A derivative of rhodamine 110 has been designed and assessed as a probe for cytochrome P450 activity. This probe is the first to utilize a "trimethyl lock" that is triggered by cleavage of an ether bond. In vitro, fluorescence was manifested by the CYP1A1 isozyme with k cat /K M = 8.8 × 10 3 M −1 s −1 and K M = 0.09 µM. In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol. KeywordsCarcinogen; CYP1A1 Isozyme; Cytochrome P450; Dioxin; Fluorogenic Substrate; Prodrug; Resveratrol; Rhodamine 110; Trimethyl LockThe cytochrome P450 (P450) family of enzymes is responsible for the oxidative metabolism of a wide variety of compounds, including chemotherapeutic agents and environmental toxins. 1,2 The catalytic activity of P450 enzymes controls the rate of xenobiotic metabolism, and can produce undesirable byproducts. 3 Originally, this activity had been assessed by using HPLC or other methods to separate and quantify metabolites. In the 1970's, 7-ethoxycoumarin and 7-ethoxyresorufin were introduced as the first fluorogenic substrates for assays of P450 activity. 4 Although these and other fluorogenic substrates have been used to assay P450 activity in vitro and enable assays in cellulo ,5,6 they suffer from background fluorescence. 7 For example, alkoxycoumarins exhibit moderate fluorescence and are used frequently as fluorophores in peptidase substrates based on Forster resonance energy transfer (FRET). 8 In addition, both 7-ethoxyresorufin and resorufin fluoresce brightly. 6 This problem arises because O-alkylation of the hydroxyl group of fluorophores such as coumarin and resorufin does little to deter the oxygen electrons from participating in the resonance that gives rise to fluorescence.Here, we report on a superior small-molecule probe for assessing P450 activity. Our probe employs the trimethyl lock. 9-11 The trimethyl lock is an o-hydroxycinnamic acid derivative in which severe crowding of three methyl groups induces rapid lactonization to form a hydrocoumarin. 12 In this strategy, the phenolic oxygen of the o-hydroxycinnamic acid is modified to create a functional group that is a substrate for a designated enzyme, and the carboxyl group is condensed with the amino group of a dye. Unmasking of the phenolic oxygen leads to rapid lactonization with concomitant release of the dye. An important attribute of this The human genome contains 27 genes encoding P450 isozymes, along with many pseudogenes. 13 Of these isozymes, cytochrome P450 1A1 (CYP1A1) is known to be especially important in the metabolism of xenobiotics. 14 Unlike most P450 isoforms, which are found primarily in the liver, CYP1A1 is present mainly in the lungs, where it plays an important role in the metabolic activation of chemical carcinogens. 1,15 The lung is a primary site of exposure for inhaled toxins along with carcinogens that can ultimately yield lung carcinomas. 16 CYP1A1 is strongly induced by cigarette smo...

Cells -[synthesis, analysis and use of fluorogenic probe (I)]. -(YATZECK, M. M.; LAVIS, L. D.; CHAO, T.-Y.; CHANDRAN, S. S.; RAINES*, R. T.; Bioorg. Med.