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

Article abstract of DOI:10.1016/j.bmcl.2008.06.015

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 × 10³ 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.

Full text of DOI:10.1016/j.bmcl.2008.06.015

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5864–5866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A highly sensitive fluorogenic probe for cytochrome P450 activity in live cells
Melissa M. Yatzeck a, Luke D. Lavis a, Tzu-Yuan Chao b, Sunil S. Chandran b, Ronald T. Raines a,b,
*
a Department of Chemistry, University of Wisconsin—Madison, 1101 University Avenue, Madison, WI 53706-1322, USA
b Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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,
Received 8 May 2008
Revised 3 June 2008
Accepted 4 June 2008
Available online 10 June 2008
fluorescence was manifested by the CYP1A1 isozyme with kcat/KM = 8.8 ꢀ 103 Mꢁ1 sꢁ1 and KM = 0.09
lM.
In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetra-
chlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Carcinogen
CYP1A1 isozyme
Cytochrome P450
Dioxin
Fluorogenic substrate
Prodrug
Resveratrol
Rhodamine 110
Trimethyl lock
The 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 xeno-
biotic 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
1970s, 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 fre-
quently as fluorophores in peptidase substrates based on Förster
resonance energy transfer (FRET).8 In addition, both 7-ethoxyres-
orufin and resorufin fluoresce brightly.6 This problem arises be-
cause O-alkylation of the hydroxyl group of fluorophores such
as coumarin and resorufin does little to deter the oxygen elec-
trons from participating in the resonance that gives rise to
fluorescence.
Here, we report on a superior small-molecule probe for assess-
ing 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 lactoniza-
tion to form a hydrocoumarin.12 In this strategy, the phenolic oxy-
gen 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
strategy is that the fluorescence/absorbance of the dye is masked
completely by amidic resonance and the resulting lactonization
within the rhodamine moiety.9,10
The human genome contains 27 genes encoding P450 iso-
zymes, along with many pseudogenes.13 Of these isozymes, cyto-
chrome 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 smoking.15 Many compounds, including some found
in cigarette smoke, are not hazardous until metabolized by
CYP1A1.16,17 Accordingly, CYP1A1 levels could be correlated with
human disease.
* Corresponding author. Address: Department of Biochemistry, University of
Wisconsin—Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA. Tel.: +1
608 262 8588; fax: +1 608 262 3453.
E-mail address: rtraines@wisc.edu (R.T. Raines).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.015

M. M. Yatzeck et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5864–5866
5865
We suspected that the trimethyl lock could provide the basis for
a useful probe for CYP1A1 activity. As a dye, we chose a morpho-
lino–urea derivative of rhodamine 110 (Rh110) that is bright
adenocarcinoma cell line A549, which is especially well suited
for studying the expression of the pulmonary CYP system.16,19
low but observable level of CYP1A1 was apparent after a 1-h incu-
A
(e
ꢀ
U
= 2.38 ꢀ 104 Mꢁ1 cmꢁ1) but has no measurable fluorescence
bation with fluorogenic probe 1 (Fig. 2B).
after N-acylation.10 We installed an ethyl group on the phenolic
oxygen of the trimethyl lock because ethyl ethers are especially
effective substrates for CYP1A1.15 The synthetic route to fluoro-
genic probe 1 is shown in Figure 1. Briefly, known intermediate
218 was alkylated with diethyl sulfate to give ethyl ether 3. Re-
moval of the silyl group followed by Jones oxidation afforded car-
boxylic acid 5. Condensation with urea–rhodamine
fluorogenic probe 1 in 5% overall yield.
6 gave
Fluorogenic probe 1 was first assayed as a substrate for human
CYP1A1 in vitro. Fluorogenesis was rapid, with kcat/KM
=
8.8 ꢀ 103 Mꢁ1 sꢁ1 and KM = 0.09
lM (Fig. 2A). These values are
Then, fluorogenic probe 1 was evaluated as a means to detect an
increase in CYP1A1 levels. To do so, A549 cells were incubated with
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM), which is the
notorious contaminant in the herbicide Agent Orange and the most
potent known inducer of CYP1A1.20 The effect of TCDD on fluoro-
genesis within A549 cells was dramatic (Fig. 2C).
comparable to the highest values obtained with other fluorogenic
substrates.1,2 These data are the first to demonstrate that the
trimethyl lock can be activated by the cleavage of an ether bond.
Next, fluorogenic probe 1 was assayed as a substrate for CYP1A1
in live human cells. These experiments employed human lung
Finally, fluorogenic probe 1 was used to reveal a more com-
plex modulation of P450 activity. Levels of P450 are highly var-
iable in individuals, and there are many known P450
polymorphisms.21 Inhibitors of P450 activity have potential as
chemotherapeutic agents.22 For example, resveratrol (3,5,40-tri-
Et2SO4
NaH
Et2O
OH
O
OTBS
OR
57%
hydroxystilbene), which is
a natural phytoalexin present in
2
3: R=TBS
4: R=H
AcOH, 98%
grapes and other foods, has been proposed to have a chemopro-
tective effect against lung cancer by virtue of its ability to de-
crease CYP1A1 activity.23 To test this hypothesis with
fluorogenic probe 1, live A549 cells were treated with both TCDD
and resveratrol, along the probe. After a 1-h incubation, cells
exhibited a dramatic decrease in fluorescence compared with
cells treated with TCDD (Fig. 2D). The levels appeared to be even
lower than those in untreated cells. These and other data23 pro-
Jones
O
34%
O
H
acetone
H2N
O
N
N
O
OH
—O2C
O
5
6
6, EDC
26%
pyr, DMF
O
O
H
N
H
N
A
O
N
2
O
O
O
O
1
not fluorescent
1
0
cytochrome P450
O—
O
O
H
N
H
O
N
N
O
0
2
4
O
O
[1] (µM)
O
O
B
C
D
fast
O
H
N
+
H2N
O
N
O
Figure 2. Fluorogenesis from fluorogenic probe 1 in vitro and in cellulo. (A) Data for
the in vitro cleavage of fluorogenic probe 1 by human CYP1A1 (5.0 pM) in PBS
containing NADPH (8 mM) and MgCl2 (8 mM). (B–D) Images of the in cellulo
cleavage of fluorogenic probe 1. A549 cells were incubated with fluorogenic probe 1
—O2C
6
fluorescent
(10
additive. (C) TCDD (10 nM). (D) TCDD (10 nM) and resveratrol (50
10 m.
l
M) and an additive for 1 h and counterstained with Hoechst 33342. (B) No
l
M). Scale bars:
l
Figure 1. Scheme for the synthesis and use of fluorogenic probe 1.

5866
M. M. Yatzeck et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5864–5866
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vide direct and conclusive evidence that resveratrol decreases
CYP1A1 activity in cellulo.
In conclusion, fluorogenic probe 1 is the first to utilize a ‘tri-
methyl lock’ that is triggered by cleavage of an ether bond. This
probe has numerous desirable attributes. Its chemical and photo-
physical properties allow for real-time imaging of P450 levels in
cellulo. The modularity of this probe enables its extension to en-
zymes throughout the P450 family, and its success indicates that
the trimethyl lock strategy can be applied to P450-activated pro-
drugs. Finally, appending the urea group with a trichloromethyl
ketone or other weak electrophile would allow the probe to react
with an intracellular thiol and enable its retention within a cell,
providing additional utility.24
Acknowledgments
This letter is dedicated to Professor Benjamin F. Cravatt on the
occasion of his winning the 2008 Tetrahedron Young Investigator
Award. This work was supported by Grant CA073808 (NIH). NMR-
FAM was supported by grant P41 RR02301 (NIH). M.M.Y. was a
Pfizer Undergraduate Summer Fellow in Chemistry and a Hilldale
Undergraduate/Faculty Research Fellow. L.D.L was supported by
Biotechnology Training Grant 08349 (NIH) and an ACS Division of
Organic Chemistry Graduate Fellowship sponsored by the Genen-
tech Foundation. T.-Y.C. was supported by the Dr. James Chieh-Hsia
Mao Wisconsin Distinguished Graduate Fellowship.
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Supplementary data
Detailed procedures for the synthesis, analysis, and use of fluo-
rogenic probe 1 can be found, in the online version, at doi:10.1016/
j.bmcl.2008.06.015.
References and notes
1. Cytochrome P450: Structure, Mechanism, and Biochemistry; Ortiz de Montellano,
P. R., Ed.; Kluwer Academic/Plenum Publishers: New York, 2005.
2. (a) Gungerich, F. P. Chem. Res. Toxicol. 2008, 21, 70; (b) Johnson, W. W. Drug
Metab. Rev. 2008, 40, 101.