scholarly journals FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay

2016 ◽  
Vol 21 (7) ◽  
pp. 701-712 ◽  
Author(s):  
Andrea Caporale ◽  
Fabiola Mascanzoni ◽  
Biancamaria Farina ◽  
Mattia Sturlese ◽  
Gianluigi Di Sorbo ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Cyclophilin A ◽  
Reaction Rates ◽  
Docking Studies ◽  
Isomerase Activity ◽  
Shape Complementarity ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds ◽  
Micromolar Range

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.

scholarly journals Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D

2021 ◽  
Vol 22 (2) ◽  
pp. 594
Author(s):  
Yi-Hsun Ho ◽  
Lan Chen ◽  
Rong Huang
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
High Throughput Screening ◽  
Coenzyme A ◽  
Therapeutic Potential ◽  
Biological Functions ◽  
Histone H4 ◽  
Fluorescent Signal ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

scholarly journals Development of a continuous fluorescence-based assay for N-terminal acetyltransferase D

2020 ◽  
Author(s):  
Yi-Hsun Ho ◽  
Lan Chen ◽  
Rong Huang
Keyword(s):  
Lung Cancer ◽  
Cancer Progression ◽  
High Throughput Screening ◽  
Coenzyme A ◽  
Therapeutic Potential ◽  
Biological Functions ◽  
Histone H4 ◽  
Fluorescent Signal ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

AbstractN-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z’-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

scholarly journals Pilot-Scale Compound Screening against RNA Editing Identifies Trypanocidal Agents

2014 ◽  
Vol 20 (1) ◽  
pp. 92-100 ◽  
Author(s):  
Houtan Moshiri ◽  
Vaibhav Mehta ◽  
Chun Wai Yip ◽  
Reza Salavati
Keyword(s):  
Rna Editing ◽  
High Throughput Screening ◽  
Pilot Scale ◽  
Messenger Rnas ◽  
Unique Type ◽  
Compound Screening ◽  
Parasite Viability ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Most mitochondrial messenger RNAs in trypanosomatid pathogens undergo a unique type of posttranscriptional modification involving insertion and/or deletion of uridylates. This process, RNA editing, is catalyzed by a multiprotein complex (~1.6 MDa), the editosome. Knockdown of core editosome proteins compromises mitochondrial function and, ultimately, parasite viability. Hence, because the editosome is restricted to trypanosomatids, it serves as a unique drug target in these pathogens. Currently, there is a lack of editosome inhibitors for antitrypanosomatid drug development or that could serve as unique tools for perturbing and characterizing editosome interactions or RNA editing reaction stages. Here, we screened a library of pharmacologically active compounds (LOPAC1280) using high-throughput screening to identify RNA editing inhibitors. We report that aurintricarboxylic acid, mitoxantrone, PPNDS, and NF449 are potent inhibitors of deletion RNA editing (IC50 range, 1–5 µM). However, none of these compounds could specifically inhibit the catalytic steps of RNA editing. Mitoxantrone blocked editing by inducing RNA-protein aggregates, whereas the other three compounds interfered with editosome-RNA interactions to varying extents. Furthermore, NF449, a suramin analogue, was effective at killing Trypanosoma brucei in vitro. Thus, new tools for editosome characterization and downstream RNA editing inhibitor have been identified.

scholarly journals Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology

2016 ◽  
Vol 22 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Jing Wang ◽  
Pengfei Fang ◽  
Peter Chase ◽  
Sagi Tshori ◽  
Ehud Razin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
High Throughput Screening ◽  
Treatment Resistance ◽  
Dna Interaction ◽  
Pigment Formation ◽  
Assay Performance ◽  
Microphthalmia Transcription Factor ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z′ factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

scholarly journals Structure of Mpro from COVID-19 virus and discovery of its inhibitors

2020 ◽  
Author(s):  
Zhenming Jin ◽  
Xiaoyu Du ◽  
Yechun Xu ◽  
Yongqiang Deng ◽  
Meiqin Liu ◽  
...  
Keyword(s):  
Drug Design ◽  
High Throughput ◽  
High Throughput Screening ◽  
Treatment Options ◽  
Screening Strategy ◽  
Drug Candidates ◽  
Approved Drugs ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds ◽  
Drug Leads

SUMMARYA new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1–4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

scholarly journals Fluorescence polarization immunoassay for the determination of diclofenac in wastewater

2020 ◽  
Author(s):  
Anna Raysyan ◽  
Robin Moerer ◽  
Bianca Coesfeld ◽  
Sergei A. Eremin ◽  
Rudolf J. Schneider
Keyword(s):  
Fluorescence Polarization ◽  
Limit Of Detection ◽  
Reference Method ◽  
Fluorescence Polarization Immunoassay ◽  
Pre Treatment ◽  
Wastewater Samples ◽  
Pharmacologically Active ◽  
Tracer Molecules ◽  
Pharmacologically Active Compounds

AbstractPharmacologically active compounds are often detected in wastewater and surface waters. The nonsteroidal anti-inflammatory drug diclofenac (DCF) was included in the European watch list of substances that requires its environmental monitoring in the member states. DCF may harmfully influence the ecosystem already at concentrations ≤ 1 μg L−1. The fast and easy quantification of DCF is becoming a subject of global importance. Fluorescence polarization immunoassay (FPIA) is a homogeneous mix-and-read method which does not require the immobilization of reagents. FPIA can be performed in one phase within 20–30 min, making it possible to analyse wastewater without any complicated pre-treatment. In this study, new tracer molecules with different structures, linking fluorophores to derivatives of the analyte, were synthesized, three homologous tracers based on DCF, two including a C6 spacer, and one heterologous tracer derived from 5-hydroxy-DCF. The tracer molecules were thoroughly assessed for performance. Regarding sensitivity of the FPIA, the lowest limit of detection reached was 2.0 μg L−1 with a working range up to 870 μg L−1. The method was validated for real wastewater samples against LC-MS/MS as reference method with good agreement of both methods.

Electrochemical Detectors in Liquid Chromatography: Recent Trends in Pharmaceutical and Biomedical Analysis

2018 ◽  
Vol 25 (33) ◽  
pp. 4050-4065 ◽  
Author(s):  
Jean-Michel Kauffmann ◽  
Nurgul K. Bakirhan ◽  
Burcin Bozal-Palabiyik ◽  
Bengi Uslu ◽  
Rocio Rodriguez Gomez ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Drug Stability ◽  
Biomedical Analysis ◽  
Optical Detectors ◽  
Scope Of Application ◽  
Recent Trends ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds ◽  
Detector Design

Liquid chromatography (LC) coupled to an electrochemical (EC) detector is a complementary analytical tool compared to LC coupled with optical or mass spectrometry detectors (LC-MS). LC-EC can be applied to the determination of molecules difficult to be analyzed by other commercially available detectors. New EC detector design and new working electrode material have extended the scope of application in the field of pharmaceutical compounds analysis. Combining EC with LC-MS offers additional advantages compared to optical detectors in terms of drug stability and drug metabolism mimicry studies. Selected literature devoted to pharmacologically active compounds in their dosage forms, herbal drugs in natural products, drug residues in feed and/or in biological samples are reported in this review.

scholarly journals Insilico Molecular Docking Studies of Volatile Compounds Identified by GC-MS from Tagetes Species Against Mamestra brassicae (Linnaeus, 1758)

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
S.R. Krishna Motukuri ◽  
D. Vijaya Nagini ◽  
J. Nallamothu ◽  
S. Karthikeyan
Keyword(s):  
Molecular Docking ◽  
Volatile Compounds ◽  
Cost Effective ◽  
Docking Studies ◽  
Gas Chromatography Mass Spectrometry ◽  
Mamestra Brassicae ◽  
Natural Origin ◽  
Molecular Docking Studies ◽  
Pharmacologically Active ◽  
Pharmacologically Active Compounds

Plants evolved to be a potential source of pharmacologically active compounds that are being widely accepted as insect repellent compounds for generations. Products of natural origin are mostly preferred over synthetic compounds because of fewer side effects on human health and the environment, have the potential to be produced locally, cost-effective, and are proved to be more efficient. They are best suited in organic food production and can play a much greater role in developing countries as a new class of eco-friendly products for controlling pests. In turn, the development of repellents is desirable alternatives to synthetic chemical insecticides for controlling pests. In the process of continual search for insect-based repellents of natural origin, a wide number of Tagetes species have been archived and all parts of this plant from root to seed possess a range of phytochemicals that are responsible for the repellent activity. The present study concentrates on the identification of active volatile compounds from Tageteserecta leaves by Gas chromatography-mass spectrometry (GC-MS) analysis and further evaluation through molecular docking studies of identified compounds against Mamestra brassicae.

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