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

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.

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

10.1101/153106 ◽

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.

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

10.21203/rs.3.rs-900405/v1 ◽

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.

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Drug Discovery

Advances in Medical Technologies and Clinical Practice - Computer Applications in Drug Discovery and Development ◽

10.4018/978-1-5225-7326-5.ch001 ◽

2019 ◽

pp. 1-46

Author(s):

S. Lakshmana Prabu

Keyword(s):

Drug Discovery ◽

High Throughput ◽

Combinatorial Chemistry ◽

High Throughput Screening ◽

Development Stage ◽

Synthetic Chemistry ◽

Success Rates ◽

Herbal Plants ◽

Pharmaceutical Industries ◽

The 1960S

Modern chemistry foundations were made in between the 18th and 19th centuries and have been extended in 20th century. R&D towards synthetic chemistry was introduced during the 1960s. Development of new molecular drugs from the herbal plants to synthetic chemistry is the fundamental scientific improvement. About 10-14 years are needed to develop a new molecule with an average cost of more than $800 million. Pharmaceutical industries spend the highest percentage of revenues, but the achievement of desired molecular entities into the market is not increasing proportionately. As a result, an approximate of 0.01% of new molecular entities are approved by the FDA. The highest failure rate is due to inadequate efficacy exhibited in Phase II of the drug discovery and development stage. Innovative technologies such as combinatorial chemistry, DNA sequencing, high-throughput screening, bioinformatics, computational drug design, and computer modeling are now utilized in the drug discovery. These technologies can accelerate the success rates in introducing new molecular entities into the market.

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High Throughput Screening 2002: Moving Toward Increased Success Rates

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710200700402 ◽

2002 ◽

Vol 7 (4) ◽

pp. 313-316 ◽

Cited By ~ 13

Author(s):

Sandra Fox ◽

Helen Wang ◽

Lynne Sopchak ◽

Shauna Farr-Jones

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Success Rates

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Simple Colorimetric Inhibition Assay of Heme Crystallization for High-Throughput Screening of Antimalarial Compounds

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00985-06 ◽

2006 ◽

Vol 51 (1) ◽

pp. 350-353 ◽

Cited By ~ 28

Author(s):

Nguyen Tien Huy ◽

Dinh Thanh Uyen ◽

Atsushi Maeda ◽

Dai Thi Xuan Trang ◽

Tatsuo Oida ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Colorimetric Assay ◽

Tween 20 ◽

Inhibition Assay ◽

Microplate Reader ◽

Antimalarial Compounds

ABSTRACT Current assays for screening new antimalarials need initiators of β-hematin formation that require laborious preparation, special devices, and substrates. In this study, based on reduction of heme absorption in β-hematin formation, we developed a simple colorimetric assay using Tween 20 as an initiator and a microplate reader for high-throughput screening of inhibitors of β-hematin formation.

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