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PubMed2004

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure-activity relationships of the 4-aryl group.

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.

Antineoplastic Agents↗
PubMed2004

High-throughput screening of genome fragments bound to differentially acetylated histones.

Although acetylation-deacetylation of histones contributes to regulation of gene expression, few methods have been available to determine the whole-genome histone acetylation profile in specific cells or tissues. We have now developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genome fragments embedded in histones subject to differential acetylation. This DCS screening was applied to a human gastric cancer cell line incubated with or without an inhibitor of histone deacetylase (HDAC) activity, resulting in the rapid identification of more than 250 genome fragments. Interestingly, a number of cancer-related genes were revealed to be the targets of HDAC in the cancer cells, including those for tumour protein 73 and cell division cycle 34. Such differential acetylation of histone was also shown to be linked to the regulation of transcriptional activity of the corresponding genes. Among the isolated genome fragments, 94% (32/34) of them were confirmed to be bound to differentially acetylated histones, and the genes corresponding to 78% (7/9) of them exhibited differential transcriptional activity consistent with the level of histone acetylation. With its high fidelity, the DCS method should open a possibility to rapidly compare the genome-wide histone acetylation profiles and to provide novel insights into molecular carcinogenesis.

Acetylation↗
PubMed2003

High-throughput screening of selectivity of melt polymerization catalysts using fluorescence spectroscopy and two-wavelength fluorescence imaging.

A new general approach for rapid assessment of polymerization catalysts is introduced. Native fluorescence emission of solid polymers is measured directly in combinatorial 96-microreactor arrays and polymers produced in a laboratory-scale validation reactor. Fluorescence features collected with a CCD-based spectrofluorometer are correlated with chemical properties of interest such as polymer molecular weight, amount of branching, and catalyst selectivity. The approach is illustrated by screening of selectivity of melt polymerization catalysts used in synthesis of an aromatic bisphenol A polycarbonate. Selectivity of catalysts correlated with the ratio of fluorescence intensities at 400 and 500 nm at 340-nm excitation. The relative standard deviation (RSD) in spectroscopic serial measurements was 1-12.5%. This spread included instrument variability (< or = 1% RSD) and sample inhomogeneity. Parallel quantitative screening of catalyst selectivity in combinatorial 96-microreactor arrays was performed as a two-wavelength ratiometric fluorescence imaging through 400- and 500-nm interference filters and showed a good correlation (R2 = 0.994) with serial screening. Our approach is an attractive alternative to traditional separation-based techniques if speed and nondestructive nature of analysis are critical and when the high cross-linking or solvent resistance of polymers complicates traditional analysis.

Journal Article↗
PubMed2005

Simple strategies for reducing sample loads in in vitro metabolic stability high-throughput screening experiments: a comparison between traditional, two-time-point and pooled sample analyses.

Higher-throughput ADME programs in early drug discovery are becoming common throughout the pharmaceutical industry as companies strive to reduce their compound attrition in later-stage development. Many of the ADME assays developed into higher-throughput formats rely on LC/MS analyses. Since the biological aspects of the assay are amenable to parallel processes using dense plate formats, the number of samples generated from these assays produce a large analysis load for serial LC/MS. Presented in this report are two novel strategies, including a sample pooling method and a two time-point method, that could be used in drug discovery to reduce the number of samples generated during multiple time-point in-vitro ADME assays. One hundred and sixty-three compounds were subjected to human microsomal incubations with full time-point method samples taken at t = 0, 5, 15, 30, and 45 min. The ER data correlation (R(2)) between the full time-point method and the pooling method and two time-point methods were 0.98 and 0.97, respectively. Both methods have the potential to: 1. produce data of similar quality to traditional high throughput ADME assays, 2. be easily implemented, 3. shorten analytical run times, and 4. be reproducible and robust.

Algorithms↗
PubMed2007

High-throughput screening for small-molecule activators of neutrophils: identification of novel N-formyl peptide receptor agonists.

We screened a chemolibrary of drug-like molecules for their ability to activate reactive oxygen species (ROS) production in murine phagocytes, and we identified 26 novel compounds with potent neutrophil activating properties. We used substructure screening, fragment-focusing, and structure-activity relationship analyses to further probe the parent library and defined at least two groups of activators of ROS production in murine neutrophils: t-butyl benzene and thiophene-2-amide-3-carboxylic ester derivatives. Further studies of the active compounds revealed 11 compounds that activated ROS production in human neutrophils, and six of these compounds also activated intercellular Ca(2+) mobilization and chemotaxis in human neutrophils. Of the latter compounds, compound 14 (1,3-benzodioxolane-5-carboxylic acid 4'-benzyloxy-3'-methoxybenzylidene-hydrazide) activated neutrophils at nanomolar concentrations, and Ca(2+) mobilization was inhibited by pertussis toxin and N-t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc-2), an antagonist of formyl peptide receptors (FPR/FPRL1). Likewise, activation by compound 14 was desensitized after N-formyl-Met-Leu-Phe pretreatment. Similar biological activities were found for compound 104 (1,3-benzodioxolane-5-carboxylic acid 3'-bromo-5'-ethoxy-4'-hydroxybenzylidene-hydrazide), an analog of compound 14. Furthermore, conformational analysis of the activators of chemotaxis and Ca(2+) mobilization showed a high degree of similarity in distances between pharmacophore points of compounds 14 and 104 with a model of FPR published by Edwards et al. (Mol Pharmacol 68:1301-1310, 2005), indicating that conformational features of the agonists identified here are structurally compatible with steric constraints of the ligand-binding pocket of the receptor. Based on these results, we conclude that compounds 14 and 104 represent novel small-molecule agonists of FPR. These studies enhance our understanding of FPR ligand/receptor interactions and structure/activity relationships of phagocyte agonists.

Animals↗
PubMed2004

Analytical validation of telomerase activity for cancer early detection: TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells.

Activation of telomerase plays a critical role in unlimited proliferation and immortalization of cells. Telomerase activity has been shown to correlate with tumor progression, indicating that tumors expressing this enzyme possess aggressive clinical behavior and that telomerase activity may be a useful biomarker for early detection of cancer. However, measurements of telomerase activity by current methods such as telomeric repeat amplification protocol (TRAP)/polymerase chain reaction (PCR) or antibody-based radioimmunoassay (RIA) are low-throughput and not robust enough to easily accommodate the required statistical analysis to determine whether telomerase activity is a practical biomarker. As part of the National Cancer Institute Early Detection Research Network of analytical validation, we have developed a robot assisted TRAP assay (RApidTRAP) of telomerase, a potential biomarker for cancer early detection. Measurements of human telomerase reverse transcriptase catalytic subunit (hTERT) mRNA were performed in concert with measurement of telomerase activity. For this purpose we determined hTERT mRNA concentration and telomerase activity in human normal (RPE-28) and cancer (A549) cell lines as well as in human serum (SRM 1951A). Telomerase activity measurements were made using the TRAP/PCR capillary electrophoresis (CE) method on (50 to 1000) cells/reaction isolated from cell extracts. Measurement of hTERT mRNA was made using specific primers and probes on a LightCycler in the range of (10 to 7000) cells/reaction. Comparison of high-throughput telomerase activity measurements using the robot and those performed manually were consistent in sensitivity and reproducibility. Using this combination of telomerase activity and hTERT mRNA measurements, the automated system improved efficiency over traditional TRAP/PCR methods.

Biomarkers↗
PubMed2003

High throughput screening of 16 million serologically negative blood donors for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1 by nucleic acid amplification testing with specific and sensitive multiplex reagent in Japan.

Nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) of blood donated voluntarily after serological screening was implemented on July 1st 1999 for transfusion and plasma fractionation by the Japanese Red Cross blood transfusion services. From February 1st 2000, HBV, HCV and HIV-1 NAT screening of pools of 50 negative serologically screened donated blood was started and the results were reported within 1 day after blood donation. Systems were established for rapid shipment, electronic communication, automated specimen preparation, pooling and automated amplification and detection. At present, NAT screening is carried out within 1 day after donation. This report describes the blood screening system by NAT and the results obtained from over 16 million blood samples using simultaneous screening for HBV, HCV and HIV-1 with multiplex reagent. Between February 1, 2000 and December 31, 2002, 16012175 serologically negative units were tested by NAT. 308 units with Hepatitis B virus DNA (HBV DNA) were detected. The sensitivity of 50 pool NAT screening with input volume of 0.2 ml is significantly higher than that of highly sensitive HBsAg testing. 46 cases with HCV RNA and six cases with HIV-1 RNA were detected. These cases were not detected by HCV antibody and HIV-1 antibody screening. The false positive rate was 0.18%. The NAT system was developed from serological screening test negative non-remunerated voluntary donations. We supply blood products to medical organizations after screening by NAT for HBV, HCV and HIV-1 for transfusion and source plasma for fractionation. This is the first automated integrated system for prevention of transfusion transmitted HBV, HCV and HIV-1 infections, by NAT screening.

Antibodies, Viral↗
PubMed2006

High-throughput screening for multi-class veterinary drug residues in animal muscle using liquid chromatography/tandem mass spectrometry with on-line solid-phase extraction.

A rapid qualitative method using on-line column-switching liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for screening 13 target veterinary drugs: four macrolides - erythromycin A, josamycin (leucomycin A3), kitasamycin (leucomycin A5), and tylosin A; six (fluoro)quinolones - ciprofloxacin, danofloxacin, enrofloxacin, flumequine, oxolinic acid, and sarafloxacin; and lincomycin, virginiamycin M1, and trimethoprim in different animal muscles. Clindamycin, norfloxacin, nalidixic acid, oleandomycin, ormetoprim, and roxithromycin were used as the internal standards. After simple deproteination and analyte extraction of muscle samples using acetonitrile, the supernatant was subjected to on-line cleanup and direct analysis by LC/MS/MS. On-line cleanup with an extraction cartridge packed with hydrophilic-hydrophobic polymer sorbent followed by fast LC using a short C18 column resulted in a total analysis cycle of 6 min for 19 drugs. This screening method considerably reduced the time and the cost for the quantitative and confirmatory analyses. The application of a control point approach was also introduced and explained.

Animals↗
PubMed2002

High-throughput screening of potential inhibitors for the metabolism of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid.

By screening potential inhibitors of drug metabolism using the in vitro models, potential drug-drug interactions in vivo may be predicted with the use of appropriate pharmacokinetic principles. This study aimed to develop a rapid screening system using human liver microsomes to efficiently identify the potential inhibitors of DMXAA metabolism. Initial IC50 was estimated by using a two-point method, and then Ki values were determined if required and compared with those initial IC50 values. More than 100 compounds including known substrates and inhibitors of human uridine diphosphate glucuronosyltransferases (UGTs) and cytochrome P450 (CYP), anti-cancer drugs and xanthenone analogues were screened for their inhibitory effect on DMXAA glucuronidation and 6-methylhydroxylation in human liver microsomes. Both metabolites of DMXAA, DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA), formed in human liver microsomes were quantitated by validated HPLC methods. The results indicated that there was a significant relationship (r2 = 0.966, P < 0.001) between the two-point IC50 values and the apparent Ki values for 20 compounds showing significant inhibitory effects on DMXAA metabolism, suggesting the usefulness of the two-point determination for the initial screening of compounds. This study has been completed using a strategy for rapid HPLC analysis and thus provided early access to detailed information for potential inhibitors of DMXAA metabolism and allows for further DMXAA-drug interaction studies.

Antineoplastic Agents↗
PubMed2000

Discovery of a New Efficient Chiral Ligand for Copper-Catalyzed Enantioselective Michael Additions by High-Throughput Screening of a Parallel Library We thank the Commission of the European Union (TMR Network grant "Combinatorial catalysts" ERB-FMR XCT 96-0011) for financial support and a postdoctoral fellowship to I. Chataigner. We are grateful to Mr. Andrea Stevenazzi and Ms. Chiara Monti (Università di Milano) for technical assistance. Part of this work was presented as a plenary lecture (C. Gennari) at 11th European Symposium on Organic Chemistry (ESOC 11, Göteborg, Sweden, July 23 - 28, 1999).

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Journal Article↗
PubMed2002

Combinatorial synthesis of new cationic lipids and high-throughput screening of their transfection properties.

Here we describe the first synthesis-screening approach for the identification and optimization of new cationic lipids for gene transfer in various cell lines. Combinatorial solid-phase chemistry was used to synthesize a library of new cationic lipids based on 3-methylamino-1,2-dihydroxypropane as the polar, cationic lipid part. As the nonpolar lipid part, different hydrocarbon chains were bound to the amino group of the scaffold and the amino group was further methylated to afford constantly cationic lipids. Lipids were synthesized in both configurations and as racemates, and the counter ions were also varied. By using a fully automated transfection screening method and COS-7 cells, the cationic lipid N,N-ditetradecyl-N-methyl-amino-2,3-propanediol (KL-1-14) was identified as a candidate lipid for the development of an improved transfection reagent. Screening the transfection properties of KL-1-14 in numerous combinations with the helper lipids dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) revealed that Chol is the most suitable helper lipid and the best KL-1-14/Chol ratio is 0.5-0.7. Compared to the standard transfection lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), transfection efficiency was improved by a factor of about 40. Furthermore, by using R- and S-configured KL-1-14, it could be shown that the configuration of the lipids had no significant influence on its transfection efficiency. The highest transfection efficiencies were achieved with chloride as the counter ion. The new lipofection reagent was further tested to transfect the cell lines MDA-MB-468, MCF-7, MDCK-C7, and primary dentritic cells (DC), which are important for the development of new anticancer gene therapy strategies. Even in these cells, KL-1-14/Chol (1:0.6) had improved transfection efficiencies, which were about two to four times higher than for DOTAP.

Animals↗