Rational Design of a Two-Photon Fluorogenic Probe for Visualizing Monoamine Oxidase A Activity in Human Glioma Tissues

Article abstract of DOI:10.1002/anie.202000059

Monoamine oxidases have two functionally distinct but structurally similar isoforms (MAO-A and MAO-B). The ability to differentiate them by using fluorescence detection/imaging technology is of significant biological relevance, but highly challenging with available chemical tools. Herein, we report the first MAO-A-specific two-photon fluorogenic probe (F1), capable of selective imaging of endogenous MAO-A enzymatic activities from a variety of biological samples, including MAO-A-expressing neuronal SY-SY5Y cells, the brain of tumor-bearing mice and human Glioma tissues by using two-photon fluorescence microscopy (TPFM) with minimal cytotoxicity.

Full text of DOI:10.1002/anie.202000059

A Journal of the Gesellschaft Deutscher Chemiker
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International Edition Chemie
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Accepted Article
Title: Rational Design of Two-photon Fluorogenic Probe for Visualizing
Monoamine Oxidase A Activity in Human Glioma Tissues
Authors: Haixiao Fang, Hang Zhang, Lin Li, Yun Ni, Riri Shi, Zheng
Li, Xuekang Yang, Bo Ma, Chengwu Zhang, Qiong Wu,
Changmin Yu, Naidi Yang, Shao Q. Yao, and Wei Huang
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202000059
Angew. Chem. 10.1002/ange.202000059
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Angewandte Chemie International Edition
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COMMUNICATION
Rational Design of Two-photon Fluorogenic Probe for Visualizing
Monoamine Oxidase A Activity in Human Glioma Tissues
Haixiao Fang, Hang Zhang, Lin Li*, Yun Ni, Riri Shi, Zheng Li, Xuekang Yang, Bo Ma, Chengwu Zhang,
Qiong Wu, Changmin Yu, Naidi Yang, Shao Q. Yao* and Wei Huang*
Abstract: Monoamine oxidases have two functionally distinct but
structurally similar isoforms (MAO-A and -B). The ability to
differentiate them by using fluorescence detection/imaging
technology is of significant biological relevance, but highly
challenging with available chemical tools. Herein, we report the
first MAO-A-specific two-photon fluorogenic probe (F1), capable
of selective imaging of endogenous MAO-A enzymatic activities
from a variety of biological samples, including MAO-A expressing
neuronal SY-SY5Y cells, the brain of tumor-bearing mice and
human Glioma tissues by using two-photon fluorescence
microscopy (TPFM) with minimal cytotoxicity.
molecule MAO inhibitors, clorgyline (CL) confers good selectivity
against MAO-A, while known MAO-B selective inhibitors include
[
6]
pargyline (PA), selegiline, and rasagiline.
Some of these
compounds are already being used in the treatment of various
[6c]
neurodegerative diseases.
On the other hand, in order to
selectively detect individual MAO enzymatic activities from cells
and tissues, researchers have turned to the development of small-
[
6c,7]
molecule fluorogenic probes (SMFPs);
of special note, two-
photon fluorogenic probes (TPFPs) when combined with advanced
two-photon fluorescence microscopy (TPFM) are able to penetrate
tissues deeper and provoke less optical damage at a higher
[8]
resolution than one-photon fluorogenic probes (OPFPs). Up to
date, MAO TPFPs have been mainly designed to detect either
[
9]
MAO-B or both isoforms of MAOs. For MAO-A-specific
detection, however, only OPFPs have been reported thus far,
including three reaction- and one binding-based probes (Figure
M
onoamine oxidases (MAOs) are important mammalian enzymes
that catalyze the transformation of various neurotransmitters
[
10]
including serotonin, dopamine, phenethylamine, and others.[1] They
1A). Ma and co-workers first reported a novel MAO-A specific
AC
OPFP ( probe) for visualizing MAO-A activity in live cells based
are normally expressed on the outer membrane of mitochondria in
cells. In human, MAOs have been found to play key roles in
maintaining the homeostasis of neurotransmission. At present,
there are two known human MAOs (i.e. MAO-A and MAO-B),
which, despite high sequence homology (~70%), differ
[
10a]
on a substrate-oxidized enzymatic reaction.
subsequently disclosed a MAO-A-specific OPFP (
covalently linking resorufin to the MAO-A-specific inhibitor
The same group
Angew
probe) by
[
10b]
CL.
Furthermore, Li and colleagues constructed an “enzyme-
recognition moiety” within a NIR-fluorophore to obtain a MAO-A-
significantly in their biological roles, cell distributions and
CC
[10c]
substrate specificity.[2] Abnormal MAO-A and -B activities are
specific OPFP ( probe) suitable for in vivo imaging.
Zhu and
co-workers used another strategy by designing an environmentally
sensitive MAO-A specific OPFP, the first fluorogenic, non-
closely related to several mental illnesses and neurodegenerative
[
3]
diseases. For example, studies have found that high expression of
MAO-A is closely associated with schizophrenia and depression,
[
10d]
covalent “inhibitor-like” probe for MAO-A detection.
Notwithstanding, specific TPFPs capable of imaging MAO-A
activities directly from live mammalian cells and deep tissues with
minimal MAO-B cross-reactivity have yet to be elucidated.
We previously reported the first MAO-B specific TPFP (i.e. U1
in Figure 1B), which was successfully used to image MAO-B-
while MAO-B is abundantly expressed in the brain of patients with
Parkinson's disease (PD) and Alzheimer's disease (AD).[4,5] MAO-
A has a strong affinity for serotonin and norepinephrine, and MAO-
B mainly metabolizes benzylamine and phenethylamine.
Due to the therapeutic potential of human MAOs, chemical tools
[
9a]
specific activities from live PD models. U1 contains a MAO-
reactive propylamine warhead (WH) and two-photon
fluorescence reporter, 2-methylamino-6-acetylnaphthanlene
acedan, or “fluorophore”). Upon further inspection of molecular
capable of selectively interogating these enzymes have been
actively pursued for years.[6] Amongst the best-known small-
a
(
docking results of U1 bound to MAO-A and -B, respectively, we
found the probe interacted with each enzyme’s active site
differently (Figure 1B, right); while U1 approached MAO-B and
projected its amine group close to the enzyme’s active-site-bound
[*]
H. X. Fang, H. Zhang, Y. Ni, R. R. Shi, Z. Li, Dr. C. W. Zhang, Dr. Q.
Wu, Dr. C. M. Yu, Dr. N. D. Yang, Prof. Dr. L. Li, Prof. Dr. W. Huang
Key Laboratory of Flexible Electronics (KLOFE) & Institute of
Advanced Materials (IAM), Nanjing Tech University (NanjingTech)
[
6]
FAD (a cofactor for both MAO-A and -B ) as previously
30 South Puzhu Road, Nanjing, 211816, P. R. China.
[
9a]
reported,
the acetyl group from its acedan moiety in the
E-mail: iamlli@njtech.edu.cn
Dr. X. Yang
U1/MAO-A complex was shown to point towards FAD instead.
Department of Burns and Cutaneous Surgery, Xijing Hospital, The
Fourth Military Medical University, Xi’an, 710032, P.R. China
Dr. B. Ma
School of Pharmaceutical Sciences, Nanjing Tech University,
Nanjing 210023, P. R. China
Prof. Dr. S. Q. Yao
Department of Chemistry, National University of Singapore, 3
Science Drive 3, 117543, Singapore
E-mail: chmyaosq@nus.edu,sg
Prof. Dr. W. Huang
Shaanxi Institute of Flexible Electronics (SIFE) & Institute of
Biomedical Materials & Engineering (IBME), Northwestern
Polytechnical University (NPU), 127 West Youyi Road, Xi’an
Anticipating that the oxidation ability of FAD in MAO-A/-B
towards their respective substrates would be roughly equivalent,
we endeavored to redesign U1 with the aim of achieving a MAO-
A specific TPFP (Figure 1C/D); we first retained the structure of
the naphthalene in U1 by exchanging the position of “WH” and
[
11]
“fluorophore”, followed by combining the resulting structure with
N-alkylated tetrahydropyridine to obtain F1, as shown in Figure 1
(in blue halo). The use of tetrahydropyridine as the new WH design
in F1 was based on the well-established tetrahydropyridine/
+
pyridinium (e.g. MPTP/MPP ) conversion properties catalyzed by
[
12]
MAO-B.
Subsequent docking results of F1 with various
crystallographic structures of MAO-A/-B (Table S1) confirmed
what was earlier predicted (Figure 1B, right bottom, and Figure S1);
in sharp contrast to U1/MAO-A structure, the N-alkylated
tetrahydropyridine in the docked F1/MAO-A complex was shown
7
10072, P. R. China
E-mail: iamwhuang@nwpu.edu.cn
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10.
1002/anie.xxxxxxxxx
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Angewandte Chemie International Edition
10.1002/anie.202000059
COMMUNICATION
Figure 1. (A) Molecular structures of reported reaction-based MAO-A-specific OPFPs.[10] (B) The design principle of F1 and docking results of F1 and
U1 to the MAO-A and -B, respectively. The binding mode of F1/U1 to MAO-A and -B at flavin-containing “substrate” cavity was viewed as a colorful solid.
Both ligands and the redox-active isoalloxazine ring of flavin were shown in stick form for clarity. See Figure S1 in the Supporting Information for details.
(
3 4
C) The synthetic scheme of FD1 and F1; i) CH I, MeCN, room temperature, 24 h; ii) NaBH , MeOH, 0 °C, 10 min. For details, see Supporting Information.
OX
(
D) Overall strategy for visualizing MAO-A-specific activity with F1 in live mammalian cells and tissues by using TPFM; F1 is the oxidized form of F1,
MAOA
MAO
re
KO SH-SY5Y denotes the MAO-A knockout SH-SY5Y cells,
denotes the reduced form of MAO-A.
transfected HepG2 denotes MAO-A-overexpressed HepG2 cell lines, MAO-A
to have projected closer toward FAD, with a measured distance of
Information). The expected MAO-A-dependent two-photon
fluorescence Turn-ON strategy of F1 is shown in Figure 1D; the
initially non-fluorescent F1 would first undergo MAO-A-catalyzed
3
1
.5 Å between the pyridine nitrogen in F1 and C5 in FAD (Figure
B, right bottom and Figure S1). In the docked F1/MAO-B
OX
structure on the other hand, the N,N-dimethyl group has oriented
towards FAD, making F1 unsuitable as a MAO-B substrate. On the
basis of above design principle, the MAO-A-specific TPFP F1, as
well as FD1 (F1-oxidized product, in red halo), was successfully
synthesized and fully characterized (Figure 1C and Supporting
oxidization to F1, followed by rapid conversion to FD1 (via
[
12a]
chemical/enzymatic oxidation in the system ). The introduction
of pyridinium (a strong electron-withdrawing moiety with good
conjugation) in FD1 would result in the formation of a push-pull
(
A)1
(B) 8
(C)1
(D)
1
F1
F1+PA
F1+CL
MAO-A
MAO-B
FD1
F1
10μg/mL
6
1
MAO-A
MAO-B
MAO-A
4
2
0
0
Amplex red
0
0
0
0
5
00 550 600 650 700 750
Wavelength/nM
MAO-A MAO-B
0
20 40 60 80 100 120
Time/min
500 550 600 650 700 750
Wavelength/nM
(
E)
(F)1
(G)
V
max
δФ/ GM
k
app×10-4
M ∙min )
K
(μM)
m
k
cat
-1
-
Comp.
FD1
λ [*]
Φ[**]
[***]
(nmol∙mg
min )
-1
-1
(
(min )
1
-1
∙
10
R2=0.994
0
430/618
0.097
35.6
--
--
--
--
F1
430/555
0.011
1.2
35.11
11.96
0.042
0.42
0
0
1/F1
0.5
0
0
3
h
50
F1/μM
100
Figure 2. (A) One-photon excited fluorescence spectra with 1.0 μM of F1 and FD1 in PBS buffer. (B) Fluorescence changes of F1 (1.0 µM) in MAO-A or
-
1
-
B (10 µg mL ; in PBS buffer) at 37 °C upon 2-h incubation, with or w/o an inhibitor (1 µM) where indicated. (Inset): RFU of enzyme/Amplex Red Kit upon
-h incubation with the enzyme. Full spectra are shown in Figure S3. (C) Time-dependent (0-120 min) fluorescence intensity profiles of F1 (1.0 μM) upon
1
-
1
incubation with MAO-A or -B (10 µg mL ), respectively. (D) Fluorescence spectra of F1 (1.0 µM) upon 2-h incubation with various concentrations of
-
1
o
MAO-A in PBS buffer at 37 °C. (E) HPLC profiles of MAO-A (10 µg mL )-catalyzed oxidation of F1 (1.0 μM) to FD1. Reactions were carried out at 37 C
in PBS buffer and monitored by analytical HPLC-MS (equipped with a fluorescence detector) from 0 to 3 h (ex/em = 310/450 and 430/620 nm for F1 and
FD1, respectively). Peaks were unambiguously assigned based on their molecular weights. (F) Kinetic studies of (F1 + MAO-A) enzymatic reaction over
-
1
a F1-concentration range (0-100 µM) with MAO-A (10 µg mL ) in PBS buffer at 37 °C. (Inset): fitted Linewearver-Burk plot. (G) The corresponding
photophysical properties and kinetic data of F1 and FD1; [*] peak position of the emission band; [**] one-photon fluorescence quantum yield (Φ) upon
the absorption maximum of FD1, using coumarin-6 (Φ = 0.85) as a reference for measurement; [***] the maxima two-photon action cross section values
upon excitation wavelength of fluorophore from 750 to 840 nm in GM (1 GM = 10- cm s photon ); --: not determined. λex/em = 430/620 nm; PBS buffer
50
4
-1
(
pH 7.4) supplemented with 0.4% Triton X-100.
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COMMUNICATION
(A)
(B)
1
*
**
(C)
*
**
Lysate only
Lysate+PA
Lysate+CL
Clorgyline (CL)
SH-SY5Y
SH-SY5Y+CL
HepG2
kDa
MAO-A
β-actin
MAO-B
β-actin
6
0
Pargline (PA)
42
59
42
0
SH-SY5Y
HepG-2
*
**
(
D) 1
(E)
Ctrl gRNA
Ctrl gRNA
gRNA1
gRNA2
gRNA3
MAO-A gRNA1
MAO-A gRNA2
MAO-A gRNA3
kDa
60
MAO-A
β-actin
42
0
SH-SY5Y
(
H)MAO-A
kDa
60
(
F)1
(G)
*
**
MAO-A Puro
MAO-A POE
β-actin
MAO-A Puro
MAO-A POE
MAO-A POE+CL
42
MAO-A POE+CL
kDa
60
1
MAO-A
Western Blot
β-actin
42
59
42
Enzymatic assay
MAO-B
β-actin
0
0
HepG-2
Figure 3. Fluorescence detection and bioimaging of MAO-A activity by F1 in live mammalian cells. (A) Structure of irreversible MAO-A-selective inhibitor
-
1
clorgyline (CL) and MAO-B-selective inhibitor pargyline (PA). (B) Fluorescence changes of F1 (1.0 μM) in SH-SY5Y lysates (500 µg mL ) in assay buffer
with or w/o an inhibitor (10 μM CL/PA), where indicated; HepG2 cell lysates were used as negative controls. (C) One-photon confocal fluorescence
images of live SH-SY5Y (with or w/o 1-h pretreatment of 50 μM CL) incubated with F1 (10 μM) for 2 h. F1-treated HepG2 cells were used as negative
-
1
controls. (D) RFU of F1 (1.0 μM) + lysates (500 µg mL ) in assay buffer from MAO-A-knocked down SH-SY5Y cells. (Inset): corresponding WB results
of the lysates. Cells treated with different gRNA (Ctrl & gRNA/1/2/3) were used. (E) One-photon confocal fluorescence images of live MAO-A-knocked
-
1
down SH-SY5Y cells upon incubation with F1 (10 μM) for 2 h. (F) RFU of F1 (1.0 μM) + lysates (500 µg mL ) in assay buffer with MAO-A-expressing
HepG2 cells, with inhibitor (10 μM CL) where indicated. (Inset): corresponding WB results of the lysates. Cells treated with Puro and POE (negative
control) were used. (G) One-photon confocal fluorescence images of live MAO-A-expressing HepG2 cells (with 1-h pretreatment of 50 μM CL where
indicated) upon incubation with F1 (10 μM) for 2 h. (H) RFU calculated from the fluorescence assay of proteome lysates from eight different mammalian
cell lines upon incubation with F1 (in red). For comparison, the relative WB signals of endogenous MAO-A expression from the same cells were quantified
and plotted (in black). The WB results are shown (on top). Error bars represent standard error of the mean (s.e.m). (n = 3), the linear regression fitting
data is shown in Figure S12. ***P < 0.001, n = 3, Student’s t-test, two-tailed in (B/D/F). Differential interference contrast (DIC) images of related confocal
fluorescence images for (C/E/G) are shown in the bottom-left of the corresponding image panels; scale bar = 25 μm.
system around the naphthalene fluorophore, thus ensuring F1-to-
FD1 conversion to be highly fluorogenic (that is, Turned-ON) and
detectable by TPFM.[8a] The subsequent evaluation of F1 against a
variety of biological samples, including recombinant MAO
proteins, mammalian cell lysates, live mammalian cells, and mouse
brain/tumor tissues, with both in vitro fluorescence-based
enzymatic assay and one/two-photon fluorescence imaging,
indicated that F1 was indeed highly selective towards MAO-A even
at high MAO-B expression levels, as shown in Figures 2/3/4.
First, we evaluated the photophysical properties of F1 and FD1
under physiological conditions (PBS buffer supplemented with 0.4%
Triton X-100, pH 7.4). As shown in Figure S2, F1 and FD1 had
absorption/emission maxima at 305/450 nm and 430/620 nm,
respectively. Upon excitation at 430 nm (maximum absorption
wavelength of the fluorophore), FD1 exhibited a strong red
fluorescence which peaked at ~620 nm, with a one-photon
fluorescence quantum yield of 0.097 and a two-photon action cross
section of 35.6 (Figure 2A/G). Meanwhile, the fluorescence signal
of F1 was very weak at 555 nm, with a lower one-photon
fluorescence quantum yield of 0.011 and a two-photon action cross
section of ~1.2. Such properties thus made the F1-to-FD1
conversion system and therefore F1 a suitable fluorogenic probe for
two-photon excited (~800 nm) fluorescence imaging in deep
assay was carried out concurrently with F1 (1.0 µM ; in PBS buffer,
-1
pH 7.4) and the corresponding enzyme (10 µg mL ) at 37 °C, and
fluorescence measurement was recorded after h. The
2
commercially available Amplex Red MAO Assay kit was used as
a reference to normalize the relative MAO-A/B activity under
[
9a,10]
similar conditions (Figure 2B, inset);
both enzymes produced
significant increases in fluorescence, with MAO-A showing a
slightly higher activity than that of MAO-B. In sharp contrast,
significant increases in F1 fluorescence were detected ONLY in
MAO-A (i.e. F1 + MAO-A) but not MAO-B (i.e. F1 + MAO-B)
enzymatic reactions under identical conditions, and they were
nearly completely abolished in the presence of a MAO-A specific
inhibitor, CL (i.e. F1 + CL + MAO-A). Addition of a MAO-B
specific inhibitor, PA, to the same reaction (i.e. F1 + MAO-A + PA)
only caused a slight suppression in F1 fluorescence increase.
Concurrently, time-dependent fluorescence measurements were
carried out for both enzymatic reactions (i.e. F1 + MAO-A and F1
+ MAO-B; Figure 2C); a concomitant fluorescence increase was
observed in the former, but not in the latter, and the resulting
sigmoidal curve could be used to provide the corresponding
4
-1
-1
reaction constant of kapp = 35.11 × 10 M min for (F1 + MAO-A)
reaction (summarized in Figure 2G). We next carried out
concentration-dependent reactions for both enzymatic reactions
[
8]
-1
tissues. Next, we assessed the enzymatic activity of F1 with both
recombinant human MAOs, and unequivocally confirmed that F1
was exclusively MAO-A-responsive (Figure 2B-G). The in vitro
(Figures 2D & S4); for assays carried out at 0-10 µg mL enzyme
concentrations and 2-h incubation time, only the (F1 + MAO-A)
reactions produced significant fluorescence increases (Figure 2D).
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COMMUNICATION
(
A)Mouse Brain (B)Tumor
(C)
1
(D)Human Glioma Tissue
F1
F1+CL
F1
F1+CL
F1
F1+CL
F1
F1+CL
0
Brain
Tumor
0
(
F)
(G)
(
E) Paracancerous Tissue
F1
F1
MAO-A
1
Z/μm
MAO-A
60kDa
42kDa
β-actin
2
20
0
Glioma Paracancerous
Figure 4. TPFM imaging of endogenous MAO-A activity with F1-treated (50 μM, 3 h) fresh mice brain (A) and SH-SY5Y tumor slices (B). CL-pretreatment (200
μM, 1 h) was done where indicated. Images were scanned once every 20 μm for a total depth of 220 μm; scale bar = 100 μm. (C) Average fluorescence intensity profiles
of reconstructed tissues image in (A/B). (D) TPFM images and the correspnding quantified plots (right) of F1-treated (50 μM, 3 h) fresh human glioma tissue (50 μm
depth). Results from the corresponding CL-pretreated (200 μM, 1 h) samples were shown. (E) TPFM image (left) and the corresponding quantified plot (right) of F1-
treated (50 μM, 3 h) fresh paracancerous tissue slices (50 μm depth). scale bar = 100 μm. (F) WB results for (D/E). (G) Comparison of average fluorescence intensity
profiles of images from (D/E; red bars) and relative endogenous MAO-A expression levels determined by the WB results from (F; black bars) of the tissues; error bars
represent standard error of the mean (s.e.m). (n = 3).
No obvious fluorescence increase was observed in the (F1 + MAO-
B) reaction (Figure S4). The reaction parameters were further
optimized under physiological conditions (pH 7.4 and 37 °C , see
Figure S5). Next, by using analytical HPLC-MS equiped with a
fluorescence detector, we unequivocally confirmed the successful
concentrations up to 50 µM (12-h incubation; Figure S7).
Subsequent live-cell imaging of F1-treated SH-SY5Y cells by
using fluorescence microscopy demonstrated
a progressive
increase in CL-sensitive fluorescence signals (Figures 3C & S8/9),
but not in live HepG2 cells. Meanwhile, F1-treated SH-SY5Y
lysates/live cells (pretreated with different concentrations of CL
from 0 to 50 µM) showed dose-dependent fluorescence decreases
(Figure S10/11). To further validate the MAO-A selectivity of F1,
endogenous MAO-A-knocked-down SH-SY5Y and stably MAO-
A-overexpressed HepG2 cells were created (Figure 3D and 3F,
respectively); as shown in the WB results of Figure 3D (inset), by
using the corresponding lentiviral Crispr/Cas9 system (gRNA1/2/3,
with Ctrl gRNA as a non-targeting gRNA vector control), we
obtained live SH-SY5Y cells in which endogenous MAO-A was
successfully knocked down.As shown in Figure 3E, in the absence
of endogenous MAO-A expression, the corresponding F1-treated
SH-SY5Y cells failed to generate detectable fluorescence signals.
In contrast, with MAO-A-expressing HepG2 cells treated with F1
(Figure 3F; POE, with Puro as an empty vector control; see
F1-to-FD1 conversion catalyzed by MAO-A, presumably through
OX
the formation of the highly unstable
F1 intermediate (not
observed in HPLC; Figure 2E). We also studied the selectivity of
the (F1 + MAO-A) reaction with a variety of potential interfering
species (Figure S6); F1 showed a high selectivity toward MAO-A
over all other species tested, which was attributed to the specific
oxidation reaction of this fluorogenic substrate catalyzed
exclusively by MAO-A. Finally, we performed a detailed kinetic
study on the (F1 + MAO-A) reaction and obtained the
corresponding Michaelis-Menten constants (K
0
m
= 11.96 μM, kcat
.042 min , Vmax = 0.42 nmol∙mg ∙min ; Figure 2F/G). Of note,
the K value of F1 was similar to those of previously reported
MAO-A-specific OPFPs.
=
-1
-1
-1
m
[
10]
As the (F1 + MAO-B) reaction
produced a negligible increase in fluorescence under all tested
conditions, we were unable to obtain its kinetic data. We thus
concluded that F1 was indeed a fluorogenic probe suitable for the
sensitive and continuous reporting of MAO-A activity with
minimal cross-activity towards MAO-B, and, with its excellent
two-photon fluorescence properties (that is, upon enzymatic
conversion to FD1), might serve as a good MAO-A-specific TPFPs
in various biological applications.
Encouraged by above results, we next determined whether F1
could be used to specifically detect MAO-A activities in complex
biological samples. Human-derived SH-SY5Y and HepG2 cells are
two disease cell lines known to express elevated levels of MAO-A
and -B activities, respectively.[13] We first confirmed the over-
expressed MAO-A, but not MAO-B, activity in SH-SY5Y cell
lysates by directly incubating them with F1 in the presence of CL
or PA (Figure 3A/B); results showed that the (F1 + SH-SY5Y)
reaction produced significant levels of CL-sensitive fluorescence
signals, which were attributed to the endogenous MAO-A activity
and subsequently confirmed by Western blotting (WB) analysis of
the cell lysates (inset of Figure 3B). On the other hand, (F1 +
HepG2) reaction failed to generate any above-background
fluorescence under similar conditions, and the WB results with the
corresponding lysates also confirmed the expected endogenous
expression of MAO-B but not MAO-A activity. Concurrently, real-
time fluorescence bioimaging was used to test the capability of F1
for visualizing MAO-A activity in live cells. We first established
that F1 was minimally cytotoxic to most mammalian cells at
[
14]
Supporting Information for details),
we observed MAO-A
enzymatic activity in both the proteome lysates and live cells
(Figure 3F/G); a significant level of CL-sensitive fluorescence
signals were detected. Finally, we incubated F1 with eight different
mammalian cell lines (SH-SY5Y, HEK293, U87, T98G, HeLa,
A549, H9C2 and HepG2). Fluorescence readings and endogenous
MAO-A expression levels (based on WB results; Figure 3H, top)
were measured; there was a good correlation between the two sets
of fluorescence values (Figures 3H, bottom & S12). Unlike the
general MAO assays that require a physical separation of the MAO-
A protein, F1 provides a simple, rapid and selective method for
reporting endogenous MAO-A activities directly from crude cell
proteomes and live mammalian cells alike.
Inspired by our success in cell-based experiments, we next
determined whether F1 could be used to image MAO-A activities
from deep tissues (e.g. muse/human brain and tumor tissues) by
using TPFM. Neurons and glial cells were previously reported to
[
15]
have elevated expression levels of MAO-A. We first confirmed
+
minimal cytoxicity of MPTP, MPP , F1, and FD1 in two well-
known neuronal cells, PC12 and SH-SY5Y (Figure S13/S14); no
toxicity was observed in both cell lines with up to 50 μM of probe
treatment. We next injected F1 and MPTP intraperitoneally into
two groups of mice, after which one group underwent behavioral
observation, while the other group underwent H&E staining of
brain tissues after 2 h (Figures S14/S15); our results indicate there
was no significant apoptosis of the substantia nigral cells in F1-
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Angewandte Chemie International Edition
10.1002/anie.202000059
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injected mice (Figure S15), and their motor performance was not
significantly affected either (Figure S16 & Supplementary
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Video).
These findings thus showed that F1 did not possess
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[
(
Figures 4A/B/C & S17), we detected similar CL-sensitive
2
fluorescence signals in both F1-treated fresh tissue slices isolated
from brains of normal mice and those transplanted with tumors.
Finally, we detected strong CL-sensitive fluorescence signals in
F1-treated fresh human glioma tissues which corroborated well
with the corresponding endogenous MAO-A expression level from
WB results (Figure 4D/F), whereas in F1-treated paracancerous
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fluorescence signals were detected, consistent with their relatively
low MAO-A expression in such tissues (Figures 4E/F/G & S19).
In summary, we have successfully developed a TPFP (F1) with
excellent specificity for detecting/visualizing MAO-A activities
from various biological samples by using one- and two-photon
fluorescence spectroscopy. The two-photon performance of F1
enabled it to sensitively and selectivevly image MAO-A activities
from fresh mouse/human brain and tumor tissues. Importantly, our
results demonstrated the feasibility of using SMFPs to explore the
chemistry and biology of MAO-A at the organism level, thus
providing a useful chemical tool for future studies of MAO-A-
related diseases like glioma.
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This work was financially supported by the National Natural
Science Foundation of China (81672508, 81773802, 81671910),
Jiangsu Provincial Foundation for Distinguished Young Scholars
(
(
BK20170041), Natural Science Foundation of Shaanxi Province
2019JM-016, 2019KJXX-089), China-Sweden Joint Mobility
Project (51811530018), Fundamental Research Funds for the
Central Universities, GSK-EDB Trust Fund (R-143-000-688-592),
and the Synthetic Biology Research & Development Programme
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Conflict of interest
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The authors declare no conflict of interest.
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Keywords: monoamine oxidase A · MPTP · two-photon
fluorogenic probes · bioimaging · glioma
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Angewandte Chemie International Edition
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COMMUNICATION
Table of Contents
COMMUNICATION
Text for Table of Contents
H. Fang, H. Zhang, L. Li,* Y. Ni, R. Shi,
Z. Li, X. Yang, B. Ma, C. Zhang, Q. Wu,
C. Yu, N. Yang, S. Q. Yao* and W.
Huang*
SH-SY5Y
MAO-Atransfected HepG2
Page No. – Page No.
MAO-A
Rational Design of Two-photon
Fluorogenic Probe for Visualizing
Monoamine Oxidase A Activity in
Human Glioma Tissues
Two-photon
Human glioma Tissue
F1
FD1
Picking MAO-A: By using two-photon fluorescence microscopy, a novel two-
photon fluorogenic probe (F1) is shown to successfully detect endogenous
MAO-A activities from a variety of biological samples, including live mammalian
cells, fresh mouse/human brain and tumor tissues, with minimal cytotoxicity and
cross-reactivity toward MAO-B.
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