Novel non-steroidal/non-anilide type androgen antagonists with an isoxazolone moiety

3-Substituted (Z)-4-(4-N,N-dialkylaminophenylmethylene)-5(4H)-isoxazolones and related compounds were designed and prepared as candidates for structurally novel androgen antagonists. Several compounds showed potent anti-androgenic activity as assessed by nuclear androgen receptor binding assay and growth inhibition assay using androgen-dependent Shionogi carcinoma cells SC-3. They were approximately 10--220 times more potent than flutamide in these assay systems. They also showed anti-androgenic activity toward prostate tumor cell line LNCaP, which has an aberrant nuclear androgen receptor.     

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.     

Data concordance from a comparison between filter binding and fluorescence polarization assay formats for identification of ROCK-II inhibitors

The Rho-associated coiled-coil-containing protein serine/threonine kinases ROCK-I and ROCK-II are thought to play a major role in cytoskeletal dynamics by serving as downstream effectors of the Rho/Rac family of cytokine- and growth factor-activated small GTPases. As such, the ROCK family members are attractive intervention targets for a variety of pathologies, including cancer and cardiovascular disease. The authors developed a high-throughput screen to identify ROCK-II inhibitors and report results from a direct comparison of 2 screening campaigns for ROCK-II inhibitors using fluorescence polarization (FP) and filter binding (FB). Screening protocols to identify inhibitors of ROCK-II were developed in FB and FP formats under similar assay and kinetic conditions. A 30,000-member compound library was screened using FB ((33)P) and FP detection systems, and compounds that were active in either assay were retested in 5-point curve confirmation assays. Analysis of these data showed an approximate 95% agreement of compounds identified as active in both assay formats. Also, compound potency determinations from FB and FP had a high degree of correlation and were considered equivalent. These data suggest that the assay methodology has little impact on the quality and productivity of the screen, provided that the assays are developed to standardize kinetic conditions.     

Using clustering techniques to improve hit selection in high-throughput screening

A typical modern high-throughput screening (HTS) operation consists of testing thousands of chemical compounds to select active ones for future detailed examination. The authors describe 3 clustering techniques that can be used to improve the selection of active compounds (i.e., hits). They are designed to identify quality hits in the observed HTS measurements. The considered clustering techniques were first tested on simulated data and then applied to analyze the assay inhibiting Escherichia coli dihydrofo-late reductase produced at the HTS laboratory of McMaster University.     

Detection of oligonucleotide-ligand complexes by ESI-MS (DOLCE-MS) as a component of high throughput screening

With the advent of combinatorial chemistry and high throughput screening, a major bottleneck in the pharmaceutical industry has changed from quickly finding active compounds to limiting them to a manageable number for proper follow-up. With hundreds to thousands of active compounds identified by a multitude of biological screens, there need to be rapid and unambiguous methods for eliminating false positive, toxic, or otherwise difficult compounds from further scrutiny. We have used electrospray ionization mass spectrometry as a rapid screening method to identify compounds from viral screens that yield a positive assay response by interaction with DNA rather than inhibiting the target enzyme. Both the sample preparation and data acquisition have been automated, allowing the screening of all hits from relevant biological screens (up to 1,000/week). The assay was validated using several known DNA intercalators and minor groove binders. These "standards" and many but not all of our "active compounds" were shown to form noncovalent complexes with a variety of different DNA:DNA and DNA:RNA duplexes.     

Evaluation of the antioxidant activity of flavonoids by "ferric reducing antioxidant power" assay and cyclic voltammetry

Flavonoids, naturally occurring phenolic compounds, have recently been studied extensively for their antioxidant properties. The structure-antioxidant activity relationships (SAR) of flavonoids have been evaluated against different free radicals, but "ferric reducing antioxidant power" (FRAP) assay, which determines directly the reducing capacity of a compound, has not been used for this purpose. In this study, the antioxidant activities of 18 structurally different flavonoids were evaluated by FRAP assay modified to be used in 96-well microplates. Furthermore, their oxidation potentials were also measured, which were in the range of +0.3 V (myricetin) to +1.2 V (5-hydroxy flavone) and were in good agreement with FRAP assay results. Quercetin, fisetin and myricetin had the lowest oxidation potentials and appeared the most active compounds in FRAP assay and were 3.02, 2.52 and 2.28 times more active than Trolox, respectively. Indications were found that the o-dihydroxy structure in the B ring and the 3-hydroxy group and 2,3-double bond in the C ring give the highest contribution to the antioxidant activity.     

"Plate cherry picking": a novel semi-sequential screening paradigm for cheaper, faster, information-rich compound selection

This work describes a novel semi-sequential technique for in silico enhancement of high-throughput screening (HTS) experiments now employed at Novartis. It is used in situations in which the size of the screen is limited by the readout (e.g., high-content screens) or the amount of reagents or tools (proteins or cells) available. By performing computational chemical diversity selection on a per plate basis (instead of a per compound basis), 25% of the 1,000,000-compound screening was optimized for general initial HTS. Statistical models are then generated from target-specific primary results (percentage inhibition data) to drive the cherry picking and testing from the entire collection. Using retrospective analysis of 11 HTS campaigns, the authors show that this method would have captured on average two thirds of the active compounds (IC(50) < 10 microM) and three fourths of the active Murcko scaffolds while decreasing screening expenditure by nearly 75%. This result is true for a wide variety of targets, including G-protein-coupled receptors, chemokine receptors, kinases, metalloproteinases, pathway screens, and protein-protein interactions. Unlike time-consuming "classic" sequential approaches that require multiple iterations of cherry picking, testing, and building statistical models, here individual compounds are cherry picked just once, based directly on primary screening data. Strikingly, the authors demonstrate that models built from primary data are as robust as models built from IC(50) data. This is true for all HTS campaigns analyzed, which represent a wide variety of target classes and assay types.     

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.     

Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors

The mode of binding of four active-site directed inhibitors to human thrombin has been determined by x-ray crystallographic analysis. The inhibitors studied are benzamidine, PPACK, NAPAP, and MD-805, of which the last three are compounds evolved specifically to inhibit thrombin. Crystal structures were determined in the presence of both the inhibitor and the undecapeptide [des-amino Asp55]hirudin(55-65) which binds distant from the active site. Despite having significantly different chemical structures, NAPAP and MD-805 bind to thrombin in a very similar "inhibitor binding mode" which is not that expected by direct analogy with the binding of substrate. Both inhibitors bind to thrombin in a similar way as to trypsin, but thrombin has an extra loop, the "Tyr-Pro-Pro-Trp loop," not present in trypsin, which gives further binding interactions and is seen to move somewhat to accommodate binding of the different inhibitors. The fact that NAPAP and MD-805 require different stereochemistry for potent inhibition is demonstrated, and its structural basis clarified. The wealth of data on analogs and variants of these lead compounds is shown to be compatible with this inhibitor binding mode.     

Sex genes for genomic analysis in human brain: internal controls for comparison of probe level data extraction.

Background

Genomic studies of complex tissues pose unique analytical challenges for assessment of data quality, performance of statistical methods used for data extraction, and detection of differentially expressed genes. Ideally, to assess the accuracy of gene expression analysis methods, one needs a set of genes which are known to be differentially expressed in the samples and which can be used as a "gold standard". We introduce the idea of using sex-chromosome genes as an alternative to spiked-in control genes or simulations for assessment of microarray data and analysis methods.

Results

Expression of sex-chromosome genes were used as true internal biological controls to compare alternate probe-level data extraction algorithms (Microarray Suite 5.0 [MAS5.0], Model Based Expression Index [MBEI] and Robust Multi-array Average [RMA]), to assess microarray data quality and to establish some statistical guidelines for analyzing large-scale gene expression. These approaches were implemented on a large new dataset of human brain samples. RMA-generated gene expression values were markedly less variable and more reliable than MAS5.0 and MBEI-derived values. A statistical technique controlling the false discovery rate was applied to adjust for multiple testing, as an alternative to the Bonferroni method, and showed no evidence of false negative results. Fourteen probesets, representing nine Y- and two X-chromosome linked genes, displayed significant sex differences in brain prefrontal cortex gene expression.

Conclusion

In this study, we have demonstrated the use of sex genes as true biological internal controls for genomic analysis of complex tissues, and suggested analytical guidelines for testing alternate oligonucleotide microarray data extraction protocols and for adjusting multiple statistical analysis of differentially expressed genes. Our results also provided evidence for sex differences in gene expression in the brain prefrontal cortex, supporting the notion of a putative direct role of sex-chromosome genes in differentiation and maintenance of sexual dimorphism of the central nervous system. Importantly, these analytical approaches are applicable to all microarray studies that include male and female human or animal subjects.

     

Synthesis and properties of novel lipopeptides and lipid mimetics

Lipid mimetics, synthetic molecules that resemble natural lipids either structurally or functionally, have been developed as potential medicinal substances. They have been successfully applied in the development of drug and peptide delivery systems and for the development of inhibitors or lipid metabolizing enzymes. Phospholipase A2 is considered to be involved as the rate‒limiting step in the production of lipid mediators of inflammatory responses and, as such, it has been a target for drug design. A series of lipid mimetics including lipopeptides, amides and alcohols of lipidic α‒amino acids, have been tested by bulk and monolayer assay techniques. The findings suggested the direct interaction of the tested compounds with porcine pancreatic phospholipase A2. The inactivation of the enzyme occurred in a competitive manner. The most active compound 1 (2-amino-N-hexadecyl-L -hexanamide) showed an apparent IC₅₀ of 12 μ M and inhibitory power Z=13 in the monolayer assay. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity, such as 13-keto-9(Z),11(E),15(Z)-octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly, this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms.     

Reconciling sexual selection to species recognition: a process-based model of mating decision

Mating signals often encode information important for both species recognition and mate quality assessment and endure selection pressures that combine both stabilizing and directional components. Here, we present a family of models of mate preference for multiple-message signals. Our models are process based rather than purely normative, they assume the existence of one (or more) "utility function" that order signals along a scale of perceived appropriateness, and interpret preferences either as the differential probability of signals recognition or as the combined effect of differential recognition and direct comparison between signal alternatives. These models show the critical role played by the proximate mechanisms of information processing in influencing the ultimate function of female mate choice. They show that if preferences are an emergent property of the way animals recognize signals then species recognition and mate quality assessment are expected to constrain each other severely and to limit the overall discrimination power of the system. In contrast, if preferences result from two computational processes, recognition and comparison, the constraining effects of species recognition and mate quality assessment are sensibly reduced. In these cases, females may improve discrimination in mate quality by adopting permissive recognition rules and limiting the risks of heterospecific mating.     

A nonradioactive high-throughput/high-content assay for measurement of the human serotonin reuptake transporter function in vitro

Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC(50) values close to those reported in the literature for tritiated serotonin uptake. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Z of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a "classical" assay for serotonin uptake are all reported. The assay can also be read in "high-content" mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.     

Studies on the use of uncertainty factors in deriving RfDs

Risk assessment of exposure to chemicals having a toxic end point routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. RfD model can be used with widely different databases. However, the quality of individual risk assessment is unequal among chemicals, often resulting in either an over‐ or underestimation of adverse health risk. The purpose of this investigation was to evaluate whether the magnitude of the 10X uncertainty factors has scientific merit against data from recent human and animal experimental studies. Although we assessed the use of uncertainty factors for representative chemicals from various classes of compounds, such as volatile organics, alcohols, gasoline components, and pesticides, we are presenting our findings for 24 chemicals.

A compilation and comparison of ratios between LOAEL/NOAEL (Lowest Observed Adverse Effect Level/No Observed Adverse Effect Level), and subchronic/chronic values were made. Although a 10X uncertainty factor is most commonly used in the risk assessment processes, an examination of the datasets which have been used to calculate RfDs suggests different values which are scientifically justifiable.     

In vitro bone marrow granulocyte-macrophage progenitor cultures in the assessment of hematotoxic potential of the new drugs

In pharmaceutical research, in vitro toxicity tests, for assessing the potential toxicity of new chemical entities are necessary in the early stages of the developmental process, when no information is available about the metabolism or even the target organ toxicity of the compounds to be tested. In vitro specific organ toxicity tests, such as the granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay, are useful tools for predicting the adverse effects of new compounds on the blood-forming system, provided that some reference points are available, e.g., toxicological information about compounds belonging to the same chemical class and structure-activity relationship data. Furthermore, when no information is available about metabolism, the in vitro system should cover as many possibilities as possible, to avoid false positive or false negative results. In fact, while many compounds are metabolized to a variety of inactive chemical species, some undergo bioactivation to form more active metabolites. The addition of a metabolic activation system to the CFU-GM assay enables assessment of direct and metabolism-mediated toxicity. The regulatory agencies and industry value the concept of assays performed with and without metabolic activation, since they often have to take decisions about compounds with unknown mechanisms of action. CFU-GM assay, designed in this way, is an example of such a mechanism-naive assay. It has been suggested that, for new compounds, metabolites should be generated and tested both in the presence and in the absence of the parent compound itself, to identify the possible contribution of metabolites to the hematotoxicity observed, and to determine whether there is any synergistic or antagonistic effect between metabolites and the parent compound that might affect hematotoxicity in vivo. Various approaches can be used to obtain such information.     

Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In na?ve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.     

Miniaturization and validation of a high-throughput serine kinase assay using the AlphaScreen platform

Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include low sensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility of miniaturization of a serine kinase assay using generic reagents in the AlphaScreen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-microL final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z' values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the AlphaScreen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.     

Bioactive components in the fruits of Ziziphus jujuba Mill. against the inflammatory irritant action of Euphorbia plants

Chinese jujube (also known as Chinese date) is the fruit of Ziziphus jujuba Mill. (Rhamnaceae). As a famous folk medicine, it is used as antidote in traditional Chinese formula, Shi Zao Decoction, to relieve the drastic inflammatory irritant nature of Euphorbia species. The irritant activities may cause serious adverse effects in clinical practices. This study aimed to investigate the active components of Z. jujuba through the inhibitory effects on the inflammatory cells activated by Euphorbia kansui and prostratin, a phorbol ester isolated from Euphorbia fischeriana. Peritoneal macrophage of rat and splenic lymphocyte (splenocyte) of mouse were selected to evaluate these actions in vitro. Nitric oxide (NO) release of macrophage and the proliferation of splenocyte were examined through Griess method and MTT assay. TNF-α, as an important pro-inflammatory cytokines, was detected with enzyme-linked immunosorbent assay (ELISA) method. Six fractions extracted from Z. jujuba were evaluated and fraction F (triterpene acids fraction) was demonstrated to be the most active part, and then, 21 compounds isolated from Z. jujuba were tested at the concentrations range from 1 μg/ml to 100 μg/ml. The results show that 7 compounds of them are likely to be active compounds concerning to their pronounced inhibitory action on the activated inflammatory cells. These effects might be helpful to attenuate the irritant action of Euphorbiaceae plants and protect the gastrointestinal tissue from potent inflammatory injury, which should be beneficial to some diseases, like inflammatory bowel disease.     

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.