scholarly journals Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mads Breum Larsen ◽  
Mireia Perez Verdaguer ◽  
Brigitte F Schmidt ◽  
Marcel P Bruchez ◽  
Simon C Watkins ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Gene Editing ◽  
Egf Receptor ◽  
Ph Sensitive ◽  
Fluorescence Excitation ◽  
High Throughput Analysis ◽  
Egfr Expression ◽  
High Throughput Assay

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

scholarly journals High-Throughput Screening of Novel Peptide Inhibitors of an Integrin Receptor from the Hexapeptide Library by Using a Protein Microarray Chip

2004 ◽  
Vol 9 (8) ◽  
pp. 687-694 ◽  
Author(s):  
Yoonsuk Lee ◽  
Dong-Ku Kang ◽  
Soo-Ik Chang ◽  
Moon Hi Han ◽  
In-Cheol Kang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Competitive Inhibition ◽  
Protein Microarray ◽  
Dependent Manner ◽  
High Throughput Analysis ◽  
Microarray Chip ◽  
New Drug

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, Proteo Chip, in new drug discovery. Integrin αvβ3 microarray immobilized on the Proteo Chip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin αvβ3-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner andwas inhibited not only by the syntheticRGDpeptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and highthroughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the Proteo Chip is a promising tool for highthroughput screening of lead molecules in new drug development.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3355 ◽  
Author(s):  
Wanyoung Lim ◽  
Sungsu Park
Keyword(s):  
High Throughput ◽  
Concentration Gradient ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Drug Efficacy ◽  
Cancer Drug ◽  
Spheroid Culture ◽  
Gradient Generator ◽  
Micro Channels

Three-dimensional (3D) cell culture is considered more clinically relevant in mimicking the structural and physiological conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (μFSCD) with a concentration gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concentration gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approximately 120 μm in diameter) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for three days. The number of spheroids, roundness, and cell viability, were inversely proportional to the drug concentration. These results suggest that the μFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.

scholarly journals High-Throughput Analysis of Selected Urinary Hydroxy Polycyclic Aromatic Hydrocarbons by an Innovative Automated Solid-Phase Microextraction

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1869 ◽  
Author(s):  
Stefano Dugheri ◽  
Alessandro Bonari ◽  
Matteo Gentili ◽  
Giovanni Cappelli ◽  
Ilenia Pompilio ◽  
...  
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
High Throughput ◽  
Aromatic Hydrocarbons ◽  
High Throughput Screening ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
User Friendliness ◽  
High Throughput Analysis ◽  
Polycyclic Aromatic ◽  
User Friendly

High-throughput screening of samples is the strategy of choice to detect occupational exposure biomarkers, yet it requires a user-friendly apparatus that gives relatively prompt results while ensuring high degrees of selectivity, precision, accuracy and automation, particularly in the preparation process. Miniaturization has attracted much attention in analytical chemistry and has driven solvent and sample savings as easier automation, the latter thanks to the introduction on the market of the three axis autosampler. In light of the above, this contribution describes a novel user-friendly solid-phase microextraction (SPME) off- and on-line platform coupled with gas chromatography and triple quadrupole-mass spectrometry to determine urinary metabolites of polycyclic aromatic hydrocarbons 1- and 2-hydroxy-naphthalene, 9-hydroxy-phenanthrene, 1-hydroxy-pyrene, 3- and 9-hydroxy-benzoantracene, and 3-hydroxy-benzo[a]pyrene. In this new procedure, chromatography’s sensitivity is combined with the user-friendliness of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide on-fiber SPME derivatization using direct immersion sampling; moreover, specific isotope-labelled internal standards provide quantitative accuracy. The detection limits for the seven OH-PAHs ranged from 0.25 to 4.52 ng/L. Intra-(from 2.5 to 3.0%) and inter-session (from 2.4 to 3.9%) repeatability was also evaluated. This method serves to identify suitable risk-control strategies for occupational hygiene conservation programs.

scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

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