Abstract:We have developed a fluorogenic NAD(P)(+) detection method using 2-acetylbenzofuran. The reaction of NAD(P)(+) with 2-acetylbenzofuran produced a fluorescent product, allowing the highly-sensitive and quick detection of NAD(P)(+). This method was successfully applied to the detection of P450 substrates in the microtiter-plate format.
“…24 The fact that the presented methodology can also be used to measure NADPH consumption shows that the setup is not limited to fluorogenic and chromogenic substrates. However, although NADPH oxidation has been applied as a generic method for measuring CYP activities, [25][26][27] it does not necessarily represent product formation because several substrate/CYP combinations show a high degree of uncoupling, as is the case with metabolism of alkoxyresorufins by CYP BM3 mutants. The most generic method therefore would be online detection of metabolites by MS, as has been successfully applied for analysis of products formed by CYP BM3mediated metabolism of steroids and p38α kinase inhibitors.…”
Section: Cyp Bm3-mediated Metabolism Of the Drug Amitriptylinementioning
A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3-mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.
“…24 The fact that the presented methodology can also be used to measure NADPH consumption shows that the setup is not limited to fluorogenic and chromogenic substrates. However, although NADPH oxidation has been applied as a generic method for measuring CYP activities, [25][26][27] it does not necessarily represent product formation because several substrate/CYP combinations show a high degree of uncoupling, as is the case with metabolism of alkoxyresorufins by CYP BM3 mutants. The most generic method therefore would be online detection of metabolites by MS, as has been successfully applied for analysis of products formed by CYP BM3mediated metabolism of steroids and p38α kinase inhibitors.…”
Section: Cyp Bm3-mediated Metabolism Of the Drug Amitriptylinementioning
A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3-mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.
“…31 By analogy with chelerythrine with its quaternary ammonium group that is linked by three carbons to an oxygen, we aimed to develop 8-hydroxy-2,7-naphthyridin-2-ium salts (3) as potential inhibitors of AChE and BChE. The synthesis of the highly fluorescent 8-hydroxy-2,7-naphthyridin-2ium salts, as well as their conjugated bases, the 2,7naphthyridin-1IJ7H)-ones, has already been published, [32][33][34][35] however, no distinct biological activity has been attributed to this chemotype. Instead, the formation of 8-hydroxy-2,7naphthyridin-2-ium salts was utilized as an operating principle of several fluorescence-based assay methods to detect nicotinamide and its analogues.…”
By analogy with the natural product chelerythrine, which has been identified as an inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), we prepared a small series of 8-hydroxy-2,7-naphthyridin-2-ium salts. Spectroscopic analyses allowed us to elucidate the zwitterionic nature of 2,7-naphthyridin-1(7)-ones, the neutral state of 8-hydroxy-2,7-naphthyridin-2-ium salts. Among the tested compounds, we identified dual inhibitors of AChE and BChE as well as an inhibitor showing a preferential inhibition of AChE over BChE. By characterization in combination with docking studies, we were able to identify structural features that influence the biological activity of 8-hydroxy-2,7-naphthyridin-2-ium salts.
“…Previously, therefore, we sought sensitive NAD(P) + detection reagents and platforms 34 to be used as high-throughput P450 substrate screens, and found that 2-acetylbenzofuran (2-ABF), which generates fluorescent 2,7-naphthyridinone derivatives upon reaction with NAD(P) + , is a more sensitive, efficient, and inexpensive NAD(P) + detection reagent compared with the pre-existing reagent (Fig. 1a) 35 . 2-ABF reacts with NAD + 20 times faster than acetophenone, the standard reagent, and detects NAD + with 1000-fold greater sensitivity 35 .…”
Section: Resultsmentioning
confidence: 99%
“…1a) 35 . 2-ABF reacts with NAD + 20 times faster than acetophenone, the standard reagent, and detects NAD + with 1000-fold greater sensitivity 35 . The NAD(P) + detection method using 2-ABF, termed the 2-ABF method, is thought to be well-suited for microtiter plate-based screening of P450 substrates.Figure 1Summary of the integrated P450 substrate screening system.…”
Information about substrate and product selectivity is critical for understanding the function of cytochrome P450 monooxygenases. In addition, comprehensive understanding of changes in substrate selectivity of P450 upon amino acid mutation would enable the design and creation of engineered P450s with desired selectivities. Therefore, systematic methods for obtaining such information are required. Herein, we developed an integrated P450 substrate screening system for the selection of “exemplary” substrates for a P450 of interest. The established screening system accurately selected the known exemplary substrates and also identified previously unknown exemplary substrates for microbial-derived P450s from a library containing sp3-rich synthetic small molecules. Synthetically potent transformations were also found by analyzing the reactions and oxidation products. The screening system was applied to analyze the substrate selectivity of the P450 BM3 mutants F87A and F87A/A330W, which acquired an ability to hydroxylate non-natural substrate steroids regio- and stereoselectively by two amino acid mutations. The distinct transition of exemplary substrates due to each single amino acid mutation was revealed, demonstrating the utility of the established system.
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