scholarly journals Identification and Characterization of a New Series of Ghrelin O-Acyl Transferase Inhibitors

2017 ◽  
Vol 23 (2) ◽  
pp. 154-163 ◽  
Author(s):  
Mariko Yoneyama-Hirozane ◽  
Kohei Deguchi ◽  
Takeshi Hirakawa ◽  
Tsuyoshi Ishii ◽  
Tomoyuki Odani ◽  
...  
Keyword(s):  
High Throughput ◽  
Inhibitory Activity ◽  
High Throughput Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Acyl Transferase ◽  
Lead Compound ◽  
Time Resolved ◽  
Acyl Ghrelin ◽  
Antiobesity Drugs ◽  
High Throughput Assay

Ghrelin O-acyl transferase (GOAT; MBOAT4) catalyzes O-acylation at serine-3 of des-acyl ghrelin. Acyl ghrelin is secreted by stomach X/A-like cells and plays a role in appetite and metabolism. Therefore, GOAT has been expected to be a novel antiobesity target because it is responsible for acyl ghrelin production. Here, we report homogeneous time-resolved fluorescence (HTRF) and enzyme-linked immunosorbent assay (ELISA) methods utilizing human GOAT-expressing microsomes as a novel high-throughput assay system for the discovery of hit compounds and optimization of lead compounds. Hit compounds exemplified by compound A (2-[(2,4-dichlorobenzyl)sulfanyl]-1,3-benzoxazole-5-carboxylic acid) were identified by high-throughput screening using the HTRF assay and confirmed to have GOAT inhibitory activity using the ELISA. Based on the hit compound information, the novel lead compound (compound B, (4-chloro-6-{[2-methyl-6-(trifluoromethyl)pyridin-3-yl]methoxy}-1-benzothiophen-3-yl)acetic acid) was synthesized and exhibited potent GOAT inhibition with oral bioavailability. Both the hit compound and lead compound showed octanoyl-CoA competitive inhibitory activity. Moreover, these two compounds decreased acyl ghrelin production in the stomach of mice after their oral administration. These novel findings demonstrate that GOAT is a druggable target, and its inhibitors are promising antiobesity drugs.

scholarly journals Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay

2015 ◽  
Vol 20 (7) ◽  
pp. 906-912 ◽  
Author(s):  
Yazhou Xu ◽  
Yunjie Wang ◽  
Yuan Xu ◽  
Jia Li ◽  
Hong Liao ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Whole Body ◽  
Protection Assay ◽  
Time Resolved ◽  
Compound C ◽  
Phosphatase 2A ◽  
High Throughput Assay

AMP-activated protein kinase (AMPK), a conserved heterotrimeric kinase, serves as an energy sensor maintaining energy balance at both cellular and whole-body levels and plays multiple beneficial roles in carbohydrate and lipid metabolism, which makes AMPK an attractive target for diabetes and other metabolic disorders. To date, establishment of the physiologically relevant biochemical assay for AMPK has not been reported. Here we developed a phosphorylated AMPK protection assay based on a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, using the protein phosphatase 2A (PP2A) to dephosphorylate AMPK. The partially dephosphorylated AMPK by PP2A had lower activity than phosphorylated AMPK. This specific TR-FRET assay for AMPK was optimized in the 384-well format and produced similar EC50 values for AMPK activators AMP and A769662 and a similar IC50 value for AMPK inhibitor compound C, as previously reported. Under the optimized conditions, the assay Z′ factor calculated over 160 data points has an optimal value greater than 0.5, which is suitable for high-throughput screening. In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions.

Time-Resolved Luminescent High-Throughput Screening Platform for Lysosomotropic Compounds in Living Cells

ACS Sensors ◽  
2020 ◽  
Author(s):  
Ke-Jia Wu ◽  
Chun Wu ◽  
Feng Chen ◽  
Sha-Sha Cheng ◽  
Dik-Lung Ma ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Living Cells ◽  
Time Resolved ◽  
Screening Platform

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

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.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

A high-throughput assay for HIV-1 integrase 3′-processing activity using time-resolved fluorescence

2012 ◽  
Vol 184 (1-2) ◽  
pp. 34-40 ◽  
Author(s):  
Ying-Shan Han ◽  
Peter Quashie ◽  
Thibault Mesplede ◽  
Hongtao Xu ◽  
Kevork Mekhssian ◽  
...  
Keyword(s):  
High Throughput ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Processing Activity ◽  
High Throughput Assay ◽  
Hiv 1

scholarly journals Asymmetric synthesis of host-directed inhibitors of myxoviruses

2013 ◽  
Vol 9 ◽  
pp. 197-203 ◽  
Author(s):  
Terry W Moore ◽  
Kasinath Sana ◽  
Dan Yan ◽  
Pahk Thepchatri ◽  
John M Ndungu ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Asymmetric Synthesis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Aqueous Solubility ◽  
Influenza Viruses ◽  
Lead Compound ◽  
Synthetic Strategy ◽  
Aqueous Solubilities ◽  
Nanomolar Range

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC50 values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

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.

scholarly journals Developing an Automated Enzyme Immunoassay for High-Throughput Screening of Bovine Serum Antibodies to Neospora Caninum

2003 ◽  
Vol 8 (1) ◽  
pp. 46-50
Author(s):  
John T. Y. Wu ◽  
Sally Dreger ◽  
Eva Y. W. Chow ◽  
Evelyn E. Bowlby ◽  
Lester S. Y. Wong
Keyword(s):  
Enzyme Immunoassay ◽  
High Throughput ◽  
Bovine Serum ◽  
High Throughput Screening ◽  
Neospora Caninum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Predictive Values ◽  
Automated Assay ◽  
Robotic Workstation ◽  
Elisa Data

An enzyme-linked immunosorbent assay (ELISA) for Neospora caninum antibodies was automated with a robotic workstation, the Beckman Coulter Biomek 2000, to screen 200 bovine sera. Comparing these results with manually run ELISA data, a 95.92% agreement (K = 0.9592) between the two assays was obtained. The automated assay was specific and sensitive with excellent positive and negative predictive values. The results were repeatable and reproducible. The automation flexibility was high and the operation complexity was minimal. High-throughput screening (HTS) for bovine antibodies to Neospora caninum was achieved. The assay was developed according to the internationally recognized ISO17025 standard requirements.

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