Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.

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Fluorescence-Based High Throughput Screening Technologies for Natural Chloride Ion Channel Blockers

Chemical Research in Toxicology ◽

10.1021/acs.chemrestox.8b00205 ◽

2018 ◽

Vol 31 (12) ◽

pp. 1332-1338 ◽

Cited By ~ 1

Author(s):

Rong Xu ◽

Yuan Xiao ◽

Yan Liu ◽

Bo Wang ◽

Xing Li ◽

...

Keyword(s):

Ion Channel ◽

High Throughput ◽

High Throughput Screening ◽

Chloride Ion ◽

Channel Blockers ◽

Ion Channel Blockers

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A Novel High-Throughput Screening Assay for Sickle Cell Disease Drug Discovery

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109333975 ◽

2009 ◽

Vol 14 (4) ◽

pp. 330-336 ◽

Cited By ~ 4

Author(s):

Eszter Pais ◽

John S. Cambridge ◽

Cage S. Johnson ◽

Herbert J. Meiselman ◽

Timothy C. Fisher ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

High Throughput ◽

High Throughput Screening ◽

Rapid Screening ◽

Cell Disease ◽

Screening Assay ◽

Drug Candidates ◽

Test Molecule ◽

High Throughput Screening Assay

Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD. ( Journal of Biomolecular Screening. 2009:330-336)

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A Fluorescent-Based High-Throughput Screening Assay for Small Molecules That Inhibit the Interaction of MdmX with p53

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112460729 ◽

2012 ◽

Vol 18 (2) ◽

pp. 191-198 ◽

Cited By ~ 15

Author(s):

Keiko Tsuganezawa ◽

Yukari Nakagawa ◽

Miki Kato ◽

Shigenao Taruya ◽

Fumio Takahashi ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Diffusion Time ◽

Correlation Spectroscopy ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Green Fluorescent ◽

Fluorescence Quencher

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC50 values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

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A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers

Assay and Drug Development Technologies ◽

10.1089/adt.2009.191 ◽

2009 ◽

Vol 7 (3) ◽

pp. 266-280 ◽

Cited By ~ 16

Author(s):

Francesco Belardetti ◽

Elizabeth Tringham ◽

Cyrus Eduljee ◽

Xinpo Jiang ◽

Haiheng Dong ◽

...

Keyword(s):

Calcium Channel ◽

High Throughput ◽

Calcium Channel Blockers ◽

High Throughput Screening ◽

Channel Blockers ◽

Screening Assay ◽

High Throughput Screening Assay

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Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103255988 ◽

2003 ◽

Vol 8 (4) ◽

pp. 453-462 ◽

Cited By ~ 87

Author(s):

Li Di ◽

Edward H. Kerns ◽

Yan Hong ◽

Teresa A. Kleintop ◽

Oliver J. Mc Connell ◽

...

Keyword(s):

Mass Spectrometry ◽

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Parent Compound ◽

Metabolic Stability ◽

Screening Assay ◽

Drug Candidates ◽

Increasing Demand ◽

The Stability

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.

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A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Scientific Reports ◽

10.1038/s41598-019-50533-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Jessie Neuckermans ◽

Alan Mertens ◽

Dinja De Win ◽

Ulrich Schwaneberg ◽

Joery De Kock

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Metabolic Disorders ◽

Cost Effective ◽

Dependent Manner ◽

Screening System ◽

Screening Assay ◽

E Coli ◽

High Throughput Screening Assay ◽

Hereditary Tyrosinemia

Abstract Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical β-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.

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A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

10.1101/825190 ◽

2019 ◽

Author(s):

André Alcântara ◽

Denise Seitner ◽

Fernando Navarrete ◽

Armin Djamei

Keyword(s):

Unfolded Protein Response ◽

High Throughput ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Screening Method ◽

Screening Assay ◽

Unfolded Protein ◽

High Throughput Screening Assay ◽

Protein Response

AbstractBackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signalingin planta. A set of 35 genes from a library of secreted proteins from the maize pathogenUstilago maydiswere transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress inNicotiana benthamianaplants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

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