Hepatocytes of Wistar and Sprague Dawley rats differ significantly in their central metabolism.

Wistar and Sprague-Dawley (SD) rats are most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and SD rat hepatocytes from middle (6 months) and old (23 months) age groups. Principle component analysis (PCA) on the specific uptake and production rates of amino acids, glucose, lactate and urea indicated clear differences between Wistar and SD rat hepatocytes. SD rat hepatocytes showed higher uptake rates of various essential and non-essential amino acids, particularly in early culture phases (0-12 h) compared to later phases (12-24 h). SD hepatocytes seem to be more sensitive to isolation procedure and in vitro culture requiring more amino acids for cellular maintenance and repair. Major differences between Wistar and SD rat hepatocytes were observed for glucose and branched chain amino acid metabolism. We conclude that the observed differences in the central carbon metabolism of isolated hepatocytes from these two rats should be considered when using one or the other rat type in studies on metabolic effects or diseases such as diabetes or obesity.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

A shift in paradigm towards human biology-based systems for cholestatic-liver diseases.

Cholestatic-liver diseases (CLDs) arise from diverse causes ranging from genetic factors to drug-induced cholestasis. The so-called diseases of civilization (obesity, diabetes, metabolic disorders, non-alcoholic liver disease, cardiovascular diseases, etc.) are intricately implicated in liver and gall bladder diseases. Although CLDs have been extensively studied, there seem to be important gaps in the understanding of human disease. Despite the fact that many animal models exist and substantial clinical data are available, translation of this knowledge towards therapy has been disappointingly limited. Recent advances in liver cell culture such as in vivo-like 3D cultivation of human primary hepatic cells, human induced pluripotent stem cell-derived hepatocytes; and cutting-edge analytical techniques such as 'omics' technologies and high-content screenings could play a decisive role in deeper mechanistic understanding of CLDs. This Topical Review proposes a roadmap to human biology-based research using omics technologies providing quantitative information on mechanisms in an adverse outcome/disease pathway framework. With modern sensitive tools, a shift in paradigm in human disease research seems timely and even inevitable to overcome species barriers in translation.

Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study.

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in 'omics'-based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum-supplemented and serum-free conditions. Viability and Cytochrome P450 (CYP) activity during long-term cultivation were assessed. Liver-specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum-free and serum-supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum-free conditions for 30 days without viability change and with high CYP activity. We also tested the acute (24 h) and long-term repeated-dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4 mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum-free conditions opens up the opportunity for the use of these cells in human long-term repeated-dose hepatotoxicity studies and for application in systems toxicology.

Metabolic profiling using HPLC allows classification of drugs according to their mechanisms of action in HL-1 cardiomyocytes.

Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening comprising of not only electrophysiological characterization but also cellular molecular alterations are expected to improve the cardiotoxicity prediction potential. Metabolomic approaches recently have become an important focus of research in pharmacological testing and prediction. In this study, the culture medium supernatants from HL-1 cardiomyocytes after exposure to drugs from different classes (analgesics, antimetabolites, anthracyclines, antihistamines, channel blockers) were analyzed to determine specific metabolic footprints in response to the tested drugs. Since most drugs influence energy metabolism in cardiac cells, the metabolite "sub-profile" consisting of glucose, lactate, pyruvate and amino acids was considered. These metabolites were quantified using HPLC in samples after exposure of cells to test compounds of the respective drug groups. The studied drug concentrations were selected from concentration response curves for each drug. The metabolite profiles were randomly split into training/validation and test set; and then analysed using multivariate statistics (principal component analysis and discriminant analysis). Discriminant analysis resulted in clustering of drugs according to their modes of action. After cross validation and cross model validation, the underlying training data were able to predict 50%-80% of conditions to the correct classification group. We show that HPLC based characterisation of known cell culture medium components is sufficient to predict a drug's potential classification according to its mode of action.

Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.

Gene expression profile of CD14+ blood monocytes following lifestyle-induced weight loss in individuals with metabolic syndrome.

Lifestyle-induced weight loss is regarded as an efficient therapy to reverse metabolic syndrome (MetS) and to prevent disease progression. The objective of this study was to investigate whether lifestyle-induced weight loss modulates gene expression in circulating monocytes. We analyzed and compared gene expression in monocytes (CD14+ cells) and subcutaneous adipose tissue biopsies by unbiased mRNA profiling. Samples were obtained before and after diet-induced weight loss in well-defined male individuals in a prospective controlled clinical trial (ICTRP Trial Number: U1111-1158-3672). The BMI declined significantly (- 12.6%) in the treatment arm (N = 39) during the 6-month weight loss intervention. This was associated with a significant reduction in hsCRP (- 45.84%) and circulating CD14+ cells (- 21.0%). Four genes were differentially expressed (DEG's) in CD14+ cells following weight loss (ZRANB1, RNF25, RB1CC1 and KMT2C). Comparative analyses of paired CD14+ monocytes and subcutaneous adipose tissue samples before and after weight loss did not identify common genes differentially regulated in both sample types. Lifestyle-induced weight loss is associated with specific changes in gene expression in circulating CD14+ monocytes, which may affect ubiquitination, histone methylation and autophagy.

Enhanced cellular uptake and cytotoxicity studies of organometallic bioconjugates of the NLS peptide in Hep G2 cells.

SPACE INVADERS: Organometallic fragments such as the ferrocenyl group (shown in red in the picture) help to enhance cellular entry of NLS peptides. Eventually, these nontoxic conjugates find their way to the cellular nucleus as shown by fluorescence microscopy studies in this work. Intracellular delivery to biomolecular targets is still a major challenge in molecular and cell biology. We recently found that attaching an organometallic group, namely the cobaltocenium cation, to the SV 40 large T antigen nuclear localisation signal (NLS) greatly enhances cellular uptake of the conjugate (Noor et al., Angew. Chem. Int. Ed. 2005, 45, 2429). In addition, nuclear localisation of the conjugate was observed. In this work, we present a thorough investigation of this novel cellular delivery system with respect to the nature of the metal complex and the peptide sequence. A number of ferrocene ((Fe(II)), neutral metal complex) and cobaltocenium ((Co(III)), cationic metal complex) bioconjugates with both the NLS wild-type sequence PKKKRKV and a scrambled sequence (NLS(scr), KKVKPKR) were prepared by solid-phase peptide synthesis (SPPS). Cellular and nuclear uptake of these bioconjugates was studied by fluorescence microscopy on living Hep G2 cells. In addition, cytotoxicity screening on the conjugates was carried out, as the toxic effects of several simple metallocenes have been noted previously. Rapid cellular uptake as well as nuclear localisation was observed for the metal-NLS conjugates, but not for any dipeptide controls, the metal-NLS(scr) conjugates or any metal-free conjugates. It thus appears that the presence of a metallocene, but not its charge, and the correct NLS sequence is essential for cellular uptake. Fluorescence microscopy co-localisation studies did not reveal a significant endosomal entrapment of the conjugates. The metallocene not only provides a hydrophobic handle for membrane translocation but also facilitates the localisation and distribution of the conjugate in the cytoplasm. The NLS peptide on the other hand is responsible for the nuclear localisation of the bioconjugate. Finally, none of the conjugates were found to be toxic up to the highest concentrations that was tested (1 mM) against the Hep G2 cells that were used in this study. In conclusion, this work supports metallocene-NLS bioconjugates, in particular with the very robust cobaltocenium group, as a simple but potent, nontoxic system for cellular uptake and nuclear delivery. Concurrently, our finding is relevant to the still-unresolved question of cytotoxicity of metallocenes because it excludes binding and/or damage to the DNA as a mechanism of metallocene cytotoxicity. This finding is confirmed by a combined yeast cytotoxicity/genotoxicity assay, which also shows very little toxic effects for all organometal-NLS conjugates that were tested.

Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2.

Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC(50) values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs.

An integrated approach to improved toxicity prediction for the safety assessment during preclinical drug development using Hep G2 cells.

Efficient and accurate safety assessment of compounds is extremely important in the preclinical development of drugs especially when hepatotoxicity is in question. Multiparameter and time resolved assays are expected to greatly improve the prediction of toxicity by assessing complex mechanisms of toxicity. An integrated approach is presented in which Hep G2 cells and primary rat hepatocytes are compared in frequently used cytotoxicity assays for parent compound toxicity. The interassay variability was determined. The cytotoxicity assays were also compared with a reliable alternative time resolved respirometric assay. The set of training compounds consisted of well known hepatotoxins; amiodarone, carbamazepine, clozapine, diclofenac, tacrine, troglitazone and verapamil. The sensitivity of both cell systems in each tested assay was determined. Results show that careful selection of assay parameters and inclusion of a kinetic time resolved assay improves prediction for non-metabolism mediated toxicity using Hep G2 cells as indicated by a sensitivity ratio of 1. The drugs with EC(50) values 100 microM or lower were considered toxic. The difference in the sensitivity of the two cell systems to carbamazepine which causes toxicity via reactive metabolites emphasizes the importance of human cell based in-vitro assays. Using the described system, primary rat hepatocytes do not offer advantage over the Hep G2 cells in parent compound toxicity evaluation. Moreover, respiration method is non invasive, highly sensitive and allows following the time course of toxicity. Respiration assay could serve as early indicator of changes that subsequently lead to toxicity.

The Gut Microbiota and Hematopoietic Stem Cell Transplantation: Challenges and Potentials.

The human gut microbiota gained tremendous importance in the last decade as next-generation technologies of sequencing and multiomics analyses linked the role of the microbial communities to host physiology and pathophysiology. A growing number of human pathologies and diseases are linked to the gut microbiota. One of the main mechanisms by which the microbiota influences the host is through its interactions with the host immune system. These interactions with both innate and adaptive host intestinal and extraintestinal immunity, although usually commensalistic even mutualistic with the host, in some cases lead to serious health effects. In the case of allogenic hematopoietic stem cell transplantation (allo-HSCT), the disruption of the intestinal microbiota diversity is associated with acute graft-versus-host disease (GvHD). Causing inflammation of the liver, skin, lungs, and the intestine, GvHD occurs in 40-50% of patients undergoing allo-HSCT and results in significant posttransplantation mortality. In this review, we highlight the impact of the gut microbiota on the host immunity in GvHD and the potential of microbiota in alleviation or even prevention of GvHD.

Increased Circulating VAP-1 Levels Are Associated with Liver Fibrosis in Chronic Hepatitis C Infection.

Vascular adhesion protein-1 (VAP-1) is a multifunction protein. While membrane-bound VAP-1 is an adhesion protein, soluble VAP-1 catalyzes the deamination of primary amines through its semicarbazide-sensitive amino oxidase (SSAO) activity. VAP-1 supports the transmigration of leukocytes and increases oxidative stress. In chronic liver diseases, it plays a role in leukocyte infiltration and fibrogenesis. Here, we measured VAP-1 plasma concentration and its SSAO activity in 322 patients with chronic hepatitis C infection and evaluated the association of VAP-1 with fibrosis stages. VAP-1 concentration strongly correlated with liver stiffness and was the second strongest influencing variable after gamma-glutamytransferase (GGT) for liver stiffness in regression analysis. The VAP-1 concentration increased with advancing fibrosis stages and the highest concentrations were found in patients with cirrhosis. According to the receiver operating characteristic (ROC) analysis, a VAP-1 cut-off value of 541 ng/mL predicted histologically confirmed cirrhosis (sensitivity 74%; specificity 72%). SSAO activity correlated only moderately with liver stiffness, showing a relatively small increase in advanced fibrosis. To our knowledge, this is the first study on VAP-1 in chronic hepatitis C infection showing its association with progressive fibrosis. In conclusion, VAP-1 plasma concentration, rather than its SSAO activity, may represent a non-invasive biomarker for monitoring fibrogenesis in patients with chronic hepatitis C infection.

Synthesis and pharmacological activities of 7-azaindole derivatives.

As a part of our program to discover novel analogues of 7-azaindole (1H-Pyrrolo[2,3-b] pyridine) having useful biological activities, some derivatives have been synthesized and evaluated for their analgesic and hypotensive activity. Compounds evaluated by thermal stimuli (tail immersion method) at the dose of 50 mg/kg of body weight revealed significant analgesic activity. Pethidine was used as reference drug. Same compounds tested at the dose of 75 mg/kg of body weight showed toxicity. Compounds tested for their effect on blood pressure in normotensive rat produced slight fall in blood pressure.

Accessing 3D microtissue metabolism: Lactate and oxygen monitoring in hepatocyte spheroids.

3D hepatic microtissues, unlike 2D cell cultures, retain many of the in-vivo-like functionalities even after long-term cultivation. Such 3D cultures are increasingly applied to investigate liver damage due to drug exposure in toxicology. However, there is a need for thorough metabolic characterization of these microtissues for mechanistic understanding of effects on culture behaviour. We measured metabolic parameters from single human HepaRG hepatocyte spheroids online and continuously with electrochemical microsensors. A microsensor platform for lactate and oxygen was integrated in a standard 96-well plate. Electrochemical microsensors for lactate and oxygen allow fast, precise and continuous long-term measurement of metabolic parameters directly in the microwell. The demonstrated capability to precisely detect small concentration changes by single spheroids is the key to access their metabolism. Lactate levels in the culture medium starting from 50µM with production rates of 5µMh-1 were monitored and precisely quantified over three days. Parallel long-term oxygen measurements showed no oxygen depletion or hypoxic conditions in the microwell. Increased lactate production by spheroids upon suppression of the aerobic metabolism was observed. The dose-dependent decrease in lactate production caused by the addition of the hepatotoxic drug Bosentan was determined. We showed that in a toxicological application, metabolic monitoring yields quantitative, online information on cell viability, which complements and supports other methods such as microscopy. The demonstrated continuous access to 3D cell culture metabolism within a standard setup improves in vitro toxicology models in replacement strategies of animal experiments. Controlling the microenvironment of such organotypic cultures has impact in tissue engineering, cancer therapy and personalized medicine.

Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations.

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

In Silico Modeling for the Prediction of Dose and Pathway-Related Adverse Effects in Humans From In Vitro Repeated-Dose Studies.

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG cells, are used for toxicity screening. Extrapolation of in vitro toxicity to in vivo effects is possible by reverse dosimetry using pharmacokinetic modeling. We assessed long-term repeated-dose toxicity of bosentan and valproic acid (VPA) in HepaRG cells under serum-free conditions. Upon 28-day exposure, the EC50 values for bosentan and VPA decreased by 21- and 33-fold, respectively. Using EC(10) as lowest threshold of toxicity in vitro, we estimated the oral equivalent doses for both test compounds using a simplified pharmacokinetic model for the extrapolation of in vitro toxicity to in vivo effect. The model predicts that bosentan is safe at the considered dose under the assumed conditions upon 4 weeks exposure. For VPA, hepatotoxicity is predicted for 4% and 47% of the virtual population at the maximum recommended daily dose after 3 and 4 weeks of exposure, respectively. We also investigated the changes in the central carbon metabolism of HepaRG cells exposed to orally bioavailable concentrations of both drugs. These concentrations are below the 28-day EC(10) and induce significant changes especially in glucose metabolism and urea production. These metabolic changes may have a pronounced impact in susceptible patients such as those with compromised liver function and urea cycle deficiency leading to idiosyncratic toxicity. We show that the combination of modeling based on in vitro repeated-dose data and metabolic changes allows the prediction of human relevant in vivo toxicity with mechanistic insights.

Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro.

Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis.

Antibacterial activity of 1-methyl-7-methoxy-beta-carboline and its phenacyl and coumarine analogues.

Antibacterial activity of 1-methyl-7-methoxy-beta-carboline (harmaline) and its phenacyl and coumarine analogues 1-(3-nitro-phenyl)-(2-(7-methoxy-1-methyl-1,3,4,9-tetrahydro-beta-carbolin-2-yl)-ethanone (II), 1-(3,4-dihydroxy-phenyl)-2-(7-methoxy-1-methyl-1,3,4,9-tetrahydro-beta-carbolin-2-yl)-ethanone (III) 7-(methoxy-beta-carboline),15-24,de-hydro(19,20-dimethoxy)coumarine (IV), 7-(methoxy-beta-carboline)15-24,dehydro(20-methoxy)coumarine (V) were studied by disc diffusion method. All compounds were tested against three gram positive and four gram-negative bacteria. Parent compound showed good activity. All compounds revealed better results against gram positive as compared to gram-negative bacteria. 1-(3,4-Dihydroxy-phenyl)-2-(7-methoxy-1-methyl-1,3,4,9-tetrahydro-beta-carbolin-2-yl)-ethanone (III) was found most potent compound showing broad spectrum activity when compared with all synthesized analogues. Coumarine analogues showed more or less same activity indicating that number and position of methoxy groups are not important regarding antimicrobial activity.