scholarly journals Oncology Drug Discovery Applications Using the FMAT™ 8100 HTS System

2003 ◽  
Vol 8 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Jennifer Y. Lee ◽  
Sheri Miraglia ◽  
Xiongwei Yan ◽  
Elana Swartzman ◽  
Susan Cornell-Kennon ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
High Throughput Screening ◽  
Enzyme Assay ◽  
Annexin V ◽  
Enzyme Assays ◽  
Binding Assays ◽  
Src Tyrosine Kinase ◽  
Apoptosis Assay ◽  
Peptide Interaction ◽  
Homogeneous Assays

High-throughput screening (HTS) for potential anticancer agents requires a broad portfolio of assay platforms that may include kinase enzyme assays, protein-protein binding assays, and functional cell-based apoptosis assays. The authors have explored the use of fluorometric microvolume assay technology (the FMAT™ 8100 HTS System) in three distinct homogeneous HTS assays: (1) a Src tyrosine kinase enzyme assay, (2) a Grb2-SH2 protein-peptide interaction assay, and (3) an annexin V binding apoptosis assay. Data obtained from all three assays suggest that the FMAT system should facilitate the implementation of homogeneous assays for a wide variety of molecular targeted and cell-based screens. ( Journal of Biomolecular Screening 2003:81-88)

scholarly journals Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

1999 ◽  
Vol 4 (4) ◽  
pp. 193-204 ◽  
Author(s):  
Sheri Miraglia ◽  
Elana E. Swartzman ◽  
Julia Mellentin-Michelotti ◽  
Lolita Evangelista ◽  
Christopher Smith ◽  
...  
Keyword(s):  
Ligand Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Laser Scanner ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Using Data ◽  
Immunological Assays ◽  
The Individual ◽  
Homogeneous Assays

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm2 digitized image of a 100-μm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twister™ (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Comparison of 3 AT1 Receptor Binding Assays: Filtration Assay, ScreenReady™ Target, and WGA Flashplate®

2005 ◽  
Vol 10 (2) ◽  
pp. 118-126 ◽  
Author(s):  
Regine M. van der Hee ◽  
Tanja Deurholt ◽  
Cindy C. Gerhardt ◽  
Els M. de Groene
Keyword(s):  
Angiotensin Ii ◽  
Receptor Binding ◽  
High Throughput Screening ◽  
Cell Membranes ◽  
Radioligand Binding ◽  
Nonspecific Binding ◽  
Binding Assays ◽  
Homogeneous Assay ◽  
Homogeneous Assays

In this article, the study of 3 different angiotensin II type 1 (AT1) receptor binding assays in terms of reproducibility, robustness, and feasibility for high-throughput screening (HTS) is described. The following methods were used: a nonhomogeneous filtration assay in a 96-well format using CHO-AT1 cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady™ Target for the AT1 receptor and the wheat germ agglutinin (WGA) Flashplate®, which was coated “in-house” with the CHO-AT1 cell membranes. Receptors were labeled with [125I]-Sar1-Ile8-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible Kd, Bmax, and Ki values and good total binding/nonspecific binding (TB/NSB) ratios were obtained with both the ScreenReady™ Targets and the filtration assay, whereas the WGA Flashplates® showed unacceptably high nonspecific binding and high variation when applied as a homogeneous assay. However, when applied as a heterogeneous assay (i.e., when a wash step at the end of the assay is included), the results were significantly better. Interestingly, ligand affinities were consistently lower in Flashplate®-based assays than in the filtration assay. This may be due to the immobilization of the receptors onto the solid surface of the plate, affecting their conformation. In terms of reproducibility, robustness, and feasibility for HTS, the authors conclude that the ScreenReady™ Target plates are most suitable for AT1 receptor binding screening.

Design, Synthesis, Anti-Proliferative Evaluation and Cell Cycle Analysis of Hybrid 2-Quinolones

2019 ◽  
Vol 19 (9) ◽  
pp. 1132-1140
Author(s):  
Heba A.E. Mohamed ◽  
Hossa F. Al-Shareef
Keyword(s):  
Cell Cycle ◽  
Anticancer Agents ◽  
Biological Activities ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Cytotoxic Activities ◽  
Significant Group ◽  
Design Synthesis ◽  
Standard Drug ◽  
Wide Range

Background: Quinolones are a significant group of nitrogen heterocyclic compounds that exist in therapeutic agents, alkaloids, and synthetic small molecules that have important biological activities. A wide range of quinolones have been used as antituberculosis, antibacterial, anti-malarial, antifungal, anticonvulsant, anticancer agents and urease inhibitors. Methods: Ethyl 3,3-disubstituted-2-cyano propionates containing hybride quinolones derivatives were synthesized by the reaction of 1-amino-7-hydroxy-4-methylquinolin-2(1H)-one and its dibromo derivative with α, β-unsaturated carbonyl in ethanol. Results: A novel series of hybrid 2-quinolone derivatives was designed and synthesized. The compounds structures were confirmed using different spectroscopic methods and elemental analysis. The cytotoxic activities of all the compounds were assessed against HepG2 cell line in comparison with doxorubicin as a standard drug. Conclusion: Most compounds revealed superior anti-proliferative activity than the standard. Compound 4b, is the most active compound (IC50 = 0.39mM) compared with doxorubicin (IC50 = 9.23mM). DNA flow cytometric analysis of compound 4b showed cell cycle arrest at G2/M phase with a concomitant increase of cells in apoptotic phase. Dual annexin-V/ propidium iodide staining assay of compound 4b revealed that the selected candidate increased the apoptosis of HepG-2 cells more than control.

scholarly journals Research fronts of Chemical Biology

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Shanshan Lv
Keyword(s):  
High Throughput Screening ◽  
Drug Targets ◽  
Chemical Biology ◽  
Rational Design ◽  
Future Research ◽  
Design Strategies ◽  
Binding Assays ◽  
Cellular Mechanisms ◽  
Diagnostic Technologies

Abstract Over the past decades, researchers have witnessed substantially increasing and ever-growing interests and efforts in Chemical Biology studies, thanks to the development of genome and epi-genome sequencing (revealing potential drug targets), synthetic chemistry (producing new medicines), bioorthogonal chemistry (chemistry in living systems) and high-throughput screening technologies (in vitro cell systems, protein binding assays and phenotypic assays). This report presents literature search results for current research in Chemical Biology, to explore basic principles, summarize recent advances, identify key challenges, and provide suggestions for future research (with a focus on Chemical Biology in the context of human health and diseases). Chemical Biology research can positively contribute to delivering a better understanding of the molecular and cellular mechanisms that accompany pathology underlying diseases, as well as developing improved methods for diagnosis, drug discovery, and therapeutic delivery. While much progress has been made, as shown in this report, there are still further needs and opportunities. For instance, pressing challenges still exist in selecting appropriate targets in biological systems and adopting more rational design strategies for the development of innovative and sustainable diagnostic technologies and medical treatments. Therefore, more than ever, researchers from different disciplines need to collaborate to address the challenges in Chemical Biology.

Anticancer Drug Action on Poly(A) Polymerase Activity and Isoforms during Hela and Wish Cell Apoptosis

2000 ◽  
Vol 15 (2) ◽  
pp. 171-178 ◽  
Author(s):  
N.A.A. Balatsos ◽  
M. Havredaki ◽  
C.M. Tsiapalis
Keyword(s):  
Anticancer Agents ◽  
Enzyme Assay ◽  
Polymerase Activity ◽  
Activity Levels ◽  
Immunoblotting Analysis ◽  
A Cell ◽  
Lower Mobility ◽  
Cell Commitment ◽  
Diploid Cells ◽  
Mrna Polyadenylation

Poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes mRNA polyadenylation. Its activity and isoform levels vary during cell cycle transformation and apoptosis. It has become widely accepted that cell death after DNA damage by anticancer agents is primarily the result of apoptosis and that cells able to evade apoptosis will be resistant to cell killing. The therapeutic agents interferon (IFN), 5-fluorouracil (5-FU) and tamoxifen (Tam) with different mechanisms of action mediate both partial dephosphorylation and inactivation of PAP, detected by immunoblotting analysis and PAP enzyme assay, respectively. We examined the apoptotic tendencies of HeLa and WISH cell lines caused by one of the drugs used, 5-FU. The trend in the cells examined, observed by DAPI and/or DNA fragmentation assay, was found to be accompanied by and reversibly related to PAP activity levels and PAP lower mobility phosphorylated forms of 106 and 100 kDa isoforms. Moreover, a cell type-modulated, differential response of HeLa (chemosensitive cells) versus WISH (drug-resistant diploid cells) has been revealed. This finding yields information on the possible use of PAP as a tumor marker involved in cell commitment and/or induction of apoptosis and may help to improve our understanding of tumor cell sensitivity to anticancer agents.

Serum Glutamic—Oxaloacetic Transaminase: Evaluation of a Coupled-Reaction Enzyme Assay by Means of Kinetic Theory

1971 ◽  
Vol 17 (11) ◽  
pp. 1114-1122 ◽  
Author(s):  
C D Russell ◽  
E Cotlove
Keyword(s):  
Kinetic Theory ◽  
Enzyme Assay ◽  
Rate Equations ◽  
Enzyme Assays ◽  
Glutamic Oxaloacetic Transaminase ◽  
Malic Dehydrogenase ◽  
Serum Glutamic Oxaloacetic Transaminase ◽  
Coupled Reaction ◽  
Analytical Errors ◽  
Substrate Concentrations

Abstract Rate equations and rate parameters are determined for the coupled-reaction assay for serum glutamic-oxaloacetic transaminase in which malic dehydrogenase is used. These are used to interpret observed curves of absorbance vs. time, to select optimum substrate concentrations, and to estimate nonrandom analytical errors. A simple, systematic, general approach is presented, which can readily be applied to other enzyme assays in which two or more reactions are coupled. It depends for its simplicity on access to an adequate computing facility.

scholarly journals Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy

2001 ◽  
Vol 6 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Martin Rudiger ◽  
Ulrich Haupts ◽  
Keith J. Moore ◽  
Andrew J. Pope
Keyword(s):  
Single Molecule ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Binding Assays ◽  
Fluorescence Intensity Distribution Analysis ◽  
Fluorescent Ligands ◽  
G Protein Coupled

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

scholarly journals A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

2009 ◽  
Vol 2 ◽  
pp. JCD.S3660
Author(s):  
Hang Fai Kwok ◽  
Julie A. Gormley ◽  
Christopher J. Scott ◽  
James A. Johnston ◽  
Shane A. Olwill
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
False Negative ◽  
Death Receptor ◽  
Annexin V ◽  
Fas Receptor ◽  
Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

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