EC50 Determination Research Articles

Некоторые цианозамещённые спиросочленённые пиразолины и пирролидины, обладающие антимикробной активностью

This work summarizes one-pot synthetic approaches to spiro-fused (di)azoles, which are promising in terms of studying biological activity. Proposed approaches involve condensation reactions of (hetero)cyclic ketones, methylene active moieties, and N-nucleophiles, occurring under mild conditions by both stepwise and concerted mechanisms. The antimicrobial activity of signals from the proposed approaches of cyanosubstituted spiro-membered pyrazolines and pyrrolidines against gram-negative (P. aeruginosa, E. coli) and gram-positive (S. aureus) losses has been studied (EC50 determination (μg/ml)).The well-known component of broad-spectrum antimicrobial drugs, ciprofloxacin, has been used as a control. It has been found that 3-amino-2-(4-nitrophenyl)-1,2-diazaspiro[4.5]dec-3-ene-4-carbonitrile exhibits the greatest inhibitory effect against selected gram-negative bacteria. In this case, the phenyl substituent at the second nitrogen atom of the pyrazoline ring plays an important role, since its replacement with an aroyl fragment reduces the studied effect by several tens of times, regardless of the type of the second ring. Substituted spiroindolinopyrrolizidinedicarbonitriles, containing cyano groups at the 2nd carbon atom of the pyrrolizidine ring and a phenyl substituent at the first, also exhibit a moderate and high inhibitory effect. For pairs with the same type of substituent position, compounds containing a 3,4 Cl2 substituent in the phenyl fragment have the greatest antimicrobial activity.

Evaluating anthelmintic activity through Caenorhabditis elegans egg hatching assay

The Caenorhabditis elegans egg hatching methodology is a valuable tool for assessing the anthelmintic activity of drugs and compounds and evaluating anthelmintic drug efficacy.Isolated eggs from gravid adults are exposed to different concentrations of selected drugs and the percentage of egg hatching is determined with respect to the control condition. The assay allows the construction of concentration-response curves and determination of EC50 or EC90 values for egg hatching inhibition. Also, it allows measurements of inhibition as a function of time of exposure.This approach addresses the urgent need for new anthelmintics, as resistance to current treatments poses a significant challenge in parasitic nematode infection. This resistance not only affects humans but also animals and plants, causing significant economic losses in livestock farming and agriculture.By using the free-living nematode C. elegans as a parasitic model organism, researchers can efficiently screen for potential treatments and assess drug combinations for synergistic effects. Importantly, this assay offers a cost-effective and accessible alternative to traditional methods, eliminating the need for specialized infrastructure, hosts, and trained animal maintenance personnel. Additionally, the methodology closely mimics natural conditions, providing insights into egg development and potential therapeutic targets.This method allows for evaluating the direct negative impact of drugs on egg hatching, which correlates with long-term anthelmintic effects, offering advantages in preventing or reducing the transmission and spread of worm infections by eggs.Overall, this approach represents a significant advancement for anthelmintic discovery, offering both practical applications and avenues for further scientific research.•The C. elegans egg hatching assay is a robust and effective method for assessing the anthelmintic potential of various drugs and compounds, allowing the generation of concentration-response curves.•By leveraging the free-living nematode C. elegans as a parasitic model organism, this method facilitates efficient screening of potential treatments and evaluation of drug combinations.•The method addresses the urgent need for new anthelmintics, offering a cost-effective and accessible alternative to traditional approaches.

Evaluating drug-drug interaction risk associated with peptide analogues using advanced in vitro systems.

Drug-drug interaction (DDI) assessment of therapeutic peptides is an evolving area. The industry generally follows DDI guidelines for small molecules, but the translation of data generated with commonly used in vitro systems to in vivo is sparse. In the current study, we investigated the ability of advanced human hepatocyte in vitro systems namely HepatoPac, spheroids, and Liver-on-a-chip to assess potential changes in regulation of CYP1A2, CYP2B6, CYP3A4, SLCO1B1 and ABCC2 in the presence of selected therapeutic peptides, proteins, and small molecules. The peptide NN1177, a glucagon and GLP-1 receptor co-agonist, did not suppress mRNA expression or activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac, spheroids, or Liver-on-a-chip; these findings were in contrast to the data obtained in sandwich cultured hepatocytes. No effect of NN1177 on SLCO1B1 and ABCC2 mRNA was observed in any of the complex systems. The induction magnitude differed across the systems (e.g., rifampicin induction of CYP3A4 mRNA ranged from 2.8-fold in spheroids to 81.2-fold in Liver-on-a-chip). Small molecules, obeticholic acid and abemaciclib, showed varying responses in HepatoPac, spheroids and Liver-on-a-chip, indicating a need for EC50 determinations to fully assess translatability data. HepatoPac, the most extensively investigated in this study (3 donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential. This study establishes an excellent foundation towards the establishment of more clinically-relevant in vitro tools for evaluation of potential DDIs with therapeutic peptides. Significance Statement At present, there are no guidelines for drug-drug interaction (DDI) assessment of therapeutic peptides. Existing in vitro methods recommended for assessing small molecule DDIs do not appear to translate well for peptide drugs, complicating drug development for these moieties. Here, we establish evidence that complex cellular systems have potential to be used as more clinically-relevant tools for the in vitro DDI evaluation of therapeutic peptides.

Reduced Anticancer Potency of Carboplatin in the Presence of Sialic Acid in SH-SY5Y Cells

Background: Neuroblastoma (NB) is the most common solid childhood tumor with aggressive behavior and a high mortality rate. Multiple therapeutic approaches have been developed and applied to treat neuroblastoma, but resistance to therapies is inevitable, leading to treatment failure and cancer relapse. Therefore, perceiving the mechanisms of reduced drug sensitivity seems appropriate and promising in NB treatment to determine synergistic effects strategies like combination therapy. Objectives: The present study aimed to investigate the modulation effect of the therapeutic efficacy of carboplatin (a chemotherapeutic agent) by co-administration of sialic acid (an alpha-keto acid sugar with a 9-carbon backbone located at the outermost end of N-linked and O-linked carbohydrate chains and in lipid-associated glycoconjugates showing aberrant expression in tumor cells caused tumor-genesis) in NB cells. Methods: In the present study, the effects of sialic acid, carboplatin, and Sia-Carbo (sialic acid in combination with carboplatin) were evaluated on viability and apoptosis using the determination of EC50 and IC50. Also, the capability of metastasis and apoptosis were assessed using real-time PCR. Moreover, the expression of MRP-1 encoded by ABCC1 (as a target for therapeutic suppression in high-risk neuroblastoma) was studied in different treatment groups. Conclusions: Regarding IC50 increasing carboplatin in the presence of sialic acid, the results showed that sialic acid treatment significantly modulated carboplatin's effect on cell apoptosis and induced ABCC1 expression. These findings show that sialic acid metabolic engineering can be a good approach to neuroblastoma therapy. It was suggested that targeting aberrant sialylation in combination with carboplatin exerts more profound apoptotic and anticancer effects on the neuroblastoma SH-SY5Y cell line than carboplatin monotherapy.

In vitro evaluation of the efficacy of 8-hydroxyquinoline derivatives for the control of Phaeomoniella chlamydospora, the causative agent of Petri disease in grapevines.

This study evaluates the in vitro efficacy of 8-hydroxyquinoline (8HQ) derivatives in controlling the phytopathogenic fungus Phaeomoniella chlamydospora. The in vitro tests assessed the susceptibility to the minimum inhibitory concentration (MIC), checkerboard assay, mycelial growth (MG) inhibition, and EC50 determination. Among the seven agricultural fungicides tested, tebuconazole (TEB) displayed the lowest MIC, 1.01µg mL-1, followed by captan (CAP), thiophanate methyl (TM), and mancozeb with MICs of 4.06, 5.46, and 10.62µg mL-1, respectively. The 8HQ derivatives used in this study were clioquinol and PH 151 (PH) with MICs of 1.09 and 2.02µg mL-1, respectively. PH associated with TEB and CAP showed synergism and inhibited 95.8% of MG at the highest dose. TEB inhibited 100% of MG at the three highest doses, while associated with PH exhibited the lowest EC50 (0.863+0.0381µg mL-1). We concluded that the 8HQ derivatives tested controlled effectively the P. chlamydospora in vitro. PH associated with CAP and TEB exhibited a synergistic effect. The association between PH and TM was considered indifferent. This study expands the list of active ingredients tested against P. chlamydospora, with the PH 151 and clioquinol derivatives being tested for the first time. The in vitro efficacy and synergistic action with other fungicides suggest a potential use as a grapevine wound protectant. This association makes it possible to reduce doses and increase the potency of both drugs, reducing the risk of resistance development and harm to humans and the environment.

Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms.

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

Evaluation of FOXO1 Target Engagement Using a Single-Cell Microfluidic Platform.

The cellular thermal shift assay (CETSA) has been used extensively since its introduction to study drug-target engagement within both live cells and cellular lysate. This has proven to be a useful tool in early stage drug discovery and is used to study a wide range of protein classes. We describe the application of a single-cell CETSA workflow within a microfluidic affinity capture (MAC) chip. This has enabled us to quantitatively determine the active FOXO1 single-molecule count and observe FOXO1 stabilization and destabilization in the presence of three small molecule inhibitors, including demonstrating the determination of EC50. The successful use of the MAC chip for single-cell CETSA paves the way for the study of precious clinical samples owing to the low number of cells needed by the chip. It also provides a useful tool for studying any underlying population heterogeneity that exists within a cellular system, a feature that is usually masked when conducting ensemble measurements.

A Novel High-Content Screening-Based Method for Anti-Trypanosoma cruzi Drug Discovery Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Chagas disease is caused by Trypanosoma cruzi infection and remains a relevant cause of chronic heart failure in Latin America. The pharmacological arsenal for Chagas disease is limited, and the available anti-T. cruzi drugs are not effective when administered during the chronic phase. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to accelerate the process of drug discovery for Chagas disease, through predictive preclinical assays in target human cells. Here, we aimed to establish a novel high-content screening- (HCS-) based method using hiPSC-CMs to simultaneously evaluate anti-T. cruzi activity and cardiotoxicity of chemical compounds. To provide proof-of-concept data, the reference drug benznidazole and three compounds with known anti-T. cruzi activity (a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B) were evaluated in the assay. hiPSC-CMs were infected with T. cruzi and incubated for 48 h with serial dilutions of the compounds for determination of EC50 and CC50 values. Automated multiparametric analyses were performed using an automated high-content imaging system. Sublethal toxicity measurements were evaluated through morphological measurements related to the integrity of the cytoskeleton by phalloidin staining, nuclear score by Hoechst 33342 staining, mitochondria score following MitoTracker staining, and quantification of NT-pro-BNP, a peptide released upon mechanical myocardial stress. The compounds showed EC50 values for anti-T. cruzi activity similar to those previously described for other cell types, and GT5B showed a pronounced trypanocidal activity in hiPSC-CMs. Sublethal changes in cytoskeletal and nucleus scores correlated with NT-pro-BNP levels in the culture supernatant. Mitochondrial score changes were associated with increased cytotoxicity. The assay was feasible and allowed rapid assessment of anti-T. cruzi action of the compounds, in addition to cardiotoxicity parameters. The utilization of hiPSC-CMs in the drug development workflow for Chagas disease may help in the identification of novel compounds.

COMBINATION IMPACT OF pH AND TEMPERATURE ON THE TOXICITY OF LEAD ON ZOOPLANKTON IN THE CONTEXT OF GLOBAL WARMING

Heavy metal pollution in urban lakes of Hanoi has become one of the major environmental issues in the last decades. Research on the toxicity of Pb is necessary due to the extent of its harm to aquatic ecosystems. The toxicity of Pb to aquatic organisms (EC50 values) depends on different environmental conditions. The study was conducted to evaluate the effect of temperature on the toxicity of Pb on the test organism Moina Dubia. Moina Dubia belongs to the Cladocera group (Cladocera) which is an indigenous creature collected at Lake Hanoi. Moina Dubia was cultured under laboratory conditions and has been used to conduct EC50 determination for Pb. Moina Dubia grew and developed well in the laboratory and was suitable as a test organism in toxicological studies. A survey from 24oC to 28oC water temperature conditions showed that the EC50 value decreases from 1402 ug/l to 775 ug/l. The higher water temperature, the higher toxicity level of Pb on Moina Dubia. Clear impact of water temperature on toxicity of heavy metal as Pb showed that global warming may gradually lead to potential biodegradation in freshwater ecology.

Dose-response analysis of toxic effect from palm oil mill effluent (POME) by-products on biohydrogen producing bacteria – A preliminary study on microbial density and determination of EC50

The stimulant and toxicity effects of reported organic (acetic acid, propionic acid, butyric acid, formic acid, oil & grease) and inorganic (copper) by-products presented in palm oil mill effluent on anaerobic bacterial population were examined in this paper. The toxicity test had shown that acetic, propionic and butyric acids tend to stimulate the bacterial density level (survival rate more than 50%), while formic acid, copper, oil and grease were shown to have suppressed the density level (survival rate less than 50%). The highest biomass recorded was 1.66 mg/L for the concentration of acetic acid at 216 mg/L and lowest biomass concentration, 0.90 mg/L for copper at 1.40 mg/L. Biohydrogen-producing bacteria have a favourable growth rate around pH 5.5. The comparison of half maximal effective concentration (EC50) values between two test duration on the effects of organic and inorganic by-products postulate that bacteria had a higher tolerance towards volatile fatty acids. While acetic, butyric and propionic acids had exhibited higher tolerance EC50 values for bacteria, but the opposite trend was observed for formic acid, copper and oil & grease.

Not merely noxious? Time-dependent hormesis and differential toxic effects systematically induced by rare earth elements in Escherichia coli.

Progressive rare earth element (REE) enrichment in aquatic environments worldwide and their resulting anthropogenic anomalies have highlighted the need for a better understanding of their biological effects, with a special emphasis on microbial cells since they play a crucial role in good ecosystem functioning. Therefore, the primary aim of this work was to achieve simultaneous characterization of the 16 REE toxicity effects on the growth kinetics of the commonly found Gram-negative bacterium E. coli (BW25113 strain). Bacterial growth curve modelling showed hormetic effects in the presence of REEs, while EC50 determination (in the mid-log phase) indicated that the four HREEs from Er to Lu in addition to Y were the most toxic metals (EC50 in the range of 8.3 to 3μM), just after Sc (EC50 of 1.1μM). Additional subcellular parameter assessment revealed cell membrane lipid peroxidation as well as enhanced membrane depolarization and permeability in the presence of La, Gd, or Yb as representatives of LREEs and HREEs. These subcellular effects appeared to be more intense with Gd and Yb compared with La-exposed cells, in relation to the overall higher toxicity potential reported for HREEs on bacterial growth. Also, the cellular ATP production decreased after REE exposure at their EC50.Finally, these results emphasize the importance of growth kinetic consideration as well as the complexity of REE biological effect mechanisms towards bacteria.

Endothelial capture using antibodies and nanoparticles in human tissues: Antigen identification and liver segment imaging

Background: The unique phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. The potential of this phenomenon has already been demonstrated in animal models. In the present study, the translational potential of several human endothelium-specific antibodies for their use in the endocapt-based approach was analysed. Methods: The binding of different endothelium-surface specific monoclonal antibody clones was analysed in human tissue and in endothelial cells using image-based immunofluorescence and the determination of half-maximal effective concentration (EC50). The potential of endocapt-based locoregional application of antibodies or antibody-coated liposomes was analysed ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumour models. Results: Eight out of ten antibody clones were assigned to the group of “fast binding antibodies”. Different antibody clones showed various binding kinetics to the same endothelial marker whereas the binding kinetics of single antibody clones was independent from the tissue type. Anti-CD49e, anti-CD31, anti-CD34 and anti-CD102 antibodies showed the lowest EC50 of antibody binding concentration and constant results in EC50 determination of antibody binding to cells and human tissue. Experimental studies using anti-mouse CD49e antibody and coated immunoliposomes confirmed their effective capture by endothelial cells in vitro and in vivo by which fluorescent liver segment labelling was achieved. Conclusions: Our findings identify the high potential of several human antibody clones, especially anti-CD49e, -CD31, -CD34 and -CD102, for endocapt technology. We also propose important translational implications of these antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes. Toxicological studies are indispensable for further translational development of new antibodies for endocapt. Statement of SignificanceThe phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. This phenomenon broadly prevents systemic circulation of the antibody or antibody-drug conjugates. In the present study, our findings identify several human antibody clones promising for endothelial capture technology.This study provided the experimental demonstration of liver segment labelling ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumor models. In addition, this study proposed the important translational implications of selected antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes.

Antifungal Activity of Pelargonium graveolens Essential Oil and its Fractions Against Wood Decay Fungi

In order to valorize Moroccan aromatic and medicinal plants, this study focused on both the chemical characterization and the study of the antifungal activity of Pelargonium graveolens essential oil and its fractions against four fungi that are responsible for wood decay (Coniophora puteana, Coriolus versicolor, Poria placenta and Gloeophyllum trabeum). The essential oils were analyzed by Gas chromatography/Mass spectrometry (GC/MS) and evaluated for antifungal activity, using the macrodilution method for the determination of EC50. The GC/MS essential oil analysis has led to the identification of 54 components. Citronellol (25.24 %), geraniol (23.36 %), citronellyl formate (8.35 %), linalool (7.11 %), β-eudesmol (6.13 %), geranyl formate (4.26 %) and iso-menthone (3.37 %), were identified as the major components. Fractions 1, 2 and 3 essential oils have displayed qualitative similarities with the EO sample, and quantitative differences among the relative percentages of major compounds were observed. A significant difference (p < 0.0001) was shown in main compounds of fraction 3 obtained by fractional distillation. P. graveolens essential oil has inhibited all fungi with a value of EC50 between 0.255 to 1.022 mg/mL. The EC50 values of fractions 1 and 2 are ranging from 0.292 to 1.022 mg/mL. The fraction 3 exhibited a great antifungal activity against all tested fungi. These results support the potential use of P. graveolens essential oils and its fractions for wood protection against wood decay fungi.

Rapid EC50 determination of hydrophobic toxicants in continuous droplet biomicrofluidics

Abstract Droplet-based bioassays are attractive for increasing throughput while minimizing reagent consumption. However, the choice of continuous oil phase within the bioassay can substantially alter assay outcomes when hydrophobic reagents and analytes dynamically partition between the oil and aqueous phases. Fluorinated continuous phases have been recognized as a better alternative to hydrocarbons to prevent leakage from droplets but variations among them has not been quantitatively addressed and analysis is complicated by the presence of surfactants. Herein, surface modification strategies to avoid surfactants are demonstrated with fluorocarbon oils for the first time. Perfluorocarbons, hydrofluoroether and hexadecane are quantitatively compared based on their aqueous distribution coefficient with a model industrial toxicant, pentachlorophenol (PCP). PCP partition in the oil phase skews cytotoxicity evaluation, wherein a bacterial inhibition assay displays a wide range of EC50 values for the same toxicant. Prevention of PCP extraction in a real-time viability assay reveals a 40-fold difference in LD50 toxicity vs. hexadecane control. Theoretical and empirical approaches to limit bioassay partitioning are established for expansion outside cell viability analyses employed herein. Structure activity relationships of surface modification and oil-water partition are related to Hamaker constants and Hansen solubility parameters towards translation on disposable plastics beside poly(methyl acrylate). The results provide a theoretical basis towards design and selection of continuous phase oils and microfluidic substrates to achieve a range of aqueous/oil partitioning for droplet microfluidic bioassays.

AgNPs Change Microbial Community Structures of Wastewater.

Due to their strong antimicrobial activity, silver nanoparticles (AgNPs) are massively produced, applied, consumed and, as a negative consequence, released into wastewater treatment plants. Most AgNPs are assumed to be bound by sludge, and thus bear potential risk for microbial performance and stability. In this lab-scale study, flow cytometry as a high-throughput method and 16S rRNA gene amplicon Illumina MiSeq sequencing were used to track microbial community structure changes when being exposed to AgNPs. Both methods allowed deeper investigation of the toxic impact of chemicals on microbial communities than classical EC50 determination. In addition, ecological metrics were used to quantify microbial community variations depending on AgNP types (10 and 30 nm) and concentrations. Only low changes in α- and intra-community β-diversity values were found both in successive negative and positive control batches and batches that were run with AgNPs below the EC50 value. Instead, AgNPs at EC50 concentrations caused upcoming of certain and disappearance of formerly dominant subcommunities. Flavobacteriia were among those that almost disappeared, while phylotypes affiliated with Gammaproteobacteria (3.6-fold) and Bacilli (8.4-fold) increased in cell abundance in comparison to the negative control. Thus, silver amounts at the EC50 value affected community structure suggesting a potential negative impact on functions in wastewater treatment systems.

Toxicity of Ochratoxin to Early Life Stages of Zebrafish (Danio rerio).

Ochratoxin A (OTA) is a known contaminant in fish feed but its effect on fish health remains rather unknown. A study was conducted to investigate the effects of different concentrations of ochratoxin on early life stages of zebrafish (Danio rerio). The tests with ochratoxin A showed a correlation between the exposure to mycotoxin and the amount of damage. The mortality rate and the incidents of embryonal damage was increased by increasing ochratoxin concentrations. The calculations resulted in a lethal concentration for 50% of the embryos (LC50) of 0.29 mg/L and a concentration at which 50% of the animals showed impairment (EC50) of 0.36 mg/L after 96 h of exposure. During the test, reduced heart rates were also observed revealing a clear dose-response relationship. The EC50 determination for this endpoint was 1.26 mg/L after 72 h of exposure. The measurement of oxidative stress was proven to be the most sensitive system to indicate OTA effects on the zebrafish embryos with an EC50 value of 0.067 mg/L after 72 h of exposure. The test validity was given because the control test with 3,4-Dichloroaniline (3,4-D) showed a LC50 value of 2.88 mg after 96 h of exposure which is comparable to the available reference values. According to the current knowledge, these experimental doses did not exceed the environmental concentrations of this ochratoxin A. However, this study raises concerns about the effects of ochratoxin on fish.

Batch-injection analysis with amperometric detection of the DPPH radical for evaluation of antioxidant capacity

This work proposes the application of batch-injection analysis with amperometric detection to determine the antioxidant capacity of real samples based on the measurement of DPPH radical consumption. The efficient concentration or EC50 value corresponds to the concentration of sample or standard required to scavenge 50% DPPH radicals. For the accurate determination of EC50, samples were incubated with DPPH radical for 1h because many polyphenolic compounds typically found in plants and responsible for the antioxidant activity exhibit slow kinetics. The BIA system with amperometric detection using a glassy-carbon electrode presented high precision (RSD=0.7%, n=12), low detection limit (1μmolL−1) and selective detection of DPPH (free of interferences from antioxidants). These contributed to low detection limits for the antioxidant (0.015 and 0.19μmolL−1 for gallic acid and butylated hydroxytoluene, respectively). Moreover, BIA methods show great promise for portable analysis because battery-powered instrumentation (electronic micropipette and potentiostats) is commercially available.

Determination of antibiotic EC50 using a zero-flow microfluidic chip based growth phenotype assay.

Current existing assay systems for evaluating antimicrobial activity suffer from several limitations including excess reagent consumption and inaccurate concentration gradient preparation. Recently, microfluidic systems have been developed to provide miniaturized platforms for antimicrobial susceptibility assays. However, some of current microfluidic based assays require continuous flows of reagents or elaborate preparation steps during concentration preparation. In this study, we introduce a novel microfluidic chip based growth phenotype assay that automatically generates a logarithmic concentration gradient and allows observing the growth of pathogenic bacteria under different concentrations of antibiotics in nanoliter batch culture reactors. We chose pathogen bacterium Pseudomonas aeruginosa as a model strain and evaluated the inhibitory effects of gentamicin and ciprofloxacin. We determined the EC50 values and confirmed the validity of the present system by comparing the EC50 values obtained through conventional test tube method. We demonstrated that the EC50 values acquired from present assay are comparable to those obtained from conventional test tube cultures. The potential application of present assay system for investigating combinatorial effects of antibiotics on multidrug resistant pathogenic bacteria is discussed and it can be further used for systematic evaluation of antifungal or antiviral agents.

The CERT antagonist HPA-12: First practical synthesis and individual binding evaluation of the four stereoisomers

The first unified synthetic route to the four enantiopure HPA-12 stereoisomers in multi-gram scale is reported based on Crystallization-Induced Asymmetric Transformation (CIAT) technology. This preparative stereoselective synthesis allowed the unprecedented comparative evaluation of HPA-12 stereoisomers regarding their interaction with the CERT START domain. In vitro binding assay coupled to in silico docking approach indicate a possible interaction for the four derivatives. The first TR-FRET homogeneous-phase assay was developed to quantify their binding to the START domain, allowing complete determination of HPA-12 EC50. Results indicate that not only the (1R,3S) lead to the strongest binding, but that both 1R and 3S stereocenters similarly contribute to extent of recognition This automated homogenous assay further opens up promising prospect for the identification of novel potential CERT antagonist by means of high throughput screening.

Residence time and kinetic efficiency analysis of extracellular signal-regulated kinase 2 inhibitors

The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of Ki, Kd, IC50, and/or EC50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the koff constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing koff constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.