Technological advances for interrogating the human kinome

2017 ◽  
Vol 45 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Akanksha Baharani ◽  
Brett Trost ◽  
Anthony Kusalik ◽  
Scott Napper
Keyword(s):  
High Throughput ◽  
Therapeutic Intervention ◽  
Therapeutic Efficacy ◽  
Kinase Inhibitors ◽  
Cost Effective ◽  
Disease Prognosis ◽  
Technological Advances ◽  
Human Kinome ◽  
High Throughput Manner

There is increasing appreciation among researchers and clinicians of the value of investigating biology and pathobiology at the level of cellular kinase (kinome) activity. Kinome analysis provides valuable opportunity to gain insights into complex biology (including disease pathology), identify biomarkers of critical phenotypes (including disease prognosis and evaluation of therapeutic efficacy), and identify targets for therapeutic intervention through kinase inhibitors. The growing interest in kinome analysis has fueled efforts to develop and optimize technologies that enable characterization of phosphorylation-mediated signaling events in a cost-effective, high-throughput manner. In this review, we highlight recent advances to the central technologies currently available for kinome profiling and offer our perspectives on the key challenges remaining to be addressed.

scholarly journals Identification and Characterization of a Small-Molecule Rabies Virus Entry Inhibitor

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Venice Du Pont ◽  
Christoph Wirblich ◽  
Jeong-Joong Yoon ◽  
Robert M. Cox ◽  
Matthias J. Schnell ◽  
...  
Keyword(s):  
G Protein ◽  
Small Molecule ◽  
High Throughput ◽  
Viral Entry ◽  
Cost Effective ◽  
Molecular Probe ◽  
Entry Inhibitor ◽  
Protein Fusion ◽  
Life Saving

ABSTRACT Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry. IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

scholarly journals High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells

2017 ◽  
Vol 8 (4) ◽  
pp. 1101-1111 ◽  
Author(s):  
Matteo D'Antonio ◽  
Grace Woodruff ◽  
Jason L. Nathanson ◽  
Agnieszka D'Antonio-Chronowska ◽  
Angelo Arias ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Cost Effective ◽  
Induced Pluripotent

scholarly journals A High-Throughput NanoBiT-Based Serological Assay Detects SARS-CoV-2 Seroconversion

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 807
Author(s):  
Taha Azad ◽  
Reza Rezaei ◽  
Ragunath Singaravelu ◽  
Taylor R. Jamieson ◽  
Mathieu J. F. Crupi ◽  
...  
Keyword(s):  
High Throughput ◽  
Cost Effective ◽  
Post Vaccination ◽  
Serological Assay ◽  
High Throughput Detection ◽  
Speed Stability ◽  
The Stability ◽  
Detection Strategies ◽  
High Throughput Manner ◽  
Vaccination Period

High-throughput detection strategies for antibodies against SARS-CoV-2 in patients recovering from COVID-19, or in vaccinated individuals, are urgently required during this ongoing pandemic. Serological assays are the most widely used method to measure antibody responses in patients. However, most of the current methods lack the speed, stability, sensitivity, and specificity to be selected as a test for worldwide serosurveys. Here, we demonstrate a novel NanoBiT-based serological assay for fast and sensitive detection of SARS-CoV-2 RBD-specific antibodies in sera of COVID-19 patients. This assay can be done in high-throughput manner at 384 samples per hour and only requires a minimum of 5 μL of serum or 10 ng of antibody. The stability of our NanoBiT reporter in various temperatures (4–42 °C) and pH (4–12) settings suggests the assay will be able to withstand imperfect shipping and handling conditions for worldwide seroepidemiologic surveillance in the post-vaccination period of the pandemic. Our newly developed rapid assay is highly accessible and may facilitate a more cost-effective solution for seroconversion screening as vaccination efforts progress.

High-throughput genotyping assay for the large-scale genetic characterization ofCryptosporidiumparasites from human and bovine samples

Parasitology ◽  
2013 ◽  
Vol 141 (4) ◽  
pp. 491-500 ◽  
Author(s):  
J. L. ABAL-FABEIRO ◽  
X. MASIDE ◽  
J. LLOVO ◽  
X. BELLO ◽  
M. TORRES ◽  
...  
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
Large Scale ◽  
Genetic Characterization ◽  
Low Cost ◽  
Cost Effective ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Identification Of Species

SUMMARYThe epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of commongp60subtypes of fourCryptosporidiumspecies that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) andgp60subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four differentCryptosporidiumspecies (C. hominis, C. parvum, C. meleagridisandC. felis) were detected in humans; the most common ones wereC. hominissubtype Ib, andC. parvumIIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

scholarly journals Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection

2018 ◽  
Vol 24 (3) ◽  
pp. 284-294 ◽  
Author(s):  
Riyo Maruki Imamura ◽  
Kazuo Kumagai ◽  
Hirofumi Nakano ◽  
Takayoshi Okabe ◽  
Tetsuo Nagano ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Kinase Inhibitors ◽  
Coupling Reaction ◽  
Cost Effective ◽  
Adenosine Diphosphate ◽  
Fluorescence Assay ◽  
Biological Research ◽  
Chemical Library

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

Kinetic Solvent Effects in Organic Reactions

2017 ◽  
Author(s):  
Belinda Slakman ◽  
Richard West
Keyword(s):  
Solvent Effect ◽  
Reaction Kinetics ◽  
Computational Methods ◽  
High Throughput ◽  
Kinetic Modeling ◽  
Solvent Effects ◽  
Reaction Rates ◽  
Prior Work ◽  
Solvent Phase ◽  
High Throughput Manner

<div> <div> <div> <p>This article reviews prior work studying reaction kinetics in solution, with the goal of using this information to improve detailed kinetic modeling in the solvent phase. Both experimental and computational methods for calculating reaction rates in liquids are reviewed. Previous studies, which used such methods to determine solvent effects, are then analyzed based on reaction family. Many of these studies correlate kinetic solvent effect with one or more solvent parameters or properties of reacting species, but it is not always possible, and investigations are usually done on too few reactions and solvents to truly generalize. From these studies, we present suggestions on how best to use data to generalize solvent effects for many different reaction types in a high throughput manner. </p> </div> </div> </div>

Combinatorial Fabrication and High-Throughput Characterization of Thin Film Metal Oxide Libraries for Solar water Splitting

2018 ◽  
Author(s):  
Alfred Ludwig ◽  
Mona Nowak ◽  
Swati Kumari ◽  
Helge S. Stein ◽  
Ramona Gutkowski ◽  
...  
Keyword(s):  
Thin Film ◽  
Metal Oxide ◽  
Water Splitting ◽  
High Throughput ◽  
Solar Water ◽  
Solar Water Splitting

Monitoring of Petroleum-Related Environmental Contamination Using Fluorescence Fingerprinting

2006 ◽  
Vol 1 (2) ◽  
Author(s):  
P. Literathy ◽  
M. Quinn
Keyword(s):  
Environmental Contamination ◽  
Statistical Evaluation ◽  
Oil Pollution ◽  
Emission Spectra ◽  
Cost Effective ◽  
Petroleum Products ◽  
Danube River ◽  
Aromatic Components ◽  
Analytical Approaches

Petroleum and its refined products are considered the most complex contaminants frequently impacting the environment in significant quantities. They have heterogeneous chemical composition and alterations occur during environmental weathering. No single analytical method exists to characterize the petroleum-related environmental contamination. For monitoring, the analytical approaches include gravimetric, spectrometric and chromatographic methods having significant differences in their selectivity, sensitivity and cost-effectiveness. Recording fluorescence fingerprints of the cyclohexane extracts of the water, suspended solids, sediment or soil samples and applying appropriate statistical evaluation (e.g. by correlating the concatenated emission spectra of the fingerprints of the samples with arbitrary standards (e.g. petroleum products)), provides a powerful, cost-effective analytical tool for characterization of the type of oil pollution and detecting the most harmful aromatic components of the petroleum contaminated matrix. For monitoring purposes, the level of the contamination can be expressed as the equivalent concentration of an appropriate characteristic standard, based on the fluorescence intensities at the relevant characteristic wavelengths. These procedures are demonstrated in the monitoring of petroleum-related pollution in the water and suspended sediment in the Danube river basin

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