scholarly journals High-throughput screening and characterization of xylose-utilizing, ethanol-tolerant thermophilic bacteria for bioethanol production

2011 ◽  
Vol 110 (6) ◽  
pp. 1584-1591 ◽  
Author(s):  
X. Qi ◽  
Y. Zhang ◽  
R. Tu ◽  
Y. Lin ◽  
X. Li ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Thermophilic Bacteria ◽  
Bioethanol Production

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins

2019 ◽  
Vol 60 (5) ◽  
pp. 1082-1097 ◽  
Author(s):  
Panneerselvam Krishnamurthy ◽  
Yukiko Fujisawa ◽  
Yuya Takahashi ◽  
Hanako Abe ◽  
Kentaro Yamane ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Density ◽  
Biosynthesis Pathway ◽  
Mutant Library ◽  
Triterpenoid Saponins

scholarly journals Discovery of Selective Cannabinoid CB2 Receptor Agonists by High-Throughput Screening

2017 ◽  
Vol 23 (4) ◽  
pp. 375-383 ◽  
Author(s):  
Lisa M. Ogawa ◽  
Neil T. Burford ◽  
Yu-Hsien Liao ◽  
Caitlin E. Scott ◽  
Ashley M. Hine ◽  
...  
Keyword(s):  
Side Effects ◽  
High Throughput ◽  
High Throughput Screening ◽  
Immune Modulation ◽  
Cannabinoid Receptors ◽  
Endocannabinoid System ◽  
Allosteric Modulators ◽  
Peripheral Tissues ◽  
Selective Agonists

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.

scholarly journals Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

2004 ◽  
Vol 383 (3) ◽  
pp. 551-559 ◽  
Author(s):  
Sheraz GUL ◽  
Richard BROWN ◽  
Earl MAY ◽  
Marie MAZZULLA ◽  
Martin G. SMYTH ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dna Ligase ◽  
Protection Assay ◽  
Dna Ligases ◽  
Enzyme Substrate ◽  
Ic50 Values ◽  
Acridinium Ester

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Characterization of an in vitro placental transfer assay for high-throughput screening

2017 ◽  
Vol 280 ◽  
pp. S263-S264
Author(s):  
Caroline Gomes ◽  
Barbara Birk ◽  
Eric Fabian ◽  
Julian Doersam ◽  
Catharina Wilhelmina van Dongen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Placental Transfer

scholarly journals Applications of Biophysics in High-Throughput Screening Hit Validation

2014 ◽  
Vol 19 (5) ◽  
pp. 707-714 ◽  
Author(s):  
Christine Clougherty Genick ◽  
Danielle Barlier ◽  
Dominique Monna ◽  
Reto Brunner ◽  
Céline Bé ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Lessons Learned ◽  
Label Free ◽  
Lead Discovery ◽  
Binding Interaction ◽  
Differential Scanning Fluorimetry ◽  
Types Of Information ◽  
Hit Validation

For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include dynamic light scattering, turbidometry, resonance waveguide, surface plasmon resonance, differential scanning fluorimetry, mass spectrometry, and others. Each technology can provide different types of information to enable the characterization of the binding interaction. Thus, these technologies can be incorporated in a hit-validation strategy not only according to the profile of chemical matter that is desired by the medicinal chemists, but also in a manner that is in agreement with the target protein’s amenability to the screening format. Here, we present the results of screening strategies using biophysics with the objective to evaluate the approaches, discuss the advantages and challenges, and summarize the benefits in reference to lead discovery. In summary, the biophysics screens presented here demonstrated various hit rates from a list of ~2000 preselected, IC50-validated hits from HTS (an IC50 is the inhibitor concentration at which 50% inhibition of activity is observed). There are several lessons learned from these biophysical screens, which will be discussed in this article.

scholarly journals Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin

2020 ◽  
Vol 3 (4) ◽  
pp. 81
Author(s):  
Caitlin N. Suire ◽  
Monica K. Brizuela ◽  
Malcolm A. Leissring
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Peptide Hormone ◽  
Proteolytic Degradation ◽  
Insulin Degrading Enzyme ◽  
Islet Amyloid ◽  
Health And Disease ◽  
Pancreatic Peptide

Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications.

Sign in / Sign up

Export Citation Format

Share Document