Measuring and Statistically Testing the Size of the Effect of a Chemical Compound on a Continuous In-Vitro Pharmacological Response Through a New Statistical Model of Response Detection Limit.

Biomolecular screening research frequently searches for the chemical compounds that are most likely to make a biochemical or cell-based assay system produce a strong continuous response. Several doses are tested with each compound and it is assumed that, if there is a dose-response relationship, the relationship follows a monotonic curve, usually a version of the median-effect equation. However, the null hypothesis of no relationship cannot be statistically tested using this equation. We used a linearized version of this equation to define a measure of pharmacological effect size, and use this measure to rank the investigated compounds in order of their overall capability to produce strong responses. The null hypothesis that none of the examined doses of a particular compound produced a strong response can be tested with this approach. The proposed approach is based on a new statistical model of the important concept of response detection limit, a concept that is usually neglected in the analysis of dose-response data with continuous responses. The methodology is illustrated with data from a study searching for compounds that neutralize the infection by a human immunodeficiency virus of brain glioblastoma cells.

Bioactivity Profiling of Plant Biodiversity of Panama by High Throughput Screening.

We report relative bioactivities of extracts prepared from a large collection of plants from three national parks in Panama. Over 181 plants were collected, taxonomically identified and their detannified dichloromethane (DCM)-methanolic extracts were used for profiling selected bioactivities. Assays were performed to evaluate the antioxidant activity of the extracts for Antioxidant Response Element (ARE) induction, total non-enzymatic antioxidant potential, anti-inflammatory and anticancer properties. The high throughput analysis of 280 extracts resulted in identification of 57.5% of the extracts that could induce ARE at one or more concentrations tested, 93.5% that harbored total antioxidant capacity, and 2.1% of the extracts that showed lung cancer cell line-specific cytotoxicity. Data from our profiling experiments indicate that a large number of extracts could be a source for further isolation and chemical identification of compounds that could serve as leads for discovery of antioxidant, anticancer and anti-inflammatory agents to prevent or treat complex diseases like cancer and neurodegenerative disorders.

A cell-based high-throughput screen for novel chemical inducers of fetal hemoglobin for treatment of hemoglobinopathies.

Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to ß-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase ß-globin promoter-Renilla luciferase ß-globin yeast artificial chromosome (γ-luc ß-luc ß-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no ß-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type ß-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.

Targeting inhibitor 2 of protein phosphatase 2A as a therapeutic strategy for prostate cancer treatment.

Inhibitor 2 of protein phosphatase 2A (I2PP2A), a biological inhibitor of the cellular serine/threonine protein phosphatase PP2A, is associated with numerous cellular processes that often lead to the formation and progression of cancer. In this study we hypothesized that targeting the inhibition of I2PP2A's multiple functions in prostate cancer cells might prevent cancer progression. We have investigated the effect of the small chain C6-ceramide, known to be a bioactive tumor suppressor lipid, on I2PP2A function, thereby affecting c-Myc signaling and histone acetylation in cells. Our data indicated that C6-ceramide treatment of prostate cancer cells induces cell death in PC-3, DU145, and LNCaP cells, but not normal prostate epithelial cells. C6-ceramide was able to disrupt the association between PP2A and I2PP2A. C6-ceramide inhibits I2PP2A's upregulation of c-Myc and downregulation of histone acetylation in prostate cancer cells. Our data indicated that targeting cancer related signaling pathways through I2PP2A using ceramide as an anti-I2PP2A agent could have beneficial effects as a therapeutic approach to prevent prostate cancer.

Compound ranking based on a new mathematical measure of effectiveness using time course data from cell-based assays.

The half maximal inhibitory concentration (IC50) has several limitations that make it unsuitable for examining a large number of compounds in cytotoxicity studies, particularly when multiple exposure periods are tested. This article proposes a new approach to measure drug effectiveness, which allows ranking compounds according to their toxic effects on live cells. This effectiveness measure, which combines all exposure times tested, compares the growth rates of a particular cell line in the presence of the compound with its growth rate in the presence of DMSO alone. Our approach allows measuring a wider spectrum of toxicity than the IC50 approach, and allows automatic analyses of a large number of compounds. It can be easily implemented in linear regression software, provides a comparable measure of effectiveness for each investigated compound (both toxic and non-toxic), and allows statistically testing the null hypothesis that a compound is non-toxic versus the alternative that it is toxic. Importantly, our approach allows defining an automated decision rule for deciding whether a compound is significantly toxic. As an illustration, we describe the results of a cellbased study of the cytotoxicity of 24 analogs of novobiocin, a C-terminal inhibitor of heat shock protein 90 (Hsp90); the compounds were ranked in order of cytotoxicity to a panel of 18 cancer cell lines and 1 normal cell line. Our approach may also be a good alternative to computing the half maximal effective concentration (EC50) in studies searching for compounds that promote cell growth.

Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01-30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.

The Screening Asia Conference 2010: an update.

The conference was the first of its kind in a series of trend-setting conferences conceived and organized by Select Biosciences. The conference was held in the state-of-the-art Matrix Building, Metropolis. The topics covered were grouped into the following half-day sessions: high-content screening, high-throughput screening, RNAi screening, novel drug-discovery techniques and assay development, and screening natural extracts and small molecules. Petr Bartunek (Group Leader, Institute of Molecular Genetics, Prague, Czech Republic), Rathnam Chaguturu (Director, High-Throughput Screening Laboratories, University of Kansas, USA) and Paul Orange (Business Leader, Bio-Discovery, PerkinElmer) were the keynote speakers. The conference attendees were from academia and the biotechnology and pharmaceutical sectors. The Screening Asia 2010 was designed to address, from a regional and international perspective, how best to approach the one issue that has been at the forefront of the drug-discovery endeavors: who is best suited among academia, biotechnology and pharmaceutics to discover chemical probes against key therapeutic targets, and how best one could implement the processes.

K-Screen: a free application for high throughput screening data analysis, visualization, and laboratory information management.

High throughput screening (HTS) has emerged as an important technique for allowing researchers to rapidly profile very large numbers of chemicals against drug targets. As recent and future advances make HTS cheaper to perform on even larger scales, the amount of data that has to be processed, analyzed, and searched will only grow larger in size and harder for researchers to manually sift through. It is therefore an unavoidable requirement that institutions utilizing HTS technology will need to begin looking for effective solutions in the maturing area of laboratory information management systems like many other types of labs have already done. K-Screen is one such solution. Our initial goal with K-Screen was to have an integrated application environment that supported data analysis, management, and presentation so we could efficiently perform client requested screens and searches as well as generate detailed reports on the results of those. Previously, we had attempted but failed to locate an existing software suite that sufficiently addressed all our requirements. K-Screen is a web accessible application that offers the ability to host a large chemical structure library, process and store single-dose (primary) and dose response (secondary) screening data, perform searches based on screening results, plate coordinates, and structure, substructure and structure similarity. It uses heat maps and histograms to visualize screen or plate level statistics. Interfaces to external searches against PubChem and ZINC databases are also provided. We feel that these features make K-Screen a practical and effective alternative to other commercial or academic HTS LIMS systems.

The University of Kansas High-Throughput Screening laboratory. Part I: meeting drug-discovery needs in the heartland of America with entrepreneurial flair.

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core applies pharmaceutical industry project-management principles in an academic setting by bringing together multidisciplinary teams to fill critical scientific and technology gaps, using an experienced team of industry-trained researchers and project managers. The KU HTS proactively engages in supporting grant applications for extramural funding, intellectual-property management and technology transfer. The KU HTS staff further provides educational opportunities for the KU faculty and students to learn cutting-edge technologies in drug-discovery platforms through seminars, workshops, internships and course teaching. This is the first instalment of a two-part contribution from the KU HTS laboratory.

The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology.

The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory.

Conference report: update on Probe Discovery World Congress 2009.

The conference was the first of its kind in a series of trend-setting conferences conceived and organized by Select Biosciences. The word 'probe' is a broad term that can be interpreted to mean any one of a wide variety of agents. These include active chemistries discovered in academic screening laboratories or in government organizations (Molecular Libraries Probe Production Centers Network or the US NIH), commercially available probes (i.e., dyes, antibodies or fluorescent proteins), failed drug candidates from the pharmaceutical industry, whole-body or cellular imaging agents, specific biomarkers or tool molecules from chemogenomics and/or systems biology efforts. The conference attendees represented academia, biotech and pharma, the speaker list was stellar, and the conference organizers succeeded in bringing together all of the various incarnations of probe hunters in order to share their experiences and to network with a common purpose.