scholarly journals A cell based, high throughput assay for quantitative analysis of Hedgehog pathway activation using a Smoothened phosphorylation sensor

2017 ◽  
Author(s):  
Eugene A. Albert ◽  
Christian Bökel
Keyword(s):  
High Throughput ◽  
Fluorescent Sensor ◽  
Population Level ◽  
Pathway Activation ◽  
Molecule Inhibitor ◽  
Graded Response ◽  
Biochemical Validation ◽  
Cell Culture Assay ◽  
High Throughput Assay ◽  
Signalling Cascade

AbstractThe Hedgehog (Hh) signalling cascade is conserved across evolution and plays an important role in development and disease. In the absence of Hh, activity of the key signal transducer Smoothened (Smo) is downregulated by the Hh receptor Patched (Ptc). However, the mechanisms underlying this inhibition, and especially its release upon ligand stimulation, are still poorly understood, in part because tools for directly following Smo activation at the subcellular level were long lacking. Here we present a high throughput, cell culture assay based on a fluorescent sensor for Drosophila Smo phosphorlyation. Using this approach we could first demonstrate that the graded response to increasing Hh levels observed at the population level can be traced back to threshold responses of individual cells exhibiting differential Hh sensitivity. Second, we screened a small molecule inhibitor library for regulators of Smo phosphorylation. We observed increased Smo sensor fluorescence with compounds aimed at two major target groups, the MAPK signalling cascade and polo and aurora kinases. Biochemical validation confirmed the screen results for selected inhibitors (dobrafenib, tak-733, volasertib) and revealed differences in the mode of Smo activation, demonstrating that the assay is in principle suitable for dissecting the cell biological basis of Hh pathway activation.

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 INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5054
Author(s):  
Qianxin Wu ◽  
Chenqu Suo ◽  
Tom Brown ◽  
Tengyao Wang ◽  
Sarah A. Teichmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Population Level ◽  
Reaction Products ◽  
Testing Strategy ◽  
Asymptomatic Patients ◽  
Testing Stage ◽  
Population Scale

We present INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing–based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

scholarly journals Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Michael D. Scholle ◽  
Zachary A. Gurard-Levin
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
Ionization Mass Spectrometry ◽  
Ionization Mass ◽  
Label Free ◽  
Desorption Ionization ◽  
Arginase 1 ◽  
Self Assembled ◽  
High Throughput Assay

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid–metabolizing enzymes.

High-throughput extracellular pH monitoring and antibiotics screening by polymeric fluorescent sensor with LCST property

Methods ◽  
2019 ◽  
Vol 168 ◽  
pp. 51-61 ◽  
Author(s):  
Tingting Pan ◽  
Cheng Yang ◽  
Jiaze Li ◽  
Jiapei Jiang ◽  
Jiaxing Wen ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Sensor ◽  
Ph Monitoring ◽  
Extracellular Ph

A high-throughput assay for HIV-1 integrase 3′-processing activity using time-resolved fluorescence

2012 ◽  
Vol 184 (1-2) ◽  
pp. 34-40 ◽  
Author(s):  
Ying-Shan Han ◽  
Peter Quashie ◽  
Thibault Mesplede ◽  
Hongtao Xu ◽  
Kevork Mekhssian ◽  
...  
Keyword(s):  
High Throughput ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Processing Activity ◽  
High Throughput Assay ◽  
Hiv 1
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