Target Enzyme-Activated Two-Photon Fluorescent Probes: A Case Study of CYP3A4 Using a Two-Dimensional Design Strategy

Article abstract of DOI:10.1002/anie.201903683

The rapid development of fluorescent probes for monitoring target enzymes is still a great challenge owing to the lack of efficient ways to optimize a specific fluorophore. Herein, a practical two-dimensional strategy was designed for the development of an isoform-specific probe for CYP3A4, a key cytochrome P450 isoform responsible for the oxidation of most clinical drugs. In first dimension of the design strategy, a potential two-photon fluorescent substrate (NN) for CYP3A4 was effectively selected using ensemble-based virtual screening. In the second dimension, various substituent groups were introduced into NN to optimize the isoform-selectivity and reactivity. Finally, with ideal selectivity and sensitivity, NEN was successfully applied to the real-time detection of CYP3A4 in living cells and zebrafish. These findings suggested that our strategy is practical for developing an isoform-specific probe for a target enzyme.

Full text of DOI:10.1002/anie.201903683

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Targeted Enzyme Activated Two-Photon Fluorescent Probes:
A Case Study of CYP3A4 Using a Two-Dimensional Design
Strategy
Authors: Jing Ning, Wei Wang, Guangbo Ge, Peng Chu, Feida Long,
Yongliang Yang, Yulin Peng, Lei Feng, Xiaochi Ma, and Tony
D. James
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903683
Angew. Chem. 10.1002/ange.201903683
Link to VoR: http://dx.doi.org/10.1002/anie.201903683
http://dx.doi.org/10.1002/ange.201903683

10.1002/anie.201903683
Angewandte Chemie International Edition
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Targeted Enzyme Activated Two-Photon Fluorescent Probes: A
Case Study of CYP3A4 Using a Two-Dimensional Design Strategy
Jing Ning, Wei Wang, Guangbo Ge, Peng Chu, Feida Long, Yongliang Yang,* Yulin Peng, Lei Feng,*
Xiaochi Ma*, and Tony D. James
Abstract: Nowadays, the rapid development of fluorescent probe for
regarded as the most important CYP isoform in human, due to its
monitoring target enzyme is still a great challenge, due to the lack of high abundance in liver as well as broad substrate spectrum which
efficient way to optimize a specific fluorophore. Herein, a practical
two-dimension molecular strategy was designed to construct an
isoform-specific probe for CYP3A4, a key cytochrome P450 isoform
responsible for oxidize most of clinical drugs. In first dimension of the
molecular design, using ensemble-based virtual screening, the
potential two-photon fluorophore substrate (NN) for CYP3A4 was
effectively filtered. For second dimension by chemical modification,
various substituent groups were introduced to candidate NN for
optimizing the isoform-selectivity and reactivity. Finally, with ideal
selectivity and sensitivity, NEN was successfully applied to real-time
detect CYP3A4 in living cells and zebrafish, thereby providing an
efficient tool for quantitative tracking of CYP3A4 activity in complex
biological systems. These findings suggested that our strategy is
practical to develop an isoform-specific probe for a target enzyme.
contributes to that metabolism of more than 50% of clinical drugs.[2,3]
Unfortunately, CYP3A4 activity can be modulated by many clinical
drugs, thereby frequently causing unfavorable drug-drug interactions
(DDI), further leading to altered clinical outcomes or even life-
threatening adverse reactions.[4] Additionally,
a significant inter-
individual variability of CYP3A4 activity is frequently reported, arising
from a number of sources including genetic polymorphism and the
response to environmental influences.[5] These factors have greatly
limited the understanding of the precise role of CYP3A4 in drug
metabolism and DDI, as
a result, negatively impacted clinical
medication safety and effectiveness.
To accurately characterize CYP3A4 activity, sensitive
technologies capable of real-time monitoring CYP3A4 activity are
urgently needed. Traditional detection methods mainly rely upon
mass spectrometry or high-performance liquid chromatography,[6,7]
while they are not compatible with living cell or in vivo applications.
Two-photon (TP) fluorescence microscopy, by virtue of its higher
sensitivity, real-time spatial high-resolution imaging, and amenability
to deep-tissue bioimaging, has shed new light on monitoring target
enzyme activity in complex systems.[8,9] However, there is still no TP
fluorescent probe developed for real-time and selective imaging of
endogenous CYP3A4 activity in living systems.
Cytochrome P450 monooxygenase (CYP) is
a superfamily of
oxidative enzymes that metabolizes thousands of endogenous and
exogenous substances through alkyl carbon and aromatic ring
hydroxylation, O- and N-dealkylation, and epoxidation.[1] CYP3A4 is
[ ] Dr. J. Ning,[†] P. Chu,[†] Y. L. Peng, Dr. L. Feng, Prof. X. C. Ma
*
Previous attempts to develop a fluorescent probe for CYP3A4
were based on a dealkylation mechanism to release a detectable
moiety. The O-alkyl derivatives of coumarin, resorufin, and
fluorescein were designed and synthesized, but met with limited
success in isoform selectivity.[10] The leading cause for the poor
College of Integrative Medicine, The National & Local Joint
Engineering Research Center for Drug Development of
Neurodegenerative Disease, College of Pharmacy
Dalian Medical University
Dalian 116044, China
E-mail: leifeng@mail.dlut.edu.cn (L. Feng); maxc1978@163.com (X.
C. Ma)
specificity of these probes is that dealkylation usually is
a
preferential reaction for CYP1A1 and CYP1A2 rather than CYP3A4,
especially with polycyclic aromatic compounds.[11] In the light of the
extraordinary ability of CYP3A4 to mediate the aromatic
hydroxylation,[12] we hypothesized that the high specific probe of
CYP3A4 might be obtained by tuning the fluorescence-generation
using site specific hydroxylation to ‘Switch-ON’ the fluorophore.
Nowadays, the greatest challenge in designing an isoform-
selective and sensitive activity-based fluorescent probe is how to
P. Chu,[†] F. D. Long, Prof. Y. L. Yang, Dr. L. Feng
Center for Molecular Medicine, School of Life Science and
Biotechnology, State Key Laboratory of Fine Chemicals
Dalian University of Technology
Dalian 116024, China
E-mail: everbright99@foxmail.com (Y. L. Yang)
Prof. X. C. Ma
Jiangsu Key Laboratory of New Drug Research and Clinical
Pharmacy
Xuzhou Medical University
Xuzhou 221004, China.
effectively find a specific fluorophore and optimize the structure.[13]
A
simple ‘lock and key’ method to acquire a rational fluorophore is
urgently needed. Herein, we developed a two-dimensional design
strategy to achieve an overall "best-fit" molecular tool for selective
detection of CYP3A4, which would provide the molecular design
strategy from scratch virtual screening of putative two-proton
fluorescent substrates (Scheme 1 and Figure S1).[14] For the first
design dimension, an in-house docking program FIPSDock was
used to perform the constrained ensemble-based hydroxylation of
the specific sites on fluorophore. For sensitive fluorescence
detection, the introduction of a hydroxyl to fluorophore substrates
producing significant fluorescence response, was set as the
restrictive reaction and thus was limited to the catalytic site. To
ensemble the conformational changes of CYP3A4 upon binding with
the different sizes of substrates, we obtained twelve crystallographic
human CYP3A4 structures from the Protein Data screening against
the Bank (PDB code: 1TQN, 1W0F, 1W0G, 2J0D, 2V0M, 3TJS,
E-mail: maxc1978@163.com (X. C. Ma)
Prof. W. Wang[†]
School of Pharmacy
Hunan University of Chinese Medicine
Changsha 410208, China
Prof. G. B. Ge[†]
Institute of Interdisciplinary Integrative Medicine Research
Shanghai University of Traditional Chinese Medicine
Shanghai, 201203, China
Prof. T. D. James
Department of Chemistry
University of Bath
Bath BA2 7AY, United Kingdom
[†] These authors contributed equally.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201903683
Angewandte Chemie International Edition
COMMUNICATION
4I4G, 4K9T, 4K9U, 4K9V, 5TE8 and 6BD8) as the receptor
ensemble for virtual screening during which the given catalytic sites
of these substrates were constrained to 4.5 Å from the Fe atom of
the heme (C-Fe). For each lead fluorophore, the receptor ensemble
will generate a binding spectrum with twelve binding scores. The
mean value of the binding scores were recorded and used to rank
these substrates of CYP3A4. We selected top 5 ranked CYP3A4
substrates which were then subjected to further constrained
molecular dynamics simulation and free energy calculation using the
Poisson–Boltzmann surface area continuum solvation (MM/PBSA)
methods (Table S1). Lastly, N-n-butyl-1,8-naphthalimide with the
hydroxylation of C-4, stands out as the optimal candidate with the
best binding free energy in the screening campaign (Figure 1). N-
butyl and N-phenyl derivatives of naphthalimide (NN) were
synthesized to verify the virtual screening results. It found that
CYP3A4 could hydroxylate the two derivatives and caused
remarkable fluorescence enhancement at 558 nm (Figure S2, S3).
This result suggested that the first step in molecular design for
rapidly screening fluorophore structures was very efficient.
Scheme 1. Two-dimensional strategy to develop
a
two-photon
fluorescent probe for CYP3A4. A. In the first dimension, an in-house
docking program was used to perform the constrained virtual screening
of 58 putative two-proton fluorophores with the curated CYP3A4 receptor
ensemble, and NN was identified as the most promising fluorescent
substrate for CYP3A4. B. In the second dimensional design, the chemical
modification at a nitrogen atom of NN was carried out to optimize the
reaction rate and isoform-selectivity of NN activation by 4-hydroxylation.
The CYP isoform-selectivity of 18 NN derivatives was shown as an insert.
The brightness of green represents the rate of activation of NN
derivatives by different subtypes of CYPs. The derivative 2 (NN 2, NEN)
displayed the great reactivity and high selectivity toward CYP3A4 over
other CYPs isoforms.
Finally, NN was selected as the fluorophore in the present study,
because it possesses CYP3A4 preference and desirable
photophysical properties, such as high photostability and significant
two-photon absorbability at the near infrared wavelengths.[15]
Hydroxylation at C-4 triggers significant change in the fluorescence
spectra, which makes NN
a desirable OFF-ON two-photon
fluorescent probe based on the mechanism of internal charge
transfer (ICT).[16] CYP1As and isoforms of other subfamily did not
participate in the hydroxylation with the disappearance of emission
at 558 nm. However, it can still be hydroxylated by CYP3A5, a highly
homologous isoform of CYP3A4.[17]
Subsequently, according to the structural features of NN and
CYP3A4 catalytic cavity, we introduced various substituent groups to
non-reaction site of NN, to adjust the reactive rate and isoform-
selectivity (Figure S2), all of which construct our secondary design
dimension. Briefly, the nitrogen atom of NN was selected as the
modification site to introduce the substituents including steric bulk,
hydrophilic and hydrophobic groups. A library of NN derivatives (NN
1-18) were synthesized to evaluate their potential as selective
substrates for CYP3A4 by reaction phenotyping-based experimental
screening (Figure S2). When introducing the benzene and its
analogues to nitrogen atom of NN (NN 10-13), the reactive rates and
isoform-selectivity were decreased. Introducing the hydrophobic
substituents including alkanes with different lengths, some short-
chain alkyl derivatives such as NN 1 (methyl) and NN 2 (ethyl)
exhibited good reactivity and high specificity toward CYP3A4, rather
than other derivatives with long-chain alkanes (NN 3-9). Additionally,
the hydrophilic substituents (NN 15-18) also decreased the reaction
rates. N-ethyl-1,8-naphthalimide (NEN, NN 2) displayed a good
reactivity and high selectivity toward CYP3A4 over the other
isoforms (Figure S3). Subsequently, we explore the feasibility and
practicability of this activatable fluorescence method in the complex
biological systems, and thereby examine our two-dimension
molecular design strategy.
Figure 1. Computational screening and discovery of fluorescent
substrates for CYP3A4. (A) Overlay of twelve structurally diverse
CYP3A4 structures as the receptor ensemble for virtual screening. (B)
The heatmap derived from the virtual screening against the receptor
ensemble consisting of 12 binding scores for each of 58 substrates. (C)
The distance of C4 atom (catalytic site) as well as C5 atom of S39 with
Fe of heme (C-Fe) along the time trajectory during 10 ns position
constrained MD simulations. (D) S39 displayed in red stick binding in the
catalytic site of CYP3A4 structure derived from constrained MD
simulation.
The specificity of NEN for CYP3A4 was firstly investigated. As
shown in Figure 2B, CYP3A4 caused remarkable fluorescence
enhancement at 558 nm, whereas there was no obvious
fluorescence changes upon addition of other CYP isoforms.
Moreover, NEN exhibits high selectivity for CYP3A4 over various
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10.1002/anie.201903683
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us to use this probe for quantitative measurement of CYP3A4
activity.
Using NEN, the catalytic activity of CYP3A4 in a panel of 13
individual HLM can be efficiently determined (Figure 3A). More than
11-fold variation in the catalytic activity was observed. These data
were compared with catalytic activity and protein content of CYP3A4
in the corresponding HLM, and a strong positive correlation was
observed (Figure 3B, S9).[6] Moreover, the CYP3A4 activity of
different tissue homogenates was consistent with the corresponding
protein levels of CYP3A4 (Figure S10). These findings demonstrated
that NEN could serve as a novel molecular tool for in vitro detecting
CYP3A4 in biospecimens.
The high specificity and two-photon absorption properties of NEN
make it possible to directly detect CYP3A4 activity in intact cells and
tissues. To explore the feature, human primary hepatocytes, which
express various CYP isoenzymes, were used as model cells. Human
primary hepatocytes were loaded with NEN (50 µM) at 37 °C for 1 h,
and an obvious fluorescence enhancement in cells was observed
(Figure 4). CYP3A4 inhibition and induction assays were also
performed, where the cells were treated with TKZ (50 µM) or
rifampin (10 µM, a inducer of CYP3A4).[19] TKZ pretreated cells
Figure 2. Fluorescence response of NEN towards CYP3A4. A) Proposed
mechanism of CYP3A4 triggering the fluorescence response of NEN. B)
Fluorescence response of NEN (10 μM) following incubation with various
human CYPs. C) Fluorescence spectra of NEN (10 μM) following
incubation with increasing concentrations of CYP3A4. CYP3A4
dependent fluorescence intensity change at 558 nm is shown as an inset.
potential interfering species (Figure S4). To further verify the
selectivity of NEN, inhibition assays were conducted in human liver
microsome (HLM) using some selective inhibitors of CYP isoforms.
The hydroxylation of NEN can be strongly inhibited by CYP3cide and
ketoconazole (TKZ), which are selective and potent inhibitors of
CYP3A4 (Figure S5).[18] These results indicated that NEN was highly
selective for CYP3A4.
exhibited
a largely suppressed fluorescence, while the cells
pretreated with rifampin exhibited an enhanced fluorescence. The
HPLC analysis for detecting cell lysates showed that the production
of NEHN was well corresponding to the fluorescence intensity in the
cells of different treated groups (Figure S12). Additionally, the TP
imaging of a rat liver slice showed the practicability of NEN for
imaging CYP3A4 in deep tissues (Figure S13). The images at the
various depths showed the distribution of CYP3A4. All these results
demonstrated that NEN displayed good cell permeability and deep
tissue penetration capability which were suitable for direct detecting
endogenous CYP3A4 activity of cell and tissue.
The metabolite of NEN was identified as N-ethyl-4-hydroxyl-1,8-
naphthalimide (NEHN, Φfl = 0.199 in PBS:ACN, v/v = 1:1) by
CYP3A4, by comparison of LC retention times, UV and MS spectra
with those of a standard compound (Figure S6). NEN showed
extremely weak fluorescence over a wide pH range, while NEHN
showed strong fluorescence with maxima at 558 nm over pH range
of 7-12 (Figure S7). As a result, CYP3A4-mediated 4-hydroxylation
of NEN led to discernible changes in the fluorescence emission
(Figure 2A). The fluorescence intensity increased linearly when
CYP3A4 was progressively added from 0 to 0.1 mg/mL (R2 = 0.997,
Y = 2.22 × 105 * x + 539.9) (Figure 2C). To understand the
interaction between NEN and CYP3A4, the kinetics was assayed. As
shown in Table S2 and Figure S8, NEN 4-hydroxylation in CYP3A4
and HLM followed the classic Michaeli−Menten kinetics, and
displayed high affinity and good reactivity. However, the catalytic
efficiency of CYP3A5 was very low, with at least 55-fold lower
inherent clearance (Vm/Km) than CYP3A4. The preferred
characteristics and ideal kinetic behavior of probe reaction prompting
Understanding and predicting whether new drug candidates will
induce drug-drug interactions is a critical hurdle in pharmaceutical
development.[20] Encouraged by the above findings, we investigate
the utility of NEN for high-thoughput screening of CYP3A4
modulators in vitro and in vivo. One known chemical inhibitor of
CYP3A4 (TKZ) was used to evaluate its inhibitory effects toward
CYP3A4 in HLM and recombinant CYP3A4. TKZ inhibited NEN 4-
hydroxylation in a dose-dependent manner with similar inhibitory
tendency as determined in the two enzyme sources (Figure S15).
Additionally, a known CYP3A4 inducer (dexamethasone, DEX) was
used to evaluate its effects on the expression and activity of
CYP3A4 in rat primary hepatocytes.[19] After co-incubation with DEX
for 3 days, cells showed a dose-dependent increase of fluorescence,
corresponding to the elevated formation rate of NEHN and CYP3A4
content in the cell lysates (Figure S16). Hence, NEN could be used
to screen and characterize the CYP3A4 modulators. Unlike general
CYP3A4-detecting assays such as the previous probe reaction or
antibody-based enzyme-linked immunosorbent assay, NEN provides
a simple and rapid approach to selectively and real-time detect
CYP3A4 activity in cells or crude proteomes.[20]
Zebrafish is a relatively new model system for drug metabolism
and toxicity studies, offering whole organism screening coupled with
the potential for high-throughput screening.[21] In our study, zebrafish
Figure 3. A) The catalytic activity of CYP3A4 in 13 individual HLM using
NEN as the probe. B) Correlation between reactivity and the CYP3A4
protein content in HLM. The activity for NEN 4-hydroxylation was
expressed by the formation rate of NEHN. λex/λem = 450/558 nm.
larvae display
a clear and strong fluorescence region, which
precisely corresponding to liver area of zebrafish (Figure 5A, a-c).
Meanwhile, the inhibitory effect of TKZ demonstrated that NEN
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10.1002/anie.201903683
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COMMUNICATION
S17). Similarly, the phenomenon was observed in the TKZ- and
nilotinib-treated group. Therefore, NEN displayed well tracking ability
for CYP3A4 and faithfully reported the drug-induced inhibition of
CYP3A4 in living zebrafish. NEN facilitates the rapid screening of
CYP3A4 modulators and characterization of CYP3A4-mediated
clinical drug interactions to assess risk of DDI caused by CYP3A4
inhibition.
In summary, using a two-dimensional molecular design strategy,
we have successfully developed the first two-photon fluorescent
probe for the selective detection of CYP3A4. NEN has been
successfully applied for real-time imaging of CYP3A4 in various
living systems, thereby providing a light-up readout to quantitatively
characterize endogenous CYP3A4 activity in vitro and in vivo. Our
practical strategy provides a novel approach for developing enzyme-
activatable and isoform-specific fluorescent probes of target
enzymes.
Figure 4. Two-photon confocal fluorescence images of CYP3A4 in
human primary hepatocytes. Cells incubated with NEN (a-c); pretreated
with rifampin (d-f); TKZ (g-i). (a, d, g) fluorescence images; (b, e, h) bright
field images; (c, f, i) overlay images. Excitation: 800 nm, emission
window: 520-560 nm.
measure the specific activity of CYP3A4 expressed in liver of
zebrafish (Figure 5A, d-f). To examine whether NEN could faithfully
report the drug-induced inhibition of CYP3A4 in living zebrafish,
eight commonly used drugs (among which TKZ, indinavir and
nilotinib are widely recognized CYP3A4 inhibitors, whereas others
are not) are selected to co-incubate with NEN in zebrafish culture
media.[22] As shown in Figure 5B, the fluorescence intensity was
evidently suppressed by three CYP3A4 inhibitors. Meanwhile, there
was nearly no effect of known non-modulators of CYP3A4.[23] When
pretreated with 1 and 10 µM indinavir, the fluorescence signal
decreased to 37.1% and 16.8% of the control, implying the dose-
dependent inhibitory effect of indinavir towards CYP3A4 (Figure
Acknowledgements
The authors thank the National Natural Science Foundation of
China (81622047, 81503201, 81874301, 81473334 and
21572029), the Fundamental Research Funds for Central
University (DLUT15QY43), the National Key R&D program of
China (2018YFC1705900), the Distinguished Professor of
Liaoning Province program, the Liaoning Revitalization Talents
Program and State Key Laboratory of Fine Chemicals (KF1803)
for financial support. T.D.J. wishes to thank the Royal Society for
a Wolfson Research Merit Award.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Analytical methods • Cytochrome P450 3A4 •
Enzyme-activatable probe • Fluorescent probe • Molecular
design strategy
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This article is protected by copyright. All rights reserved.

10.1002/anie.201903683
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Selective two-photon CYP3A4
probe: A highly sensitive two-photon
fluorescent probe specific for CYP3A4
was designed based on a two-
dimension design strategy that
combined ensemble-based virtual
screening with rationally structure
modification. Facilitating the detection
of CYP3A4 activity in living cell, tissue
and zebrafish, allowing for the
evaluation of risks from CYP3A4-
mediated drug-drug interactions in the
clinic.
J. Ning, W. Wang, G. B. Ge, P. Chu, F.
D. Long, Y. L. Yang,* Y. L. Peng, L.
Feng,* X. C. Ma* and T. D. James
Page No. – Page No.
Targeted Enzyme Activated Two-Photon
Fluorescent Probes: A Case Study of
CYP3A4 Using
Design Strategy
a
Two-Dimensional
This article is protected by copyright. All rights reserved.