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 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 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 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 Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

2021 ◽  
Vol 22 (9) ◽  
pp. 4417
Author(s):  
Lester J Lambert ◽  
Stefan Grotegut ◽  
Maria Celeridad ◽  
Palak Gosalia ◽  
Laurent JS De Backer ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Synaptic Function ◽  
Label Free ◽  
Protein Tyrosine

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing

APOPTOSIS ◽  
2014 ◽  
Vol 19 (9) ◽  
pp. 1411-1418 ◽  
Author(s):  
Obaid Aftab ◽  
Madiha Nazir ◽  
Mårten Fryknäs ◽  
Ulf Hammerling ◽  
Rolf Larsson ◽  
...  
Keyword(s):  
Signal Processing ◽  
High Throughput ◽  
High Throughput Screening ◽  
Time Lapse ◽  
Label Free ◽  
Time Lapse Microscopy

Photonic crystals: A platform for label-free and enhanced fluorescence biomolecular and cellular assays

2008 ◽  
Vol 1133 ◽  
Author(s):  
Brian T. Cunningham ◽  
Leo Chan ◽  
Patrick C. Mathias ◽  
Nikhil Ganesh ◽  
Sherine George ◽  
...  
Keyword(s):  
Photonic Crystal ◽  
Photonic Crystals ◽  
High Throughput Screening ◽  
High Sensitivity ◽  
Excitation Wavelength ◽  
Label Free ◽  
Crystal Surfaces ◽  
Enhanced Fluorescence ◽  
Preferred Direction ◽  
Label Free Detection

Abstract Photonic crystal surfaces represent a class of resonant optical structures that are capable of supporting high intensity electromagnetic standing waves with near-field and far-field properties that can be exploited for high sensitivity detection of biomolecules and cells. While modulation of the resonant wavelength of a photonic crystal by the dielectric permittivity of adsorbed biomaterials enables label-free detection, the resonance can also be tuned to coincide with the excitation wavelength of common fluorescent tags - including organic molecules and semiconductor quantum dots. Photonic crystals are also capable of efficiently channeling fluorescent emission into a preferred direction for enhanced extraction efficiency. Photonic crystals can be designed to support multiple resonant modes that can perform label free detection, enhanced fluorescence excitation, and enhanced fluorescence extraction simultaneously on the same device. Because photonic crystal surfaces may be inexpensively produced over large surface areas by nanoreplica molding processes, they can be incorporated into disposable labware for applications such as pharmaceutical high throughput screening. In this talk, the optical properties of surface photonic crystals will be reviewed and several applications will be described, including results from screening a 200,000-member chemical compound library for inhibitors of protein-DNA interactions, gene expression microarrays, and high sensitivity of protein biomarkers.

scholarly journals Computational Method for Simulating Thermoset Polymer Curing and Prediction of Thermophysical Properties

2020 ◽  
Author(s):  
Jeffrey Sanders ◽  
Carla E. Estridge ◽  
Matthew B Jackson ◽  
Thomas JL Mustard ◽  
Samuel J. Tucker ◽  
...  
Keyword(s):  
High Throughput ◽  
Thermophysical Properties ◽  
High Throughput Screening ◽  
Simulation Analysis ◽  
Low Cost ◽  
Polymer Network ◽  
Thermomechanical Properties ◽  
Computational Method ◽  
Cross Linking ◽  
Automated Simulation

Thermoset polymers are an area of intense research due to their low cost, ease of processing, environmental resistance, and unique physical properties. The favorable properties of this class of polymers have many applications in aerospace, automotive, marine, and sports equipment industries. Molecular simulations of thermosets are frequently used to model formation of the polymer network, and to predict the thermomechanical properties. These simulations usually require custom algorithms that are not easily accessible to non-experts and not suited for high throughput screening. To address these issues, we have developed a robust cross-linking algorithm that can incorporate different types of chemistries and leverage GPU-enabled molecular dynamics simulations. Automated simulation analysis tools for cross-linking simulations are also presented. Using four well known epoxy/amine formulations as a foundational case study and benzoxazine as an example of how additional chemistries can be modeled, we demonstrate the power of the algorithm to accurately predict curing and thermophysical properties. These tools are able to streamline the thermoset simulation process, opening up avenues to in-silico high throughput screening for advanced material development.

scholarly journals Computational High-Throughput Screening of Polymeric Photocatalysts: Exploring the Effect of Composition, Sequence Isomerism and Conformational Degrees of Freedom

2018 ◽  
Author(s):  
isabelle Heath-Apostolopoulos ◽  
Liam Wilbraham ◽  
Martijn Zwijnenburg
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Degrees Of Freedom ◽  
Chemical Space ◽  
Low Cost ◽  
Computational Screening ◽  
Computational Workflow ◽  
Conformational Degrees Of Freedom

We discuss a low-cost computational workflow for the high-throughput screening of polymeric photocatalysts and demonstrate its utility by applying it to a number of challenging problems that would be difficult to tackle otherwise. Specifically we show how having access to a low-cost method allows one to screen a vast chemical space, as well as to probe the effects of conformational degrees of freedom and sequence isomerism. Finally, we discuss both the opportunities of computational screening in the search for polymer photocatalysts, as well as the biggest challenges.

Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening

2021 ◽  
pp. 247263032110478
Author(s):  
Nicolás M. Morato ◽  
MyPhuong T. Le ◽  
Dylan T. Holden ◽  
R. Graham Cooks
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Desorption Electrospray Ionization ◽  
Ambient Ionization ◽  
Machine Learning Algorithms ◽  
Small Scale ◽  
Label Free ◽  
Minimal Sample ◽  
Secondary Droplets ◽  
Automated Platform

The Purdue Make It system is a unique automated platform capable of small-scale in situ synthesis, screening small-molecule reactions, and performing direct label-free bioassays. The platform is based on desorption electrospray ionization (DESI), an ambient ionization method that allows for minimal sample workup and is capable of accelerating reactions in secondary droplets, thus conferring unique advantages compared with other high-throughput screening technologies. By combining DESI with liquid handling robotics, the system achieves throughputs of more than 1 sample/s, handling up to 6144 samples in a single run. As little as 100 fmol/spot of analyte is required to perform both initial analysis by mass spectrometry (MS) and further MSn structural characterization. The data obtained are processed using custom software so that results are easily visualized as interactive heatmaps of reaction plates based on the peak intensities of m/ z values of interest. In this paper, we review the system’s capabilities as described in previous publications and demonstrate its utilization in two new high-throughput campaigns: (1) the screening of 188 unique combinatorial reactions (24 reaction types, 188 unique reaction mixtures) to determine reactivity trends and (2) label-free studies of the nicotinamide N-methyltransferase enzyme directly from the bioassay buffer. The system’s versatility holds promise for several future directions, including the collection of secondary droplets containing the products from successful reaction screening measurements, the development of machine learning algorithms using data collected from compound library screening, and the adaption of a variety of relevant bioassays to high-throughput MS.

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