scholarly journals Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

10.3791/61146 ◽  
2020 ◽  
Author(s):  
Karine Nozeret ◽  
Aurélia Pernin ◽  
Nienke Buddelmeijer
Keyword(s):  
Click Chemistry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Characterization ◽  
Fluorometric Assay

Rapid build-up of high-throughput screening microarrays with biochemistry gradients via light-induced thiol–ene “click” chemistry

2021 ◽  
Vol 9 (13) ◽  
pp. 3032-3037
Author(s):  
Hongye Hao ◽  
Junjie Huang ◽  
Ping Liu ◽  
Yunfan Xue ◽  
Jing Wang ◽  
...  
Keyword(s):  
Click Chemistry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Behaviors

Microarrays with biochemistry gradients were rapidly fabricated via light-induced thiol–ene “click” chemistry and showed great applicability in cell behaviors screening.

scholarly journals A click chemistry-based microRNA maturation assay optimized for high-throughput screening

2016 ◽  
Vol 52 (53) ◽  
pp. 8267-8270 ◽  
Author(s):  
Daniel A. Lorenz ◽  
Amanda L. Garner
Keyword(s):  
Click Chemistry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Diels Alder ◽  
Inverse Electron Demand ◽  
Electron Demand

A catalytic enzyme-linked click chemistry assay (cat-ELCCA) for Dicer-catalyzed pre-microRNA maturation was optimized to employ inverse-electron demand Diels–Alder (IEDDA) chemistry affording high-throughput screening capability.

SuFEx-Enabled High-Throughput Medicinal Chemistry

2019 ◽  
Author(s):  
Seiya Kitamura ◽  
Qinheng Zheng ◽  
Jordan L. Woehl ◽  
angelo solan ◽  
Emily Chen ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Small Molecule ◽  
High Throughput ◽  
Cysteine Protease ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Biologically Active ◽  
Drug Candidates ◽  
Biological Probes ◽  
Small Molecule Probes

<p>Optimization of small-molecule probes or drugs is a lengthy, challenging and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible SuFEx click chemistry. A modest high-throughput screening hit against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN=S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products directly screened to yield drug-like inhibitors with 300-fold higher potency. We showed that the improved molecule is drug-like and biologically active in a bacteria-host coculture. Since these reactions can be performed on a picomole scale to conserve reagents, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.</p>

scholarly journals Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Lakshmi Ganesan ◽  
Peyton Shieh ◽  
Carolyn R. Bertozzi ◽  
Ilya Levental
Keyword(s):  
Click Chemistry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Platform

SuFEx-Enabled High-Throughput Medicinal Chemistry

2019 ◽  
Author(s):  
Seiya Kitamura ◽  
Qinheng Zheng ◽  
Jordan L. Woehl ◽  
angelo solan ◽  
Emily Chen ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Small Molecule ◽  
High Throughput ◽  
Cysteine Protease ◽  
High Throughput Screening ◽  
Medicinal Chemistry ◽  
Biologically Active ◽  
Drug Candidates ◽  
Biological Probes ◽  
Small Molecule Probes

<p>Optimization of small-molecule probes or drugs is a lengthy, challenging and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible SuFEx click chemistry. A modest high-throughput screening hit against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN=S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products directly screened to yield drug-like inhibitors with 300-fold higher potency. We showed that the improved molecule is drug-like and biologically active in a bacteria-host coculture. Since these reactions can be performed on a picomole scale to conserve reagents, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.</p>

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

2011 ◽  
Vol 412 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Ruo-Yu Zhang ◽  
Ye Qin ◽  
Xiao-Qun Lv ◽  
Pei Wang ◽  
Tian-Ying Xu ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Fluorometric Assay ◽  
Nicotinamide Phosphoribosyltransferase

Discovery of novel USP8 inhibitors via Ubiquitin-Rho-110 fluorometric assay based high throughput screening

2020 ◽  
Vol 101 ◽  
pp. 103962
Author(s):  
Jie Han ◽  
Yucheng Tian ◽  
Liang Yu ◽  
Qilin Zhang ◽  
Xi Xu ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Fluorometric Assay

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.

Sign in / Sign up

Export Citation Format

Share Document