Label-Free High-Throughput Screening Assay for Inhibitors of Alzheimer’s Amyloid-β Peptide Aggregation Based on MALDI MS

AbstractThe human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET—norepinephrine (NE), dopamine (DA) and epinephrine (EP)—were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z′ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

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Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors

10.21203/rs.3.rs-134611/v1 ◽

2021 ◽

Author(s):

Hubert Sijben ◽

Wieke van Oostveen ◽

Peter Hartog ◽

Laura Stucchi ◽

Giovanna Maresca ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Norepinephrine Transporter ◽

Functional Assay ◽

Substrate Uptake ◽

Label Free ◽

Screening Assay ◽

Fluorescent Substrate ◽

High Throughput Screening Assay ◽

Wide Range

Abstract The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z’ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

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An efficient high-throughput screening assay against nuclear trans-port

Hereditas (Beijing) ◽

10.3724/sp.j.1005.2012.00927 ◽

2012 ◽

Vol 34 (7) ◽

pp. 927-934

Author(s):

Min LUO ◽

Quan-Cang ZHANG ◽

Zhi-Gang LU

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Assay ◽

High Throughput Screening Assay

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High-throughput screening assay for groups of effective components extracted from Xiaoxuming Recipe

Journal of Chinese Integrative Medicine ◽

10.3736/jcim20060117 ◽

2006 ◽

Vol 4 (1) ◽

pp. 64-67 ◽

Cited By ~ 7

Author(s):

Yue-Hua Wang

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Assay ◽

Effective Components ◽

High Throughput Screening Assay

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High Throughput Screening Assay to Identify Modulators of IL-17 Expression

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207320666170602091308 ◽

2018 ◽

Vol 20 (9) ◽

pp. 804-819 ◽

Cited By ~ 2

Author(s):

Mohamed Boudjelal ◽

Ana Maria Ruiz-Avendano ◽

Gonzalo Colmenarejo ◽

Sergio A. Senar-Sancho ◽

Ashley Barnes ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Assay ◽

High Throughput Screening Assay

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A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

BMC Microbiology ◽

10.1186/s12866-021-02212-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Sadaf Kalsum ◽

Blanka Andersson ◽

Jyotirmoy Das ◽

Thomas Schön ◽

Maria Lerm

Keyword(s):

Mycobacterium Tuberculosis ◽

High Throughput ◽

High Throughput Screening ◽

Live Cell Imaging ◽

Cell Imaging ◽

Imaging System ◽

Aggregate Size ◽

Live Cell ◽

Screening Assay ◽

High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

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

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211000652 ◽

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.

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Blood-Brain Barrier Disruption Increases Amyloid-Related Pathology in TgSwDI Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22031231 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1231

Author(s):

Ihab M. Abdallah ◽

Kamal M. Al-Shami ◽

Euitaek Yang ◽

Amal Kaddoumi

Keyword(s):

Mouse Model ◽

Blood Brain Barrier ◽

High Throughput Screening ◽

Amyloid Β ◽

Brain Barrier ◽

Amyloid Angiopathy ◽

Cancer Resistance ◽

Screening Assay ◽

Blood Brain ◽

High Throughput Screening Assay

In Alzheimer’s disease (AD), several studies have reported blood-brain barrier (BBB) breakdown with compromised function. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are transport proteins localized at the BBB luminal membrane and play an important role in the clearance of amyloid-β (Aβ). The purpose of this study was to investigate the effect of pharmacological inhibition of Aβ efflux transporters on BBB function and Aβ accumulation and related pathology. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that modulate the integrity of a cell-based BBB model, which identified elacridar as a disruptor of the monolayer integrity. Elacridar, an investigational compound known for its P-gp and BCRP inhibitory effect and widely used in cancer research. Therefore, it was used as a model compound for further evaluation in a mouse model of AD, namely TgSwDI. TgSwDI mouse is also used as a model for cerebral amyloid angiopathy (CAA). Results showed that P-gp and BCRP inhibition by elacridar disrupted the BBB integrity as measured by increased IgG extravasation and reduced expression of tight junction proteins, increased amyloid deposition due to P-gp, and BCRP downregulation and receptor for advanced glycation end products (RAGE) upregulation, increased CAA and astrogliosis. Further studies revealed the effect was mediated by activation of NF-κB pathway. In conclusion, results suggest that BBB disruption by inhibiting P-gp and BCRP exacerbates AD pathology in a mouse model of AD, and indicate that therapeutic drugs that inhibit P-gp and BCRP could increase the risk for AD.

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Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽

10.3390/antibiotics9110808 ◽

2020 ◽

Vol 9 (11) ◽

pp. 808

Author(s):

Maurice Steenhuis ◽

Corinne M. ten Hagen-Jongman ◽

Peter van Ulsen ◽

Joen Luirink

Keyword(s):

Outer Membrane ◽

High Throughput ◽

Stress Responses ◽

High Throughput Screening ◽

Structural Integrity ◽

Cell Envelope ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Compound Screening ◽

Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

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