scholarly journals Detecting Drug-Target Binding in Cells using Fluorescence Activated Cell Sorting Coupled with Mass Spectrometry Analysis

2017 ◽  
Author(s):  
Kris Wilson ◽  
Scott P Webster ◽  
John P Iredale ◽  
Xiaozhong Zheng ◽  
Natalie Z Homer ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Targets ◽  
Mass Spectrometry Analysis ◽  
Protein Targets ◽  
Target Proteins ◽  
Spectrometry Analysis ◽  
Cellular Permeability ◽  
Target Binding ◽  
Hit Validation

AbstractThe assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity - the Toxicity-Affinity-Permeability-Selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

scholarly journals Phytochemicals of Minthostachys diffusa Epling and Their Health-Promoting Bioactivities

Foods ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 144
Author(s):  
Immacolata Faraone ◽  
Daniela Russo ◽  
Lucia Chiummiento ◽  
Eloy Fernandez ◽  
Alka Choudhary ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Radical Scavenging ◽  
Ethyl Acetate Fraction ◽  
Mass Spectrometry Analysis ◽  
Antioxidant Power ◽  
Spectrometry Analysis ◽  
In Silico Docking ◽  
Aerial Parts ◽  
Health Promoting

The genus Minthostachys belonging to the Lamiaceae family, and is an important South American mint genus used commonly in folk medicine as an aroma in cooking. The phytochemical-rich samples of the aerial parts of Minthostachys diffusa Epling. were tested for pharmacological and health-promoting bioactivities using in vitro chemical and enzymatic assays. A range of radical scavenging activities of the samples against biological radicals such as nitric oxide and superoxide anion and against synthetic 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, the ferric reducing antioxidant power and the lipid peroxidation inhibition were determined and ranked using the ‘relative antioxidant capacity index’ (RACI). The ethyl acetate fraction showed the highest RACI of +1.12. Analysis of the various fractions’ inhibitory ability against enzymes involved in diabetes (α-amylase and α-glucosidase), and against enzymes associated with Parkinson’s or Alzheimer’s diseases (acetylcholinesterase and butyrylcholinesterase) also suggested that the ethyl acetate fraction was the most active. Liquid chromatography–tandem mass spectrometry analysis of the ethyl acetate fraction showed more than 30 polyphenolic compounds, including triterpenes. The inhibitory cholinesterase effects of the triterpenes identified from M. diffusa were further analysed by in silico docking of these compounds into 3D-structures of acetylcholinesterase and butyrylcholinesterase. This is the first study on pharmacological activities and phytochemical profiling of the aerial parts of M. diffusa, showing that this plant, normally used as food in South America, is also rich in health-promoting phytochemicals.

scholarly journals A Comparative Study of the Antioxidative Effects of Helichrysum italicum and Helichrysum arenarium Infusions

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 380
Author(s):  
Katja Kramberger ◽  
Zala Jenko Pražnikar ◽  
Alenka Baruca Arbeiter ◽  
Ana Petelin ◽  
Dunja Bandelj ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
High Performance ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Morphological Type ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Spectrometry Analysis ◽  
Stress Related Genes

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

Monitoring Drug-Target Interactions through Target Engagement-Mediated Amplification on Arrays and in situ

2021 ◽  
Author(s):  
Rasel Al-Amin ◽  
Lars Johansson ◽  
Eldar Abdurakhmanov ◽  
Nils Landegren ◽  
Liza Löf ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Clinical Samples ◽  
Target Engagement ◽  
Rolling Circle Replication ◽  
Target Proteins ◽  
Dna Conjugates

Abstract Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to a panel of fixed adherent cells agreed with expectations from expression profiles of the cells. These findings were corroborated by competition experiments using kinase inhibitors with overlapping and non-overlapping target specificities, and translated to pathology tissue sections. We also introduce a proximity ligation variant of TEMA in which these drug-DNA conjugates are combined with antibody-DNA conjugates to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and tissues.

scholarly journals Phytotoxicity of Essential Oils on Selected Weeds: Potential Hazard on Food Crops

Plants ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 79 ◽  
Author(s):  
María Ibáñez ◽  
María Blázquez
Keyword(s):  
Seed Germination ◽  
Essential Oil ◽  
Essential Oils ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Food Crops ◽  
Potential Hazard ◽  
Spectrometry Analysis

The chemical composition of winter savory, peppermint, and anise essential oils, and in vitro and in vivo phytotoxic activity against weeds (Portulaca oleracea, Lolium multiflorum, and Echinochloa crus-galli) and food crops (maize, rice, and tomato), have been studied. Sixty-four compounds accounting for between 97.67–99.66% of the total essential oils were identified by Gas Chromatography-Mass Spectrometry analysis. Winter savory with carvacrol (43.34%) and thymol (23.20%) as the main compounds produced a total inhibitory effect against the seed germination of tested weed. Menthol (48.23%), menthone (23.33%), and iso-menthone (16.33%) from peppermint only showed total seed germination inhibition on L. multiflorum, whereas no significant effects were observed with trans-anethole (99.46%) from anise at all concentrations (0.125–1 µL/mL). Low doses of peppermint essential oil could be used as a sustainable alternative to synthetic agrochemicals to control L. multiflorum. The results corroborate that in vivo assays with a commercial emulsifiable concentrate need higher doses of the essential oils to reproduce previous in vitro trials. The higher in vivo phytotoxicity of winter savory essential oil constitutes an eco-friendly and less pernicious alternative to weed control. It is possible to achieve a greater in vivo phytotoxicity if less active essential oil like peppermint is included with other active excipients.

scholarly journals Data on peptides identified by mass spectrometry analysis of in vitro DYRK1A-mediated phosphorylation sites on GLI1

Data in Brief ◽  
2017 ◽  
Vol 15 ◽  
pp. 577-583 ◽  
Author(s):  
Ben K. Ehe ◽  
David R. Lamson ◽  
Michael Tarpley ◽  
Rob U. Onyenwoke ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis

scholarly journals Deubiquitination and Stabilization of PD-L1 by USP21

2021 ◽  
Author(s):  
Shuying Yang ◽  
Youqian Wu ◽  
Huanhuan Yan ◽  
Bing Shan ◽  
Dongheng Zhou ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Lung Squamous Cell Carcinoma ◽  
Mass Spectrometry Analysis ◽  
Polyubiquitin Chains ◽  
Spectrometry Analysis ◽  
Protein Levels ◽  
Programmed Death ◽  
Programmed Death Protein 1 ◽  
Novel Strategy

Abstract Background: The immunotherapy for different types of cancers that targeting programmed death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) has highlighted the importance of suppressing specific T cell responses. Recently, several studies have shown that the expression level of PD-L1 in tumor cells is positively correlated with tumor metastasis as well as recurrence rate. The potent effects of post-translational modifications (PTMs) for PD-L1, such as ubiquitination, glycosylation, phosphorylation and palmitoylation, have been reported to be related to immunosuppression. However, the regulation of PD-L1 degradation in cancers is still not well understood. In this paper, we mainly investigate the deubiquitination regulation of PD-L1. Methods: The protein levels of PD-L1 and USP21 were detected by Immunoblotting and immunohistochemistry. The interaction between PD-L1 and USP21 was determined by co-immunoprecipitation. The deubiquitination of PD-L1 was determined by in vitro deubiquitination assay. The deubiquitination sites of PD-L1 were identified by mass spectrometry analysis. The expression of mRNA in target tissues was presented by bioinformatics analysis.Results: Overexpression of USP21 significantly increased PD-L1 abundance and knockdown of USP21 induced degradation of PD-L1. In vitro deubiquitination assay showed that USP21-WT reduced polyubiquitin chains from PD-L1 while USP21-C221A did not. Furthermore, five lysines in intracellular segment of PD-L1 are potential deubiquitin sites and cancer-derived mutations of PD-L1 in Asp276 have the ability to enhance the deubiquitination of PD-L1 mediated by USP21. Finally, we found that USP21 is the frequently amplified deubiquitinase in lung cancer, especially in lung squamous cell carcinoma, and its amplification co-occurs with the upregulation of PD-L1 levels. Moreover, IHC analysis showed stronger staining of PD-L1 and USP21 in lung cancer samples than adjacent tissues. Conclusion: We identified USP21 as a novel deubiquitinase of PD-L1. Hopefully, targeting PD-L1 by inhibiting USP21 might be a potentially novel strategy for the treatment of lung cancer.

scholarly journals Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows

BioTechniques ◽  
2021 ◽  
Author(s):  
David Kotol ◽  
Andreas Hober ◽  
Linnéa Strandberg ◽  
Anne-Sophie Svensson ◽  
Mathias Uhlén ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Plasma Proteins ◽  
Mass Spectrometry Analysis ◽  
Targeted Proteomics ◽  
Plasma Samples ◽  
Blood Proteins ◽  
Proteomics Analysis ◽  
Protein Targets ◽  
Spectrometry Analysis ◽  
Absolute Concentrations

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

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