyr113His and homozygous carriers ofww gene CYP2E1 * 6; also, there was a decreasing trend in OR among heterozy- gous carriers of wp gene CYP2E1 * 6. Two variants of pair allelic combinations of genes (genotypes ww CYP1A1 * 2A/genotype wp mEPOX Tyr113His, and genotype ww CYP2E1 * 6/genotype wp mEPOX Tyr113His) were revealed, which carriers had a statistically significant increase in the odds ratio of lung can- cer. With regard to these variants of genotype combinations, OR of lung cancer was 2.01 (CI 95%: 1.04-3.91) and 2.09 (CI 95%: 1.08-4.03), correspondingly.

Magnetic techniques for the detection and determination of xenobiotics and cells in water.

Magnetic techniques based on the application of magnetic nanoparticles and microparticles and films have been successfully used for the determination and detection of different types of xenobiotics (e.g. herbicides, insecticides, fungicides, aromatic and polyaromatic hydrocarbons, pentachlorophenol and heavy metal ions) as well as viruses, microbial pathogens and protozoan parasites in water samples. Preconcentration of xenobiotics from large volumes of samples can be performed using magnetic solid-phase extraction, stir-bar sorptive extraction and related procedures. This review provides basic information about these techniques. Published examples of successful applications document the importance of these simple and efficient procedures employing magnetic materials.

Biogenic substrate benefits activated sludge in acclimation to a xenobiotic.

Activated sludge that originated from a biogenic fed-batch reactor under steady-state was re-cultivated with the same biogenic substrates to test the changes in the sludge's performance in acclimation and degradation of a xenobiotic. Re-cultivations with varying biogenic concentrations were conducted at time points ranging from 16 d before to 4 d after the acclimation reactions. Biogenic re-cultivation energizes sludge cells thereby benefiting the re-cultivated biomass by shortening its acclimation lag time. Lag time increases on both sides of the re-cultivation time where lag has been shortened the most: (1) in short re-cultivation times before and after acclimation reactions, high concentrations of new or unfinished biogenic substrates cause diauxic growth that delays acclimation; (2) in long re-cultivation times, the re-cultivated biomass loses its energy-rich advantage. Both these lag lengthening situations have their worst cases in which acclimation lag times become longer than that of the original sludge, thus counterbalancing the benefits.

Xenobiotic removal efficiencies in wastewater treatment plants: residence time distributions as a guiding principle for sampling strategies.

The effect of mixing regimes and residence time distribution (RTD) on solute transport in wastewater treatment plants (WWTPs) is well understood in environmental engineering. Nevertheless, it is frequently neglected in sampling design and data analysis for the investigation of polar xenobiotic removal efficiencies in WWTPs. Most studies on the latter use 24-h composite samples in influent and effluent. The effluent sampling period is often shifted by the mean hydraulic retention time assuming that this allows a total coverage of the influent load. However, this assumption disregards mixing regime characteristics as well as flow and concentration variability in evaluating xenobiotic removal performances and may consequently lead to biased estimates or even negative elimination efficiencies. The present study aims at developing a modeling approach to estimate xenobiotic removal efficiencies from monitoring data taking the hydraulic RTD in WWTPs into consideration. For this purpose, completely mixed tanks-in-series were applied to address hydraulic mixing regimes in a Luxembourg WWTP. Hydraulic calibration for this WWTP was performed using wastewater conductivity as a tracer. The RTD mixing approach was coupled with first-order biodegradation kinetics for xenobiotics covering three classes of biodegradability during aerobic treatment. Model simulations showed that a daily influent load is distributed over more than one day in the effluent. A 24-h sampling period with an optimal time offset between influent and effluent covers less than the half of the influent load in a dry weather scenario. According to RTD calculations, an optimized sampling strategy covering four consecutive measuring days in the influent would be necessary to estimate the full-scale elimination efficiencies with sufficient accuracy. Daily variations of influent flow and concentrations can substantially affect the reliability of these sampling results. Commonly reported negative removal efficiencies for xenobiotics might therefore be a consequence of biased sampling schemes. In this regard, the present study aims at contributing to bridge the gap between environmental chemistry and engineering practices.

Comparison of PPCPs removal on a parallel-operated MBR and AS system and evaluation of effluent post-treatment on vertical flow reed beds.

The presence in the aquatic environment of xenobiotics such as Pharmaceutical and Personal Care Products (PPCPs) has emerged as an issue of concern. Upgrading sewage treatment quality with modern technologies such as Membrane Bioreactors (MBRs) and/or implementing a further posttreatment might mitigate the release of xenobiotics into surface waters. The performance of two processes treating municipal sewage, a MBR and an Activated Sludge (AS) unit, have been compared in terms of PPCPs removal. Moreover, their effluents were treated using vertical flow reed beds. Both systems were operated under similar conditions, more specifically Hydraulic Retention Time (HRT), maintained at 8 hours, and Sludge Retention Time (SRT) set at 6 and 20 days. Pharmaceuticals belong to therapeutic groups such as antiepileptics (carbamazepine) and analgesics (ibuprofen, naproxen, diclofenac), whereas the personal care products are musk fragrances (galaxolide and tonalide). Xenobiotics removals achieved in the MBR showed better results, particularly for the acidic drugs ibuprofen (87% vs. 50%) and naproxen (56% vs. 6%) operating at low SRT. After filtration through vertical flow reed-beds, PPCPs content in effluents was decreased, below 1 ppb in most cases, improving the effluent quality and confirming reed-beds as an interesting low cost alternative in order to attenuate xenobiotics contamination.

High-performance liquid chromatography-mass spectrometry (HPLC-MS)-based drug metabolite profiling.

The identification of drug metabolites in biofluids such as urine, plasma, and bile, as well as in in vitro systems, is an important step in drug discovery and development. Mass spectrometry, particularly when combined with high-performance liquid chromatography (HPLC-MS), can enable detailed structural information to be obtained on the metabolites of a drug or xenobiotic as a result of metabolism. The successful identification of drug metabolites by HPLC-MS-based techniques requires careful optimisation of a number of factors. First, the chromatographic separation should provide good resolution of the individual xenobiotic metabolites present in the sample. There is also the need to minimise the interference caused by the presence of endogenous metabolites, which may interfere with MS detection. Ideally, untreated samples should be profiled to reduce the likelihood of missing important metabolites due to losses during sample processing, but, depending upon the matrix, some degree of sample cleanup/extraction and concentration of the metabolites may be required using liquid-liquid or liquid-solid extraction. Second, the MS conditions must be carefully selected in order to maximise the potential of detecting the separated metabolites, which may have a very different character to the parent compound. The use of radiolabelled drugs in metabolism experiments greatly aids in the detection (and quantification) of metabolites, directing the investigator towards peaks that need to be characterised by MS. The presence of characteristic isotope patterns from either the incorporation of stable isotopes (e.g. (13)C, (15)N) or naturally occurring isotope patterns from substituents on the molecule (e.g. (35/37)Cl, (79/81)Br) can also provide a useful handle on the drug and its metabolites for the purposes of detection and spectrometric interpretation. This chapter provides guidelines for, and examples of, HPLC-MS-based drug metabolite profiling.

Biological reactive intermediates (BRIs) formed from botanical dietary supplements.

The use of botanical dietary supplements is increasingly popular, due to their natural origin and the perceived assumption that they are safer than prescription drugs. While most botanical dietary supplements can be considered safe, a few contain compounds, which can be converted to biological reactive intermediates (BRIs) causing toxicity. For example, sassafras oil contains safrole, which can be converted to a reactive carbocation forming genotoxic DNA adducts. Alternatively, some botanical dietary supplements contain stable BRIs such as simple Michael acceptors that react with chemosensor proteins such as Keap1 resulting in induction of protective detoxification enzymes. Examples include curcumin from turmeric, xanthohumol from hops, and Z-ligustilide from dang gui. Quinones (sassafras, kava, black cohosh), quinone methides (sassafras), and epoxides (pennyroyal oil) represent BRIs of intermediate reactivity, which could generate both genotoxic and/or chemopreventive effects. The biological targets of BRIs formed from botanical dietary supplements and their resulting toxic and/or chemopreventive effects are closely linked to the reactivity of BRIs as well as dose and time of exposure.

Occurrence of xenobiotics in gray water and removal in three biological treatment systems.

Eighteen selected xenobiotics related to personal care and household chemicals (UV-filters, fragrances, preservatives, biocides, surfactants) were measured in gray water from 32 houses and in effluents of three different biological treatment systems (aerobic, anaerobic, and combined anaerobic+aerobic). All selected xenobiotics were detected in gray water samples in the low microg L(-1) range. Generally, lower concentrations were measured after biological treatment and removal efficiencies were higher under aerobic conditions than under anaerobic conditions. However, most of the xenobiotics were still detected in biologically treated gray water. The most persistent compounds were the fragrance tonalide and the UV-filters 2-phenyl-5-benzimidazolesulfonic acid and ethylhexyl methoxycinnamate. Estimated estrogenic potential of the effluent ranged between 0.07 and 0.72 ng L(-1) of 17beta-estradiol equivalents. Depending on the application of the effluent and its environmental risk, physical-chemical processes might be required to increase the removal efficiency of these compounds from gray water.

High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation.

The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.