scholarly journals A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase

2020 ◽  
Vol 132 (24) ◽  
pp. 9688-9696 ◽  
Author(s):  
Matthew G. Alteen ◽  
Christina Gros ◽  
Richard W. Meek ◽  
David A. Cardoso ◽  
Jil A. Busmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Activity Assay ◽  
Glcnac Transferase

scholarly journals A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase

2020 ◽  
Vol 59 (24) ◽  
pp. 9601-9609 ◽  
Author(s):  
Matthew G. Alteen ◽  
Christina Gros ◽  
Richard W. Meek ◽  
David A. Cardoso ◽  
Jil A. Busmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Activity Assay ◽  
Glcnac Transferase

scholarly journals A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

2021 ◽  
Author(s):  
Gang Liu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Direct Method ◽  
Total Concentration ◽  
Colorimetric Method ◽  
Industrial Applications ◽  
Assay Method ◽  
Activity Assay ◽  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity

Abstract Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

scholarly journals Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors

2012 ◽  
Vol 18 (4) ◽  
pp. 388-399 ◽  
Author(s):  
Syed Ahmad ◽  
Mark A. Hughes ◽  
Gary L. Johnson ◽  
John E. Scott
Keyword(s):  
Atpase Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Selective Inhibition ◽  
Mass Spectrometry Analysis ◽  
Activity Assay ◽  
Spectrometry Analysis ◽  
Compound Libraries ◽  
Atpase Assay

The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small-molecule inhibitors of MEKK2 activity.

Homogeneous single-label tyrosine kinase activity assay for high throughput screening

2015 ◽  
Vol 897 ◽  
pp. 96-101 ◽  
Author(s):  
Natalia Tong-Ochoa ◽  
Kari Kopra ◽  
Markku Syrjänpää ◽  
Nicolas Legrand ◽  
Harri Härmä
Keyword(s):  
Tyrosine Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Activity Assay ◽  
Tyrosine Kinase Activity ◽  
Kinase Activity Assay

scholarly journals Development of a Novel β-Secretase Binding Assay Using the AlphaScreen Platform

2013 ◽  
Vol 18 (6) ◽  
pp. 695-704 ◽  
Author(s):  
Zhao Ren ◽  
Danny Tam ◽  
Ying-Zi Xu ◽  
David Wone ◽  
Shendong Yuan ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Binding Assay ◽  
Parent Compound ◽  
High Signal ◽  
Activity Assay ◽  
Compound A ◽  
Nickel Chelate ◽  
Assay Conditions

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z′ values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization–based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened.

scholarly journals High-Throughput Scintillation Proximity Assay for Stearoyl-CoA Desaturase-1

2011 ◽  
Vol 16 (5) ◽  
pp. 506-517 ◽  
Author(s):  
Paul Tawa ◽  
Jean-Pierre Falgueyret ◽  
Sebastien Guiral ◽  
Elise Isabel ◽  
David A. Powell ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Activity Assay ◽  
Enzyme Activity Assay ◽  
Scintillation Proximity Assay ◽  
Binding Characteristics ◽  
Obesity And Diabetes ◽  
High Throughput Screening Assay ◽  
Stearoyl Coa Desaturase

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([3H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z′ factor (0.8). A screening collection of 1.6 million compounds was screened at 11 µM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [3H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.

scholarly journals High-Throughput Fluorescence-Based Activity Assay for Arginase-1

2020 ◽  
Vol 25 (9) ◽  
pp. 1018-1025
Author(s):  
Yvonne Grobben ◽  
Nicole Willemsen-Seegers ◽  
Joost C. M. Uitdehaag ◽  
Jos de Man ◽  
Jan van Groningen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Target ◽  
Rank Order ◽  
Activity Assay ◽  
Arginase 1 ◽  
Cell Immunity ◽  
Small Molecule Library ◽  
Kinetics Of

Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z′-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.

A tetravalent sialic acid-coated tetraphenylethene luminogen with aggregation-induced emission characteristics: design, synthesis and application for sialidase activity assay, high-throughput screening of sialidase inhibitors and diagnosis of bacterial vaginosis

2018 ◽  
Vol 54 (76) ◽  
pp. 10691-10694 ◽  
Author(s):  
Guang-jian Liu ◽  
Beihan Wang ◽  
Yuan Zhang ◽  
Guo-wen Xing ◽  
Xiaoli Yang ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Bacterial Vaginosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Emission Characteristics ◽  
Activity Assay ◽  
Design Synthesis ◽  
Aggregation Induced Emission ◽  
Sialidase Activity

A sialic acid-coated tetraphenylethene luminogen with excellent hydrophilicity was reported for sialidase activity assay and diagnosis of bacterial vaginosis.

scholarly journals Adaptation of nitrate reductase activity assay for high throughput screening of crops

2020 ◽  
Vol 49 (6) ◽  
pp. 23-33
Author(s):  
G I. Karlov ◽  
D. Y. Litvinov ◽  
P. N. Kharchenko ◽  
P. Yu. Krupin ◽  
S. Yu. Shirnin ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
High Throughput ◽  
Total Protein ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Potassium Nitrate ◽  
Activity Assay ◽  
Freeze Dried ◽  
The Difference ◽  
Nr Activity

The  possibility of freeze drying of plant material and its grinding in a shaking bead mill to determine the activity of nitrate reductase (NR) was studied. The effectiveness of applying this approach to high throughput mass screening of crops was shown. The assay was carried out on seedlings of common wheat (Triticum aestivum) of the following cultivars: Altigo, Vassa, Grom, Doka, Soberbash, Starshina, Fisht and spring wheat Novosibirskaya 67. The crops were grown during 4-5 weeks on substrate without nitrogen and on substrate supplemented with 50 millimol / l (mM) of potassium nitrate. Nitrate reductase in plants retained its activity after lyophilization and grinding of dried leaves in a mill. The proposed protocol for NR activity assay is suitable for plant lysates with an NR activity suffi cient to form nitrite in the range of 5–120 micromoles / l (μM) in 800 μl of reaction mix (for instance, freeze-dried sample originated from 100 mg of wheat seedling leaves).  Centrifugation of a plant lysate at 20,000 g almost did not change NR activity compared to 12,000 g that is achievable for most lab centrifuges. Lysates from fresh leaves contained signifi cantly more total protein than lysates from lyophilized leaves (with an equal amount of starting wet material). The difference in the nitrate-reducing activity in lysates from fresh and lyophilized leaves was not as high as the difference in protein concentration. Thus, the activity of NR calculated per g of total protein was higher in lyophilized leaves than in fresh leaves. The activity of NR was signifi cantly induced by nitrate for all cultivars. The basal and nitrate-induced NR activity varied widely between the cultivars, and the induction ranged from 2.5 fold for Novosibirskaya 67 variety and 2.7 fold for Vassa to 5.4 for Altigo and 5.7 fold for Grom.

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