scholarly journals Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods

2020 ◽  
Vol 21 (23) ◽  
pp. 9150
Author(s):  
Yoshitomo Shiroma ◽  
Go Fujita ◽  
Takuya Yamamoto ◽  
Ryou-u Takahashi ◽  
Ashutosh Kumar ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclear Factor Κb ◽  
Screening Methods ◽  
Biological Functions ◽  
Mobility Shift ◽  
Dna Strand Exchange ◽  
Mobility Shift Assay ◽  
Dna Strand

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA–DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

scholarly journals Design and Implementation of High Throughput Screening Assays for Drug Discoveries

2021 ◽  
Author(s):  
Fawzi Faisal Bokhari ◽  
Ashwag Albukhari
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Screening Methods ◽  
Distribution Analysis ◽  
Time Resolved ◽  
Speed Up ◽  
Pharmaceutical Industries

The process of drug discovery is challenging and a costly affair. It takes about 12 to 15 years and costs over $1 billion dollars to develop a new drug and introduce the finished product in the market. With the increase in diseases, virus spread, and patients, it has become essential to invent new medicines. Consequently, today researchers are becoming interested in inventing new medicines faster by adopting higher throughput screening methods. One avenue of approach to discovering drugs faster is the High-Throughput Screening (HTS) method, which has gained a lot of attention in the previous few years. Today, High-Throughput Screening (HTS) has become a standard method for discovering drugs in various pharmaceutical industries. This review focuses on the advancement of technologies in High-Throughput Screening (HTS) methods, namely fluorescence resonance energy transfer (FRET), biochemical assay, fluorescence polarization (FP), homogeneous time resolved fluorescence (HTRF), Fluorescence correlation spectroscopy (FCS), Fluorescence intensity distribution analysis (FIDA), Nuclear magnetic resonance (NMR), and research advances in three major technology areas including miniaturization, automation and robotics, and artificial intelligence, which promises to help speed up the discovery of medicines and its development process.

scholarly journals Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase

2018 ◽  
Vol 23 (9) ◽  
pp. 974-981
Author(s):  
Yu-Chi Juang ◽  
Xavier Fradera ◽  
Yongxin Han ◽  
Anthony William Partridge
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Histidine Decarboxylase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gastric Ulcers ◽  
Statistical Parameters ◽  
Time Resolved ◽  
Neurological Response

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z′ score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure–activity relationships.

High-throughput screening of TRPV1 ligands in the light of the Bioluminescence Resonance Energy Transfer technique

2021 ◽  
pp. MOLPHARM-AR-2021-000271
Author(s):  
Yann Chappe ◽  
Pauline Michel ◽  
Alexandre Joushomme ◽  
Solène Barbeau ◽  
Sandra Pierredon ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer

scholarly journals DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange

2007 ◽  
Vol 189 (12) ◽  
pp. 4502-4509 ◽  
Author(s):  
Syam P. Anand ◽  
Haocheng Zheng ◽  
Piero R. Bianco ◽  
Sanford H. Leuba ◽  
Saleem A. Khan
Keyword(s):  
Dna Recombination ◽  
Reca Protein ◽  
Resonance Energy ◽  
Dna Helicase ◽  
Strand Exchange ◽  
Single Pair ◽  
Helicase Activity ◽  
Dna Strand Exchange ◽  
Dna Strand

ABSTRACT PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

scholarly journals Assay Concordance between SPA and TR-FRET in High-Throughput Screening

2006 ◽  
Vol 11 (6) ◽  
pp. 606-616 ◽  
Author(s):  
Oliver Von Ahsen ◽  
Anne Schmidt ◽  
Monika Klotz ◽  
Karsten Parczyk
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Statistical Significance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Detection Technology ◽  
Significant Difference ◽  
Set Up ◽  
Detection Technologies

High-throughput screening (HTS) of large chemical libraries has become the main source of new lead compounds for drug development. Several specialized detection technologies have been developed to facilitate the cost- and time-efficient screening of millions of compounds. However, concerns have been raised, claiming that different HTS technologies may produce different hits, thus limiting trust in the reliability of HTS data. This study was aimed to investigate the reliability of the authors most frequently used assay techniques: scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET). To investigate the data concordance between these 2 detection technologies, the authors screened a large subset of the Schering compound library consisting of 300,000 compounds for inhibitors of a nonreceptor tyrosine kinase. They chose to set up this study in realistic HTS scale to ensure statistical significance of the results. The findings clearly demonstrate that the choice of detection technology has no significant impact on hit finding, provided that assays are biochemically equivalent. Data concordance is up to 90%. The little differences in hit findings are caused by threshold setting but not by systematic differences between the technologies. The most significant difference between the compared techniques is that in the SPA format, more false-positive primary hits were obtained.

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