High-throughput screening based on label-free detection of small molecule microarrays

2017 ◽  
Author(s):  
Chenggang Zhu ◽  
Yiyan Fei ◽  
Xiangdong Zhu
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Label Free ◽  
Label Free Detection

scholarly journals Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

2017 ◽  
Vol 22 (10) ◽  
pp. 1203-1210 ◽  
Author(s):  
Katrin Beeman ◽  
Jens Baumgärtner ◽  
Manuel Laubenheimer ◽  
Karlheinz Hergesell ◽  
Martin Hoffmann ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Maldi Ms ◽  
Kinase Assay ◽  
Label Free ◽  
Screening Process ◽  
Label Free Detection ◽  
Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for 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.

scholarly journals Expanding the Toolbox for Label-Free Enzyme Assays: A Dinuclear Platinum(II) Complex/DNA Ensemble with Switchable Near-IR Emission

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4390 ◽  
Author(s):  
Moustafa T. Gabr ◽  
F. Christopher Pigge
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Dnase I ◽  
Dna Assembly ◽  
Label Free ◽  
Serum Samples ◽  
Near Ir ◽  
Dinuclear Platinum ◽  
Label Free Detection

Switchable luminescent bioprobes whose emission can be turned on as a function of specific enzymatic activity are emerging as important tools in chemical biology. We report a promising platform for the development of label-free and continuous enzymatic assays in high-throughput mode based on the reversible solvent-induced self-assembly of a neutral dinuclear Pt(II) complex. To demonstrate the utility of this strategy, the switchable luminescence of a dinuclear Pt(II) complex was utilized in developing an experimentally simple, fast (10 min), low cost, and label-free turn-on luminescence assay for the endonuclease enzyme DNAse I. The complex displays a near-IR (NIR) aggregation-induced emission at 785 nm in aqueous solution that is completely quenched upon binding to G-quadruplex DNA from the human c-myc oncogene. Luminescence is restored upon DNA degradation elicited by exposure to DNAse I. Correlation between near-IR luminescence intensity and DNAse I concentration in human serum samples allows for fast and label-free detection of DNAse I down to 0.002 U/mL. The Pt(II) complex/DNA assembly is also effective for identification of DNAse I inhibitors, and assays can be performed in multiwell plates compatible with high-throughput screening. The combination of sensitivity, speed, convenience, and cost render this method superior to all other reported luminescence-based DNAse I assays. The versatile response of the Pt(II) complex to DNA structures promises broad potential applications in developing real-time and label-free assays for other nucleases as well as enzymes that regulate DNA topology.

scholarly journals A novel high-throughput scanning microscope for label-free detection of protein and small-molecule chemical microarrays

2008 ◽  
Vol 79 (1) ◽  
pp. 013708 ◽  
Author(s):  
Y. Y. Fei ◽  
J. P. Landry ◽  
Y. S. Sun ◽  
X. D. Zhu ◽  
J. T. Luo ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Label Free ◽  
Scanning Microscope ◽  
Label Free Detection

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

scholarly journals A small molecule UPR modulator for diabetes identified by high throughput screening

2021 ◽  
Author(s):  
Valeria Marrocco ◽  
Tuan Tran ◽  
Siying Zhu ◽  
Seung Hyuk Choi ◽  
Ana M. Gamo ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening
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