scholarly journals High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine Decarboxylase

2007 ◽  
Vol 12 (5) ◽  
pp. 628-634 ◽  
Author(s):  
Chris D. Forbes ◽  
Joshuaine G. Toth ◽  
Can C. Özbal ◽  
William A. Lamarr ◽  
Jennifer A. Pendleton ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Catalytic Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Lipid Synthesis ◽  
High Quality ◽  
Compound Libraries ◽  
Z Value ◽  
Large Compound ◽  
Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFire™ platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z′ value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target. ( Journal of Biomolecular Screening 2007:628-634)

Solution-Based Indirect Affinity Selection Mass Spectrometry—A General Tool For High-Throughput Screening Of Pharmaceutical Compound Libraries

2014 ◽  
Vol 86 (15) ◽  
pp. 7413-7420 ◽  
Author(s):  
Thomas N. O’Connell ◽  
Jason Ramsay ◽  
Steven F. Rieth ◽  
Michael J. Shapiro ◽  
Justin G. Stroh
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Affinity Selection ◽  
Pharmaceutical Compound ◽  
Compound Libraries ◽  
General Tool

scholarly journals Screening for Antibacterial Inhibitors of the UDP-3-O-(R-3-Hydroxymyristoyl)- N-Acetylglucosamine Deacetylase (LpxC) Using a High-Throughput Mass Spectrometry Assay

2009 ◽  
Vol 15 (1) ◽  
pp. 52-61 ◽  
Author(s):  
Erik F. Langsdorf ◽  
Asra Malikzay ◽  
William A. Lamarr ◽  
Dayna Daubaras ◽  
Cynthia Kravec ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Screening Tool ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)- Nacetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire™ mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 µg/mL). The results show that mass spectrometry—based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

scholarly journals Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

1999 ◽  
Vol 4 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Ingrid Schmid ◽  
Isabel Sattler ◽  
Susanne Grabley ◽  
Ralf Thiericke
Keyword(s):  
Drug Discovery ◽  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Structural Diversity ◽  
High Quality ◽  
Synthetic Compound ◽  
Natural Origin ◽  
Compound Libraries

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

2021 ◽  
pp. 247255522110232
Author(s):  
Michael D. Scholle ◽  
Doug McLaughlin ◽  
Zachary A. Gurard-Levin
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Rhinovirus ◽  
Binding Potential ◽  
Affinity Selection ◽  
Response Curves ◽  
Desorption Ionization ◽  
Self Assembled

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

scholarly journals Evaluation of ambient mass spectrometry tools for assessing inherent postharvest pepper quality

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Tyler J. Mason ◽  
Harmonie M. Bettenhausen ◽  
Jacqueline M. Chaparro ◽  
Mark E. Uchanski ◽  
Jessica E. Prenni
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Direct Analysis ◽  
Quality Characteristics ◽  
Ambient Mass Spectrometry ◽  
Postharvest Quality ◽  
Ionization Mass ◽  
Ambient Conditions ◽  
Assess Quality

AbstractHorticulturists are interested in evaluating how cultivar, environment, or production system inputs can affect postharvest quality. Ambient mass spectrometry approaches enable analysis of minimally processed samples under ambient conditions and offer an attractive high-throughput alternative for assessing quality characteristics in plant products. Here, we evaluate direct analysis in real time (DART-MS) mass spectrometry and rapid evaporative ionization-mass spectrometry (REIMS) to assess quality characteristics in various pepper (Capsicum annuum L.) cultivars. DART-MS exhibited the ability to discriminate between pod colors and pungency based on chemical fingerprints, while REIMS could distinguish pepper market class (e.g., bell, lunchbox, and popper). Furthermore, DART-MS analysis resulted in the putative detection of important bioactive compounds in human diet such as vitamin C, p-coumaric acid, and capsaicin. The results of this study demonstrate the potential for these approaches as accessible and reliable tools for high throughput screening of pepper quality.

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