scholarly journals High-Throughput Screening Fluorescence Polarization Assay for Tumor-Specific Hsp90

2007 ◽  
Vol 12 (7) ◽  
pp. 915-924 ◽  
Author(s):  
Yuhong Du ◽  
Kamalika Moulick ◽  
Anna Rodina ◽  
Julia Aguirre ◽  
Sara Felts ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Small Cell Lung Carcinoma ◽  
Heat Shock Protein 90 ◽  
Cost Effective ◽  
Cell Lung Carcinoma ◽  
Lung Carcinoma Cells ◽  
Fluorescence Polarization Assay ◽  
Molecular Libraries

Heat shock protein 90 (Hsp90) is a molecular chaperone that has emerged as an important target in cancer and several other diseases, such as neurodegenerative diseases, nerve injuries, inflammation, and infection. Discovery of novel agents that inhibit Hsp90 and have druglike properties is therefore a major focus in several academic and industrial laboratories. In this study, the authors describe the development and optimization in a 384-well format of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization, which measures competitive binding of red-shifted fluorescently labeled geldanamycin (GM-cy3B) to Hsp90 found in the NCI-N417 small-cell lung carcinoma cells. The authors demonstrate that GMcy3B binds with high affinity and specificity to cellular Hsp90. The assay results in excellent signal-to-noise ratios (>10) and Z′ values (>0.75) at tracer concentrations greater than 4 nM and 1 µg/well of total NCI-N417 protein, indicating a robust assay. It also equilibrates after 5 h of incubation at room temperature and remains stable for up to 24 h. Furthermore, it is a simple mix-and-read format that is cost-effective and uses only low amounts of fluorophore and cell lysates. A study using more than 15,000 compounds from the National Institutes of Health Molecular Libraries Screening Center Network was performed to validate its performance in a high-throughput screening format. ( Journal of Biomolecular Screening 2007:915-924)

scholarly journals High-Throughput Fluorescence Polarization Assay for Chemical Library Screening against Anti-Apoptotic Bcl-2 Family Member Bfl-1

2011 ◽  
Vol 17 (3) ◽  
pp. 350-360 ◽  
Author(s):  
Dayong Zhai ◽  
Paulo Godoi ◽  
Eduard Sergienko ◽  
Russell Dahl ◽  
Xochella Chan ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Response Analysis ◽  
Chemical Library ◽  
Time Resolved ◽  
Chemical Inhibitors ◽  
Fluorescence Polarization Assay

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

scholarly journals Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

2012 ◽  
Vol 17 (8) ◽  
pp. 1030-1040 ◽  
Author(s):  
Glenda Alquicer ◽  
David Sedlák ◽  
Youngjoo Byun ◽  
Jiří Pavlíček ◽  
Marigo Stathis ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Fluorescence Probe ◽  
Glutamate Carboxypeptidase Ii ◽  
Neurologic Disorders ◽  
Small Molecule Libraries ◽  
Fluorescence Polarization Assay ◽  
Covalently Linked ◽  
Glutamate Carboxypeptidase

Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000–compound library. The novel assay presented here is robust, highly reproducible (Z′ = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.

scholarly journals Development of a Fluorescence Polarization Assay for the Molecular Chaperone Hsp90

2004 ◽  
Vol 9 (5) ◽  
pp. 375-381 ◽  
Author(s):  
Joungnam Kim ◽  
Sara Felts ◽  
Laura Llauger ◽  
Huazhong He ◽  
Henri Huezo ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Cancer Therapeutics ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitors ◽  
Amino Terminal ◽  
High Affinity ◽  
Measured Polarization ◽  
Fluorescence Polarization Assay

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90α (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90α with high affinity. Even at low Hsp90α concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z′ values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90α with EC50s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.

scholarly journals A High-Throughput Fluorescence Polarization Assay for Inhibitors of Gyrase B

2011 ◽  
Vol 16 (2) ◽  
pp. 230-238 ◽  
Author(s):  
Bryan T. Glaser ◽  
Jeremiah P. Malerich ◽  
Sarah J. Duellman ◽  
Julie Fong ◽  
Christopher Hutson ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Antibacterial Drug ◽  
Gyrase A ◽  
Fluorescence Polarization Assay ◽  
Well Assay ◽  
Antibacterial Drug Target ◽  
Texas Red ◽  
Kinetics Of

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A2B2 heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin–Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration–approved compounds. The assay performed with an average Z′ factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

scholarly journals Development of a Plasmepsin II Fluorescence Polarization Assay Suitable for High Throughput Antimalarial Drug Discovery

2002 ◽  
Vol 7 (4) ◽  
pp. 367-371 ◽  
Author(s):  
Horst Flotow ◽  
Chung-Yan Leong ◽  
Antony D. Buss
Keyword(s):  
Natural Products ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rapid Screening ◽  
Screening Assay ◽  
Plasmepsin Ii ◽  
Fluorescence Polarization Assay

Despite decades of research, malaria remains the world's most deadly parasitic disease. New treatments with novel mechanisms of action are urgently needed. Plasmepsin II is an aspartyl protease that has been validated as an antimalarial therapeutic target enzyme. Although natural products form the basis of most modern antimalarial drugs, no systematic high-throughput screening has been reported against this target. We have designed an effective strategy for carrying out high-throughput screening of an extensive library of natural products that uses a fluorescence resonance energy transfer primary screening assay in tandem with a fluorescence polarization assay. This strategy allows rapid screening of the library coupled with effective discrimination and elimination of false-positive samples and selection of true hits for chemical isolation of inhibitors of plasmepsin II.

scholarly journals High-Throughput Fluorescence Polarization Method for Identification of FKBP12 Ligands

2002 ◽  
Vol 7 (6) ◽  
pp. 526-530 ◽  
Author(s):  
S. Bollini ◽  
J. J. Herbst ◽  
G. T. Gaughan ◽  
T. A. Verdoorn ◽  
J. Ditta ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Metal Ion ◽  
Gel Filtration ◽  
Immunosuppressive Drug ◽  
Fluorescence Polarization Assay ◽  
High Throughput Screens

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His6-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with Ki values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

scholarly journals Development of a Novel Fluorescence Polarization–Based Assay for Studying the β-Catenin/Tcf4 Interaction

2011 ◽  
Vol 17 (4) ◽  
pp. 530-534 ◽  
Author(s):  
Wang Tian ◽  
Yan Xu ◽  
Xiaofeng Han ◽  
Srinivas Duggineni ◽  
Xiaobing Han ◽  
...  
Keyword(s):  
T Cell ◽  
Signaling Pathway ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Tumor Therapy ◽  
Fluorescein Isothiocyanate ◽  
T Cell Factor ◽  
Wide Range ◽  
Fluorescence Polarization Assay

Aberrant activation of the Wnt/β-catenin signaling pathway is associated with a wide range of human cancers. The interaction of β-catenin with T cell factor (Tcf) is a key step in activation of proliferative genes in this pathway. Interruption of this interaction would be a valuable strategy as a tumor therapy. In this study, we developed a novel fluorescein isothiocyanate (FITC)–labeled Tcf4-derived probe for identification of inhibitors of the β-catenin/Tcf4 interaction using a fluorescence polarization assay. This assay shows high potential for use in high-throughput screening for the discovery of inhibitors of the β-catenin/Tcf4 interaction.

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