Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence

2006 ◽  
Vol 351 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Koji Enomoto ◽  
Tohru Nagasaki ◽  
Akira Yamauchi ◽  
Junji Onoda ◽  
Katsunori Sakai ◽  
...  
Keyword(s):  
High Throughput ◽  
Spermidine Synthase ◽  
Activity Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved

A high-throughput assay for HIV-1 integrase 3′-processing activity using time-resolved fluorescence

2012 ◽  
Vol 184 (1-2) ◽  
pp. 34-40 ◽  
Author(s):  
Ying-Shan Han ◽  
Peter Quashie ◽  
Thibault Mesplede ◽  
Hongtao Xu ◽  
Kevork Mekhssian ◽  
...  
Keyword(s):  
High Throughput ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Processing Activity ◽  
High Throughput Assay ◽  
Hiv 1

scholarly journals High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by Surrogate Ligand Competition

2003 ◽  
Vol 8 (3) ◽  
pp. 332-339 ◽  
Author(s):  
Elizabeth B. Gottlin ◽  
R. Edward Benson ◽  
Scott Conary ◽  
Brett Antonio ◽  
Kellie Duke ◽  
...  
Keyword(s):  
High Throughput ◽  
Binding Sites ◽  
Biosynthetic Pathway ◽  
Enzyme Assay ◽  
High Throughput Screen ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Key Enzyme ◽  
Nadph Oxidation

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. ( Journal of Biomolecular Screening 2003:332-339)

scholarly journals High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

2018 ◽  
Vol 23 (10) ◽  
pp. 1018-1029 ◽  
Author(s):  
Ken Katsuya ◽  
Yuji Hori ◽  
Daisuke Oikawa ◽  
Tomohisa Yamamoto ◽  
Kayo Umetani ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Mass Spectrometry Analysis ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Time Resolved Fluorescence ◽  
Ubiquitin Chain ◽  
Time Resolved ◽  
Gene Assay

The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography–mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.

P4-342 High throughput screening of β-amyloid secretion inhibitors using homogenous time-resolved fluorescence

2004 ◽  
Vol 25 ◽  
pp. S572
Author(s):  
Bruno P. Imbimbo ◽  
Hugo Albrecht ◽  
Peter Zbinden ◽  
Andrea Rizzi ◽  
Ginno Villetti ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Β Amyloid
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