scholarly journals A high-throughput assay to identify robust inhibitors of dynamin GTPase activity

2017 ◽  
Author(s):  
Aparna Mohanakrishnan ◽  
Triet Vincent M. Tran ◽  
Meera Kumar ◽  
Hong Chen ◽  
Bruce A. Posner ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Gtpase Activity ◽  
Response Curves ◽  
Coated Pits ◽  
Major Pathway ◽  
Gtp Hydrolysis ◽  
Extensive Search ◽  
Highly Sensitive ◽  
High Throughput Assay

AbstractClathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin’s GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin’s stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin’s basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several “hits” that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin’s basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for robust dynamin inhibitors.

scholarly journals A highly-sensitive high throughput assay for dynamin's basal GTPase activity

PLoS ONE ◽  
2017 ◽  
Vol 12 (9) ◽  
pp. e0185639 ◽  
Author(s):  
Aparna Mohanakrishnan ◽  
Triet Vincent M. Tran ◽  
Meera Kumar ◽  
Hong Chen ◽  
Bruce A. Posner ◽  
...  
Keyword(s):  
High Throughput ◽  
Gtpase Activity ◽  
Highly Sensitive ◽  
High Throughput Assay

scholarly journals Restoration of the GTPase activity of Gαo mutants by Zn2+ in GNAO1 encephalopathy models

2021 ◽  
Author(s):  
Vladimir Katanaev ◽  
Yonika Larasati ◽  
Mikhail Savitsky ◽  
Alexey Koval ◽  
Gonzalo Solis
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Motor Dysfunction ◽  
Cellular Interactions ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
The Core ◽  
Gtp Binding

Abstract GNAO1 encephalopathy is a rare pediatric disease characterized by motor dysfunction, developmental delay, and epileptic seizures1-3. De novo point mutations in the gene encoding Gαo, the major neuronal G protein, lie at the core of this dominant genetic malady4. Half of the clinical case mutations fall on codons Gly203, Arg209, or Glu246 near the GTP binding/hydrolysis pocket of Gαo1-3. We here show that these pathologic mutations strongly speed up GTP uptake and inactivate GTP hydrolysis by Gαo, resulting in constitutive GTP binding by the G protein. Molecular dynamics simulations indicate that the mutations cause displacement of Gln205, the key to GTP hydrolysis. Decreased interactions with cellular partners including RGS19 suggest that despite the enhanced GTP residence, the mutants fail to fully adopt the activated conformation and thus transmit the signal. Through a high-throughput screening of approved drugs aiming at correction of this core biochemical dysfunction, we identify zinc pyrithione and Zn2+ ions as agents restoring the active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with a negligible effect on wild type Gαo. We describe a Drosophila model of GNAO1 encephalopathy and show that dietary zinc supplementation restores the motor function and longevity of the mutant flies. With zinc supplements frequently recommended for diverse human neurological conditions, our work spanning from identification of the core biochemical defect in Gαo mutants and cellular interactions analysis to high-throughput screening and animal validation of the deficiency-correcting drug defines the potential therapy for GNAO1 encephalopathy patients.

scholarly journals Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB)

2019 ◽  
Vol 24 (5) ◽  
pp. 597-605 ◽  
Author(s):  
John Veloria ◽  
Minhye Shin ◽  
Ashwini K. Devkota ◽  
Shelley M. Payne ◽  
Eun Jeong Cho ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Ferrous Iron ◽  
Direct Calculation ◽  
Tween 20 ◽  
Gtpase Activity ◽  
Success Rates ◽  
Gtp Hydrolysis ◽  
Iron Transporter ◽  
Protein B

Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.

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 Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

2021 ◽  
pp. 247255522110232
Author(s):  
Michael D. Scholle ◽  
Doug McLaughlin ◽  
Zachary A. Gurard-Levin
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Rhinovirus ◽  
Binding Potential ◽  
Affinity Selection ◽  
Response Curves ◽  
Desorption Ionization ◽  
Self Assembled

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

Automatic 3D Cell Analysis in High-Throughput Microarray Using Micropillar and Microwell Chips

2015 ◽  
Vol 20 (9) ◽  
pp. 1178-1184 ◽  
Author(s):  
Dong Woo Lee ◽  
Moo-Yeal Lee ◽  
Bosung Ku ◽  
Do-Hyun Nam
Keyword(s):  
Standard Deviation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Doubling Time ◽  
Cultured Cells ◽  
Measurement Methods ◽  
Cell Analysis ◽  
Response Curves ◽  
U251 Cell ◽  
Doubling Times

Area-based and intensity-based 3D cell viability measurement methods are compared in high-throughput screening in order to analyze their effects on the assay results (doubling time and IC50) and their repeatability. Many other 3D cell-based high-throughput screening platforms had been previously introduced, but these had not clearly addressed the effects of the two methods on the assay results and assay repeatability. In this study, the optimal way to analyze 3D cultured cells is achieved by comparing day-to-day data of doubling times and IC50 values obtained from the two methods. In experiments, the U251 cell line is grown in chips. The doubling time, based on the area of the 3D cells, was 27.8 ± 1.8 h (standard deviation: 6.6%) and 27.8 ± 3.8 h (standard deviation: 13.7%) based on the intensity of the 3D cells. The doubling time calculated by area shows a smaller standard deviation than one calculated by intensity. IC50 values calculated by both methods are very similar. The standard deviations of IC50 values for the two methods were within ±3-fold. The IC50 variations of the 12 compounds were similar regardless of the viability measurement methods and were highly related to the shape of the dose–response curves.

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.

Application of the BD ACTOne™ Technology for the High-Throughput Screening of Gs-Coupled Receptor Antagonists

2007 ◽  
Vol 12 (8) ◽  
pp. 1068-1073 ◽  
Author(s):  
András Visegrády ◽  
András Boros ◽  
Zsolt Némethy ◽  
Béla Kiss ◽  
György M. Keserű
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Calcium Influx ◽  
Live Cells ◽  
Cyclic Nucleotide Gated Channels ◽  
Novel Technology ◽  
Adenosine Cyclic Monophosphate ◽  
Compound Screening ◽  
High Throughput Assay ◽  
Act One

A novel technology for monitoring the changes of 3,′5′-adenosine cyclic monophosphate (cAMP) in live cells suitable for drug screening relies on the use of cyclic nucleotide-gated channels as biosensors coexpressed with the appropriate target receptor. The technique (termed BD ACT One™) offers measurement of cAMP-dependent calcium influx or membrane depolarization with conventional fluorescent methods both in kinetic and in endpoint modes, optimal for high-throughput and subsequent compound screening. The utility of the technique is reported here based on assay development and high-throughput screening for small-molecule antagonists of the peptide parathyroid hormone 2 receptor (PTH2R). The dual-signaling properties of the receptor were retained in the recombinant system, and the observed pharmacological profile corresponded to data from radiolabeled cAMP determination. The membrane-potential-based high-throughput assay produced reproducible actives and led to the identification of several chemical scaffolds with potential utility as PTH2R ligands. ( Journal of Biomolecular Screening 2007:1068-1073)

scholarly journals High-Throughput Screening of Psychotropic Compounds: Impacts on Swimming Behaviours in Artemia franciscana

Toxics ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 64
Author(s):  
Shanelle A. Kohler ◽  
Matthew O. Parker ◽  
Alex T. Ford
Keyword(s):  
High Throughput ◽  
Swimming Speed ◽  
High Throughput Screening ◽  
Artemia Franciscana ◽  
Animal Behaviour ◽  
Crustacean Species ◽  
Significant Difference ◽  
Increased Sensitivity ◽  
Light Side ◽  
High Throughput Assay

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

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