A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells

Abstract

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...

Crosstalk Between Co-cultured A549 Cells and THP1 Cells Exposed to Cigarette Smoke

“…It should be stressed that CS extract contain several toxic compounds as nicotine, acrolein, formaldehyde, hydrogen cyanide, polycyclic aromatic hydrocarbons, and nitrosamines (Moylan et al 2013) and A549 cells have low activity of cytochrome P450 enzymes, which are responsible for metabolism of several xenobiotics (Yatzeck et al 2008). Published CS toxicity data in A549 cells are highly variable aïve or CS-pretreated (24 h) A549 cells were grown in co-cultures without physical contact with naïve or CS-pretreated (24 h) THP1 cells which were grown in co-culture inserts allowing to diffuse soluble molecules to common culture medium (1:1 mixture of F12K and RPMI 1640 media).…”

Trimethyl lock: a trigger for molecular release in chemistry, biology, and pharmacology

“…17). 53 This substrate revealed the induction of activity in lung cells by a carcinogenic dioxin, as well as its repression by a chemoprotectant, resveratrol.…”

Trimethyl Lock: A Multifunctional Molecular Tool for Drug Delivery, Cellular Imaging, and Stimuli‐Responsive Materials

“…[183] The catalytic activity of P450 enzymes controls the rate of xenobiotic metabolism through hydroxylative degradation,w hich leads to renal clearance and can produce undesirable by-products. [186] They silence fluorescenceo fm orpholinourea rhodamine 97 by N-acylation with aT ML moiety bearing ap henylethyl ether to obtain compound 134 (Scheme 30). [186] They silence fluorescenceo fm orpholinourea rhodamine 97 by N-acylation with aT ML moiety bearing ap henylethyl ether to obtain compound 134 (Scheme 30).…”