3-Cyano-7-ethoxycoumarin

3-Cyano-7-ethoxycoumarin is a fluorogenic cytochrome P-450 substrate that generates blue fluorescent product upon enzyme cleavage Target: Cytochrome P450 3-Cyano-7-ethoxycoumarin is a fluorescent probe useful in microsomal dealkylase studies. 3-Cyano-7-ethoxycoumarin is a high purity and quality chemical used as molecular probe.Typical drug-drug interactions resulting from enzyme inhibition.

A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin.[Pubmed:3189781]

Anal Biochem. 1988 Aug 1;172(2):304-10.

A direct fluorometric procedure for the continuous determination of cytochrome P-450-dependent mixed function oxidases, using 3-Cyano-7-ethoxycoumarin substrate, is described. The reaction product, 3-cyano-7-hydroxycoumarin, is fluorescent at neutral pH values (excitation and emission wavelength maxima: 408 and 450 nm, respectively). Using hepatic microsomal preparations from control rats, the enzyme(s) had an apparent Km of 16 microM. Vmax values (0.5 nmol/min/mg protein) were induced 6- and 21-fold by pretreatment of rats with phenobarbitone and about 50- to 100-fold more sensitive than the ethoxyresorufin deethylase assay. Reaction rates using 3-cyano-7-pentoxycoumarin as substrate were generally much lower than with the ethoxy analog. 3-Cyano-7-ethoxycoumarin can also be used as a substrate to measure mixed function oxidases in isolated hepatocytes. However, 3-cyano-7-hydroxycoumarin shows a time- and concentration-dependent loss of fluorescence when incubated with such cells. This causes an approximately 5% underestimate of the true reaction rates.

Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes.[Pubmed:15205384]

Drug Metab Dispos. 2004 Jul;32(7):699-706.

In this study we describe a battery of fluorescence assays for rapid measurement in intact cells of the activity of nine cytochromes P450 (P450s) involved in drug metabolism. The assays are based on the direct incubation of monolayers of cells expressing individual P450 enzymes with a fluorogenic substrate followed by fluorimetric quantification of the product formed and released into incubation medium. For each individual P450 activity, different fluorescence probes were examined, and the one showing the best properties (highest metabolic rates, lowest background fluorescence) was selected: 3-Cyano-7-ethoxycoumarin for CYP1A2 and CYP2C19, coumarin for CYP2A6, 7-ethoxy-4-trifluoromethylcoumarin for CYP2B6, dibenzylfluorescein for CYP2C8, 7-methoxy-4-trifluoromethylcoumarin (MFC) for CYP2C9 and CYP2E1, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin for CYP2D6, and 7-benzyloxy-4-trifluoromethylcoumarin for CYP3A4. Fluorescence-based assays are highly sensitive and allow the simultaneous measurement of a large number of samples using plate readers, thus enhancing sample throughput. Major advantages over high-throughput assays in subcellular fractions are that, as living cells are used, manual handling and enzyme damage are minimized, the endoplasmic reticulum of the cells remains intact, exogenous cofactors or NADPH-regenerating systems are not required, and transport processes are maintained. These assays can be applied to preliminary screening of inhibitory effects of new drugs on individual P450 enzymes. After comparison of the results obtained using the fluorescent probes in intact P450-expressing cells and those obtained using the high-performance liquid chromatography-based selective assays in the same cells, in primary human hepatocytes or in human liver microsomes, a fairly good agreement was found.

3-Cyano-7-ethoxycoumarin is a fluorogenic cytochrome P-450 substrate that generates blue fluorescent product upon enzyme cleavage Target: Cytochrome P450 3-Cyano-7-ethoxycoumarin is a fluorescent probe useful in microsomal dealkylase studies.

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