scholarly journals High Throughput Screen for Inhibitors of Bacterial DNA Topoisomerase I Using the Scintillation Proximity Assay

1996 ◽  
Vol 1 (3) ◽  
pp. 135-143 ◽  
Author(s):  
Claude G. Lerner ◽  
Anne Y. Chiang Saiki ◽  
A. Craig Mackinnon ◽  
Xiaoling Xuei
Keyword(s):  
Fusion Protein ◽  
High Throughput ◽  
Topoisomerase I ◽  
Immobilized Enzyme ◽  
Single Step ◽  
High Throughput Screen ◽  
Dna Topoisomerase ◽  
Scintillation Proximity Assay ◽  
E Coli

We report the development of a rapid method to detect binding of supercoiled DNA to Escherichia coli topoisomerase I using the scintillation proximity assay (SPA). Streptavidin-SPA beads were coated with biotinylated topoisomerase I produced in vivo as a chimeric fusion protein. The hybrid biotinyl-fusion protein was overproduced in E. coli and purified in a single step by monomeric avidin affinity chromatography. The assay signal originates from both covalent and noncovalent binding of [3H]DNA to the SPA bead-immobilized enzyme. About 20-30% of the total [3H]DNA bound to the bead-immobilized enzyme remains associated with the bead in the presence of 0.5% SDS. The residual signal arises from the trapping of covalent [3H]DNA-enzyme complexes. The assay was employed in a high throughput screen that identified two general classes of topoisomerase inhibitors: agents that (1) inhibit DNA binding or (2) stabilize a covalent enzyme-DNA intermediate.

scholarly journals Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting

The Analyst ◽  
2018 ◽  
Vol 143 (19) ◽  
pp. 4747-4755 ◽  
Author(s):  
Joanna C. Sadler ◽  
Andrew Currin ◽  
Douglas B. Kell
Keyword(s):  
Monoamine Oxidase ◽  
Directed Evolution ◽  
High Throughput ◽  
Cell Sorting ◽  
Oxidase Activity ◽  
Functional Enrichment ◽  
High Throughput Screen ◽  
Fluorescence Activated Cell Sorting ◽  
E Coli

A novel ultra-high throughput screen forin vivodetection of oxidase activity inE. colicells and its application to directed evolution.

scholarly journals Escherichia coli DNA Topoisomerase I Copurifies with Tn5 Transposase, and Tn5 Transposase Inhibits Topoisomerase I

1999 ◽  
Vol 181 (10) ◽  
pp. 3185-3192 ◽  
Author(s):  
Hesna Yigit ◽  
William S. Reznikoff
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Topoisomerase I ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Dna Relaxation ◽  
Tn5 Transposase ◽  
Derivatives Of

ABSTRACT Tn5 transposase (Tnp) overproduction is lethal toEscherichia coli. Genetic evidence suggested that this killing involves titration of E. coli topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn5 Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Δ37Tnp and Δ55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Δ37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a topA mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp.

ISOLATION OF AN E. COLI DNA TOPOISOMERASE I MUTANT

1980 ◽  
pp. 833-837 ◽  
Author(s):  
Rolf Sternglanz ◽  
Stephen DiNardo ◽  
James C. Wang ◽  
Y. Nishimura ◽  
Y. Hirota
Keyword(s):  
Topoisomerase I ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
E Coli

scholarly journals A Duplexed High-Throughput Screen to Identify Allosteric Modulators of the Glucagon-Like Peptide 1 and Glucagon Receptors

2014 ◽  
Vol 19 (6) ◽  
pp. 847-858 ◽  
Author(s):  
Lindsey C. Morris ◽  
Emily L. Days ◽  
Maxine Turney ◽  
Dehui Mi ◽  
Craig W. Lindsley ◽  
...  
Keyword(s):  
High Throughput ◽  
Allosteric Modulators ◽  
High Throughput Screen ◽  
Glucagon Receptor ◽  
Three Phase ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Orally Bioavailable ◽  
Glucagon Receptors

Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.

scholarly journals Nucleosomes represent a physical barrier for cleavage activity of DNA topoisomerase I in vivo

2008 ◽  
Vol 409 (3) ◽  
pp. 651-656 ◽  
Author(s):  
Francesca Di Felice ◽  
Francesco Chiani ◽  
Giorgio Camilloni
Keyword(s):  
Topoisomerase I ◽  
Basic Question ◽  
Dna Topoisomerase I ◽  
Physical Barrier ◽  
Dna Topoisomerase ◽  
Histone Octamer ◽  
Cleavage Activity ◽  
Cellular Dna

DNA topoisomerase I together with the other cellular DNA topoisomerases releases the torsional stress from DNA caused by processes such as replication, transcription and recombination. Despite the well-defined knowledge of its mechanism of action, DNA topoisomerase I in vivo activity has been only partially characterized. In fact the basic question concerning the capability of the enzyme to cleave and rejoin DNA wrapped around a histone octamer remains still unanswered. By studying both in vivo and in vitro the cleavage activity of DNA topoisomerase I in the presence of camptothecin on a repeated trinucleotide sequence, (TTA)35, lying in chromosome XIII of Saccharomyces cerevisiae, we can conclude that nucleosomes represent a physical barrier for the enzyme activity.

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

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