scholarly journals A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

2011 ◽  
Vol 16 (5) ◽  
pp. 486-493 ◽  
Author(s):  
Adam B. Shapiro ◽  
Ann E. Eakin ◽  
Grant K. Walkup ◽  
Olga Rivin
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Fluorescence Resonance

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

scholarly journals Comparison of Miniaturized Time-Resolved Fluorescence Resonance Energy Transfer and Enzyme-Coupled Luciferase High-Throughput Screening Assays to Discover Inhibitors of Rho-Kinase II (ROCK-II)

2007 ◽  
Vol 13 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Thomas Schröter ◽  
Dmitriy Minond ◽  
Amiee Weiser ◽  
Chinh Dao ◽  
Jeff Habel ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Rho Kinase ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 ± 27 nM and 336 ± 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 ± 7.8 nM and 150 ± 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z′ values during the HTS campaign (0.84 ± 0.03; 0.72 ± 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available. ( Journal of Biomolecular Screening 2008:17-28)

scholarly journals Homogeneous TR-FRET High-Throughput Screening Assay for Calcium-Dependent Multimerization of Sorcin

2007 ◽  
Vol 12 (6) ◽  
pp. 842-848 ◽  
Author(s):  
Heidi Appelblom ◽  
Jussi Nurmi ◽  
Tero Soukka ◽  
Michael Pasternack ◽  
Kai E. Penttilä ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Calcium Dependent ◽  
High Throughput Screening Assay ◽  
Fluorescence Resonance

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions. ( Journal of Biomolecular Screening 2007:842-848)

scholarly journals Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer

2000 ◽  
Vol 5 (5) ◽  
pp. 319-328 ◽  
Author(s):  
Michael D. Boisclair ◽  
Christopher McClure ◽  
Serene Josiah ◽  
Susan Glass ◽  
Steve Bottomley ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Wave Length ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Fluorescence Resonance ◽  
Lower Ratio

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu3+, and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu3+ and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wave-length of APC, almost entirely free of nonspecific fluorescence from Eu3+ and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 pxg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.

scholarly journals High-Throughput Screening of Low Molecular Weight NS3-NS4A Protease Inhibitors Using a Fluorescence Resonance Energy Transfer Substrate

2005 ◽  
Vol 16 (6) ◽  
pp. 385-392 ◽  
Author(s):  
Kenji Sudo ◽  
Kayo Yamaji ◽  
Kouich Kawamura ◽  
Tomoko Nishijima ◽  
Naoko Kojima ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Compound Libraries ◽  
Fluorescence Resonance ◽  
Potent Inhibitory Activity

Hepatitis C virus (HCV) NS3-NS4A protease is an attractive target for anti-HCV agents because of its important role in replication. An optimized fluorescence resonance energy transfer (FRET) substrate for NS3-NS4A protease, based on the sequence of the NS5A-5B cleavage site, was designed and synthesized. High-throughput screening of in-house compound libraries was performed using a FRET substrate FS10 (MOCAc-DKIVPC-SMSYK-Dnp) and MBP-NS3-NS4A fusion protein. Several hit compounds were found, including YZ-9577 (2-oxido-1,2,5-oxadiazole-3,4-diyl) bis (phenylmethanone) with potent inhibitory activity (IC50=1.6 μM) and good selectivity against other human serine proteases.

scholarly journals A High-Throughput, Homogeneous, Fluorescence Resonance Energy Transfer-Based Assay for Phospho-N-acetylmuramoyl-pentapeptide Translocase (MraY)

2012 ◽  
Vol 17 (5) ◽  
pp. 662-672 ◽  
Author(s):  
Adam B. Shapiro ◽  
Haris Jahić ◽  
Ning Gao ◽  
Laurel Hajec ◽  
Olga Rivin
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Integral Membrane Protein ◽  
Antibacterial Drugs ◽  
Peptidoglycan Biosynthesis ◽  
Fluorescence Resonance

Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP- N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product–derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer–based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.

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