scholarly journals Identification of an Arginase II Inhibitor via RapidFire Mass Spectrometry Combined with Hydrophilic Interaction Chromatography

2018 ◽  
Vol 24 (4) ◽  
pp. 457-465 ◽  
Author(s):  
Wataru Asano ◽  
Yu Takahashi ◽  
Motoaki Kawano ◽  
Yoshiji Hantani
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Hydrophilic Interaction Chromatography ◽  
Label Free ◽  
Hydrophilic Interaction ◽  
High Concentration ◽  
Allosteric Site ◽  
Arginase Ii

Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-scale high-throughput screening to identify other structural types of Arg II inhibitors.

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery

2021 ◽  
pp. 247255522110281
Author(s):  
Roman P. Simon ◽  
Tim T. Häbe ◽  
Robert Ries ◽  
Martin Winter ◽  
Yuting Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Method Development ◽  
Accurate Determination ◽  
Adenosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Label Free

Acoustic droplet ejection (ADE)–open port interface (OPI)–mass spectrometry (MS) has recently been introduced as a versatile analytical method that combines fast and contactless acoustic sampling with sensitive and accurate electrospray ionization (ESI)–MS-based analyte detection. The potential of the technology to provide label-free measurements in subsecond analytical cycle times makes it an attractive option for high-throughput screening (HTS). Here, we report the first implementation of ADE-OPI-MS in a fully automated HTS environment, based on the example of a biochemical assay aiming at the identification of small-molecule inhibitors of the cyclic guanosine monophosphate–adenosine monophosphate (GMP-AMP) synthase (cGAS). First, we describe the optimization of the method to enable sensitive and accurate determination of enzyme activity and inhibition in miniaturized 1536-well microtiter plate format. Then we show both results from a validation single-concentration screen using a test set of 5500 compounds, and the subsequent concentration–response testing of selected hits in direct comparison with a previously established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) readout. Finally, we present the development of an in-line OPI cleaning procedure aiming to match the instrument robustness required for large-scale HTS campaigns. Overall, this work points to critical method development parameters and provides guidance for the establishment of integrated ADE-OPI-MS as HTS-compatible technology for early drug discovery.

scholarly journals MALDIViz: A Comprehensive Informatics Tool for MALDI-MS Data Visualization and Analysis

2017 ◽  
Vol 22 (10) ◽  
pp. 1246-1252 ◽  
Author(s):  
Kishore Kumar Jagadeesan ◽  
Simon Ekström
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Web Application ◽  
Low Cost ◽  
Maldi Ms ◽  
Ionization Mass ◽  
Label Free ◽  
Label Free Detection ◽  
R Shiny

Recently, mass spectrometry (MS) has emerged as an important tool for high-throughput screening (HTS) providing a direct and label-free detection method, complementing traditional fluorescent and colorimetric methodologies. Among the various MS techniques used for HTS, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides many of the characteristics required for high-throughput analyses, such as low cost, speed, and automation. However, visualization and analysis of the large datasets generated by HTS MALDI-MS can pose significant challenges, especially for multiparametric experiments. The datasets can be generated fast, and the complexity of the experimental data (e.g., screening many different sorbent phases, the sorbent mass, and the load, wash, and elution conditions) makes manual data analysis difficult. To address these challenges, a comprehensive informatics tool called MALDIViz was developed. This tool is an R-Shiny-based web application, accessible independently of the operating system and without the need to install any program locally. It has been designed to facilitate easy analysis and visualization of MALDI-MS datasets, comparison of multiplex experiments, and export of the analysis results to high-quality images.

scholarly journals Assay Development and Screening of Human DGAT1 Inhibitors with an LC/MS-Based Assay

2010 ◽  
Vol 15 (6) ◽  
pp. 695-702 ◽  
Author(s):  
Ji-Hu Zhang ◽  
Thomas P. Roddy ◽  
Pei-I Ho ◽  
Christopher R. Horvath ◽  
Chad Vickers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Biological Response ◽  
Fluorescent Labeling ◽  
Assay Development ◽  
Label Free ◽  
Chemical Library ◽  
Label Free Detection

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.

Large-scale plasmonic microarrays for label-free high-throughput screening

Lab on a Chip ◽  
2011 ◽  
Vol 11 (21) ◽  
pp. 3596 ◽  
Author(s):  
Tsung-Yao Chang ◽  
Min Huang ◽  
Ahmet Ali Yanik ◽  
Hsin-Yu Tsai ◽  
Peng Shi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Label Free

scholarly journals High-Throughput Screening by Mass Spectrometry: Comparison with the Scintillation Proximity Assay with a Focused-File Screen of AKT1/PKBα

2007 ◽  
Vol 12 (4) ◽  
pp. 473-480 ◽  
Author(s):  
Andrea K. Quercia ◽  
William A. Lamarr ◽  
Jayhyuk Myung ◽  
Can C. Özbal ◽  
James A. Landro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Label Free ◽  
False Negatives ◽  
Scintillation Proximity Assay ◽  
Therapy Target ◽  
Hit Identification ◽  
Selection Of

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire™) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBα was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited ≤ 50% inhibition in the primary screen was chosen as controls to confirm inactives. The selection of compounds is expected to identify common actives, common inactives, false positives, and false negatives. Agreement is found between HTMS and SPA in terms of primary hit identification and hit confirmation. ( Journal of Biomolecular Screening 2007:473-480)

scholarly journals A Chemoinformatics Analysis of Hit Lists Obtained from High-Throughput Affinity-Selection Screening

2005 ◽  
Vol 11 (2) ◽  
pp. 123-130 ◽  
Author(s):  
Nathan Brown ◽  
Hartmut Zehender ◽  
Kamal Azzaoui ◽  
Ansgar Schuffenhauer ◽  
Lorenz M. Mayr ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Size Exclusion ◽  
Label Free ◽  
Affinity Selection ◽  
High Performing ◽  
Exclusion Chromatography ◽  
Screening Platform ◽  
Screening Technology

The high-throughput affinity-selection screening platform SpeedScreen was recently reported by the Novartis Institutes for BioMedical Research as a homogeneous, label-free screening technology with mass-spectrometry readout. SpeedScreen relies on the screening of compound mixtures with various target proteins and uses fast size-exclusion chromatography to separate target-bound from unbound substances. After disintegration of the target-binder complex, the binder molecules are identified by their molecular masses using liquid chromatography/mass spectrometry. The authors report an analysis of the molecular properties of hits obtained with SpeedScreen on 26 targets screened within the past few years at Novartis using this technology. Affinity-based SpeedScreen is a robust high-throughput screening technology that does not accumulate frequent hitters or potential covalent binders. The hits are representative of the most commonly identified scaffold classes observed for known drugs. Validated SpeedScreen hits tend to be enriched on more lipophilic and larger-molecular-weight compounds compared to the whole library. The potential for a reduced SpeedScreen screening set to be used in case only limited protein quantities are available is evaluated. Such a reduced compound set should also maximize the coverage of the high-performing regions of the chemical property and class spaces; chemoinformatics methods including genetic algorithms and divisive K-means clustering are used for this aim.

scholarly journals Use of High-Throughput Mass Spectrometry to Reduce False Positives in Protease uHTS Screens

2014 ◽  
Vol 20 (2) ◽  
pp. 212-222 ◽  
Author(s):  
Gregory C. Adam ◽  
Juncai Meng ◽  
Joseph M. Rizzo ◽  
Adam Amoss ◽  
Jeffrey W. Lusen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Dyes ◽  
False Positives ◽  
Indirect Detection ◽  
Detection Methods ◽  
Label Free ◽  
Fluorescent Compound ◽  
Time Resolved

As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)–MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5–7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.

scholarly journals The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond

2015 ◽  
Vol 21 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Carl Haslam ◽  
John Hellicar ◽  
Adrian Dunn ◽  
Arne Fuetterer ◽  
Neil Hardy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Direct Detection ◽  
Sample Volume ◽  
Model Systems ◽  
Analysis Time ◽  
Label Free ◽  
Maldi Tof

Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction–based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography–coupled MS, an analysis time of 8–10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays.

Sign in / Sign up

Export Citation Format

Share Document