Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro

Abstract

<jats:p>in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric,and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescentproduct from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays areusually highly sensitive and specific, allowing measurements on small specimens of tissues withlow enzyme activities. Fluorescence assays are also amenable to miniaturization of the reactionmixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives arewidely used as fluorophores due to their desirable photophysical properties. They possess a large -conjugated system with electron-rich and charge transfer properties. This conjugated structure leadsto applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe inthis review historical highlights and current use of coumarins and their derivatives in evaluatingactivities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarinsubstrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For thispurpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYPforms. With the aid of molecular modeling, we have recently described several new coumarin-basedsubstrates for measuring activities of CYP and conjugating enzymes with improved selectivity.</jats:p>

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

“…The use of other substrates would require de novo organic synthesis, which is beyond the scope of our study. Furthermore, umbelliferyl substrates are commonly used for multiple enzymes (xylanase, glucose oxidase, glycosidase, cellulase, deacetylase, phosphatase, and others), providing additional justification for attempting to make FACS-based screening compatible with hydrophobic substrates [ 38 , 39 , 40 ]. We therefore selected the synthetic fluorogenic substrate 4MUTC for the detection of chitinase activity.…”

Trending Topics on Coumarin and Its Derivatives in 2020

“…In addition to the interest of coumarins as a versatile scaffold in drug design, the important role that this scaffold plays as fluorescent probes to detect metals, enzymes, and biomaterials, among others, should be highlighted [ 56 , 57 , 58 , 59 ]. These fluorescent probes have a great imaging potential for the diagnosis of several pathologies.…”

“…Other reviews report on the use of 7-hydroxycoumarin and its derivatives in determining the activity of cytochromes P450 (CYP) enzymes [ 74 ] as well as 7-aminocoumarin derivatives in determining amino acids from serine or cysteine proteases [ 58 ]. In other cases, they are used to detect the metabolism of mitochondrial cysteines, the oxidation of which is a measure of cellular oxidative stress ( Figure 8 , compound LI ) [ 75 ].…”

Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling

“…Regarding the first application, fluorescence-based methods with coumarin substrates are sensitive, fast, reliable, simple, and low cost. 14 Determination of CYP1 activity is integrated into modern toxicological concepts and testing guidelines, emphasizing the importance of this enzyme for risk assessment and regulation of chemicals. 57 The second application arises from the need to detect CYP-mediated drug–drug interaction liability of drug candidates early in the drug discovery process.…”

“…The main challenge with profluorescent substrates is often their poor selectivity for the multiple CYP forms present in human and animal tissues. 14 The classical substrate for CYP1 enzymes, 7-ethoxyresorufin, is profluorescent. However, its oxidation to fluorescent resorufin is almost equally well catalyzed by all three CYP1 enzymes.…”