scholarly journals Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hannah M. Work ◽  
Sylvie E. Kandel ◽  
Jed N. Lampe
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antiviral Drug ◽  
Dehydroepiandrosterone Sulfate ◽  
Fluorescent Compound ◽  
Critical Function ◽  
Drug Candidates ◽  
High Throughput Assay ◽  
Fetus And Neonate

AbstractCYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.

scholarly journals Colorimetric LPMO assay with direct implication for cellulolytic activity

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Søren Brander ◽  
Stine Lausten ◽  
Johan Ø. Ipsen ◽  
Kristoffer B. Falkenberg ◽  
Andreas B. Bertelsen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Biochemical Characterization ◽  
Cellulose Degradation ◽  
Microtiter Plate ◽  
Enzyme Characterization ◽  
Cellulolytic Activity ◽  
Polysaccharide Monooxygenases ◽  
High Throughput Assay

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes known for their catalytic degradation of recalcitrant polymers such as cellulose or chitin. Their activity can be measured by lengthy HPLC methods, while high-throughput methods are less specific. A fast and specific LPMO assay would simplify screening for new or engineered LPMOs and accelerate biochemical characterization. Results A novel LPMO activity assay was developed based on the production of the dye phenolphthalein (PHP) from its reduced counterpart (rPHP). The colour response of rPHP oxidisation catalysed by the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A), was found to increase tenfold by adding dehydroascorbate (DHA) as a co-substrate. The assay using a combination of rPHP and DHA was tested on 12 different metallo-enzymes, but only the LPMOs catalysed this reaction. The assay was optimized for characterization of TaAA9A and showed a sensitivity of 15 nM after 30 min incubation. It followed apparent Michaelis–Menten kinetics with kcat = 0.09 s−1 and KM = 244 µM, and the assay was used to confirm stoichiometric copper–enzyme binding and enzyme unfolding at a temperature of approximately 60 °C. DHA, glutathione and fructose were found to enhance LPMO oxidation of rPHP and in the optimized assay conditions these co-substrates also enabled cellulose degradation. Conclusions This novel and specific LPMO assay can be carried out in a convenient microtiter plate format ready for high-throughput screening and enzyme characterization. DHA was the best co-substrate tested for oxidation of rPHP and this preference appears to be LPMO-specific. The identified co-substrates DHA and fructose are not normally considered as LPMO co-substrates but here they are shown to facilitate both oxidation of rPHP and degradation of cellulose. This is a rare example of a finding from a high-throughput assay that directly translate into enzyme activity on an insoluble substrate. The rPHP-based assay thus expands our understanding of LPMO catalysed reactions and has the potential to characterize LPMO activity in industrial settings, where usual co-substrates such as ascorbate and oxygen are depleted.

scholarly journals A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria

2014 ◽  
Vol 58 (12) ◽  
pp. 7264-7272 ◽  
Author(s):  
Jun Fan ◽  
Boudewijn L. M. de Jonge ◽  
Kathy MacCormack ◽  
Shubha Sriram ◽  
Robert E. McLaughlin ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Green Fluorescence Protein ◽  
Dna Gyrase ◽  
Dna Metabolism ◽  
Bacterial Biosensor ◽  
Content Type ◽  
Biosensor Strain ◽  
High Throughput Assay

ABSTRACTBacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between therecApromoter and green fluorescence protein gene was introduced into anEscherichia coliΔtolCstrain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

scholarly journals Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer

2014 ◽  
Vol 19 (8) ◽  
pp. 1193-1200 ◽  
Author(s):  
Eric Brooks ◽  
Xiang Wu ◽  
Art Hanel ◽  
Shaun Nguyen ◽  
Jing Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Isocitrate Dehydrogenase ◽  
High Throughput Screening ◽  
Wild Type ◽  
Isocitrate Dehydrogenase 1 ◽  
Multiple Tumor ◽  
Idh1 R132h ◽  
Tumor Types ◽  
High Throughput Assay

Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire–mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6–300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (Km =110 µM) compared with the R132H homodimer (Km = 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM).

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 and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins

2019 ◽  
Vol 60 (5) ◽  
pp. 1082-1097 ◽  
Author(s):  
Panneerselvam Krishnamurthy ◽  
Yukiko Fujisawa ◽  
Yuya Takahashi ◽  
Hanako Abe ◽  
Kentaro Yamane ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Density ◽  
Biosynthesis Pathway ◽  
Mutant Library ◽  
Triterpenoid Saponins

scholarly journals EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

2020 ◽  
Author(s):  
T.A. Hartjes ◽  
J.A. Slotman ◽  
M.S. Vredenbregt ◽  
N. Dits ◽  
R. Van der Meel ◽  
...  
Keyword(s):  
Protein Content ◽  
Size Distribution ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Ideal Liquid ◽  
Extracellular Vesicle ◽  
Cell Of Origin ◽  
Clinical Implementation ◽  
High Throughput Assay

AbstractExtracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

scholarly journals Development of a Fluorescent Intensity Assay Amenable for High-Throughput Screening for Determining 15-Lipoxygenase Activity

2010 ◽  
Vol 15 (6) ◽  
pp. 671-679 ◽  
Author(s):  
Märta Dahlström ◽  
Daniel Forsström ◽  
Malin Johannesson ◽  
Yasmin Huque-Andersson ◽  
Marie Björk ◽  
...  
Keyword(s):  
Fatty Acids ◽  
High Throughput ◽  
High Throughput Screening ◽  
Lipid Hydroperoxide ◽  
Lipoxygenase Activity ◽  
Lipid Hydroperoxides ◽  
Fluorescent Compound ◽  
Fluorescent Intensity ◽  
Cellular Assays ◽  
Z Value

15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z′ value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC50 determinations.

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