Evaluation of the use of imaging parameters for the detection of compound-induced hepatotoxicity in 384-well cultures of HepG2 cells and cryopreserved primary human hepatocytes.

Drug-induced liver injury (DILI) is a major cause of failed drug development, withdrawal and restricted usage. Therefore screening assays which aid selection of candidate drugs with reduced propensity to cause DILI are required. We have investigated the toxicity of 144 drugs, 108 of which caused DILI, using assays identified in the literature as having some predictivity for hepatotoxicity.

1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor.

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target.

Functional characterization and high-throughput screening of positive allosteric modulators of α7 nicotinic acetylcholine receptors in IMR-32 neuroblastoma cells.

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs).

Structure-activity studies on a series of a 2-aminopyrimidine-containing histamine H4 receptor ligands.

A series of 2-aminopyrimidines was synthesized as ligands of the histamine H4 receptor (H4R). Working in part from a pyrimidine hit that was identified in an HTS campaign, SAR studies were carried out to optimize the potency, which led to compound 3, 4- tert-butyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-ylamine. We further studied this compound by systematically modifying the core pyrimidine moiety, the methylpiperazine at position 4, the NH2 at position 2, and positions 5 and 6 of the pyrimidine ring.

Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans.

Prioritization of compounds based on human hepatotoxicity potential is currently a key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. The authors report the validation and implementation of a high-content multiparametric cytotoxicity assay based on simultaneous measurement of 8 key cell health indicators associated with nuclear morphology, plasma membrane integrity, mitochondrial function, and cell proliferation. Compounds are prioritized by (a) computing an in vitro safety margin using the minimum cytotoxic concentration (IC(20)) across all 8 indicators and cell-based efficacy data and (b) using the minimal cytotoxic concentration alone to take into account concentration of drug in tissues.

Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase.

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable.

Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC12 cells.

Activation of the Rho kinase (ROCK) pathway has been associated with inhibition of neurite regeneration and outgrowth in spinal cord injury. Growth-inhibitory substances present in the glial scar such as chondroitin sulfate proteoglycans (CSPGs) have been shown to create a nonpermissive environment for axon regeneration that results in growth cone collapse. In this study, an in vitro model was developed in nerve growth factor-differentiated PC12 cells where the Rho/ROCK pathway was modulated by CSPG.

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation.

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations.

A high-throughput soft agar assay for identification of anticancer compound.

A 384-well soft agar assay was developed to identify potential novel anticancer compounds. Normally used to detect cell transformation, the assay is used here to quantitate cell proliferation in a 3-dimensional (3-D) anchorage-independent format. HCC827 cells, which are highly sensitive to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors, were used to develop the method and a set of 9600 compounds used to validate the assay.

Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease.

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised.

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources.

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC).

An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR.

Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca(2+) with an EC(50) value of 0.

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms.

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented.

Functional analysis of large conductance Ca2(+)-activated K(+) channels: ion flux studies by atomic absorption spectrometry.

Although techniques such as (86)Rb(+) flux provide a sensitive measure of K(+) channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK(Ca) alpha subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established.

Screening for inhibitors of histone deacetylase by incorporating a spraying method to micro-arrayed compound screening.

We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold.

Utilization of microarrayed compound screening (microARCS) to identify inhibitors of p56lck tyrosine kinase.

Protein tyrosine kinases play critical roles in cell signaling and are considered attractive targets for drug discovery. The authors have applied microARCS (microarrayed compound screening) technology to develop a high-throughput screen for finding inhibitors of the p56lck tyrosine kinase. Initial assay development was performed in a homogeneous time-resolved (LANCE) format in 96-well microplates and then converted into the gel-based microARCS format.

A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen.

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact.

Microarrayed compound screening (microARCS) to identify activators and inhibitors of AMP-activated protein kinase.

A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (microARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM trade mark ).

Putting thought to paper: a microARCS protease screen.

In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity.

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors.

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening microARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the G alpha(q) protein can readily be assessed by fluorescence-based Ca(2+) release measurements, many GPCRs that are coupled to G alpha(s) or G alpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner.

Identification and characterization of novel antagonists of the CCR3 receptor.

Eotaxin, an inducer of eosinophil migration and activation, exerts its activity by binding to CCR3, the C-C chemokine receptor 3. An inhibitor of the eotaxin-CCR3 binding interaction may have potential as an anti-inflammatory drug for treatment of asthma, parasitic infections, and allergic disorders. A radioligand binding assay was developed using HEK cells transfected with CCR3, with (125)I eotaxin as the ligand.

Use of SAM2 biotin capture membrane in microarrayed compound screening (muARCS) format for nucleic acid polymerization assays.

Microarrayed compound screening format (muARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the muARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase.

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles, a novel class of immunomodulators.

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen.

Application of Micro Arrayed Compound Screening (microARCS) to identify inhibitors of caspase-3.

Micro Arrayed Compound Screening (microARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8,640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher.